Lack of absolute selectivity against cancer cells is a major limitation for current cancer therapies. In the previous study, we developed a prodrug strategy for selective cancer therapy using a masked cytotoxic agent puromycin [Boc-Lys(Ac)-Puromycin], which can be sequentially activated by histone deacetylases (HDACs) and cathepsin L (CTSL) to kill cancer cells expressing high levels of both enzymes. Despite the promise as a selective cancer therapy, its requirement of relatively high dosage could be a potential issue in the clinical setting. To address this issue, we aimed to further improve the overall efficacy of our prodrug strategy. Since the proteolytic cleavage by CTSL is the rate-limiting step for the drug activation, we sought to improve the substrate structure for CTSL activity by modifying the α-amino protecting group of lysine. Here we show that protection with Fmoc [Fmoc-Lys(Ac)-Puromycin] exhibits a marked improvement in overall anticancer efficacy compared to the original Boc-Lys(Ac)-Puromycin and this is mainly due to the highly efficient cellular uptake besides its improved substrate structure. Furthermore, to address a concern that the improved drug efficacy might direct high toxicity to the normal cells, we confirmed that Fmoc-Lys(Ac)-Puromycin still retains excellent cancer selectivity in vitro and no obvious systemic off-target toxicity in vivo. Thus our preclinical evaluation data presented here demonstrate that the Fmoc-Lys(Ac)-Puromycin exhibits substantially improved anticancer efficacy, further supporting our approach for the selective cancer therapy.

Rationale: Atherosclerosis is characterized by lipid accumulation, plaque formation, and artery stenosis. The pharmacological treatment is a promising therapy for atherosclerosis, but this approach faces major challenges such as targeted drug delivery, controlled release, and non-specific clearance. Methods: Based on the finding that the cathepsin k (CTSK) enzyme is enriched in atherosclerotic lesions, we constructed an integrin αvβ3 targeted and CTSK-responsive nanoparticle to control the release of rapamycin (RAP) locally. The targeted and responsive nanoparticles (T/R NPs) were engineered by the self-assembly of a targeting polymer PLGA-PEG-c(RGDfC) and a CTSK-sensitive polymer PLGA-Pep-PEG. PLGA-Pep-PEG was also modified with a pair of FRET probe to monitor the hydrolysis events. Results: Our results indicated that RAP@T/R NPs accelerated the release of RAP in response to CTSK stimulation in vitro, which significantly inhibited the phagocytosis of OxLDL and the release of cytokines by inflammatory macrophages. Additionally, T/R NPs had prolonged blood retention time and increased accumulation in the early and late stage of atherosclerosis lesions. RAP@T/R NPs significantly blocked the development of atherosclerosis and suppressed the systemic and local inflammation in ApoE-/- mice. Conclusions: RAP@T/R NPs hold a great promise as a drug delivery system for safer and more efficient therapy of atherosclerosis.

Despite the common use of lipid-lowering medications, cardiovascular diseases continue to be a significant health concern. Atherosclerosis, one of the most frequent causes of cardiovascular morbidity, involves extensive inflammatory activity and remodeling of the vascular endothelium. This relentless inflammatory condition can ultimately give rise to clinical manifestations, such as ischemic heart disease or stroke. Accumulating evidence over the past decades implicates cysteine protease cathepsins in cardiovascular disorders. In particular, Cathepsins B, L, and S are over-expressed during vascular inflammation, and their activity is associated with impaired clinical outcomes. Here we took advantage of these molecular events to introduce a non-invasive detection and treatment approach to modulate vascular inflammation using a Photosensitizing quenched Activity-Based Probed (PS-qABP) that targets these proteases. Methods: We tested the application of this approach in LDL receptor-deficient mice and used non-invasive imaging and heart cross-section staining to assess the theranostic efficacy of this probe. Moreover, we used fresh human endarterectomy tissues to analyze cathepsin signals on gel, and verified cathepsin identity by mass spectrometry. Results: We showed that our PS-qABP can rapidly accumulate in areas of inflammatory atheromas in vivo, and application of light therapy profoundly reduced lesional immune cell content without affecting smooth muscle cell and collagen contents. Lastly, using human tissue samples we provided proof-of-concept for future clinical applications of this technology. Conclusions: Photodynamic therapy guided by cysteine cathepsin activity is an effective approach to reduce vascular inflammation and attenuate atherosclerosis progression. This approach could potentially be applied in clinical settings.

