•1R/3S- and 1S/3S-1-methyltetrahydro-β-carboline-3-carboxylic acids were designed.•As GPIIb/IIIa inhibitor the 1R/3S was more active than 1S/3S in vitro and in vivo.•As the lead of GPIIb/IIIa inhibitors the 1R/3S should be superior to the 1S/3S.In GPIIb/IIIa mediated arterial thrombosis platelet activation plays a central role. To discover platelet activation inhibitor the pharmacophores of GPIIb/IIIa receptor inhibitors and anti-thrombotic agents were analyzed. This led to the design of (1R,3S)- and (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acids as GPIIb/IIIa inhibitors. Comparing to (1S,3S)-isomer (1R,3S)-isomer had lower cdocker interaction energy. AFM image showed that the minimal effective concentration of (1S,3S)-isomer and (1R,3S)-isomer inhibiting platelet activation were 10−5 M and 10−6 M, respectively. In vivo 1 μmol/kg of oral (1S,3S)-isomer effectively inhibited the rats to form arterial thrombus and down regulated GPIIb/IIIa expression, but the activities were significantly lower than those of 1 μmol/kg of oral (1R,3S)-isomer. Both (1S,3S)-isomer and (1R,3S)-isomer can be safely used for structural modifications, but (1R,3S)-isomer should be superior to (1S,3S)-isomer.Download high-res image (106KB)Download full-size image

By use of carboxylbutyryl as a linker, adriamycin (ADR) and cholyl-L-Lys (an anti-inflammatory agent) were covalently conjugated and Nα-cholyl-Nε-(N-carbonylpropionoadriamycin)-L-Lys (BCBALys) was constructed as a liver-targeting nano-delivery system to release cholyl-L-Lys and protect the liver from CCl4-induced injury. In ultrapure water and rat plasma, 10−6 M BCBALys formed nanoparticles of 42–231 nm in diameter and ∼116 nm in height. In a CCl4-injured mouse model, however, only 2 µmol kg−1 of BCBALys effectively protected the liver of the mice from injury, and the mouse liver histology showed no hepatic architecture loss and inflammatory cell infiltration. BCBALys selectively accumulated in the liver of CCl4-injured mice, but not in other vital organs, and released cholyl-L-Lys. These data demonstrated that BCBALys exhibited high efficacy for treating CCl4-induced liver injury in a targeted manner. The chemical mechanism of BCBALys nanoparticle formation and the pharmacological mechanism of BCBALys mouse liver protection from CCl4-induced injury were also revealed by experiments.

Thrombosis is a serious threat to human health worldwide. Tetrahydroisoquinoline-3-carboxylic acid (IQCA) is an antithrombotic agent, while Thr-Ala-Arg-Gly-Asp(Ser)-Ser (TASS) can target thrombus. Herein, tetrahydro-isoquinoline-3-carbonyl-Thr-Ala-Arg-Gly-Asp(Ser)-Ser (IQCA-TASS) was designed with the aim towards the discovery of a nano-delivery system for targeting thrombus. In vitro, IQCA-TASS acted on P-selectin and down-regulated P-selectin expression. The IC50 values of IQCA-TASS against the platelet aggregation induced by four aggregators were less than 0.45 nM. In vivo, IQCA-TASS targeted thrombus, released IQCA and TASS inside the thrombus, showed dose-dependent anti-thrombotic action, of which the minimal effective dose was 1 nmol kg−1, and showed anti-inflammatory action. Even with the dose up to 1 μmol kg−1, a dose of 1000 times the minimal effective dose, IQCA-TASS still induced no toxic reaction. In rat plasma, IQCA-TASS formed nanoparticles with diameters of less than 41 nm. The interactions of the nanoparticles with both resting and activated platelets were imaged. IQCA-TASS should be a safe nano-medicine capable of targeting thrombus and releasing anti-thrombotic/anti-inflammatory pharmacophores in disease sites.

To improve the therapeutic efficacy of cancer patients a novel conjugate of thymopentin (TP5) and (1S,3S)-1-methyl-tetrahydro-β-carboline-3-carboxylic acid (MTC) was presented. In water and mouse plasma MTCTP5 forms the nanoparticles of 14–139 nm in diameter, the suitable size for delivery in blood circulation. In mouse plasma MTCTP5 releases MTC, while in the presence of trypsin MTCTP5 releases MTC and TP5. On mouse and rat models the MTCTP5 dose dependently slows down the tumor growth, inhibits inflammatory response and blocks thrombosis. The anti-tumor activity as well as the anti-inflammation activity and anti-thrombotic activity of MTCTP5 are 100 fold and 10 fold higher than those of MTC, respectively, which are attributed to the fact that it down-regulates the plasma levels of TNF-α and IL-8 of the treated animals. The immunology enhancing activities in vitro and in vivo of MTCTP5 are similar to those of TP5, which is attributed to the fact that MTCTP5 up-regulates the plasma levels of IL-2 and CD4 as well as down-regulates the plasma level of CD8 of the treated animals. The plasma alanine transaminase, aspartate transaminase and creatinine assays indicate that MTCTP5 therapy does not injure the liver and the kidney of the animals. The survival time of MTCTP5 treated mice is significantly longer than that of TP5 treated mice.

