Polymeric fluorescent heparin as one-step FRET substrate of human heparanase.

Heparanase, an endo-ß-D-glucuronidase, cleaves cell surface and extracellular matrix heparan sulfate (HS) chains and plays important roles in cellular growth and metastasis. Heparanase assays reported to-date are labor intensive, complex and/or expensive. A simpler assay is critically needed to understand the myriad roles of heparanase. We reasoned that fluorescent heparin could serve as an effective probe of heparanase levels. Following synthesis and screening, a heparin preparation labeled with DABCYL and EDANS was identified, which exhibited a characteristic increase in signal following cleavage by human heparanase. This work describes the synthesis of this heparin substrate, its kinetic and spectrofluorometric properties, optimization of the heparanase assay, use of the assay in inhibitor screening, and elucidation of the state of heparanase in different cell lines. Our FRET-based assay is much simpler and more robust than all assays reported in the literature as well as a commercially available kit.

Hierarchical self-assembly of heparin-PEG end-capped porous silica as a redox sensitive nanocarrier for doxorubicin delivery.

Porous nanosilica (PNS) has been attracting a great attention in fabrication carriers for drug delivery system (DDS). However, unmodified PNS-based carriers exhibited the initial burst release of loaded bioactive molecules, which may limit their potential clinical application. In this study, the surface of PNS was conjugated with adamantylamine (A) via disulfide bonds (PNS-SS-A) which was functionalized with cyclodextrin-heparin-polyethylene glycol (CD-HPEG) for redox triggered doxorubicin (DOX) delivery. The modified PNS was successfully formed with spherical shape and diameter around 50nm determined by transmission electron microscopy (TEM). DOX was efficiently trapped in the PNS-SS-A@CD-HPEG and slowly released in phosphate buffered saline (PBS) without any initial burst effect. Importantly, the release of DOX was triggered due to the cleavage of the disulfide bonds in the presence of dithiothreitol (DTT). In addition, the MTT assay data showed that PNS-SS-A@CD-HPEG was a biocompatible nanocarrier and reduced the toxicity of DOX. These results demonstrated that PNS-SS-A@CD-HPEG has great potential as a novel nanocarrier for anticancer drug in cancer therapy.

Antitumor and Antimetastasis Activities of Heparin-based Micelle Served As Both Carrier and Drug.

Effective treatments for tumors are not easy to achieve due to the existence of metastases, which are responsible for most tumor death. Hence, a new drug delivery system is a pressing need, which should be biocompatible, stimuli-responsive, and multifunctional, including antitumor, antimetastasis, and antiangiogenesis effects. However, it is challenging to achieve all of these properties in one drug delivery system. Here, we developed a system of drug DOX and heparin into one self-assemble nanoparticle via pH-sensitive hydrazone bond and hydrophobic groups, deoxycholate. In the process, heparin itself was not only as the hydrophilic segments of the carrier, but also processed multiple biological functions such as antiangiogenesis and antimetastasis effect. The micelle nanoparticle HD-DOX processed good stability and acidic pH-triggered drug release property. After systemic administration, heparin-based micelle nanoparticle showed longer half-time and enhanced accumulation of DOX in tumors through the enhanced permeability and retention effect, leading to more efficient antitumor effects. In addition, heparin could hinder platelet-induced tumor cells epithelial-mesenchymal transition (EMT) and partially affect cell actin cytoskeletal arrangement, resulting in the disorganization of the actin cytoskeleton. Therefore, HD-DOX exhibited significant inhibitory effect on the metastasis in melanoma animal model in C57BL/6 mouse. Meanwhile, benefited from the antiangiogenesis effect of heparin, tube formations in endothelial cells were effectively inhibited and tumor vascular density was decreased by HD-DOX. Taken together, our study developed a self-assembly nanoplatform that both the drug and carrier had therapeutic effects with ideal antitumor efficacy.

Single-step synthesis of heparin-doped polypyrrole nanoparticles for delivery of angiogenic factor.

AIM: To perform one-pot synthesis of heparin-immobilized polypyrrole (PPy) nanoparticles and evaluate the use of these nanoparticles for the delivery of VEGF. MATERIALS & METHODS: Heparin-stabilized synthesis of PPy nanoparticles was performed via oxidative polymerization. VEGF-bound PPy-heparin nanoparticles were delivered to endothelial cells and bioactivity of VEGF was assessed by Matrigel tube formation. RESULTS: Size-controllable synthesis of heparin-doped PPy nanoparticles was achieved, and heparin promoted the conjugation of VEGF. Angiogenic activity of the VEGF-conjugated PPy nanoparticles was verified. CONCLUSION: Heparin-doped PPy nanoparticles can be synthesized using one-pot reaction and provide a delivery platform by which VEGF can be conjugated onto.