Cysteine cathepsins often contribute to cancer progression due to their overexpression in the tumour microenvironment and therefore present attractive targets for non-invasive diagnostic imaging. However, the development of highly selective and versatile small molecule probes for cathepsins has been challenging. Here, we targeted tumour-associated cathepsin B using designed ankyrin repeat proteins (DARPins). The selective DARPin 8h6 inhibited cathepsin B with picomolar affinity (Ki = 35 pM) by binding to a site with low structural conservation in cathepsins, as revealed by the X-ray structure of the complex. DARPin 8h6 blocked cathepsin B activity in tumours ex vivo and was successfully applied in in vivo optical imaging in two mouse breast cancer models, in which cathepsin B was bound to the cell membrane or secreted to the extracellular milieu by tumour and stromal cells. Our approach validates cathepsin B as a promising diagnostic and theranostic target in cancer and other inflammation-associated diseases.

Cysteine-type cathepsins such as cathepsin B are involved in various steps of inflammatory processes such as antigen processing and angiogenesis. Here, we uncovered the role of cysteine-type cathepsins in the effector phase of T cell-driven cutaneous delayed-type hypersensitivity reactions (DTHR) and the implication of this role on therapeutic cathepsin B-specific inhibition. Methods: Wild-type, cathepsin B-deficient (Ctsb-/-) and cathepsin Z-deficient (Ctsz-/-) mice were sensitized with 2,4,6-trinitrochlorobenzene (TNCB) on the abdomen and challenged with TNCB on the right ear to induce acute and chronic cutaneous DTHR. The severity of cutaneous DTHR was assessed by evaluating ear swelling responses and histopathology. We performed fluorescence microscopy on tissue from inflamed ears and lymph nodes of wild-type mice, as well as on biopsies from psoriasis patients, focusing on cathepsin B expression by T cells, B cells, macrophages, dendritic cells and NK cells. Cathepsin activity was determined noninvasively by optical imaging employing protease-activated substrate-like probes. Cathepsin expression and activity were validated ex vivo by covalent active site labeling of proteases and Western blotting. Results: Noninvasive in vivo optical imaging revealed strong cysteine-type cathepsin activity in inflamed ears and draining lymph nodes in acute and chronic cutaneous DTHR. In inflamed ears and draining lymph nodes, cathepsin B was expressed by neutrophils, dendritic cells, macrophages, B, T and natural killer (NK) cells. Similar expression patterns were found in psoriatic plaques of patients. The biochemical methods confirmed active cathepsin B in tissues of mice with cutaneous DTHR. Topically applied cathepsin B inhibitors significantly reduced ear swelling in acute but not chronic DTHR. Compared with wild-type mice, Ctsb-/- mice exhibited an enhanced ear swelling response during acute DTHR despite a lack of cathepsin B expression. Cathepsin Z, a protease closely related to cathepsin B, revealed compensatory expression in inflamed ears of Ctsb-/- mice, while cathepsin B expression was reciprocally elevated in Ctsz-/- mice. Conclusion: Cathepsin B is actively involved in the effector phase of acute cutaneous DTHR. Thus, topically applied cathepsin B inhibitors might effectively limit DTHR such as contact dermatitis or psoriasis. However, the cathepsin B and Z knockout mouse experiments suggested a complementary role for these two cysteine-type proteases.