By docking 126 derivatives of β-carboline-3-carboxylic acid, tetrahydro-β-carboline-3-carboxylic acid and indoloquinolizine into the active pocket of P-selectin (2-(3-(hydroxymethyl)-9H-pyrido[3,4-b]indol-1-yl)ethyl)-l-phenylalanine (HMCEF) was assigned a novel inhibitor. ELISA and flow cytometry experiments showed that HMCEF effectively down-regulated P-selectin expression and supported the rationality of the computer assistant screening, while UV spectrum experiments demonstrated that HMCEF directly bound to P-selectin. In vivo HMCEF dose dependently inhibited the rats and mice to form thrombus and had a minimal effective dose of 20 nmol/kg, dose dependently inhibited inflammatory response of mice and had a minimal effective dose of 20 nmol/kg. The decrease of serum TNFα and IL-8 of the treated mice was proposed to be the action mechanism of HMCEF inhibiting thrombosis and inflammation. All data imply that HMCEF is a novel lead of P-selectin inhibitor.

Based on the knowledge that cyclohexane-1,4-dione, piperazine and β-carboline are the essential building blocks of DNA intercalators, β-carboline-3-carboxylic acid is a π–π stack-like DNA intercalator, and β-carboline derivatives can form nanoparticles, this paper hypothesized that (2′S,5′S)-tetrahydropyrazino[1′,2′:1,6]- di{2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole}-1′,4′-dione (THPDTPI) would be a π–π stacking lead nano-intercalator. The docking investigation found that THPDTPI can intercalate into DNA in a π–π stacking manner. The simple condensation of 3S-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid provided THPDTPI in good yield and high purity. The TEM, SEM and AFM imaging visualized that THPDTPI formed nanoparticles in ultrapure water, in the solid state and in rat plasma. The Faraday–Tyndall effect proved that THPDTPI exhibited nano-properties in pH 2.0 and pH 7.0 water. Spectrophotometric assays suggested that the interaction model of THPDTPI and CT DNA was π–π stacking intercalation. In vivo THPDTPI dose-dependently slowed the tumor growth of S180 mice with a minimal effective dose of 0.01 μmol kg−1 per day. In vitro THPDTPI exhibited anti-proliferation activities on S180 and HeLa cells with IC50 values of 0.39 and 3.5 μM, respectively. Even when the single dose was raised up to 10000 fold of the minimal effective dose, i.e. 100 μmol kg−1, THPDTPI still did not show liver, kidney and systemic toxicity in mice. These findings provide a strategy for designing THPDTPI-like π–π stacking nano-intercalators.

Recurrent ischemic events in Chinese patients with symptomatic extracranial or intracranial stenosis caused by aspirin or clopidogrel resistance are well known. We aimed to identify the contribution of genetic variants to the events.Patients with symptomatic extracranial or intracranial stenosis receiving dual antiplatelet treatment for at least 5 days were enrolled in this study. The primary endpoint was a composite of ischemic events, including recurrent transient ischemic attack, stroke, myocardial infarction, and vascular-related mortality. Twenty-four single nucleotide polymorphisms (SNPs) were assessed and genotyped. The clinical characteristics of enrolled patients were collected from medical records. The influence of genetic polymorphisms on the recurrent ischemic events of the patients was examined.A total of 377 patients were included. During a 12-month follow-up, the composite primary endpoint was observed in 64 patients. The CYP2C19*3 (rs4986893) may increase the occurrence of the primary composite endpoint (OR = 2.56, 95 % CI = 1.29–5.10, P = 0.007), and the mutation of CES1 rs8192950 was associated with the decreased recurrence of ischemic events (OR = 0.53, 95 % CI = 0.30–0.94, P = 0.029). The other SNPs that were tested did not have statistically significant associations with the composite endpoint.For Chinese patients with symptomatic extracranial or intracranial stenosis treated with clopidogrel, CYP2C19*3 mutation was associated with an increased risk of ischemic events, and the mutation of rs8192950 in CES1 is associated with a decreased risk of recurrent ischemic events. Testing these two SNPs could be of value in the identification of patients at risk for recurrent ischemic events.

A nonviral tumor targeting vector for siRNA transfer is of importance. Here, a novel delivery system consisting of a covalent conjugate of NDCO-RGDS and VEGF-siRNA, NDCO-RGDS/VEGF-siRNA, was presented. In vitro, NDCO-RGDS/VEGF-siRNA released and transferred VEGF-siRNA in a long-acting manner. Compared to the control, NDCO-RGDS/VEGF-siRNA decreased the expression of VEGF mRNA and protein in HeLa cells by 88.41 ± 3.49% and 83.94 ± 2.00%, respectively. In vivo, NDCO-RGDS/VEGF-siRNA exhibited gene silencing and slowed tumor growth. FT-MS spectrum analysis revealed that NDCO-RGDS/VEGF-siRNA mainly distributed in tumor tissue of the treated S180 mice. Therefore NDCO-RGDS could be considered a promising nonviral tumor-targeting vector for siRNA transfer in tumor therapy.

