CD44-Targeted Photoactivatable Polymeric Nanosystem with On-Demand Drug Release as a "Photoactivatable Bomb" for Combined Photodynamic Therapy-Chemotherapy of Cancer.

Nowadays, the combined use of chemotherapy and photodynamic therapy (PDT) remains the most popular strategy for cancer treatment with high theraprutic efficacy. However, targeted therapy with the on-demand release of drugs is what most clinical treatments lack, leading to heavy side effects. Herein, a new CD44-targeted and red-light-activatable nanosystem, Ru-HA@DOX nanoparticles (NPs), was developed by conjugating hydrophilic biodegradable hyaluronic acid (HA) and hydrophobic photoresponsive ruthenium (Ru) complexes, which could encapsulate the chemotherapeutic drug doxrubicin (DOX). Ru-HA@DOX NPs can selectively accumulate at the tumor through the enhanced permeability and retention (EPR) effect and CD44-mediated endocytosis, thus avoiding off-target toxicity during circulation. After 660 nm of irradiation at the tumor site, Ru-HA@DOX NPs, as a "photoactivatable bomb", was split via the photocleavable Ru-N coordination bond to fast release DOX and produce singlet oxygen (1O2) for PDT. In general, Ru-HA@DOX NPs retained its integrity before irradiation and possessed minimal cytotoxicity, while under red-light irradiation, Ru-HA@DOX NPs showed significant cytotoxicity due to the release of DOX and production of 1O2 at the tumor. Chemotherapy-PDT of Ru-HA@DOX NPs resulted in a significant inhibition of tumor growth in A549-tumor-bearing mice and reduced the cardiotoxicity of DOX. Therefore, this study offers a novel CD44-targeted drug-delivery system with on-demand drug release for synergistic chemotherapy-PDT.

All-in-one: Accurate quantification, on-site detection, and bioimaging of sulfite using a colorimetric and ratiometric fluorescent probe in vitro and in vivo.

SO2 and its derivatives (SO32-/HSO3-) are used widely in food, beverages, and pharmaceutical production. However, they could induce multiple diseases in respiratory, nervous, and cardiovascular systems. Although several fluorescent probes have been developed for detecting SO32-/HSO3-, reports on rapid fluorescent probes for the on-site detection of SO2 derivatives are scarce. Herein, a colorimetric and ratiometric fluorescent probe 1 based on the intramolecular charge transfer (ICT) was reported. Probe 1 resulted in a 122 nm blue-shift in fluorescent emission and decrement of absorbance at 500 nm upon the addition of sulfite. Therefore, probe 1 could quantify SO32-/HSO3- using both UV-Vis and fluorescent methods (LOD: UV-Vis method 34 nM; fluorescent method 51 nM). Importantly, probe 1 was used for a rapid (60 s) and convenient (1 step, on-site) measurement of the SO2 derivatives in real samples (LOD: 0.47 µM) using smartphone based on the colorimetric method. The SO32-/HSO3--sensing mechanism was confirmed as the Michael addition reaction. Furthermore, the probe was used for the real-time monitoring of SO32-/HSO3- in A549 cells and zebrafish. In summary, an all-in-one fluorescent probe was successfully developed for the accurate quantification, on-site detection, and bioimaging of SO32-/HSO3-.

A Carrier-Free Folate Receptor-Targeted Ursolic Acid/Methotrexate Nanodelivery System for Synergetic Anticancer Therapy.