Two important features are required for promising radiosensitizers: one is selective tumor cell targeting to enhance the therapeutic outcome via lethal DNA damage and the other is rapid clearance to enable excellent biocompatibility for potential clinical application. Herein, ultrasmall gold nanoparticles (Au NPs) with diameter smaller than 5 nm were prepared and covered with a multifunctional peptide to endow them with selective tumor cell uptake capability. Combined with X-ray irradiation, the responsive Au NPs demonstrated superior radio-sensitizing toxicity and rapid renal clearance in vivo. Methods: A responsive peptide (Tat-R-EK) consists of three build blocks were used: a cell and even nuclear penetrating block derived from human immunodeficiency virus-1 transactivator of transcription protein (Tat), an cathepsin B cleavable linker, and a zwitterionic antifouling block. Ultrasmall Au NPs were prepared and then covered by the peptide via the Au-S bonds between gold and thiol groups from cysteine. The morphology, colloidal stability and the responsiveness of obtained Au@Tat-R-EK NPs were studied using transmittance electron microscopy and dynamic laser scattering. The selective cancer cell uptake and accumulation of Au@Tat-R-EK NPs in cancer tissue were studied via ICP-MS in vitro and in vivo, respectively. The cytotoxicity of Au@Tat-R-EK NPs on HepG2 cancer cells was evaluated in terms of cell viability, DNA damage, intracellular reactive oxygen species generation, and apoptosis analysis. Finally, the biocompatibility and tumor destruction ability against orthotopic LM3 liver cancers were verified in vivo. Results: Multifunctional peptide modified ultrasmall Au NPs were successfully prepared. The Au NPs exhibited enough colloidal stability and cathepsin B-responsive surface change, leading to selectively uptake by cancer cells in vitro and accumulation to tumor sites in vivo. Combined with X-ray irradiation, the responsive Au NPs demonstrated superior radio-sensitizing cytotoxicity in vitro and therapeutic outcome on mouse liver cancer in vivo. The ultrasmall size enables rapid clearance of the Au NPs, guarantees the biocompatibility in vivo for potential clinical applications. Conclusion: Some obstacles faced by the Au NPs-based radiotherapy, such as short circulation half-life, non-specific distribution, slow clearance and low radio-sensitizing effect, were effective solved through rational design of the smart nanomedicine. This work provides new insight in designing tumor microenvironment-responsive nanomedicine in cancer radiotherapy.

Antibody-drug conjugates (ADCs) are being developed worldwide with the potential to revolutionize current cancer treatment strategies. Developing novel theranostic ADCs with therapeutic utility and imaging capability is an attractive and challenging subject that promises advances in the field of personalized medicine. In this work, we propose a bifunctional molecule-based strategy for the development of theranostic ADCs. Methods: We developed a theranostic ADC consisting of the anti-Her2 antibody Mil40, monomethyl auristatin E (MMAE) as the active payload, and a 7-amino-3-hydroxyethyl-coumarin (7-AHC)-based dipeptide linker, which functions as a novel bifunctional fluorescence probe that allows self-elimination cleavage in the presence of cathepsin B for payload release and fluorophore activation. The on-off fluorescence properties and the antitumor effect in vitro and in vivo were investigated. Results: A 48-fold fluorescence enhancement was observed within 1 h when the 7-AHC-based linker was exposed to cathepsin B. Cleavage upon exposure to cathepsin B allows MMAE and fluorophore intracellular release and the monitoring of MMAE distribution using confocal microscopy. Additionally, the newly developed ADC retains the advantages of traditional p-aminobenzyloxycarbonyl-containing ADCs, such as good stability (t1/2 > 7 days) and high activity in vitro (IC50 = 0.09-3.74 nM). Importantly, the theranostic ADC exhibited the equivalent antitumor efficacy to the marketed ADC T-DM1 in the classic breast cancer model. Conclusion: We suggest that the present strategy can be universally applied in all p-aminobenzyloxycarbonyl-containing ADCs. Overall, theranostic ADCs may play a role in developing new theranostic systems and promoting personalized medicine research.