Dexamethasone (Dex) is one of the most effective anti-inflammatory glucocorticoids, while the side effect, osteoporosis seriously limits its clinical use. Cell adhesion is involved in the onset of inflammation and osteoporosis, and RGD-peptides are well known as anti-adhesion peptides. To enhance the anti-inflammatory efficacy and limit the osteoporotic risk of Dex three novel conjugates of RGDV, RGDS and RGDF covalently modified Dex are presented here. For the xylene-induced ear edema model the ear edema of the mice treated with the conjugates was significantly lower than that of the mice treated with Dex. Receiving 15 day therapy the total volumetric bonemineral density, the peripheral quantitative CT and the femur weight of the mice treated with the conjugates were significantly higher than those of the mice treated with Dex. Therefore covalently modifying Dex with RGDV, RGDS and RGDF not only increased the anti-inflammatory activity but also decreased the osteoporotic risk of Dex. In addition, the enhanced anti-inflammatory activity was correlated with the down-regulated DNA expression of the conjugates.

Compound 6, a novel isoquinoline comprising two isoquinoline-3-carboxylic acids and a benzoic acid conjugated together using tris(2-aminoethyl)amine, was synthesized and tested for anti-tumor activity. In vivo evaluations found 6 to be well tolerated, of high therapeutic efficacy and of low systemic toxicity, at effective doses. The results suggest 6 to be a promising lead for future study, and the use of multiple isoquinoline-3-carboxylic acid moieties as pharmacophores in the same molecule to be a useful strategy for the design of anti-tumor drugs.Compound 6, a novel isoquinoline comprising two isoquinoline-3-carboxylic acids and a benzoic acid conjugated together using tris(2-aminoethyl)amine, was synthesized and tested for anti-tumor activity. In vivo evaluations found 6 to be well tolerated, of high therapeutic efficacy and of low systemic toxicity, at effective doses. The results suggest 6 to be a promising lead for future study, and the use of multiple isoquinoline-3-carboxylic acid moieties as pharmacophores in the same molecule to be a useful strategy for the design of anti-tumor drugs.

Inflammation has a critical role in the tumor progression, free radical damage can worse the status of patients in cancer condition. The anti-cancer agents capable of inhibiting inflammation and scavenging free radicals attract a lot of our interest. Aimed at the discovery of such anti-tumor agent, a novel intercalator, benzyl 1-[4-hydroxy-3-(methoxycarbonyl)-phenyl-9H-pyrido[3,4-b]indole-3-carboxylate (BPIC) was presented. The docking investigation of BPIC and doxorubicin towards the DNA (PDB ID: 1NAB) gave equal score and similar feature. The anti-proliferation assay of 8 cancer cells identified S180 cells had equal sensitivity to BPIC and doxorubicin. The anti-tumor assay defined the efficacy of BPIC been 2 folds higher than that of doxorubicin. At 1 μmol/kg of dose BPIC effectively inhibited xylene-induced ear edema and decreased the plasma TNF-α and IL-8 of the mice. BPIC scavenged OH, O2− and NO free radicals in a concentration dependent manner and NO free radicals had the highest sensitivity. BPIC could be a novel anti-tumor lead capable of simultaneously inhibiting inflammation and scavenging free radicals.

The inhibition of urokinase-PA (u-PA) can block a series of pathological events, such as adhesion, migration and growth of malignant cells at the metastatic site. To discover new inhibitors the SS, RR, RS and SR of 3-(4-aminobutyl)-6-(1H-indole-3-ylmethyl)piperazine-2,5-dione (AIPZ) were designed, synthesized and evaluated. HPLC with a chiral column shows that their purity approaches 100%. The docking of these analogs toward the active site of u-PA resulted in high scores (118–134). The FT-MS spectra indicate that in aqueous solution the SS, RR, RS and SR of AIPZ exist as dimer, tetramer, trimer and trimer, respectively. The ROESY 2D NMR spectra give dimer, tetramer, trimer and trimer distinct conformations. The transmission electron microscopy and scanning electron microscopy show that the SS, RR, RS and SR can form distinct nano-species. The conversion of the plasminogen towards plasmin induced by UK is configuration-dependent, blocked by the SS, RR, RS and SR. The invasion and migration of HCCLM3 cells are configuration-dependent, inhibited by the SS, RR, RS and SR. The RR was selected as a representative for in vivo evaluation. At 5 μmol kg−1 of oral dose, RR not only blocked the metastasis of Lewis lung carcinoma cells towards the lung, but also slowed the growth of the primary solid tumors initiated either by S180 ascite tumor cells or by Lewis lung carcinoma cells, and it also prevented the mice from developing xylene-induced ear edema.

