Preparation of sodium deoxycholate (DOC) conjugated heparin derivatives for inhibition of angiogenesis and cancer cell growth

We describe new DOC (sodium deoxycholate)-heparin nanoparticles for in vivo tumor targeting and inhibition of angiogenesis based on chemical conjugation and the enhanced permeability and retention (EPR) effect. Heparin has been used as a potent anticoagulant agent for 70 years, and has recently been found to inhibit the activity of growth factors which stimulate the smooth muscle cells around tumor. From the results, DOC and heparin were conjugated by bonding carboxyl groups of heparin with amine groups of aminated sodium deoxycholate. Larger antitumor effects of the DOC-heparin VI (8.5 mol of DOC coupled with 1.0 mol heparin) were achieved in animal studies, compared to heparin alone. We confirmed that the conjugated heparin retained its ability to inhibit binding with angiogenic factor, showing a significant decrease in endothelial tubular formation. These results provide new insights into the nontoxic anticancer drug carrier as well as the design of multifunctional bioconjugates for targeted drug delivery.     

Preparation and characterization of heparinized multi-walled carbon nanotubes

The characteristics of heparinized multiwalled carbon nanotubes (MWNTs) were investigated in terms of the activated partial thromboplastin time (APTT) to verify the heparin activity, a carbazole assay was done to measure the content of the immobilized heparin, and the octanol-water partition coefficient was assessed to determine the lipophilicity. Two heparin-immobilized MWNTs were prepared to evaluate their differences. The first preparation method involved polymer-coated MWNTs with heparin indirectly center-point-attached. In the second approach, heparin was directly end-point-attached through its reducing end onto acid-treated MWNTs. The blood compatibility of MWNTs to which heparin was end-point-attached through its reducing end was greatly enhanced compared to that of the MWNTs onto which heparin was center-point-attached. The APTT and carbazole assay results demonstrated that heparinized MWNTs prepared through end-point attachment result in prolonged plasma-based anticoagulant activity. The blood compatibility of MWNTs heparinized by end-point attachment was not decreased up to the fourth pasteurization. Heparinized MWNTs were also studied using octanol-water partition, which should be useful for exploring heparinized MWNTs as drug carriers including delivery systems. The results of octanol/water partition on the design of heparinized MWNTs prepared by end-point attachment with a specific binding can facilitate the design of drug delivery carriers with high blood compatibility.     

Zn2+ Mediates High Affinity Binding of Heparin to the αC Domain of Fibrinogen

The nonspecific binding of heparin to plasma proteins compromises its anticoagulant activity by reducing the amount of heparin available to bind antithrombin. In addition, interaction of heparin with fibrin promotes formation of a ternary heparin-thrombin-fibrin complex that protects fibrin-bound thrombin from inhibition by the heparin-antithrombin complex. Previous studies have shown that heparin binds the E domain of fibrinogen. The current investigation examines the role of Zn²⁺ in this interaction because Zn²⁺ is released locally by platelets and both heparin and fibrinogen bind the cation, resulting in greater protection from inhibition by antithrombin. Zn²⁺ promotes heparin binding to fibrinogen, as determined by chromatography, fluorescence, and surface plasmon resonance. Compared with intact fibrinogen, there is reduced heparin binding to fragment X, a clottable plasmin degradation product of fibrinogen. A monoclonal antibody directed against a portion of the fibrinogen αC domain removed by plasmin attenuates binding of heparin to fibrinogen and a peptide analog of this region binds heparin in a Zn²⁺-dependent fashion. These results indicate that the αC domain of fibrinogen harbors a Zn²⁺-dependent heparin binding site. As a consequence, heparin-catalyzed inhibition of factor Xa by antithrombin is compromised by fibrinogen to a greater extent when Zn²⁺ is present. These results reveal the mechanism by which Zn²⁺ augments the capacity of fibrinogen to impair the anticoagulant activity of heparin.     

