Shurui Zhao

Nanosized aspirin-Arg-Gly-Asp-Val: delivery of aspirin to thrombus by the target carrier Arg-Gly-Asp-Val tetrapeptide.

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.

Cu(2+)-RGDFRGDS: exploring the mechanism and high efficacy of the nanoparticle in antithrombotic therapy.

Thrombosis disease has been the leading cause of morbidity and mortality worldwide. In the discovery of antithrombotic agents, three complexes of Cu2+ and repetitive arginine-glycine-aspartic acid (RGD) sequences, 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 previously reported, of which Cu(II)-4a and Cu(II)-4c possessed the highest in vitro and in vivo activity, respectively. Transmission electron microscopy (TEM) images visualized that Cu(II)-4a and Cu(II)-4c formed nanoaggregates and nanoparticles, respectively. However, the details of the formation of the nanospecies complexes and of the mechanism for inhibiting thrombosis remain to be clarified. For this purpose, this study designed a novel complex of Cu(II) and the RGD octapeptide, Arg-Gly-Asp-Phe-Arg-Gly-Asp-Ser (RGDFRGDS), consisting of Arg-Gly-Asp-Phe of Cu(II)-4c and Arg-Gly-Asp-Ser of Cu(II)-4a, to colligate their biological and nanostructural benefits. In contrast with Cu(II)-4a, -4b, and -4c, Cu(II)-RGDFRGDS (Cu2+-FS) had high antiplatelet and antithrombotic activities, with the formed nanoparticles having a porous surface. Additionally, this paper evidenced the dimer had the basic structural unit of Cu2+-FS in water, theoretically simulated the formation of Cu2+-FS nanoparticles, and identified that Cu2+-FS activity in decreasing glycoprotein IIb/IIIa, P-selectin, and IL-8 was responsible for the antithrombotic action. Finally, adherence onto the surface and entry into the cytoplasm were considered the steps of a two-step model for the blocking of platelet activation by Cu2+-FS nanoparticles. Findings indicated that the antiplatelet aggregation activity of Cu2+-FS was 10–52 times higher than that of RGDFRGDS, while the effective dose for antithrombotic action was 5,000 times lower than that of RGDFRGDS.

Design, synthesis and evaluation of a novel π–π stacking nano-intercalator as an anti-tumor agent

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.

A novel lead of P-selectin inhibitor: Discovery, synthesis, bioassays and action mechanism.

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 20nmol/kg, dose dependently inhibited inflammatory response of mice and had a minimal effective dose of 20nmol/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.

Small molecule PZL318: forming fluorescent nanoparticles capable of tracing their interactions with cancer cells and activated platelets, slowing tumor growth and inhibiting thrombosis

Low selectivity of chemotherapy correlates with poor outcomes of cancer patients. To improve this issue, a novel agent, N-(1-[3-methoxycarbonyl-4-hydroxyphenyl]-β-carboline-3-carbonyl)-Trp-Lys-OBzl (PZL318), was reported here. The transmission electron microscopy, scanning electron microscopy, and atomic force microscopy images demonstrated that PZL318 can form nanoparticles. Fluorescent and confocal images visualized that PZL318 formed fluorescent nanoparticles capable of targeting cancer cells and tracing their interactions with cancer cells. In vitro, 40 μM of PZL318 inhibited the proliferation of tumorigenic cells, but not nontumorigenic cells. In vivo, 10 nmol/kg of PZL318 slowed the tumor growth of S180 mice and alleviated the thrombosis of ferric chloride-treated ICR mice, while 100 μmol/kg of PZL318 did not injure healthy mice and they exhibited no liver toxicity. By analyzing Fourier transform–mass spectrometry and rotating-frame Overhauser spectroscopy (ROESY) two-dimensional nuclear magnetic resonance spectra, the chemical mechanism of PZL318-forming trimers and nanoparticles was explored. By using mesoscale simulation, a nanoparticle of 3.01 nm in diameter was predicted containing 13 trimers. Scavenging free radicals, downregulating sP-selectin expression and intercalating toward DNA were correlated with the antitumor mechanism of PZL318.

IQCA-TASS: a nano-scaled P-selectin inhibitor capable of targeting thrombus and releasing IQCA/TARGD(S)S in vivo

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.