Through a modular ROMP (ring-opening metathesis polymerization) strategy, a random copolymer with anti-thrombotic activity and imaging capability has been constructed from RGD, rhodamine B and PEG modified norbornene monomers. As we expected, these tri-component polynorbornenes exhibit significant enhancement in anti-thrombotic efficacy and bioavailability in vivo.

The mechanisms of small molecules forming nanospecies and the effect of the nanospecies of small molecules on their pharmacological actions remain to be elucidated. As one of our efforts here, a uPA inhibitor, (5aS,12S,14aS)-5,14-dioxo-12-(2-tryptophanylthreo-nylbenzylester-N-yl-ethyl-1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo[1,2:4,5]-pyrazino[1,2:1,6]pyrido[3,4-b]indole (CIPPCT) was presented. Energy-minimization, FT-MS and 2-D ROESY spectra defined CIPPCT taking -like conformation, and the intermolecular association drove CIPPCT to form a finger ring-like trimer. Images from transmission electron, scanning electron and atomic force microscopies consistently visualized that in aqueous solution at pH 6.7 and 10−10 M concentration, CIPPCT generally assembled nanoparticles of 9–67 nm in diameter. Mesoscale simulation demonstrated that a nanoparticle 9.4 nm in diameter contained 350 trimers. In vivo CIPPCT dose-dependently inhibited tumor growth in S180 mice. An ELISA assay confirmed that CIPPCT concentration-dependently downregulated serum uPA. The nanoparticles of CIPPCT are capable of occurring in mouse plasma and adhering on HeLa cells, and nanosized CIPPCT directly correlates the downregulation of uPA with inhibition of tumor growth.

In the most frequent cardiovascular events, deep vein thrombosis has been one of the most prominent causes of morbidity and mortality. Soluble P-selectin plays an essential role in the formation of thrombosis and has been considered a target of anti-thrombotic agents. With regard to molecular modeling, here the theoretical studies were performed on variant P-selectin structures. By removing SLex from PDB 1G1R or removing both SLex and PSGL-1 from PDB 1G1S, minimizing the energy of the formed structures, and performing molecular dynamics simulations, two average structures of P-selectins were obtained. A series of comparisons of the properties reflecting the quality of the two average structures, such as the backbone RMSD values, RMSF values, the change of the secondary structures, the conformation flexible regions and the correlated motions demonstrated that both of the two average structures are suitable and equally important for anti-thrombotic agent design.

The physical and chemical mechanisms of small molecules with pharmacological activity forming nanostructures are developing into a new field of nanomedicine. By using ROESY 2D NMR spectroscopy, tandem mass spectroscopy, transmission electron microscopy, and computer-assisted molecular modeling, this paper demonstrates the contribution of the folded conformation, the intra- and intermolecular π–π stacking, the intra- and intermolecular hydrogen bonds, and the receptor binding free energy of 6-dimethylaminonaph-2-yl-{N-S-[1-benzylcarba-moyl-4-(2-chloroacetamidobutyl)]-carboxamide (YW3-56) to the rapid formation of nanorings and the slow formation of nanocapsules. Thus we have developed a strategy that makes it possible to elucidate the physical and chemical mechanisms of bioactive small molecules forming nanostructures.

Resistance and nonresponse to aspirin dramatically decreases its therapeutic efficacy. To overcome this issue, a small-molecule thrombus-targeting drug delivery system, aspirin-Arg-Gly-Asp-Val (A-RGDV), is developed by covalently linking Arg-Gly-Asp-Val tetrapeptide with aspirin. The 2D ROESY NMR and ESI-MS spectra support a molecular model of an A-RGDV tetramer. Transmission electron microscopy images suggest that the tetramer spontaneously assembles to nanoparticles (ranging from 5 to 50 nm in diameter) in water. Scanning electron microscopy images and atomic force microscopy images indicate that the smaller nanoparticles of A-RGDV further assemble to bigger particles that are stable in rat blood. The delivery investigation implies that in rat blood A-RGDV is able to keep its molecular integrity, while in a thrombus it releases aspirin. The in vitro antiplatelet aggregation assay suggests that A-RGDV selectively inhibits arachidonic acid induced platelet aggregation. The mechanisms of action probably include releasing aspirin, modifying cyclic oxidase, and decreasing the expression of GPIIb/IIIa. The in vivo assay demonstrates that the effective antithrombotic dose of A-RGDV is 16700-fold lower than the nonresponsive dose of aspirin.Keywords: aspirin; cyclooxygenase; GPIIb/IIIa; nanomedicine; RGDV; thrombus