PURPOSE: To avoid undefined metabolic mechanisms and to eliminate potential side effects of traditional nanocarriers, new green carriers are urgently needed in cancer treatment. Carrier-free nanoparticles (NPs) based on ursolic acid (UA) have attracted significant attention, but the UA NPs targeting the folate receptor have never been explored. We designed a novel self-assembled UA-Methotrexate (MTX) NPs targeting the folate-receptor and its synergetic anticancer activity was studied in vitro and in vivo. METHODS: UA-MTX NPs were prepared using the solvent precipitation method. Characterization of the UA-MTX NPs preparation was performed using a size analyzer, transmission electron microscopy, and UV-vis spectrophotometry. The in vitro pH-responsive drug release capability of UA-MTX NPs was tested at different pH values. The UA-MTX NPs targeting of folates was determined by comparing the endocytosis rates of cell lines with low or overexpression of the folate receptor (A549 and MCF-7 cells). The cytotoxicity and cell apoptosis of UA-MTX NPs were also studied to determine the in vitro synergistic effects. Combination chemotherapy of UA-MTX NPs in vivo was evaluated using MCF-7 xenografted tumor models. RESULTS: Compared with free UA or MTX, the water solubility of UA-MTX NPs improved significantly. Drug-release from the UA-MTX NPs was faster at pH 5.0 than pH 7.4, suggesting MTX-UA NPs could rapidly release MTX in the acidic conditions of the tumor microenvironment. Confocal laser scanning microscopy revealed the excellent folate receptor targeting of UA-MTX NPs in MCF-7 cells. Cytotoxicity and cell apoptosis results demonstrated greater antiproliferative capacity of UA-MTX NPs than that of free drug in folate receptor overexpressing MCF-7 cells. Anticancer effects in vivo suggested MTX-UA NPs exhibited good biological safety and could enhance antitumor efficacy due to the combination therapy. CONCLUSION: Our findings indicate that the UA-MTX NPs targeting folate-receptors is an efficient strategy for combination chemotherapy.

Design and synthesis of novel 3,4-dihydrocoumarins as potent and selective monoamine oxidase-B inhibitors with the neuroprotection against Parkinson's disease.

The monoamine oxidase-B (MAO-B) inhibitors with neuroprotective effects are better for Parkinson's disease (PD) treatment, due to the complicated pathogenesis of PD. To develop new hMAO-B inhibitors with neuroprotection, a novel series of 3,4-dihydrocoumarins was designed as selective and reversible hMAO-B inhibitors to treat PD. Most compounds showed potent and selective inhibition for hMAO-B over hMAO-A with IC50 values ranging from nanomolar to sub-nanomolar. Among them, compound 4d was the most potent hMAO-B inhibitor (IC50 = 0.37 nM) being about 20783-fold more active than iproniazid, and exhibited the highest selectivity for hMAO-B (SI > 270,270). Kinetic studies revealed that compound 4d was a reversible and competitive inhibitor of hMAO-B. Neuroprotective studies indicated that compound 4d could protect PC12 cells from the damage induced by 6-OHDA and rotenone. Besides, compound 4d did not exhibit acute toxicity at a dose up to 2500 mg/kg (po), and could cross the BBB in parallel artificial membrane permeability assay. More importantly, compound 4d was able to significantly prevent the motor deficits in the MPTP-induced PD model. These results indicate that compound 4d is an effective and promising candidate against PD.

A novel mitochondria-targetted near-infrared fluorescent probe for selective and colorimetric detection of sulfite and its application in vitro and vivo.

Overdoses of SO2 and its derivatives (SO32-/HSO3-) in food or organisms are harmful to health. To detect SO32-/HSO3-, a novel NIR fluorescent probe 1, based upon the intramolecular charge transfer (ICT) mechanism, was developed. This probe was easily synthesized, and gave noticeable colorimetric and linear fluorescence changes at 690 nm after reaction with sulfite from 3.13 to 200 µM. Moreover, probe 1 displayed high sensitivity (LOD = 0.46 µM), excellent selectivity (among 13 kinds of anions and 3 kinds of biothiols) and quick response (within 30 min) towards SO32-/HSO3-. The SO32-/HSO3- sensing mechanism was confirmed as the Michael addition reaction. Furthermore, the probe showed wide applications for measuring SO32-/HSO3- in real samples, including sugar, tap water, wine and traditional Chinese medicine. The probe could also be used to detect SO32-/HSO3- in mitochondria of HepG2 cells and zebrafish, which suggested potential application for monitoring SO2 derivatives in clinical diagnostics.

Rational modulation of coumarin-hemicyanine platform based on OH substitution for higher selective detection of hypochlorite.