DHDMIQK(KAP): a novel nano-delivery system of dihydroxyl-tetrahydro-isoquinoline-3-carboxylic acid and KPAK towards the thrombus

Vascular thrombosis is a major risk of the onset of stroke and so novel therapeutic candidates have been attracting interest. In this context, here docking based computer assisted screening and mesoscale simulation were used to design N-[(S)-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-carbonyl]-Lys(Pro-Ala-Lys), DHDMIQK(KAP), for inhibiting P-selectin expression. In vitro, 1 nM of DHDMIQK(KAP) effectively down-regulated P-selectin expression. In water, in rat plasma and in the solid state DHDMIQK(KAP) formed nanoparticles of a size capable of suitable delivery in the blood circulation. FT-MS and NOESY 2D NMR spectra showed DHDMIQK(KAP) formed hexamers, identified the intermolecular interactions of the hexamer, and assigned the hexamer a butterfly like conformation. Transmission electron microscopy, scanning electron microscopy and atomic force microscopy (AFM) imaged DHDMIQK(KAP) forming size-suitable nanoparticles for safe delivery in the blood circulation. In particular, AFM images showed that the nanoparticles effectively adhered onto the surfaces of the platelets. In vivo DHDMIQK(KAP) lysed the thrombus and inhibited thrombosis with a minimal effective dose of 0.01 nmol kg-1. FT-MS spectrum analyses defined a specific distribution of DHDMIQK(KAP) in the thrombus, but not in the blood and vital organs. Therefore, DHDMIQK(KAP) should be a novel nano-delivery system of 6,7-dihydroxyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and KPAK to target the thrombus.

Tetrahydro-β-carboline-3-carboxyl-thymopentin: a nano-conjugate for releasing pharmacophores to treat tumor and complications

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.

Design and synthesis of nanoscaled IQCA-TAVV as a delivery system capable of antiplatelet activation, targeting arterial thrombus and releasing IQCA

Background Arterial thrombosis has been associated with a series of pathological conditions, and the discovery of arterial thrombosis inhibitor is of clinical importance. Methods By analyzing the pharmacophores of anti-platelet agents, thrombus targeting peptide and anti-thrombotic nano-systems 3S-1,2,3,4-tetrahydroisoquino-line-3-carbonyl-Thr-Ala-Arg-Gly-Asp(Val)-Val (IQCA-TAVV) was designed and prepared as a nano-scaled arterial thrombosis inhibitor. Results In vitro the nanoparticles of IQCA-TAVV were able to adhere onto the surface of activated platelets, attenuate activated platelets to extend pseudopodia and inhibit activated platelets to form aggregators. In vivo IQCA-TAVV targeted arterial thrombus, dose dependently inhibited arterial thrombosis with a 1 nmol/kg of minimal effective dose, and the activity waŝ1670 folds of that of aspirin. Conclusion IQCA-TAVV represented the design, preparation and application of nanomedicine capable of adhering on the surface of activated platelets, attenuating platelet activation, targeting arterial thrombus and inhibiting arterial thrombosis.

IQCA-TAVV: To explore the effect of P-selectin, GPIIb/IIIa, IL-2, IL-6 and IL-8 on deep venous thrombosis

Deep vein thrombosis (DVT) associates with considerable morbidity, functional disability and mortality. Due to the lack of suitable inhibitor the correlation of various factors in DVT onset remains unknown. In this context we analyzed the structure of anti-platelet aggregation agent, P-selectin down-regulator, GPIIb/IIIa down-regulator and anti-inflammatory agent, thereby designed N-(3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)- Thr-Ala-Arg-Gly-Asp(Val)-Val (IQCA-TAVV) as an inhibitor of DVT to receive evaluations. The docking predicted that IQCA-TAVV can target P-selectin and GPIIb/IIIa. The UV showed that IQCA-TAVV can act on P-selectin and GPIIb/IIIa. ELISA indicated that IQCA-TAVV concentration dependently inhibited activated platelets to express P-selectin and GPIIb/IIIa, and the minimal effective concentration was 1 nM. IC50 of IQCA-TAVV against platelet aggregation induced by arachidonic acid, adenosine diphosphate and platelet activating factor fell within a range of 0.13 nM to 0.30 nM. In vivo IQCA-TAVV dose-dependently inhibited venous thrombosis and the minimal effective dose was 1 nmol/kg. On ear edema model the anti-inflammation activity of 10 nmol/kg IQCA-TAVV equaled that of 1.1mmol/kg aspirin. The concentration of IL-2, IL-6 and IL-8 in the serum of the ear edema mice were also significantly decreased by 10 nmol/kg IQCA-TAVV. Even at 1 μmol/kg of dose IQCA-TAVV still did not injure the kidney, the liver, and the nerves of healthy mice. Thereby IQCA-TAVV depicts a relationship of three levels (inhibiting platelet activation, targeting externalized membrane receptor, decreasing serum inflammatory factor) for the down-regulation of P-selectin, GPIIb/IIIa, IL-2, IL-6 and IL-8 in DVT.