Rationale: Nicotine has been reported to be a strong risk factor for atherosclerosis. However, the underlying mechanism by which nicotine controls atherosclerotic plaque stability remain largely unknown. Objective: The aim of this study was to evaluate the impact of lysosomal dysfunction mediated NLRP3 inflammasome activation in vascular smooth muscle cell (VSMC) on atherosclerotic plaque formation and stability in advanced atherosclerosis at the brachiocephalic arteries (BA). Methods and Results: Features of atherosclerotic plaque stability and the markers for NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome were monitored in the BA from nicotine or vehicle-treated apolipoprotein E deficient (Apoe-/-) mice fed with Western-type diet (WD). Nicotine treatment for 6 weeks accelerated atherosclerotic plaque formation and enhanced the hallmarks of plaque instability in BA of Apoe-/- mice. Moreover, nicotine elevated interleukin 1 beta (IL-1β) in serum and aorta and was preferred to activate NLRP3 inflammasome in aortic vascular smooth muscle cells (VSMC). Importantly, pharmacological inhibition of Caspase1, a key downstream target of NLRP3 inflammasome complex, and genetic inactivation of NLRP3 significantly restrained nicotine-elevated IL-1β in serum and aorta, as well as nicotine-stimulated atherosclerotic plaque formation and plaque destabilization in BA. We further confirmed the role of VSMC-derived NLRP3 inflammasome in nicotine-induced plaque instability by using VSMC specific TXNIP (upstream regulator of NLRP3 inflammasome) deletion mice. Mechanistic study further showed that nicotine induced lysosomal dysfunction resulted in cathepsin B cytoplasmic release. Inhibition or knockdown of cathepsin B blocked nicotine-dependent inflammasome activation. Conclusions: Nicotine promotes atherosclerotic plaque instability by lysosomal dysfunction-mediated NLRP3 inflammasome activation in vascular smooth muscle cells.

Rationale: The adverse health effects of nano-particulate pollutants have attracted much attention in recent years. Carbon nanomaterials are recognized as risk factors for prolonged inflammatory responses and diffuse alveolar injury. Previous research indicated a central role of alveolar macrophages in the pathogenesis of particle-related lung disease, but the underlying mechanism remains largely unknown. Methods: C57BL/6 mice were intratracheally instilled with carbon black nanoparticles (CBNPs). Cell necrosis and the infiltrated neutrophils in the lungs were detected by flow cytometry. Release of mitochondria was observed with Mito Tracker and mitochondrial DNA (mtDNA) was quantified by qPCR via Taqman probes. TLR9-p38 MAPK signaling pathway was detected by Western blotting. The production of lipid chemoattractant leukotriene B4 (LTB4) in the supernatant and bronchoalveolar lavage fluid (BALF) was quantitated using an enzyme immunoassay (EIA). Results: In the present study, we found that a single instillation of CBNPs induced neutrophil influx in C57BL/6 mice as early as 4 h post-exposure following the rapid appearance of cell damage indicators in BALF at 30 min. Macrophages exposed to CBNPs showed necrotic features and were characterized by lysosome rupture, cathepsin B release, reactive oxygen species generation, and reduced intracellular ATP level. Necrosis was partly inhibited by a specific lysosomal cathepsin B inhibitor CA074 Me. Further analyses suggested that the resulting leakage of mtDNA from the necrotic cells activated neutrophils and triggered severe inflammation in vivo. Pulmonary neutrophilic inflammation induced by mtDNA was reduced in TLR9-/- mice. Additionally, mtDNA induced LTB4 production from macrophages, which may contribute to neutrophil recruitment. Conclusion: We demonstrated here that CBNPs induce acute cell necrosis through lysosomal rupture and that mtDNA released from necrotic cells functions as a key event mediating pulmonary neutrophilic inflammation. This study described a novel aspect of the pathogenesis of particle-induced inflammatory response and provided a possible therapeutic target for the regulation of inflammation.