To monitor delicate changes of biological HOCl in vivo, a new probe (OH-substituted coumarin-hemicyanine, probe 2) was synthesized for NIR and ratiometric HOCl detection. Selectivity studies indicated that the electron-donating group (OH) substituted on the indolium moiety enhanced the selectivity to detect HOCl. With HOCl, the probe showed a ratiometric fluorescence (I500/I650) with a low detection limit (49.1 nM) and a rapid response (within 2 min). In addition, probe 2 was successfully applied to visualize exogenous and endogenous HOCl in living cells and animals and exhibited a perfect mitochondria target ability. This probe has been further studied as a potential and powerful tool to probe HOCl in arthritis models.

Chromone and donepezil hybrids as new multipotent cholinesterase and monoamine oxidase inhibitors for the potential treatment of Alzheimer's disease.

A series of chromone and donepezil hybrids were designed, synthesized, and evaluated as multipotent cholinesterase (ChE) and monoamine oxidase (MAO) inhibitors for the potential therapy of Alzheimer's disease (AD). In vitro studies showed that the great majority of these compounds exhibited potent inhibitory activity toward BuChE and AChE and clearly selective inhibition for hMAO-B. In particular, compound 5c presented the most balanced potential for ChE inhibition (BuChE: IC50 = 5.24 µM; AChE: IC50 = 0.37 µM) and hMAO-B selectivity (IC50 = 0.272 µM, SI = 247). Molecular modeling and kinetic studies suggested that 5c was a mixed-type inhibitor, binding simultaneously to peripheral and active sites of AChE. It was also a competitive inhibitor, which occupied the substrate and entrance cavities of MAO-B. Moreover, compound 5c could penetrate the blood-brain barrier (BBB) and showed low toxicity to rat pheochromocytoma (PC12) cells. Altogether, these results indicated that compound 5c might be a hopeful multitarget drug candidate with possible impact on Alzheimer's disease therapy.

Design, synthesis and evaluation of novel ferulic acid derivatives as multi-target-directed ligands for the treatment of Alzheimer's disease.

A series of new ferulic acid derivatives were designed, synthesized and evaluated as multi-target inhibitors against Alzheimer's disease. In vitro studies indicated that most compounds showed significant potency to inhibit self-induced ß-amyloid (Aß) aggregation and acetylcholinesterase (AChE), and had good antioxidant activity. Specifically, compound 4g exhibited the potent ability to inhibit cholinesterase (ChE) (IC50, 19.7 nM for hAChE and 0.66 µM for hBuChE) and the good Aß aggregation inhibition (49.2% at 20 µM), and it was also a good antioxidant (1.26 trolox equivalents). Kinetic and molecular modeling studies showed that compound 4g was a mixed-type inhibitor, which could interact simultaneously with the catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE. Moreover, compound 4g could remarkably increase PC12 cells viability in hydrogen peroxide-induced oxidative cell damage and Aß-induced cell damage. Finally, compound 4g had good ability to cross the BBB using the PAMPA-BBB assay. These results suggested that compound 4g was a promising multifunctional ChE inhibitor for the further investigation.

A Ratiometric and Colorimetric Hemicyanine Fluorescent Probe for Detection of SO2 Derivatives and Its Applications in Bioimaging.

Based upon the intramolecular charge transfer (ICT) mechanism, a novel ratiometric fluorescent probe EB was developed to detect SO32-/HSO3-. The probe displayed both colorimetric and ratiometric responses toward SO32-/HSO3-. It displayed a quick response (within 60 s), good selectivity and high sensitivity (a detection limit of 28 nM) towards SO32-/HSO3-. The SO32-/HSO3- sensing mechanism was confirmed as the Michael addition reaction by ESI-MS. Moreover, the probe could be applied to measure the level of sulfite in real samples, like sugar and chrysanthemum, and it could also be used to detect SO32-/HSO3- in HepG2 cells through confocal fluorescence microscopy, which proved its practical application in clinical diagnosis.

A near-infrared Nile red fluorescent probe for the discrimination of biothiols by dual-channel response and its bioimaging applications in living cells and animals.