肝素在肿瘤治疗中的应用

肝素作为传统抗凝剂已众所周知。研究发现,肝素尚具有多种生物学活性,特别是抗肿瘤作用备受学者关注。尽管临床上并未将肝素疗法作为一种常规抗肿瘤手段,但是许多研究已经证明了肝素能够抑制肿瘤细胞的侵袭与转移。本文综述肝素治疗肿瘤的主要机制以及肝素结构修饰在抗癌方面的应用。     

Novel, fluorescent, magnetic, polysaccharide-based microsphere for orientation, tracing, and anticoagulation: preparation and characterization

A fluorescent, magnetic composite poly(styrene-maleic anhydride) microsphere, suitable for conjugation with polysaccharide, was synthesized using magnetite/europium phthalate particles as seeds by copolymerization of styrene and maleic anhydride. The magnetite/europium phthalate particles were wrapped up by poly(ethylene glycol), which improved the affinity between the seed particles and the monomers. The composite microspheres obtained, with a diameter of 0.15-0.7 microm, contain 586-1013 microg of magnetite/g of microsphere and 0.5-16 mmol surface anhydride groups/g of microsphere. Heparin was conjugated with the reactive surface anhydride groups on the surface of the microspheres by covalent binding to obtain a fluorescent, magnetic, polysaccharide-based microsphere. The microspheres not only retain their bioactivities but also provide magnetic susceptibility and fluorescence. They can be used as a carrier with magnetic orientation and fluorescence tracer for potent drug targeting. The orientation, tracer, and anticoagulation of the fluorescence, magnetic, polysaccharide-based microspheres were studied. The anticoagulant activity of the microspheres and heparin binding capacity reached 54,212.8 U and 607.1 mg/g of dry microspheres. The activity recovery was 50.2%. The anticoagulant activity of the microspheres increases with the increase of the conjugated heparin on the surface of the microspheres and the decrease of the microsphere size. Furthermore, The fluorescent, magnetic, polysaccharide-based microspheres can be easily transported to a given position in a magnetic field and traced via their fluorescence.     

脂质体在抗癌药物给药系统中的研究和应用

癌症,是现今威胁人类健康的一大杀手,目前常规的治疗手段之一是予以大剂量的化疗药物进行治疗。但大多数抗癌药物因具有广泛而强烈的细胞毒性,在杀伤癌细胞的同时也无选择性的杀伤了正常人体细胞,使得患者在接受治疗的同时承受了较大的痛苦,降低了癌症患者的生存质量。因而在药剂学研究中,须以提高药效、增强靶向性及降低毒副作用等为目标,合理地选择和开发抗癌药物给药系统。自脂质体作为新型药物传递技术引入癌症治疗以来,因其独特的理化性质和递药机理,高效低毒地递送抗癌药物至病灶,因而成为现今抗癌药物给药系统研究中的热点。本文结合国内外的相关资料和最新报道,综述了脂质体抗癌药物的递药优势、研究进展与存在的问题,并在分析了产业化现状的基础上,对这一新型给药系统在抗癌药物递送领域中的发展做一展望。     

Targeting of herbal bioactives through folate receptors: a novel concept to enhance intracellular drug delivery in cancer therapy

Targeted drug delivery through folate receptor (FR) has emerged as a most biocompatible, target oriented, and non-immunogenic cargoes for the delivery of anticancer drugs. FRs are highly overexpressed in many tumor cells (like ovarian, lung, breast, kidney, brain, endometrial, and colon cancer), and targeting them through conjugates bearing specific ligand with encapsulated nanodrug moiety is undoubtedly, a promising approach toward tumor targeting. Folate, being an endogenous ligand, can be exploited well to affect various cellular events occurring during the progress of tumor, in a more natural and definite way. Thus, the aim of the review lies in summarizing the advancements taken place in the drug delivery system of different therapeutics through FRs and to refine its role as an endogenous ligand, in targeting of synthetic as well as natural bioactives. The review also provides an update on the patents received on the folate-based drug delivery system.     

Folate receptor targeted NIR cleavable liposomal delivery system augment penetration and therapeutic efficacy in breast cancer

BackgroundLiposomes are predominantly used sorts of nanocarriers for active a targeted delivery through surface functionalization using targeting ligand. The folate receptors are overexpressed in various cancers including breast cancer and because of its binding aptitude specifically to folate receptors, folic acid became the attractive ligand.MethodsIn this research, we have developed a folate and Poly-l-Lysine conjugate and coated this conjugate onto the liposomes. The prepared liposomes were characterized using DLS, FTIR, NMR, SEM, TEM, XRD, AFM, stability and drug release studies. Furthermore, in vitro studies were carried out on FR overexpressed breast cancer cell line.ResultsThe FA-LUT-ABC-Lip have diameter of 183 ± 3.17 nm with positive surface charge +33.65 ± 3 mV and the drug release studies confirm the NIR responsive payload cleavage. The coated formulation (in presence of NIR light) effectively reduced the IC₅₀ values and kills breast cancer cells through FR mediated internalization and accelerated drug release. Moreover, LUT Formulation shows anticancer effect due to significant inhibition of cell migration and proliferation by regulating VEGF expression and induced apoptosis through the caspase-3 up-regulation.ConclusionIt is evident from the in vitro studies that the formulation was found to be very effective and can be explored for triggered and targeted delivery of the substances through active targeting.General significanceCombining receptor mediated drug delivery with triggered release aid in more amounts of drug reaching the target site and achieving enhanced therapeutic activity.     