Platelet surface glycoproteins P-selectin and GPIIb/IIIa are implicated in the formation of platelet–fibrin–leukocyte thrombus and platelet–fibrin–platelet thrombus, respectively. In the current study, taking N-(3S-tetrahydroisoquinoline-3-carbonyl)-Thr-Ala-Arg-Gly-Asp-(Phe)-Phe (IQCA-TAFF) as a model compound, the molecular modeling, synthesis, and an evaluation system for a novel anti-thrombotic agent were investigated. The synthesis of IQCA-TAFF was achieved by coupling 3S-tetrahydro-isoquinoline-3-carboxylic acid (IQCA) and Thr-Ala-Arg-Gly-Asp(Phe)-Phe (TAFF). The molecular modeling indicated that IQCA-TAFF was able to occupy the active site pocket of P-selectin with its IQCA moiety and to block GPIIb/IIIa fibrinogen-binding sites with its TAFF moiety, respectively. These are consistent with the dual inhibition of the expressions of P-selectin and GPIIb/IIIa, and with the in vitro anti-platelet aggregation activity of IQCA-TAFF. Besides, the dual suppression of P-selectin and GPIIb/IIIa leads to significant in vivo efficacy of IQCA-TAFF, 500-fold higher than those of IQCA and TAFF, respectively. Transmission electron microscopy (TEM) images indicated that in water, IQCA-TAFF concentration-dependently formed nano-globes. The molecular modeling, in vitro bioassay, in vivo bioassay, action mechanism investigation, and nano-image visualization together constitute a model system to characterize the anti-thrombotic agent capable of simultaneously inhibiting P-selectin and GPIIb/IIIa mediated thrombosis.

To increase the metal selectivity of polyaspartic acid, a so-called green chelant, poly-α,β-dl-aspartyl-l-methionine (PDM) was synthesized as a novel lead chelating agent. The phosphoric acid (80%) catalyzed thermal poly condensation of dl-aspartic acid provided poly succinimide, which was amidated with l-methionine to form PDM (MW: 29161). At the doses of 0.1, 1.0, and 10.0 nmol/kg, either by intraperitoneal injection (i.p.) or oral administration, PDM removed Pb from the spleens, hearts, and kidneys of mice, especially dose-dependently decreasing the accumulation of Pb in the brains, livers, and femurs of the mice, and did not interfere with the essential metals, including Cu, Fe, Mn, and Ca. Even at the dose of 0.1 nmol/kg, the i.p. injection of PDM removed Pb from the spleens, hearts, and kidneys of mice and increased the amount of urinary volume and urinary Pb, and the amount of fecal matter and the amount of fecal Pb, resulting in effective removal of Pb from the body of mice given Pb by i.p. injection. Our findings revealed that in aqueous solution PDM formed diverse nanospecies.

Poly-α,β-dl-aspartic acid is known as a green chelant of various metal ions. To provide a novel nanochelant for treating Pb(II) poisoning, poly-α,β-dl-aspartic acid was modified with l-Cys to form poly-α,β-dl-aspartyl-l-cysteine (PDC; MW, 27273). dl-Asp was converted into polysuccinimide through a thermal polycondensation, and the amidation of polysuccinimide with l-Cys provided PDC. In water, PDC formed various porous nanospecies. In the mouse lead intoxication model, both intraperitoneal and oral administration of PDC (0.1, 1.0, and 10.0 nmol/kg) dose dependently removed Pb(II) accumulated in the organ, bone, and blood. PDC did not remove the essential metals including Cu2+, Fe2+, Mn2+, Zn2+, and Ca2+ of the treated mice. The porous feature and size of the pH- and concentration-dependent nanospecies of PDC benefited the removal of Pb(II).

To clarify the relationships of the commonly used spectral measurements and the in vivo activity of β-carboline intercalators N-[2(3-carboxyl-9-benzyl-carboline-1-yl)ethyl-1-yl]-aspartic acid (3a), -tyrosine (3b), -serine (3c), and -threonine (3d) were prepared as the model compounds by using a three-step procedure. In UV measurements of calf thymus DNA (CT DNA) the in vivo active 3a–c induced both hypochromic effect and hypsochromic shift, while the in vivo inactive 3d did not. In CD measurements the in vivo active 3a–c changed the intensities of both positive and negative bands of CT DNA in same direction, while the in vivo inactive 3d induced an opposite change. In fluorescence measurements due to adding CT DNA the increase of the fluorescence intensities of 3a–c were 10–31% higher than that of 3d, and the bathochromic shifts of 3a–c were 2–6 nm higher than that of 3d. The viscositic experiments revealed that 3d possessed the lowest binding ratio. The melting curves indicate that the effect of 3a–c (ΔTm, 10.8–12.9°C) on the Tm of CT DNA was significantly higher than that of 3d (ΔTm, 4.9°C). Spectroscopic, viscositic, and melting curve investigations are not only able to provide multiple evidences for DNA intercalation mechanism, but also are able to distinguish the in vivo active and inactive N-[2(3-carboxyl-9-benzyl-carboline-1-yl)-ethyl-1-yl]-amino acids.