Biothiols, including cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and H2S, play important roles in human physiological processes. However, it is a great difficulty to distinguish biothiols from each other because of their similar chemical properties. Based on Nile red, we have designed and synthesized a near-infrared fluorescent probe for discriminating Cys/Hcy from GSH/H2S by a dual-channel detection method. Using an ether bond, near-infrared Nile red was attached to 7-nitrobenzofurazan to construct the probe. Due to the photo-induced electron transfer, the probe showed almost no fluorescence from the green to red emission band. But upon the addition of Cys (0-150 µM) or Hcy (0-200 µM), the probe exhibited a noteworthy fluorescence "turn-on" signal in two unique emission bands (Green and Red) with a fast response (within 30 min). In contrast, the probe displayed an increase in fluorescence only in the red channel when encountering GSH (0-70 µM) or H2S (0-50 µM), and GSH/H2S could be tested respectively by different response time. The limit of detection was calculated to be 0.09 µM (Cys), 0.30 µM (Hcy), 0.24 µM (GSH), and 0.04 µM (H2S), respectively (based on S/N = 3). The desirable dual-channel detection could be achieved in serum samples and living cells. Moreover, the probe could be applied for bioimaging in mice, which indicated its potential application in the clinic.

Coumarin-dithiocarbamate hybrids as novel multitarget AChE and MAO-B inhibitors against Alzheimer's disease: Design, synthesis and biological evaluation.

A series of new coumarin-dithiocarbamate hybrids were designed and synthesized as multitarget agents for the treatment of Alzheimer's disease. Most of them showed potent and clearly selective inhibition towards AChE and MAO-B. Among these compounds, compound 8f demonstrated the most potent inhibition to AChE with IC50 values of 0.0068 µM and 0.0089 µM for eeAChE and hAChE, respectively. Compound 8g was identified as the most potent inhibitor to hMAO-B, and it is also a good and balanced inhibitor to both hAChE and hMAO-B (0.114 µM for hAChE; 0.101 µM for hMAO-B). Kinetic and molecular modeling studies revealed that 8g was a dual binding site inhibitor for AChE and a competitive inhibitor for MAO-B. Further studies indicated that 8g could penetrate the BBB and exhibit no toxicity on SH-SY5Y neuroblastoma cells. More importantly, 8g did not display any acute toxicity in mice at doses up to 2500 mg/kg and could reverse the cognitive dysfunction of scopolamine-induced AD mice. Overall, these results highlighted 8g as a potential multitarget agent for AD treatment and offered a starting point for design of new multitarget AChE/MAO-B inhibitors based on dithiocarbamate scaffold.

Synthesis and Biological Evaluation of Novel Alkyl Amine Substituted Icariside II Derivatives as Potential Anticancer Agents.

A series of novel alkyl amine-substituted icariside II (ICA II) derivatives were synthesized by Mannich reactions at the 6-C position (compounds 4a⁻d) and changing the carbon chain length at the 7-OH position (compounds 7a⁻h), and their in vitro antitumor activity towards human breast cancer lines (MCF-7 and MDA-MB-231) and human hepatoma cell lines (HepG2 and HCCLM3-LUC) were evaluated by the MTT assay. Compared with ICA II, most of the twelve derivatives showed good micromole level activity and a preliminary structure-activity relationship (SAR) for the anticancer activity was obtained. Compound 7g showed the most potent inhibitory activity for the four cancer cell lines (13.28 µM for HCCLM3-LUC, 3.96 µM for HepG2, 2.44 µM for MCF-7 and 4.21 µM for MDA-MB-231), which was 2.94, 5.54, 12.56 and 7.72-fold stronger than that of ICA II. The preliminary SAR showed that the introduction of a alkyl amine substituent at 6-C was not favorable for the anticancer activity, while most of the 7-O-alkylamino derivatives exhibited good antitumor activity and the anticancer activity 7-O-alkylamino derivatives were influenced by the alkyl chain length and the different terminal amine substituents. Furthermore, the effects of compound 7g on apoptosis and cell cycle of MCF-7 cells were further investigated, which showed that compound 7g triggered apoptosis and arrested the cell cycle at the G0/G1 phase in MCF-7 cells. Our findings indicate that compound 7g may be a promising anticancer drug candidate lead.

Design, synthesis and evaluation of resveratrol-indazole hybrids as novel monoamine oxidases inhibitors with amyloid-ß aggregation inhibition.