肝素硫酸转移酶优化表达及其在动物源肝素硫酸化中的应用

肝素是一种重要的凝血药物,目前主要依赖于动物小肠粘膜的提取。动物源肝素含有的抗凝血活性五糖单位GlcNS6S-GlcA-GlcNS6S3S-Ido2S-GlcNS6S少,抗凝血活性低下。文中提出并验证了一种基于酶法催化动物源肝素,提高其硫酸化程度和抗凝血活性的方法。通过比较3种硫酸转移酶肝素-2-硫酸转移酶(Heparan sulfate-2-O-sulfotransferase,HS2ST)、肝素-6-硫酸转移酶(Heparan sulfate-6-O-sulfotransferase,HS6ST)、肝素-3-硫酸转移酶(Heparan sulfate-3-O-sulfotransferase,HS3ST)在重组大肠杆菌及重组毕赤酵母中表达,确定了毕赤酵母作为3种硫酸转移酶的表达宿主;进一步通过N端融合麦芽糖融合蛋白MBP和硫氧还蛋白Trx A,HS2ST和HS6ST的酶表达水平分别提高至(839?14) U/L和(792?23) U/L。通过3种硫酸转移酶HS2ST、HS6ST和HS3ST共同催化动物源肝素,其抗凝血活性由(76?2) IU/mg提高至(189?17) IU/mg。     

Semi-synthetic heparin derivatives: chemical modifications of heparin beyond chain length, sulfate substitution pattern and N-sulfo/N-acetyl groups

The glycosaminoglycan heparin is a polyanionic polysaccharide most recognized for its anticoagulant activity. Heparin binds to cationic regions in hundreds of prokaryotic and eukaryotic proteins, termed heparin-binding proteins. The endogenous ligand for many of these heparin-binding proteins is a structurally similar glycosaminoglycan, heparan sulfate (HS). Chemical and biosynthetic modifications of heparin and HS have been employed to discern specific sequences and charge-substitution patterns required for these polysaccharides to bind specific proteins, with the goal of understanding structural requirements for protein binding well enough to elucidate the function of the saccharide-protein interactions and/or to develop new or improved heparin-based pharmaceuticals. The most common modifications to heparin structure have been alteration of sulfate substitution patterns, carboxyl reduction, replacement N-sulfo groups with N-acetyl groups, and chain fragmentation. However, an accumulation of reports over the past 50 years describe semi-synthetic heparin derivatives obtained by incorporating aliphatic, aryl, and heteroaryl moieties into the heparin structure. A primary goal in many of these reports has been to identify heparin-derived structures as new or improved heparin-based therapeutics. Presented here is a perspective on the introduction of non-anionic structural motifs into heparin structure, with a focus on such modifications as a strategy to generate novel reduced-charge heparin-based bind-and-block antagonists of HS-protein interactions. The chemical methods employed to synthesize such derivatives, as well as other unique heparin conjugates, are reviewed.     

Blood compatible graphene/heparin conjugate through noncovalent chemistry

Blood compatible graphene/heparin conjugate is simply formulated through noncovalent interaction between chemically reduced graphene and heparin. Charge repulsion of negatively charged heparin on graphene plates renders hydrophobic graphene to be solublized in aqueous media without any precipitation or aggregation even after 6 months. Unfractioned heparin (UFH) with higher molecular weight was effective for graphene solubilization while low molecular weight heparin (LMWH) was not. Noncovalently interacting heparin chains on graphene plates preserve their anticoagulant activity after conjugation with graphene. Graphene/UFH conjugate shows much enhanced anti factor Xa (FXa) activity of 29.6 IU/mL compared with pristine graphene oxide (GO; 1.03 IU/mL).     