The synthesis and application of heparin-based smart drug carrier.

Heparin based polymer drug which could self-assemble into sphere micelle in water was firstly prepared by grafting paclitaxel (PTX) into the hydroxyl of heparin via aconitic bond as pH sensitive spacer. Positive charged drug DOX·HCl and cationic folic acid (CFA) can be further loaded into the polymer drug via electrostatic interaction in aqueous solution so as to prepare smart drug carrier. The drug carrier was able to release more PTX and DOX at pH 4.8 than that at pH 7.4, exhibiting pH sensitivity for two drugs. Furthermore, tumor cell cytotoxicity test proved it possessed significant cytotoxicity against tumor cells MDA-MB-231 as well as its active tumor targeting ability resulting from the loading of CFA. Cellular uptake and intracellular distribution were further revealed by confocal laser scanning microscopy (CLSM). In conclusion, this paper not only provided a simple strategy but also indicated heparin is a versatile platform for the design of smart drug carrier. The as-prepared drug carrier also showed promising potential in chemotherapy.

Heparina para desobstrução de cateter venoso central de inserção periférica no recém-nascido: estudo in vitro / Heparin for clearance of peripherally inserted central venous catheter in newborns: an in vitro study

Objetivo:Comparar a eficácia de duas concentrações de heparina para a desobstrução por coágulo do cateter venoso central de inserção periférica (CCIP) neonatal in vitro.Métodos:Estudo experimental in vitro quantitativo que usou 76 CCIPs neonatais de tamanho 2 French coagulados in vitro. Os cateteres foram divididos em dois grupos com 38 CCIPs cada. Ambos os grupos receberam infusão de heparina de baixo peso molecular, com dose de 25UI/mL no Grupo I e de 50UI/mL no Grupo II. Os cateteres de ambos os grupos foram submetidos à técnica de pressão negativa com cinco, 15 e 30 minutos e com quatro horas e testou-se sua permeabilidade. Usou-se a análise de sobrevivência para verificar o desfecho dos grupos conforme os intervalos de tempo.Resultados:A comparação dos dois grupos no intervalo de tempo de cinco minutos mostrou um número maior de desobstrução de cateteres no Grupo II (57,9%) em relação ao grupo 1 (21,1%). A análise de Kaplan Meier indicou menor tempo para desobstrução dos cateteres quando a heparina em maior concentração (50UI/mL) foi usada (p<0,001).Conclusões:O uso de heparina de baixo peso molecular na concentração de 50UI/mL foi mais eficaz na restauração da permeabilidade de CCIPs neonatais ocluídos in vitro por coágulo e situou-se tal concentração dentro da margem de segurança indicada na literatura científica.

Objective:To compare the efficacy of two concentrations of heparin to clear the lumen of in vitro clotted neonatal peripherally inserted central catheters (PICCs).Methods:This is an in vitro, experimental quantitative study of 76 neonatal 2.0-Fr PICCs coagulated in vitro. The catheters were divided into two groups of 38 PICCs each. In both groups an infusion of low molecular weight heparin was administered with a dose of 25IU/mL for Group 1 and 50IU/mL for Group 2. The negative pressure technique was applied to the catheters of both groups at 5, 15 and 30min and at 4h to test their permeability. Kaplan-Meier survival analysis was used to verify the outcome of the groups according to time intervals.Results:The comparison between both groups in the first 5min showed that more catheters from Group 2 were cleared compared to Group 1 (57.9 vs. 21.1%, respectively). Kaplan-Meier survival analysis showed that less time was needed to clear catheters treated with 50IU/mL of heparin (p<0.001).Conclusions:The use of low molecular weight heparin at a concentration of 50IU/mL was more effective in restoring the permeability of neonatal PICCs occluded in vitro by a clot, and the use of this concentration is within the safety margin indicated by scientific literature.

Size Controlled Heparin Fragment-Deoxycholic Acid Conjugate Showed Anticancer Property by Inhibiting VEGF165.