Rationale: Platelets are increasingly recognized as mediators of tumor growth and metastasis. Hypothesizing that activated platelets in the tumor microenvironment provide a targeting epitope for tumor-directed chemotherapy, we developed an antibody-drug conjugate (ADC), comprised of a single-chain antibody (scFv) against the platelet integrin GPIIb/IIIa (scFvGPIIb/IIIa) linked to the potent chemotherapeutic microtubule inhibitor, monomethyl auristatin E (MMAE). Methods: We developed an ADC comprised of three components: 1) A scFv which specifically binds to the high affinity, activated integrin GPIIb/IIIa on activated platelets. 2) A highly potent microtubule inhibitor, monomethyl auristatin E. 3) A drug activation/release mechanism using a linker cleavable by cathepsin B, which we demonstrate to be abundant in the tumor microenvironment. The scFvGPIIb/IIIa-MMAE was first conjugated with Cyanine7 for in vivo imaging. The therapeutic efficacy of the scFvGPIIb/IIIa-MMAE was then tested in a mouse metastasis model of triple negative breast cancer. Results: In vitro studies confirmed that this ADC specifically binds to activated GPIIb/IIIa, and cathepsin B-mediated drug release/activation resulted in tumor cytotoxicity. In vivo fluorescence imaging demonstrated that the newly generated ADC localized to primary tumors and metastases in a mouse xenograft model of triple negative breast cancer, a difficult to treat tumor for which a selective tumor-targeting therapy remains to be clinically established. Importantly, we demonstrated that the scFvGPIIb/IIIa-MMAE displays marked efficacy as an anti-cancer agent, reducing tumor growth and preventing metastatic disease, without any discernible toxic effects. Conclusion: Here, we demonstrate the utility of a novel ADC that targets a potent cytotoxic drug to activated platelets and specifically releases the cytotoxic agent within the confines of the tumor. This unique targeting mechanism, specific to the tumor microenvironment, holds promise as a novel therapeutic approach for the treatment of a broad range of primary tumors and metastatic disease, particularly for tumors that lack specific molecular epitopes for drug targeting.

Rationale: Maladaptive cardiac remodeling is a critical step in the progression of heart failure. Low-density lipoprotein receptor-related protein 6 (LRP6), a co-receptor of Wnt, has been implicated in cardiac protection. We aimed to study the role of cardiomyocyte-expressed LRP6 in cardiac remodeling under chronic pressure overload. Methods: Cardiac parameters were analyzed in inducible cardiac-specific LRP6 overexpressing and control mice subjected to transverse aortic constriction (TAC). Results: Cardiac LRP6 was increased at an early phase after TAC. Cardiomyocyte-specific LRP6 overexpression improved cardiac function and inhibited cardiac hypertrophy and fibrosis four weeks after TAC. The overexpression significantly inhibited β-catenin activation, likely contributing to the inhibitory effect on cardiac hypertrophy after TAC. LRP6 overexpression reduced the expression and secretion of Wnt5a and Wnt11 by cardiomyocytes, and knockdown of Wnt5a and Wnt11 greatly inhibited cardiac fibrosis and dysfunction under pressure overload in vitro and in vivo. Cardiomyocyte-expressed LRP6 interacted with cathepsin D (CTSD, a protease) and promoted the degradation of Wnt5a and Wnt11, inhibiting cardiac fibrosis and dysfunction induced by TAC. The protease inhibitor leupeptin attenuated the interaction between LRP6 and CTSD, enhanced the expression of Wnt5a and Wnt11, and deteriorated cardiac function and fibrosis in cardiomyocyte-specific LRP6-overexpressing mice under pressure overload. Mutants from human patients, P1427Q of LRP6 and G316R of CTSD significantly inhibited the interaction between LRP6 and CTSD and increased Wnt5a and Wnt11 expression. Conclusion: Cardiomyocyte-expressed LRP6 promoted the degradation of Wnt5a and Wnt11 by regulating CTSD and inhibited cardiac fibrosis under pressure overload. Our study demonstrated a novel role of LRP6 as an anti-fibrosis regulator.

Rationale: Cutaneous melanoma is the most aggressive and deadliest of all skin malignancies. Complete primary tumor removal augmented by advanced imaging tools and effective post-operative treatment is critical in the prevention of tumor recurrence and future metastases formation. Methods: To meet this challenge, we designed novel polymeric imaging and therapeutic systems, implemented in a two-step theranostic approach. Both are composed of the biocompatible and biodegradable poly(α,L-glutamic acid) (PGA) nanocarrier that facilitates extravasation-dependent tumor targeting delivery. The first system is a novel, fluorescent, Turn-ON diagnostic probe evaluated for the precise excision of the primary tumor during image-guided surgery (IGS). The fluorescence activation of the probe occurs via PGA degradation by tumor-overexpressed cathepsins that leads to the separation of closely-packed, quenched FRET pair. This results in the emission of a strong fluorescence signal enabling the delineation of the tumor boundaries. Second, therapeutic step is aimed to prevent metastases formation with minimal side effects and maximal efficacy. To that end, a targeted treatment containing a BRAF (Dabrafenib - mDBF)/MEK (Selumetinib - SLM) inhibitors combined on one polymeric platform (PGA-SLM-mDBF) was evaluated for its anti-metastatic, preventive activity in combination with immune checkpoint inhibitors (ICPi) αPD1 and αCTLA4. Results: IGS in melanoma-bearing mice led to a high tumor-to-background ratio and reduced tumor recurrence in comparison with mice that underwent surgery under white light (23% versus 33%, respectively). Adjuvant therapy with PGA-SLM-mDBF combined with ICPi, was well-tolerated and resulted in prolonged survival and prevention of peritoneal and brain metastases formation in BRAF-mutated melanoma-bearing mice. Conclusions: The results reveal the great clinical potential of our PGA-based nanosystems as a tool for holistic melanoma treatment management.