Rational design of urea-based glutamate carboxypeptidase II (GCPII) inhibitors as versatile tools for specific drug targeting and delivery

Glutamate carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), is an established prostate cancer marker and is considered a promising target for specific anticancer drug delivery. Low-molecular-weight inhibitors of GCPII are advantageous specific ligands for this purpose. However, they must be modified with a linker to enable connection of the ligand with an imaging molecule, anticancer drug, and/or nanocarrier. Here, we describe a structure–activity relationship (SAR) study of GCPII inhibitors with linkers suitable for imaging and drug delivery. Structure-assisted inhibitor design and targeting of a specific GCPII exosite resulted in a 7-fold improvement in K_i value compared to the parent structure. X-ray structural analysis of the inhibitor series led to the identification of several inhibitor binding modes. We also optimized the length of the inhibitor linker for effective attachment to a biotin-binding molecule and showed that the optimized inhibitor could be used to target nanoparticles to cells expressing GCPII.     

肝素6位羧基修饰对抑制P?鄄选择素介导的A375细胞粘附活性的影响

已有的研究表明,肝素可以作为P-选择素的配体,显著抑制肿瘤转移过程中P-选择素介导的肿瘤细胞与血小板间的粘附.但是,肝素被P-选择素识别所必需的确切寡糖结构信息仍很缺乏.通过选择性化学修饰方法制备了2种低抗凝血肝素衍生物,即羧基还原肝素(CR-肝素)和羧基还原后再硫酸化肝素(SCR-肝素),系统地研究了它们对P-选择素介导的A375细胞粘附的抑制.研究结果表明,显著失去抗凝血活性的CR-肝素仍能有效地抑制P-选择素介导的A375细胞粘附,说明肝素的C6羧基并不是被P-选择素识别所必需的.而SCR-肝素所发生的C6羧基向羟甲硫酸酯基的转化却显著降低了抗粘附活性,说明P-选择素对肝素的识别并不只依赖于肝素的电荷密度.研究结果为深入阐明拮抗P-选择素介导的肿瘤细胞粘附的分子机制提供了有价值的实验基础.     

核酸适配体在靶向药物传递中的研究进展

抗癌药物的毒副作用限制了其临床应用,纳米药物载体可实现药物在病灶部位的聚集而不影响正常组织,从而降低药物毒副作用.在药物载体表面修饰靶向配体,以提高药物载体主动靶向进入到细胞的能力,可有效地将药物释放到靶细胞,大大提高药效.核酸适配体(aptamer)作为一种新型的靶向分子,近几年已被运用到靶向药物传递的研究中.本文介绍了几种适配体靶向载药体系,如适配体-药物、适配体-脂质体、适配体-聚合物胶束、适配体-聚合物纳米颗粒、适配体-金属颗粒以及适配体-支化聚合物等载药体系,并对当前研究的热点以及存在的问题和不足进行了评述.     

Combination of site-directed mutagenesis and calcium ion addition for enhanced production of thermostable MBP-fused heparinase I in recombinant Escherichia coli

Heparinase I (HepI), which specifically cleaves heparin and heparan sulfate, is one of the most extensively studied glycosaminoglycan lyases. Low productivity of HepI has largely hindered its industrial and pharmaceutical applications. Loss of bacterial HepI enzyme activity through poor thermostability during its expression and purification process in production can be an important issue. In this study, using a thermostabilization strategy combining site-directed mutagenesis and calcium ion addition during its production markedly improved the yield of maltose-binding protein-fused HepI (MBP–HepI) from recombinant Escherichia coli. Substitution of Cys297 to serine in MBP–HepI offered a 30.6 % increase in the recovered total enzyme activity due to a mutation-induced thermostabilizing effect. Furthermore, upon addition of Ca²⁺ as a stabilizer at optimized concentrations throughout its expression, extraction, and purification process, purified mutant MBP–HepI showed a specific activity of 56.3 IU/mg, 206 % higher than that of the wild type obtained without Ca²⁺ addition, along with a 177 % increase in the recovered total enzyme activity. The enzyme obtained through this novel approach also exhibited significantly enhanced thermostability, as indicated by both experimental data and the kinetic modeling. High-yield production of thermostable MBP–HepI using the present system will facilitate its applications in laboratory-scale heparin analysis as well as industrial-scale production of low molecular weight heparin as an improved anticoagulant substitute.     