Heparin is a highly sulfated, long, and linear polysaccharide, which can inhibit tumor growth by interacting with growth factors such as bFGF and VEGF. Several researchers have shown the anti-angiogenic effect of heparin and its conjugates in relation to growth factor inhibition. For drug development and inhibition of growth factors using heparin conjugates, the molecular size of heparin may be crucial considering the size of the heparin binding site of growth factors. In this study, we synthesized heparin fragments and deoxycholic acid conjugated heparin fragments (HFD) to search for the optimal size-controlled conjugate that will inhibit the angiogenic effect of VEGF165. We have also shown that the HFDs could have an enhanced therapeutic effect in vitro and in vivo consequent to the molecular size control. HFDs have significant anti-angiogenic effects by blocking the angiogenic activity of VEGF165 depending on its molecular size. Among them, HFD2 was a promising candidate for oral angiogenesis inhibitor. These results suggest that size-controlled synthesis is necessary for heparin-based drug development.

Controlled release and gradient formation of human glial-cell derived neurotrophic factor from heparinated poly(ethylene glycol) microsphere-based scaffolds.

Introduction of spatial patterning of proteins, while retaining activity and releasability, is critical for the field of regenerative medicine. Reversible binding to heparin, which many biological molecules exhibit, is one potential pathway to achieve this goal. We have covalently bound heparin to poly(ethylene glycol) (PEG) microspheres to create useful spatial patterns of glial-cell derived human neurotrophic factor (GDNF) in scaffolds to promote peripheral nerve regeneration. Labeled GDNF was incubated with heparinated microspheres that were subsequently centrifuged into cylindrical scaffolds in distinct layers containing different concentrations of GDNF. The GDNF was then allowed to diffuse out of the scaffold, and release was tracked via fluorescent scanning confocal microscopy. The measured release profile was compared to predicted Fickian models. Solutions of reaction-diffusion equations suggested the concentrations of GDNF in each discrete layer that would result in a nearly linear concentration gradient over much of the length of the scaffold. The agreement between the predicted and measured GDNF concentration gradients was high. Multilayer scaffolds with different amounts of heparin and GDNF and different crosslinking densities allow the design of a wide variety of gradients and release kinetics. Additionally, fabrication is much simpler and more robust than typical gradient-forming systems due to the low viscosity of the microsphere solutions compared to gelating solutions, which can easily result in premature gelation or the trapping of air bubbles with a nerve guidance conduit. The microsphere-based method provides a framework for producing specific growth factor gradients in conduits designed to enhance nerve regeneration.

Enhanced 4T1 breast carcinoma anticancer activity by co-delivery of doxorubicin and curcumin with core-shell drug-carrier based on heparin modified poly(L-lactide) grafted polyethylenimine cationic nanoparticles.

Use of single chemotherapy agents has shown some limitations in anti-tumor treatment, such as development of drug resistance, severe adverse reactions and limited regime for therapeutic use. Combination of two or more therapeutic drugs is a feasible strategy to overcome these limitations. This paper reports study of co-delivery by core-shell nanoparticles (NPs) with hydrophobic PLLA core loaded with curcumin (Cur) and hydrophilic heparin shell adsorbing Doxorubicin (DOX). Characterizations of Cur-PEA NPs, Cur-PEA/heparin NPs and DOX adsorbing into Cur-PEA/heparin NPs (DOX-Cur NPs) were also investigated by transmission electron microscope (TEM) and Malvern Zetasizer. Studies on cellular uptake of DOX-Cur NPs demonstrated that both drugs were effectively taken up by 4T1 tumor cells. Furthermore, DOX-Cur NPs suppressed 4T1 tumor cells growth more efficiently than either DOX or Cur alone at the same concentrations, as measured by flow cytometry (FCM). We found out that intravenous injection of DOX-Cur NPs efficiently inhibited growth of subcutaneous 4T1 breast carcinoma in vivo (p < 0.01) and prolonged survival of the treated 4T1 breast carcinoma mice. Moreover, the pathological damage to the cardiac tissue in mice treated with DOX-Cur NPs was significantly less severe than that of mice treated with free DOX. This study suggested that DOX-Cur NPs may have promising applications in breast carcinoma therapy.

New heparin-indomethacin conjugate with an ester linkage: synthesis, self aggregation and drug delivery behavior.