Autophagy allows cancer cells to respond changes in nutrient status by degrading and recycling non-essential intracellular contents. Inhibition of autophagy combined with nutrient deprivation is an effective strategy to treat cancer. Pain is a primary determinant of poor quality of life in advanced cancer patients, but there is currently no satisfactory treatment. In addition, effective treatment of cancer does not efficiently relieve cancer pain, but may increase pain in many cases. Hence, few studies focus on simultaneous cancer therapy and pain relief, and made this situation even worse. Method: Ropivacaine was loaded into tumor-active targeted liposomes. The cytotoxicity of ropivacaine-based combination therapy in B16 and HeLa cells were tested. Moreover, a mice model of cancer pain which was induced by inoculation of melanoma near the sciatic nerve was constructed to assess the cancer suppression and pain relief effects of ropivacaine-based combination therapy. Results: Ropivacaine and ropivacaine-loaded liposomes (Rop-DPRL) were novelly found to damage autophagic degradation. Replicated administration of Rop-DPRL and calorie restriction (CR) could efficiently repress the development of tumor. In addition, administration of Rop-DPRL could relieve cancer pain with its own analgestic ability in a short duration, while repeated administration of Rop-DPRL and CR resulted in continuous alleviation of cancer pain through reduction of VEGF-A levels in advanced cancer mice. Further, dual inhibition of phosphorylation of STAT3 at Tyr705 and Ser727 by Rop-DPRL and CR contribute to the reduction of VEGF-A. Conclusion: Combination therapy with Rop-DPRL and nutrient deprivation simultaneously suppresses cancer growth and relieves cancer pain.

Mesoporous silica nanoparticles (MSNs) are extensively used in bone tissue regeneration and local drug delivery. However, the effects of MSNs alone on osteoclast formation and function, as well as the utilization of MSNs to deliver natural molecules against bone resorption, remain unexplored. Here, we report the development of licorice-derived bioactive flavonoid isoliquiritigenin (ISL)-encapsulated MSNs (MSNs-ISL) as a potent bone-bioresponsive nanoencapsulation system for prevention of osteoclast-mediated bone loss in vitro and in vivo. Methods: We synthesized MSNs-ISL and then investigated the drug loading and release characteristics of the resulting nanoparticles. In vitro experiments on osteoclast differentiation and bone resorption were performed using mouse primary bone marrow-derived macrophages (BMMs). In vivo animal experiments were conducted using a lipopolysaccharide (LPS)-mediated calvarial bone erosion model. Results: The resulting MSNs-ISL were spherical and highly monodispersed; they possessed a large specific surface area and superior biocompatibility, and allowed acid-sensitive sustained drug release. Compared with free ISL and MSNs alone, MSNs-ISL significantly and additively inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast generation, decreased the size and quantity of sealing zones, and reduced the osteolytic capacity of osteoclasts in vitro. MSNs-ISL treatment also downregulated RANKL-stimulated mRNA expression of osteoclast-associated genes and transcription factors. Mechanistically, MSNs-ISL remarkably attenuated the RANKL-initiated expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), phosphorylation of mitogen-activated protein kinases (MAPKs), and phosphorylation and degradation of inhibitor of κBα (IκBα), together with the nuclear translocation of nuclear factor-κB (NF-κB) p65 and the activator protein (AP)-1 component c-Fos. Moreover, MSNs-ISL almost completely restrained the expression of nuclear factor of activated T cells (NFATc1). Consistent with the in vitro results, MSNs-ISL could block osteoclast activity; relieve inflammation-related calvarial bone destruction in vivo; and suppress c-Fos, NFATc1, and cathepsin K expression levels. Conclusion: Licorice ISL-encapsulated MSNs exhibit notable anti-osteoclastogenetic effects and protect against inflammatory bone destruction. Our findings reveal the feasibility of applying MSNs-ISL as an effective natural product-based bone-bioresponsive nanoencapsulation system to prevent osteoclast-mediated bone loss.

