Chitosan-modified lipid nanovesicles for efficient systemic delivery of l-asparaginase
Graphical abstract
Introduction
Based on functional mechanisms, enzyme therapies can be classified into three main categories: enzyme replacement therapy [1], directed enzyme prodrug therapy [2] and enzyme enhancement therapy such as l-asparaginase (ASP) therapy. ASP has been extensively used in clinics for many years as a first-line therapeutic agent for acute lymphoblastic leukemias and lymphomas [3]. Recently, some new indications of ASP such as nasal type extranodal natural killer/T-cell lymphoma and ovarian cancer, have been confirmed [4] or evaluated in different clinical trial phases [5]. ASP is a tetrameric amidohydrolase able to catalyze the deamination of l-asparagine and glutamine. Since glutamine can provide the substrate (ammonia) of l-asparagine synthetase to synthesis l-asparagine, the decreased glutamine level will certainly contribute to the decreased l-asparagine level. The normal cells can synthesis l-asparagine while cancer cells can’t, so lack or reduction of l-asparagine will induce death or growth inhibition of cancer cells [6]. Being a therapeutic enzyme and essentially a protein drug, ASP had intrinsic drawbacks such as low catalytic activity under physiological situation, low stability and short circulating life. A few of delivery systems such as ASP conjugated zinc oxide nanobiocomposites [7], polyethylene glycol-conjugated ASP [5], ASP erythrocytes [8] and ASP chitosan-tripolyphosphate nanoparticles [9] had been developed to meet different needs: increase the anti-cancerous activity against MCF-7 breast cancer or advanced ovarian cancer cells, or prolong the in vitro release half-life of ASP.
Recently, lipid nanovesicles have showed attractive potential in the effective delivery of all kinds of drugs such as paclitaxel (a phytodrug for colon adenocarcinoma) [10], pentapeptide mimic 1,4-bis(9-O dihydroquinidinyl)phthalazine/hydroquinidine 1,4-phathalazinediyl diether (a cinchona alkaloid for B-precursor leukemia) [11], doxorubicin (a chemodrug for epidermal carcinoma) [12], recoverin (a protein used to treat retinal diseases) [13] and porphyrin (a multimodal photonic contrast agent) [14]. Furthermore, modification of drug delivery systems by natural polysaccharide chitosan have increased the stability and efficacy as reported in the cases of vancomycin chitosan coated liposomes [15], macrophage-targeting gene chitosan nanoparticles [16] and bovine lactoferrin chitosan-modified solid lipid particles [17]. Moreover, nanovesicles have been employed to improve the activities of the enzymes used in different enzyme therapies. For example, alkaline enzymosomes was formulated to improve the biological properties and hypouricaemic effects of uricase [1] in enzyme replacement therapy, magnetic iron oxide nanoparticles was applied to achieved the enhanced and selective delivery of β-glucosidase to 9L-glioma tumor [2] in directed enzyme prodrug therapy. Here, novel types of nanocarriers, i.e., chitosan-modified lipid nanovesicles, were fabricated to deliver therapeutic enzymes efficiently and obtain increased antitumor effects in enzyme enhancement therapy. Since l-asparaginase (ASP) was a clinically used enzyme which usage was restricted by its instability and low catalytic activity in vivo, it was chosen as a model of therapeutic enzyme in this study.
In the experiments outlined below, l-asparaginase-containing chitosan-modified lipid nanovesicles (ACLNs) were prepared to deliver the therapeutic enzymes efficiently for the first time. Their catalytic activity, physicochemical and enzymatic stabilities, anti-lung-cancer activity, in vivo kinetic property and bioavailability were investigated. Our study indicated that ACLNs markedly increased the enzymatic activity, improved the stability, and favorably changed the in vivo kinetic characteristics. Moreover, ACLNs had higher anti-lung-cancer activity than that of free ASP. The possible existence status of ASP in ACLNs and the fluorescence changes of ACLNs reflecting the conformational changes after heat treatment were preliminary explored. ACLNs might be promising nanovesicles for effective systemic delivery of therapeutic enzyme ASP.
Section snippets
Materials
l-asparaginase from Escherichia coli. (ASP, activity of 225 units/mg powder at 37 °C, purity >96.0%) was obtained from ProSpec-Tany TechnoGene Ltd. (Rehovot, Israel). l-asparagine, Cholesterol and fluorescein isothiocyanate (FITC) were obtained from Sigma (St. Louis, MO, USA). Soybean phospholipid (Lipoid S 100) was obtained from Lipoid GmbH (Ludwigshafen, Germany). Chitosan hydrochloride (deacetylation 80.0%-90.0%, viscosity 10–120 mpa.s, average molecular weight ∼50 kDa) was obtained from
Configuration and characterization of ACLNs
The spherical or sphere-like ACLNs were individually dispersed in the buffers under transmission electron microscopy observation (Fig. 1A). The schematic diagram of ACLNs was presented in Fig. 1B. The ASP-loaded lipid nanovesicles were coated by natural polysaccharide chitosan. The chitosan 50 kDa (0.2%, weight/volume) was used to prepare the ACLNs in our study. The type and amount of chitosan were chosen mainly based on previous documents and our preliminary experiments. The chitosan 50 kDa [9],
Conclusions
The physiochemical characteristics, enzymatic activities, stabilities under different temperature-pH-proteolytic conditions, pharmacokinetic behaviors and anti-lung cancer efficacies of therapeutic enzyme ASP could be markedly improved through entrapment in novel chitosan modified lipid nanovesicles. The altered fluorescence intensities suggested that after entrapment, not only the altered conformational changes but also the interactions between lipid nanovesicle membranes and ASP molecules
Acknowledgements
This research was partially supported by grants from the National Natural Science Foundation of China (30973645) and Chongqing Natural Science Foundation (csct2015jcyjBX0027).
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These authors contributed equally to this study