New heparin-indomethacin conjugate with an ester linkage was prepared by the carbodiimide-mediated condensation reaction, and then characterized by FTIR and (1)HNMR analyses. Due to its amphiphilic character, such a conjugate could self-aggregate into spherical nanoparticles in aqueous system, as confirmed by fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. By the in vitro drug release tests, the resultant conjugate nanoparticles were found to have a sustained and esterase-sensitive release behavior for conjugated indomethacin. In addition, the uptake of these conjugate nanoparticles into human nasopharyngeal carcinoma CNE1 cells was confirmed by fluorescence microscopy.

Direct synthesis of heparin-like poly(ether sulfone) polymer and its blood compatibility.

In this study, heparin-like poly(ethersulfone) (HLPES) was synthesized by a combination of polycondensation and post-carboxylation methods, and was characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance hydrogen spectrum and gel permeation chromatography. Owing to the similar backbone structure, the synthesized HLPES could be directly blended with pristine PES at any ratios to prepare PES/HLPES membranes. After the introduction of HLPES, the microscopic structure of the modified PES membranes was changed, while the hydrophilicity was significantly enhanced. Bovine serum albumin and bovine serum fibrinogen adsorption, activated partial thromboplastin time, thromb time and platelet adhesion for the modified PES membranes were investigated. The results indicated that the blood compatibility of the PES/HLPES membranes was significantly improved compared with that of pristine PES membrane. For the PES/HLPES membranes, obvious decreases in platelet activation on PF-4 level, in complement activation on C3a and C5a levels, and in leukocytes activation on CD11b levels were observed compared with those for the pristine PES membrane. The improved blood compatibility of the PES/HLPES membrane might due to the existence of the hydrophilic groups (-SO3Na, -COONa). Furthermore, the modified PES membranes showed good cytocompatibility. Hepatocytes cultured on the PES/HLPES membranes presented improved growth in terms of SEM observation, MTT assay and confocal laser scanning microscope observation compared with those on the pristine PES membrane. These results indicate that the PES/HLPES membranes present great potential in blood-contact fields such as hemodialysis and bio-artificial liver supports.

Heparosan-derived heparan sulfate/heparin-like compounds: one kind of potential therapeutic agents.

Heparan sulfate (HS) is a highly sulfated glycosaminoglycan and exists in all animal tissues. HS and heparin are very similar, except that heparin has higher level of sulfation and higher content of iduronic acid. Despite the fact that it is a century-old drug, heparin remains as a top choice for treating thrombotic disorders. Pharmaceutical heparin is derived from porcine intestine or bovine lung via a long supply chain. This supply chain is vulnerable to the contamination of animal pathogens. Therefore, new methods for manufacturing heparin or heparin-like substances devoid of animal tissues have been explored by many researchers, among which, modifications of heparosan, the capsular polysaccharide of Escherichia coli K5 strain, is one of the promising approaches. Heparosan has a structure similar to unmodified backbone of natural HS and heparin. It is feasible to obtain HS or heparin derivatives by modifying heparosan with chemical or enzymatic methods. These derivatives display different biological activities, such as anticoagulant, anti-inflammatory, anticancer, and antiviral activities. This review focuses on the recent studies of synthesis, activity, and structure-activity relationship of HS/heparin-like derivatives prepared from heparosan.

Probing structural selectivity of synthetic heparin binding to Stabilin protein receptors.

As one of the most widely used drugs worldwide, heparin is an essential anticoagulant required for surgery, dialysis, treatment of thrombosis, cancer, and general circulatory management. Stabilin-2 is a scavenger clearance receptor with high expression in the sinusoidal endothelium of liver. It is believed that Stabilin-2 is the primary receptor for the clearance of unfractionated and low molecular weight heparins in the liver. Here, we identify the modifications and length of the heparin polymer that are required for binding and endocytosis by both human Stabilin receptors: Stabilin-2 and its homolog Stabilin-1 (also found in liver endothelium). Using enzymatically synthesized (35)S-labeled heparan sulfate oligomers, we identified that sulfation of the 3-OH position of N-sulfated glucosamine (GlcNS) is the most beneficial modification for binding and endocytosis via both Stabilin receptors. In addition, our data suggest that a decasaccharide is the minimal size for binding to the Stabilin receptors. These findings define the physical parameters of the heparin structure required for efficient clearance from blood circulation. These results will also aid in the design of synthetic heparins with desired clearance rates.

Programmable one-pot glycosylation.