Rationale: As a cancer, Glioblastoma (GBM) is a highly lethal and difficult-to-treat. With the aim of improving therapies to GBM, we developed novel and target-specific theranostic nanoparticles (TNPs) that can be selectively cleaved by cathepsin B (Cat B) to release the potent toxin monomethyl auristatin E (MMAE). Methods: We synthesized TNPs composed of a ferumoxytol-based nanoparticle carrier and a peptide prodrug with a Cat-B-responsive linker and the tubulin inhibitor MMAE. We hypothesized that intratumoral Cat B can cleave our TNPs and release MMAE to kill GBM cells. The ferumoxytol core enables in vivo drug tracking with magnetic resonance imaging (MRI). We incubated U87-MG GBM cells with TNPs or ferumoxytol and evaluated the TNP content in the cells with transmission electron microscopy and Prussian blue staining. In addition, we stereotaxically implanted 6- to 8-week-old nude mice with U87-MG with U87-MG GBM cells that express a fusion protein of Green Fluorescence Protein and firefly Luciferase (U87-MG/GFP-fLuc). We then treated the animals with an intravenous dose of TNPs (25 mg/kg of ferumoxytol, 0.3 mg/kg of MMAE) or control. We also evaluated the combination of TNP treatment with radiation therapy. We performed MRI before and after TNP injection. We compared the results for tumor and normal brain tissue between the TNP and control groups. We also monitored tumor growth for a period of 21 days. Results: We successfully synthesized TNPs with a hydrodynamic size of 41 ± 5 nm and a zeta potential of 6 ± 3 mV. TNP-treated cells demonstrated a significantly higher iron content than ferumoxytol-treated cells (98 ± 1% vs. 3 ± 1% of cells were iron-positive, respectively). We also found significantly fewer live attached cells in the TNP-treated group (3.8 ± 2.0 px2) than in the ferumoxytol-treated group (80.0 ± 14.5 px2, p < 0001). In vivo MRI studies demonstrated a decline in the tumor signal after TNP (T2= 28 ms) but not control (T2= 32 ms) injections. When TNP injection was combined with radiation therapy, the tumor signals dropped further (T2 = 24 ms). The combination therapy of radiation therapy and TNPs extended the median survival from 14.5 days for the control group to 45 days for the combination therapy group. Conclusion: The new cleavable TNPs reported in this work accumulate in GBM, cause tumor cell death, and have synergistic effects with radiation therapy.

Rationale: Growing evidence indicates that intracellular reactive oxygen species (ROS) accumulation is a critical factor in the development of osteoporosis by triggering osteoclast formation and function. Pseurotin A (Pse) is a secondary metabolite isolated from Aspergillus fumigatus with antioxidant properties, recently shown to exhibit a wide range of potential therapeutic applications. However, its effects on osteoporosis remain unknown. This study aimed to explore whether Pse, by suppressing ROS level, is able to inhibit osteoclastogenesis and prevent the bone loss induced by estrogen-deficiency in ovariectomized (OVX) mice. Methods: The effects of Pse on receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis and bone resorptive function were examined by tartrate resistant acid phosphatase (TRAcP) staining and hydroxyapatite resorption assay. 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) was used to detect intracellular ROS production in vitro. Western blot assay was used to identify proteins associated with ROS generation and scavenging as well as ROS-mediated signaling cascades including mitogen-activated protein kinases (MAPKs), NF-κB pathways, and nuclear factor of activated T cells 1 (NFATc1) signaling. The expression of osteoclast-specific genes was assessed by qPCR. The in vivo potential of Pse was determined using an OVX mouse model administered with Pse or vehicle for 6 weeks. In vivo ROS production was assessed by intravenous injection of dihydroethidium (DHE) into OVX mice 24h prior to killing. After sacrifice, the bone samples were analyzed using micro-CT and histomorphometry to determine bone volume, osteoclast activity, and ROS level ex vivo. Results: Pse was demonstrated to inhibit osteoclastogenesis and bone resorptive function in vitro, as well as the downregulation of osteoclast-specific genes including Acp5 (encoding TRAcP), Ctsk (encoding cathepsin K), and Mmp9 (encoding matrix metalloproteinase 9). Mechanistically, Pse suppressed intracellular ROS level by inhibiting RANKL-induced ROS production and enhancing ROS scavenging enzymes, subsequently suppressing MAPK pathway (ERK, P38, and JNK) and NF-κB pathways, leading to the inhibition of NFATc1 signaling. Micro-CT and histological data indicated that OVX procedure resulted in a significant bone loss, with dramatically increased the number of osteoclasts on the bone surface as well as increased ROS level in the bone marrow microenvironment; whereas Pse supplementation was capable of effectively preventing these OVX-induced changes. Conclusion: Pse was demonstrated for the first time as a novel alternative therapy for osteoclast-related bone diseases such as osteoporosis through suppressing ROS level.