Novel hybrids with MAO and Aß (1-42) self-aggregation inhibitory activities were designed and synthesized with the employment of indazole moiety and resveratrol. The biological screening results indicated that most compounds displayed potent inhibitory activity for Aß (1-42) self-aggregation, and obvious selective inhibition to MAO-B. Among these compounds, compound 6e was the most potent inhibitor not only for hMAO-B (IC50 = 1.14 µM) but also for Aß (1-42) self-aggregation (58.9% at 20 µM). Molecular modeling and kinetic studies revealed that compound 6e was a competitive MAO-B inhibitor, which can occupy the active site of MAO-B, and interact with Aß (1-42) via π-π and cation-π stacking interactions. In addition, compound 6e had no toxicity on PC12 cells and could cross the BBB. Collectively, all these results suggested that compound 6e might be a promising multi-target lead compound worthy of further investigation.

A Fluorescent Coumarin-Based Probe for the Fast Detection of Cysteine with Live Cell Application.

A new coumarin-based fluorescent probe, containing an allylic esters group, has been designed and synthesized for sensing cysteine in physiological pH. In this fluorescent probe, the coumarin was applied as the fluorophore and an allylic esters group was combined as both a fluorescence quencher and a recognition unit. The probe can selectively and sensitively detect cysteine (Cys) over homocysteine, glutathione, and other amino acids, and has a rapid response time of 30 min and a low detection limit of 47.7 nM. In addition, the probe could be applied for cell imaging with low cytotoxicity.

Design, synthesis and biological evaluation of novel coumarin-N-benzyl pyridinium hybrids as multi-target agents for the treatment of Alzheimer's disease.

Combining N-benzyl pyridinium moiety and coumarin into in a single molecule, novel hybrids with ChE and MAO-B inhibitory activities were designed and synthesized. The biological screening results indicated that most of compounds displayed potent inhibitory activity for ChE and Aß (1-42) self-aggregation, and clearly selective inhibition to MAO-B over MAO-A. Of these compounds, compound 7f was the most potent inhibitor for hMAO-B, and it was also a good and balanced inhibitor to ChEs and hMAO-B (0.0373 µM for eeAChE; 2.32 µM for eqBuChE; 1.57 µM for hMAO-B). Molecular modeling and kinetic studies revealed that compound 7f was a mixed-type inhibitor, which bond simultaneously to CAS and PAS of AChE, and it was also a competitive inhibitor, which occupied the active site of MAO-B. In addition, compound 7f with no toxicity on PC12 neuroblastoma cells, showed good ability to inhibit Aß (1-42) self-aggregation and cross the BBB. Collectively, all these results suggested that compound 7f might be a promising multi-target lead candidate worthy of further pursuit.

Design, synthesis and evaluation of novel cinnamic acid derivatives bearing N-benzyl pyridinium moiety as multifunctional cholinesterase inhibitors for Alzheimer's disease.

A novel family of cinnamic acid derivatives has been developed to be multifunctional cholinesterase inhibitors against AD by fusing N-benzyl pyridinium moiety and different substituted cinnamic acids. In vitro studies showed that most compounds were endowed with a noteworthy ability to inhibit cholinesterase, self-induced Aß (1-42) aggregation, and to chelate metal ions. Especially, compound 5l showed potent cholinesterase inhibitory activity (IC50, 12.1 nM for eeAChE, 8.6 nM for hAChE, 2.6 µM for eqBuChE and 4.4 µM for hBuChE) and the highest selectivity toward AChE over BuChE. It also showed good inhibition of Aß (1-42) aggregation (64.7% at 20 µM) and good neuroprotection on PC12 cells against amyloid-induced cell toxicity. Finally, compound 5l could penetrate the BBB, as forecasted by the PAMPA-BBB assay and proved in OF1 mice by ex vivo experiments. Overall, compound 5l seems to be a promising lead compound for the treatment of Alzheimer's diseases.

Design, synthesis and biological activity of novel donepezil derivatives bearing N-benzyl pyridinium moiety as potent and dual binding site acetylcholinesterase inhibitors.