In oligosaccharide synthesis, protecting groups, possible participating groups, promoters/catalysts, reaction conditions, and donor leaving groups and acceptors must all be carefully designed in order to generate the correct regio- and stereochemistry for the new glycosidic bond. Programmable one-pot synthesis has been developed to address the above problems. This strategy is based on the sequential use of thioglycoside building blocks to form glycosidic bonds based on the reactivity difference of the building blocks. The activation of the anomeric leaving group can be attenuated through modification of the protecting group strategy and neighboring group participation. This reactivity-based strategy has been applied to one-pot glycosylation reactions where a series of building blocks with identical leaving groups react sequentially in one vessel without laborious intermediate purification steps. It provides rapid access to oligosaccharides with a wide-range of molecular diversity. In this chapter we outline the recent development of this strategy that can be applied to synthesize a wide variety of oligosaccharides and glycoconjugates that are associated with infectious diseases or carbohydrate-based cancer antigens.

Nickel-catalyzed stereoselective glycosylation with C(2)-N-substituted benzylidene D-glucosamine and galactosamine trichloroacetimidates for the formation of 1,2-cis-2-amino glycosides. Applications to the synthesis of heparin disaccharides, GPI anchor pseudodisaccharides, and α-GalNAc.

The 1,2-cis-2-amino glycosides are key components found within a variety of biologically important oligosaccharides and glycopeptides. Although there are remarkable advances in the synthesis of 1,2-cis-2-amino glycosides, disadvantages of the current state-of-the-art methods include limited substrate scope, low yields, long reaction times, and anomeric mixtures. We have developed a novel method for the synthesis of 1,2-cis-2-amino glycosides via nickel-catalyzed α-selective glycosylation with C(2)-N-substituted benzylidene D-glucosamine and galactosamine trichloroacetimidates. These glycosyl donors are capable of coupling to a wide variety of alcohols to provide glycoconjugates in high yields with excellent levels of α-selectivity. Additionally, only a substoichiometric amount of nickel (5-10 mol %) is required for the reaction to occur at 25 °C. The current nickel method relies on the nature of the nickel-ligand complex to control the α-selectivity. The reactive sites of the nucleophiles or the nature of the protecting groups have little effect on the α-selectivity. This methodology has also been successfully applied to both disaccharide donors and acceptors to provide the corresponding oligosaccharides in high yields and α-selectivity. The efficacy of the nickel procedure has been further applied toward the preparation of heparin disaccharides, GPI anchor pseudodisaccharides, and α-GluNAc/GalNAc. Mechanistic studies suggest that the presence of the substituted benzylidene functionality at the C(2)-amino position of glycosyl donors is crucial for the high α-selectivity observed in the coupling products. Additionally, the α-orientation of the C(1)-trichloroacetimidate group on glycosyl donors is necessary for the coupling process to occur.

Synthesis and evaluation of heparin immobilized "side-on" to polystyrene microspheres coated with end-group activated polyethylene oxide.

Thiol (-SH) groups were introduced into unfractionated heparin by reaction of carboxyl groups in its uronic acid residues with 3,3'dithiobis(propanoic)hydrazide. Thiolated heparin derivatives were then linked to pyridyl disulfide-activated polyethylene oxide-polypropylene oxide-polyethylene oxide triblocks, which had previously been coated onto the surfaces of 1.15 microm polystyrene microspheres. The heparin immobilization reaction was monitored spectrophotometrically as colored pyridine-2-thione was released. In addition, the zeta potentials of uncoated, triblock-coated, and heparin-containing microsphere suspensions were recorded to demonstrate the successful attachment of heparin on the surface. However this "side-on" attachment of heparin to pendant, polyethylene oxide chains did not significantly increase the anticoagulant or anti-Factor Xa activity of microsphere suspensions.

Study on syntheses and anticoagulant action of heparin/rare earth nano-oxides hybrid material.

Four hybrid materials of RE2O3-TDI-Heparin (TDI=Toluene 2,4-diisocyanate, RE=La, Eu, Nd, Sc) were prepared by the method of graft. The materials were characterized by IR, TG, and SEM, which confirmed that the heparin was grafted on the surface of TDI modified rare earth nano-oxides. The cell adhesion experiment and the anticoagulant experiment demonstrated that the materials have lower cell toxicity, better cell adhesion as well as better anticoagulant action. In addition, the clotting time of hybrid materials were shortened compared with the heparin.

Heparin-paclitaxel conjugates as drug delivery system: synthesis, self-assembly property, drug release, and antitumor activity.