{2-[1-(3-Methoxycarbonylmethyl-1H-indol-2-yl)-1-methyl-ethyl]-1H-indol-3-yl}-acetic acid methyl ester (MIAM) was provided as a DNA-intercalator. For the comprehensive evaluation of this new intercalator, an assay system consisting of cell, S180 mouse, healthy mouse, spectrum, non-spectrum, and gel electrophoresis models was constructed. On the cell (S180, K562, MCF-7, HeLa and HepG2) models, MIAM selectively inhibited the viability of HeLa. On the S180 mouse model, 0.89, 8.9, 89 and 890 μmol kg−1 of MIAM dose-dependently inhibited the tumor growth. Even at a dose of 890 μmol kg−1, MIAM did not damage the treated S180 mice. The safety of MIAM was supported by a high spleen index and an obvious increase of body weight of the treated S180 mice. On the healthy mouse model the LD50 value of MIAM is higher than 890 μmol kg−1. The ultraviolet (UV), fluorescence, circular dichroism (CD), relative viscosity, melting curve, and gel electrophoresis assays of DNA with or without MIAM consistently supported an intercalation mechanism for MIAM.

Nitronyl nitroxides are capable of preventing cells, tissues, and organs from radical-induced damage through scavenging NO˙, ˙O2− and ˙OH. In order to explore the conversions of nitronyl nitroxides in biological systems with and without NO˙, HPLC-MS aided PC12 cell systems were developed, and the conversions of 2-(3′-nitrophenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl -3-oxide (3-nitro-PTIO), 1-oxyl-2-(3′-nitrophenyl)-4,4,5,5-tetramethylimidazoline (3-nitro-PTI), and 1-hydroxyl-2-(3′-nitrophenyl)-4,4,5,5-tetramethylimidazoline (3-nitro-PTIH) were quantitatively monitored. In these systems 3-nitro-PTIO and 3-nitro-PTI were time-dependently converted to 3-nitro-PTIH, while no conversion of 3-nitro-PTIH was detected. Free radical NO˙ donors (sodium nitroprusside, SNP) accelerated the conversions, but had no effect upon the conversion product. In the in vitro and in vivo assays the 3-nitro-PTIH treated cells and mice exhibited no toxic response.

The coupling of the 1-carboxyl of DMSA with l-amino acids led to a class of novel 1-(carbonyl-l-amino-acid)-2,3-dimercaptosuccinic acids (DMSA–amino acid conjugates, DMSA-Gly, -Ser, -Val, -Leu, -Ile, -Asn, -Asp, -Gln, -Glu, -Met, -Phe, and -Trp). In the in vivo evaluation of Pb-loaded mice, 0.4 mmol/kg of the conjugates effectively decreased the Pb levels of the femur, brain, kidney, liver, and blood, greatly enhanced urination, and increased the Pb levels of both urine and feces, while causing no redistributions of Pb to the other organs, especially to the brain. With respect to lowering the bone and brain Pb, DMSA-Ile, -Asn, -Gln, and -Met were more effective than DMSA. This benefit was attributed to their high transmembrane ability. In contrast to Pb, the essential metals such as Fe, Cu, Zn, and Ca of the treated mice were not affected by the administration of the conjugates. Silico molecular modeling predicted that the conjugates had little hepatotoxicity, except possibly for DMSA-Phe.

By linking the mercapto groups with isopropyl and introducing l-amino acid into the 5-carboxyl of DMSA a class of novel 5-(1-carbonyl-l-amino-acid)-2,2- dimethyl-[1,3]dithiolane-4-carboxylic acids were prepared. Their in vivo activities were evaluated on lead loaded mice at the dose of 0.4 mmol/kg. The results showed that the lead levels of the livers, kidneys, femurs and brains in particular could be efficiently decreased by 0.4 mmol/kg of 5-(1-carbonyl-l-amino-acid)-2,2-dimethyl-[1,3]dithiolane-4-carboxylic acids. The benefit of 5-(1-carbonyl-l-amino-acid)-2,2-dimethyl-[1,3]dithiolane-4-carboxylic acids to the detoxification of the brain lead was attributed to their transmembrane ability. Compared with the lead detoxification efficacy, they did not affect the essential metals such as Fe, Cu, Zn, and Ca of the treated mice. Silico molecular modeling predicted that 5-(1-carbonyl-l-amino-acid)-2,2-dimethyl-[1,3]dithiolane-4-carboxylic acids had no hepatotoxicity.