A series of new donepezil derivatives were designed synthesized and evaluated as multifunctional cholinesterase inhibitors against Alzheimer's disease (AD). In vitro studies showed that most of them exhibited significant potency to inhibit acetylcholinesterase and self-induced ß-amyloid (Aß) aggregation, and moderate antioxidant activity. Especially, compound 5b presented the greatest ability to inhibit cholinesterase (IC50, 1.9 nM for eeAChE and 0.8 nM for hAChE), good inhibition of Aß aggregation (53.7% at 20 µM) and good antioxidant activity (0.54 trolox equivalents). Kinetic and molecular modeling studies indicated that compound 5b was a mixed-type inhibitor, binding simultaneously to the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. In addition, compound 5b could reduce PC12 cells death induced by oxidative stress and Aß (1-42). Moreover, in vivo experiments showed that compound 5b was nontoxic and tolerated at doses up to 2000 mg/kg. These results suggested that compound 5b might be an excellent multifunctional agent for AD treatment.

Synthesis and evaluation of small molecules bearing a benzyloxy substituent as novel and potent monoamine oxidase inhibitors.

A new series of small molecules bearing a benzyloxy substituent have been designed, synthesized and evaluated for hMAO inhibitory activity in vitro. Most of the compounds were potent and selective MAO-B inhibitors, and were weak inhibitors of MAO-A. In particular, compounds 9e (IC50 = 0.35 µM) and 10e (IC50 = 0.19 µM) were the most potent MAO-B inhibitors, and exhibited the highest selectivity for MAO-B (9e, SI > 285.7-fold and 10e, SI = 146.8-fold). In addition, the structure-activity relationships for MAO-B inhibition indicated that electron-withdrawing groups in the open small molecules were more suitable for MAO-B inhibition, and substitutions at the benzyloxy of the open small molecules, particularly with the halogen substituted benzyloxy, were more favorable for MAO-B inhibition. Molecular docking studies have been done to explain the potent MAO-B inhibition of the open small molecules. Furthermore, the representative compounds 9e and 10e showed low neurotoxicity in SH-SY5Y cells in vitro. So the small molecules bearing the benzyloxy substituent could be used to develop promising drug candidates for the therapy of neurodegenerative diseases.

Synthesis and pharmacological evaluation of donepezil-based agents as new cholinesterase/monoamine oxidase inhibitors for the potential application against Alzheimer's disease.

In a continuing effort to develop multitargeted compounds as potential treatment agents against Alzheimer's disease (AD), a series of donepezil-like compounds were designed, synthesized and evaluated. In vitro studies showed that most of the designed compounds displayed potent inhibitory activities toward AChE, BuChE, MAO-B and MAO-A. Among them, w18 was a promising agent with balanced activities, which exhibited a moderate cholinesterase inhibition (IC50, 0.220 µM for eeAChE; 1.23 µM for eqBuChE; 0.454 µM for hAChE) and an acceptable inhibitory activity against monoamine oxidases (IC50, 3.14 µM for MAO-B; 13.4 µM for MAO-A). Moreover, w18 could also be a metal-chelator, and able to cross the blood-brain barrier with low cell toxicity on PC12 cells. Taken together, these results suggested that w18 might be a promising multitargeted compound for AD treatment.

Design, synthesis and biological evaluation of novel donepezil-coumarin hybrids as multi-target agents for the treatment of Alzheimer's disease.

Combining N-benzylpiperidine moiety of donepezil and coumarin into in a single molecule, novel hybrids with ChE and MAO-B inhibitory activity were designed and synthesized. The biological screening results indicated that most of compounds displayed potent inhibitory activity for AChE and BuChE, and clearly selective inhibition to MAO-B. Of these compounds, 5m was the most potent inhibitor for eeAChE and eqBuChE (0.87 µM and 0.93 µM, respectively), and it was also a good and balanced inhibitor to hChEs and hMAO-B (1.37 µM for hAChE; 1.98 µM for hBuChE; 2.62 µM for hMAO-B). Molecular modeling and kinetic studies revealed that 5m was a mixed-type inhibitor, which bond simultaneously to CAS, PAS and mid-gorge site of AChE, and it was also a competitive inhibitor, which occupied the active site of MAO-B. In addition, 5m showed good ability to cross the BBB and had no toxicity on SH-SY5Y neuroblastoma cells. Collectively, all these results suggested that 5m might be a promising multi-target lead candidate worthy of further pursuit.