Multiplex genome editing of mammalian cells for producing recombinant heparin

Heparin is an essential anticoagulant used for treating and preventing thrombosis. However, the complexity of heparin has hindered the development of a recombinant source, making its supply dependent on a vulnerable animal population. In nature, heparin is produced exclusively in mast cells, which are not suitable for commercial production, but mastocytoma cells are readily grown in culture and make heparan sulfate, a closely related glycosaminoglycan that lacks anticoagulant activity. Using gene expression profiling of mast cells as a guide, a multiplex genome engineering strategy was devised to produce heparan sulfate with high anticoagulant potency and to eliminate contaminating chondroitin sulfate from mastocytoma cells. The heparan sulfate purified from engineered cells grown in chemically defined medium has anticoagulant potency that exceeds porcine-derived heparin and confers anticoagulant activity to the blood of healthy mice. This work demonstrates the feasibility of producing recombinant heparin from mammalian cell culture as an alternative to animal sources.     

Design of a tumor-homing cell-penetrating peptide

Chemotherapy is often limited by toxicity to normal cells. Therefore, an ideal anticancer drug should discriminate between normal tissue and tumors. This would require a target receptor molecule mostly present in tumors. The cyclic peptide cCPGPEGAGC (PEGA) is a homing peptide that has previously been shown to accumulate in breast tumor tissue in mice. PEGA peptide does not cross the plasma membrane per se; however, when attached to the cell-penetrating peptide pVEC, the conjugate is taken up by different breast cancer cells in vitro. Additionally, the homing capacity of the PEGA- pVEC is conserved in vivo, where the conjugate mainly accumulates in blood vessels in breast tumor tissue and, consequently is taken up. Furthermore, we show that the efficacy of the anticancer drug, chlorambucil, is increased more than 4 times when the drug is conjugated to the PEGA- pVEC chimeric peptide. These data demonstrate that combining a homing sequence with a cell-penetrating sequence yields a peptide that combines the desirable properties of the parent peptides. Such peptides may be useful in diagnostics and delivery of therapeutic agents to an intracellular location in a specific tumor target tissue.     

Fractionation of low molecular weight heparin species and their interaction with antithrombin.

Preparations of low molecular weight porcine heparin with an average specific anticoagulant activity of 94 units/mg were fractionated into "active" and "relatively inactive" forms of the mucopolysaccharide of approximately 6000 daltons each. The active fraction was further subdivided into various species with descending but significant affinities for the protease inhibitor as well as decreasing but substantial anticoagulatn potencies. "Highly active" heparin (approximately 8% of the low molecular weight pool) possesses a specific anticoagulant activity of 350 +/- 10 units/mg. The relatively inactive fraction (67% of the low molecular weight pool) exhibits a specific anticoagulant activity of 4 +/- 1 units/mg. The binding of highly active heparin to antithrombin is accurately described by a single-site binding model with a KHep-ATDISS of approximately 1 X 10(-7) M. Variations in this binding parameter secondary to changes in environmental variables indicate that charge-charge interactions as well as an increase in entropy are critical to the formation of the highly active heparin-antithrombin complex. The interaction of relatively inactive heparin with the protease inhibitor is characterized by an apparent KHep-ATDISS of 1 X 10(-4) M. In large measure, this is due to small amounts of residual active mucopolysaccharide (0.5%). The ability of the highly active heparin to accelerate the thrombin-antithrombin interaction was also examined. We were able to demonstrate that the mucopolysaccharide acts as a catalyst in this process and is able to initiate multiple rounds of enzyme-inhibitor complex formation. The rate of enzyme neutralization is increased to a maximum of 2300-fold as the concentration of heparin is raised until the inhibitor is saturated with mucopolysaccharide. Further increases in heparin concentration result in a reduction in the speed of enzyme neutralization. This appears to be due to the formation of thrombin-heparin complexes. A mathematical model is given which provides a relationship between the initial velocity of enzyme neutralization and reactant concentrations.     

肝素的抗炎作用与抗细胞粘附调节

肝素类药物具有抗凝以外的包括抗炎在内的多种生物学活性。炎症反应是多种因子、细胞参与的复杂的病生理过程,其物质基础是粘附分子介导的白细胞粘附及其粘附级联反应。近来研究证实肝素抗炎机制主要与抗细胞粘附调节有关,肝素通过竞争抑制L—、P—选择素与其配基sLe^X的结合,阻止白细胞粘附活化及调抑炎症级联反应,进而起到抗炎作用。对肝素抗炎机制的深入研究,将有助于进一步阐明抗粘附／抗炎的作用机制。