The synthesis, bioassays and nano-structure characterization of Cu(II)-RGD-octapeptide complexes Cu(II)-Arg-Gly-Asp-Ser-Arg-Gly-Asp-Ser [Cu(II)-4a], Cu(II)-Arg-Gly-Asp-Val-Arg-Gly-Asp-Val [Cu(II)-4b] and Cu(II)-Arg-Gly-Asp-Phe-Arg-Gly-Asp-Phe [Cu(II)-4c] were investigated. UV-vis, CD and CD/ESI-MS spectra suggested that the coordination of Cu(II)-4a-c met a 3 N mode. In the in vitro anti-platelet aggregation assay the IC50 values of Cu(II)-RGD-octapeptide complexes were 10 − 110 folds lower than that of RGD-octapeptides. In the in vivo anti-thrombotic assay the effective dose of Cu(II)-RGD-octapeptide complexes was 5000 folds lower than that of RGD-octapeptides. In transmission electron microscopy measurement Cu(II)-4a-c offered distinct nano-images. The effect of the sequence on the in vitro anti-platelet aggregation/in vivo anti-thrombotic activity and the nano-structure of Cu(II)-4a-c was discussed.From the Clinical EditorThis basic science paper discusses the synthesis, coordination mode, in vitro anti-platelet aggregation and in vivo anti-thrombotic evaluation of novel Cu(II)-RGD-octapeptides.Four Cu(II) complexes of RGD-octapeptides with different sequence are prepared. The coordination of Cu(II) ions with octapeptides was confirmed by UV-vis and CD spectra, while the 3 N coordination mode of Cu(II) complexes was further characterized by CD/ESI-MS spectra. Transmission electron microscopy measurements of Cu(II) complexes in water in vitro anti-platelet aggregation and in vivo anti-thrombotic assays indicate that the activities of coordination complexes Cu(II) complexes are at most 110-fold and at least 5000-fold higher than that of corresponding octapetides, respectively.

To discover the anti-tumoral indoles a series of benzyl 1,2,3,5,11,11a-hexahydro-3,3-dimethyl-1-oxo-6H-imidazo[3′,4′:1,2]pyridin[3,4-b]indole-2-substituted acetates (2a–n) are prepared via one-pot-preparation. The IC50 values of 2a–n in vitro against human lung carcinoma, prostate cancer, nasopharyngeal carcinoma, vincristine-resistant KB subline and human breast carcinoma cells range from 40 nM to 60 μM. On Sarcoma 180 (S180) tumor-bearing mouse model four of them (2e,g,h,i) significantly inhibited the tumor growth. At the dose of 0.1 mg/kg the efficacy of the most potent 2h was equal to that of 1.0 mg/kg of doxorubicin. In contrast to doxorubicin, at 1.0 mg/kg of dose 2e,g,h,i did not induce the treated S180 mice to have organ atrophy and body emaciation. The healthy mice receiving 10, 100 and 500 mg/kg of 2e,g,h,i gave no any neurotoxic response. Even up to the dose of 500 mg/kg the healthy mice occurred no death. The interaction of 2a–n with DNA was confirmed by the fluorescence quenching experiments and automated flexible ligand docking. By 3D QSAR analysis the IC50 values of 2a–n against prostate cancer cells were correlated with the structures and conformations of their side chains. To increase the data related to their physical-chemical properties the experimental Log P values were also provided.

•Lipid rafts become the novel targets of anti-tumor liposomes' endocytosis.•Physiology based cell membrane traffic models of liposomes were established.•Intracellular trafficking of doxorubicin liposomes may be predicted.•Positive charge and fusogenic lipid promotes liposomal doxorubicin into nuclei.The clathrin-mediated endocytosis is likely a major mechanism of liposomes' internalization. A kinetic approach was used to assess the internalization mechanism of doxorubicin (Dox) loaded cationic liposomes and to establish physiology-based cell membrane traffic mathematic models. Lipid rafts-mediated endocytosis, including dynamin-dependent or -independent endocytosis of noncaveolar structure, was a dominant process. The mathematic models divided Dox loaded liposomes binding lipid rafts (B) into saturable binding (SB) and nonsaturable binding (NSB) followed by energy-driven endocytosis. The intracellular trafficking demonstrated early endosome-late endosome-lysosome or early/late endosome-cytoplasm-nucleus pathways. The three properties of liposome structures, i.e., cationic lipid, fusogenic lipid, and pegylation, were investigated to compare their contributions to cell membrane and intracellular traffic. The results revealed great contribution of cationic lipid DOTAP and fusogenic lipid DOPE to cell membrane binding and internalization. The valid Dox in the nuclei of HepG2 and A375 cells treated with cationic liposomes containing 40 mol% of DOPE were 1.2-fold and 1.5-fold higher than that in the nuclei of HepG2 and A375 cells treated with liposomes containing 20 mol% of DOPE, respectively, suggesting the dependence of cell type. This tendency was proportional to the increase of cell-associated total liposomal Dox. The mathematic models would be useful to predict intracellular trafficking of liposomal Dox.Figure optionsDownload full-size imageDownload high-quality image (245 K)Download as PowerPoint slide