We have synthesized a series of novel prodrugs consisting of amphiphilic heparin-paclitaxel conjugates. Each prodrug in the series consists of a succinylated-heparin carrier conjugated to paclitaxel via a single amino acid spacer, either valine, leucine, or phenylalanine (prodrug1, prodrug2, and prodrug3, respectively). Unlike physically encapsulated drugs, these prodrugs can self-assemble to form nanoparticles in aqueous solution while still maintaining structural integrity for loading parent drug due to the dual hydrophilic/hydrophobic nature of the carrier and drug compound. The structure of prodrugs has been characterized by 1H NMR, FT-IR, and GPC. Their morphology has been investigated by SEM. Our results show that these self-assembled nanoparticles have a narrow size distribution (140-180 nm) and form an approximately spherical shape composed of a paclitaxel core and carrier shell. The anticoagulant activity of all the prodrugs is sharply decreased compared to that of heparin, as measured by activated partial thromboplastin time (aPTT), thereby reducing the risk of severe hemorrhagic complication during systemic administration. Furthermore, the prodrugs exhibit better in vitro cell inhibition for MCF-7 cells than free paclitaxel. Flow cytometric analyses (FCM) have shown that MCF-7 cells treated with prodrugs are arrested in the G(2)/M phase of the cell cycle. Meanwhile, these three prodrugs each exhibit unique hydrolysis properties under various physiological or plasma conditions. In particular, prodrug2 with leucine spacer may result in favorable hydrolysis of the ester bond between the amino acid and paclitaxel under physiological conditions. In mice, prodrug2 shows a similar ovarian tumor growth inhibition as paclitaxel and induces no obvious body weight loss. Hence, the prepared nanoscale prodrugs are expected not only to render structural integrity to the parent drug, but also enhance targeting capacity to solid tumors.

Heparin-paclitaxel conjugates using mixed anhydride as intermediate: synthesis, influence of polymer structure on drug release, anticoagulant activity and in vitro efficiency.

PURPOSE: The heparin-paclitaxel conjugates using amino acid as linker (HD2), with low anticoagulant activity, the similar anticancer activity as paclitaxel, offer great potential for further investigation. METHODS: Two types of heparin-paclitaxel conjugates (HD) have been developed, in which O-acetylated heparin as carrier conjugates with paclitaxel by direct ester bond (HD1) and by inserting different amino acids as spacers, including valine, leucine, and phenylalanine (HD2a, HD2b, and HD2c), respectively. Specifically, mixed anhydride groups of carrier as activating intermediates mediate the synthesis of prodrugs. The HD conjugates are characterized by (1)H NMR, FT-IR and GPC. The percentage weight of drug and hydrolysis rate for HD are detected by UV and HPLC. The anticoagulant activity and cell cycle of MCF-7 of HD are measured by APTT and FCM, respectively. RESULTS: HD2 conjugates show better solubility and faster hydrolysis rates than those of HD1. Meanwhile, the anticoagulant activity of HD is reduced and FCM analysis show that MCF-7 cells treated with HD are arrested in the G2/M phase of cell cycle. CONCLUSIONS: Amino acids as linkers between paclitaxel and carrier are appropriate to facilitate the release of paclitaxel from carrier. Mixed anhydrides mediate the synthesis of prodrugs and HD2 conjugates are expected to further investigate in vivo experiment.

Synthesis, characterization, and intracellular delivery of reducible heparin nanogels for apoptotic cell death.

Reducible heparin nanogels cross-linked with disulfide linkages were developed for efficient cellular uptake of therapeutic heparin to induce apoptotic cell death. The heparin nanogels were synthesized by forming nanocomplexes between thiolated heparin and poly(ethylene glycol) in a selected organic solvent, and subsequently producing intermolecular disulfide bonds between thiolated heparin molecules by ultrasonication. The resultant heparin nanogels had a stable structure with an average diameter of 248.7+/-26.8nm in aqueous solution. However, they rapidly disintegrated and released free heparin molecules under reductive environments, such as intracellular cytosol, through the cleavage of disulfide cross-links within their network structure. Confocal laser scanning microscopy and flow cytometric analysis revealed that these heparin nanogels significantly inhibited proliferation of mouse melanoma cells by inducing caspase-mediated apoptotic cell death. The present study suggested that the reducible heparin nanogels exhibiting a remarkable apoptotic activity could be potentially applied for cancer cell targeted delivery when combined with various therapeutic and diagnostic agents.