A Novel Heparin-paclitaxel Drug Delivery System: Preparation And Biological Activity

Paclitaxel is a taxoid that has aroused considerable interest on account of its promising antitumor activity. It is in clinical use for treating a variety of malignancies, including ovarian, breast, and non-small-cell lung cancers. It was observed that the side effects were caused by Cremophor EL used in the paclitaxel formulation. Therefore, it is urgent to develop a novel drug delivery system. Recent years, the use of macromolecules for the targeted delivery of anticancer agents has generated considerable interest regarding their enhanced permeability and retention (EPR) effect in tumor tissues. Macromolecules with good solubility, non-toxicity, biocompatibility, biodegradability, have been employed as carriers to deliver active drugs towards intracellular compartments. It can improve water solubility, control the release hydrolysis rate and extend in vivo half life of active drugs. The novel drug delivery systems have shown a bright future for the development of anticancer therapy.Heparin is a biocompatible, biodegradable and water-soluble natural polysaccharide and is rich in animal tissues. Heparin has attracted intense attention because it demonstrates a variety of biological activities. According to this, we design and prepare a variety of drug delivery systems, in which heparin as carrier conjugates with paclitaxel.We first develop a model ternary heparin conjugate by direct covalent bond strategy applied to the drug delivery system. Based on the model procedure, we design and synthesis two drug delivery systems (HD) by the strategy, in which O-acetylated heparin as carrier conjugates with paclitaxel by direct ester bond (HD1) and by inserting amino acids as spacers (HD2). We further investigate amphiphilic heparin-paclitaxel conjugates as a type of novel prodrug, which can self-assemble to form nanoparticles due to the different hydrophilic/hydrophobic nature of the carrier and drug compound in aqueous solution. The structures of drug delivery systems have been characterized by 1H NMR, FT-IR, and GPC. The kinetics of hydrolysis for drug delivery system has been investigated under chemical and enzymatic conditions. The anticoagulant activity, in vitro cell inhibition and in vivo antitumor activity in drug delivery system are measured. In addition, we try to examine the apoptotic mechanism of these drug delivery systems by Flow Cytometry and Cytomorphology.1. A model ternary heparin conjugate by direct covalent bond strategy applied to drug delivery systemIn this study, we have developed a model ternary heparin conjugate by direct covalent bond strategy, in which modified heparin using active mix anhydride as intermediate conjugate with model drug molecule (benzyl alcohol) and model specific ligand (butylamine), respectively. Ester bonds linked model drug and carrier facilitate the release of drug from carrier. In contrast, amido bonds between model specific ligand and heparin show greater stability.It is demonstrated that the activating mixed anhydride groups largely improved the activity and selectivity toward the following esterification. It has been found that the optimal weight percentages of introduced functional groups are 8.8 % of acetyl, 3.21 % of benzyloxy and 1.3 % in ternary heparin conjugate, respectively. The decreased trend on degree of substitution (DS) is consistent with that of introduced anticancer drug and specific ligand in drug delivery system.Their anticoagulant activity has been investigated. The results show that model ternary heparin conjugate with reduced anticoagulant activity may avoid the risk of severe hemorrhagic complication during the administration and is potential to develop a safe and effective drug delivery system on anticancer research.2. Heparin conjugates with paclitaxel by direct ester bond and by amino acid as spacers According to the model procedure, 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 amino acids with differentα-substituted group as spacers (HD2), including valine, leucine, and phenylalanine (HD2a, HD2b, and HD2c), respectively.The content of drug conjugate to O-acetylated heparin has been quantified by UV absorbance. The weight percentages of HD conjugates contained 16~25% paclitaxel. HD conjugates have been disposed to chemical and enzymatic hydrolysis to test the drug release at pH 7.4 and pH 5.0, respectively. The HD1 is highly stable under physiological and enzyme conditions. It is demonstrated that direct ester bond between carrier and drug is difficultly hydrolyzed under physiological and enzymatic condition. In the PBS, the amount of paclitaxel, which can be liberated from HD2, increased as pH increased. It is observed the hydrolysis rate of HD2a and HD2b conjugates is greatly increased under enzymatic condition.The anticoagulant activities of HD conjugates have been investigated by APTT. The anticoagulant activities of HD1 and HD2 both decreased to some extent as compared to that of heparin (150 U/mg). As a consequence, HD conjugates with reduced anticoagulant activity could be safe and effective prodrugs to further investigate.Cytotoxicity has been studied by using an MTT assay to identify cells still active in respiration. HD1 exhibites lower cytotoxicity (IC50: 3.09μg/mL) than free paclitaxel. Specifically, IC50 values of HD2a, HD2b and HD2c for MCF-7 are about 0.97μg/mL, 0.48μg/mL and 0.43μg/mL, respectively, which are about 2~3 fold lower than that of free paclitaxel. HD2 therefore demonstrated significant anticancer activity.3. Heparin-paclitaxel drug delivery system with self-assembly property In this study, we have synthesized amphiphilic heparin-paclitaxel conjugates as a type of novel prodrug, which can self-assemble to form nanoparticles due to the different hydrophilic/hydrophobic nature of the carrier and drug compound in aqueous solution. The goal to select succinylated-heparin as a carrier is to increase drug loading capacity in which hydroxyl groups of heparin react with succinic anhydride under classical acylation conditions. Amino acids are ideal linkers, which can provide a reactive carboxyl group to conjugate with paclitaxel via an ester bond and an amino group that could be easily reacted with the carboxyl groups of the carrier via N-acylation reaction.It has been found that the weight percentage of succinylated group is about 8.5 %. The content of drug conjugate to Prodrugs is 35.4 %, 37.8 % and 39.1 %, respectively. Their morphology has been investigated by SEM and DLS. The Prodrugs have a negative surface charge ( -20 mV) and their mean diameters are about 140~180 nm measured by DLS. The results demonstrate that self-assembled nanoparticles have a narrow size distribution and form an approximately spherical shape composed of a paclitaxel core and carrier shell.Prodrugs have been subjected to chemical and plasma hydrolysis to test the paclitaxel release. In the PBS, the amount of paclitaxel, which can be liberated from Prodrugs, increased as pH increased. The stability of Prodrugs in mouse plasma is lower than that in human plasma. Under physiological condition, the amount of released paclitaxel is lower than that in plasma. Apparently, Prodrug2 using leucine as spacer is appropriate to facilitate the release of pacitaxel from the carrier.The anticoagulant activity of Prodrugs has been investigated by APTT. The results show that Prodrugs with reduced anticoagulant activity may avoid the risk of severe hemorrhagic complication during the administration and is potential to develop a safe and effective drug delivery system on anticancer research.It shows that the Prodrugs exhibit higher cytotoxicity than free paclitaxel in MCF-7 cell after 48 h by using an MTT assay. IC50 values of Prodrug1, Prodrug2 and Prodrug3 were about 0.19μg/mL, 0.058μg/mL and 0.06μg/mL, respectively. In in vivo experiments, prodrug2 shows a similar ovarian tumor growth inhibition as the parent drug while inducing no obvious body weight loss. As a whole, Prodrug2 shows not only proper hydrolysis rate, but also better anticancer inhibition. It is demonstrate that Prodrug2 with self-assembled property offers promising potential for further clinical applications.