Human CLCN7 encodes voltage-gated chloride channel 7 (ClC-7); mutations of CLCN7 lead to osteopetrosis which is characterized by increased bone mass and impaired osteoclast function. In our previous clinical practice, we noticed that osteopetrosis patients with CLCN7 mutations had some special deformities in craniofacial morphology and tooth dysplasia. It is unclear whether these phenotypes are the typical features of CLCN7 involved osteopetrosis and whether ClC-7 could regulate the development of craniofacial bone and tooth in some signaling pathways. Methods: First, we collected 80 osteopetrosis cases from the literature and compared their craniofacial and dental phenotypes. Second, four osteopetrosis pedigrees with CLCN7 mutations were recruited from our clinic for gene testing and clinical analysis of their craniofacial and dental phenotypes. Third, we used a zebrafish model with clcn7 morpholino treatment to detect the effects of ClC-7 deficiency on the development of craniofacial and dental phenotypes. General observation, whole mount alcian blue and alizarin red staining, whole mount in situ hybridization, scanning electron microscope observation, lysoSensor staining, Q-PCR and western blotting were performed to observe the in vivo characteristics of craniofacial bone and tooth changes. Fourth, mouse marrow stromal cells were further primarily cultured to detect ClC-7 related mRNA and protein changes using siRNA, Q-PCR and western blotting. Results: Over 84% of osteopetrosis patients in the literature had some typical craniofacial and tooth phenotypes, including macrocephaly, frontal bossing, and changes in shape and proportions of facial skeleton, and these unique features are more severe and frequent in autosomal recessive osteopetrosis than in autosomal dominant osteopetrosis patients. Our four pedigrees with CLCN7 mutations confirmed the aforementioned clinical features. clcn7 knockdown in zebrafish reproduced the craniofacial cartilage defects and various dental malformations combined the decreased levels of col10a1, sp7, dlx2b, eve1, and cx43. Loss of clcn7 function resulted in lysosomal storage in the brain and jaw as well as downregulated cathepsin K (CTSK). The craniofacial phenotype severity also presented a dose-dependent relationship with the levels of ClC-7 and CTSK. ClC-7/CTSK further altered the balance of TGF-β/BMP signaling pathway, causing elevated TGF-β-like Smad2 signals and reduced BMP-like Smad1/5/8 signals in clcn7 morphants. SB431542 inhibitor of TGF-β pathway partially rescued the aforementioned craniofacial bone and tooth defects of clcn7 morphants. The ClC-7 involved CTSK/BMP and SMAD changes were also confirmed in mouse bone marrow stromal cells. Conclusion: These findings highlighted the vital role of clcn7 in zebrafish craniofacial bone and tooth development and mineralization, revealing novel insights for the causation of osteopetrosis with CLCN7 mutations. The mechanism chain of ClC-7/CTSK/ TGF-β/BMP/SMAD might explain the typical craniofacial bone and tooth changes in osteopetrosis as well as pycnodysostosis patients.