Thrombosis is a serious threat to human health worldwide. Tetrahydroisoquinoline-3-carboxylic acid (IQCA) is an antithrombotic agent, while Thr-Ala-Arg-Gly-Asp(Ser)-Ser (TASS) can target thrombus. Herein, tetrahydro-isoquinoline-3-carbonyl-Thr-Ala-Arg-Gly-Asp(Ser)-Ser (IQCA-TASS) was designed with the aim towards the discovery of a nano-delivery system for targeting thrombus. In vitro, IQCA-TASS acted on P-selectin and down-regulated P-selectin expression. The IC50 values of IQCA-TASS against the platelet aggregation induced by four aggregators were less than 0.45 nM. In vivo, IQCA-TASS targeted thrombus, released IQCA and TASS inside the thrombus, showed dose-dependent anti-thrombotic action, of which the minimal effective dose was 1 nmol kg−1, and showed anti-inflammatory action. Even with the dose up to 1 μmol kg−1, a dose of 1000 times the minimal effective dose, IQCA-TASS still induced no toxic reaction. In rat plasma, IQCA-TASS formed nanoparticles with diameters of less than 41 nm. The interactions of the nanoparticles with both resting and activated platelets were imaged. IQCA-TASS should be a safe nano-medicine capable of targeting thrombus and releasing anti-thrombotic/anti-inflammatory pharmacophores in disease sites.

By use of carboxylbutyryl as a linker, adriamycin (ADR) and cholyl-L-Lys (an anti-inflammatory agent) were covalently conjugated and Nα-cholyl-Nε-(N-carbonylpropionoadriamycin)-L-Lys (BCBALys) was constructed as a liver-targeting nano-delivery system to release cholyl-L-Lys and protect the liver from CCl4-induced injury. In ultrapure water and rat plasma, 10−6 M BCBALys formed nanoparticles of 42–231 nm in diameter and ∼116 nm in height. In a CCl4-injured mouse model, however, only 2 µmol kg−1 of BCBALys effectively protected the liver of the mice from injury, and the mouse liver histology showed no hepatic architecture loss and inflammatory cell infiltration. BCBALys selectively accumulated in the liver of CCl4-injured mice, but not in other vital organs, and released cholyl-L-Lys. These data demonstrated that BCBALys exhibited high efficacy for treating CCl4-induced liver injury in a targeted manner. The chemical mechanism of BCBALys nanoparticle formation and the pharmacological mechanism of BCBALys mouse liver protection from CCl4-induced injury were also revealed by experiments.

A nonviral tumor targeting vector for siRNA transfer is of importance. Here, a novel delivery system consisting of a covalent conjugate of NDCO-RGDS and VEGF-siRNA, NDCO-RGDS/VEGF-siRNA, was presented. In vitro, NDCO-RGDS/VEGF-siRNA released and transferred VEGF-siRNA in a long-acting manner. Compared to the control, NDCO-RGDS/VEGF-siRNA decreased the expression of VEGF mRNA and protein in HeLa cells by 88.41 ± 3.49% and 83.94 ± 2.00%, respectively. In vivo, NDCO-RGDS/VEGF-siRNA exhibited gene silencing and slowed tumor growth. FT-MS spectrum analysis revealed that NDCO-RGDS/VEGF-siRNA mainly distributed in tumor tissue of the treated S180 mice. Therefore NDCO-RGDS could be considered a promising nonviral tumor-targeting vector for siRNA transfer in tumor therapy.

To improve the therapeutic efficacy of cancer patients a novel conjugate of thymopentin (TP5) and (1S,3S)-1-methyl-tetrahydro-β-carboline-3-carboxylic acid (MTC) was presented. In water and mouse plasma MTCTP5 forms the nanoparticles of 14–139 nm in diameter, the suitable size for delivery in blood circulation. In mouse plasma MTCTP5 releases MTC, while in the presence of trypsin MTCTP5 releases MTC and TP5. On mouse and rat models the MTCTP5 dose dependently slows down the tumor growth, inhibits inflammatory response and blocks thrombosis. The anti-tumor activity as well as the anti-inflammation activity and anti-thrombotic activity of MTCTP5 are 100 fold and 10 fold higher than those of MTC, respectively, which are attributed to the fact that it down-regulates the plasma levels of TNF-α and IL-8 of the treated animals. The immunology enhancing activities in vitro and in vivo of MTCTP5 are similar to those of TP5, which is attributed to the fact that MTCTP5 up-regulates the plasma levels of IL-2 and CD4 as well as down-regulates the plasma level of CD8 of the treated animals. The plasma alanine transaminase, aspartate transaminase and creatinine assays indicate that MTCTP5 therapy does not injure the liver and the kidney of the animals. The survival time of MTCTP5 treated mice is significantly longer than that of TP5 treated mice.

Through a modular ROMP (ring-opening metathesis polymerization) strategy, a random copolymer with anti-thrombotic activity and imaging capability has been constructed from RGD, rhodamine B and PEG modified norbornene monomers. As we expected, these tri-component polynorbornenes exhibit significant enhancement in anti-thrombotic efficacy and bioavailability in vivo.