Structure-biocompatibility relationship of dendritic polyglycerol derivatives
Introduction
Dendritic polymers are unique because they can be prepared by controlled sequential processes with defined structural, molecular-level size, shape, surface chemistry, topology, and flexibility from individual building blocks to three dimensional nanoarchitectures [1]. These polymers' exceptional structural precisions has led to a variety of applications in gene therapy, catalysis, micro arrays, and targeted drug delivery systems [2]. Dendritic polyglycerols (dPGs) are structurally defined, consist of an aliphatic polyether backbone, and possess multiple functional end groups [3], [4], [5]. Since dPGs are synthesized in a controlled manner to obtain definite molecular weight and narrow molecular polydispersity, they have been evaluated for a variety of biomedical applications [5], [6], [7]. Hyperbranched polyglycerol (PG) analogs have similar properties as perfect dendritic structures with the added advantage of defined mono- and multi-functionalization [3], [5].
Nevertheless, delivery carrier systems are expected to possess suitable physicochemical properties for improved bioavailability, cellular dynamics, and targetability [8], [9]. This is particularly true if the polymeric architectures have high surface charge, molecular weight, and a tendency to interact with biomacromolecules in blood due to their surface properties. While most of the hyperbranched polymeric architectures consisting of bioactive therapeutic agents deliver drugs by a systemic route, their fate in blood and interactions with the plasma proteins need to be further evaluated. Therefore, some studies have been recently undertaken to establish the molecular and physiological interactions of the dendritic polymers with plasma components [10]. Interactions of delivery carrier with serum albumins (SA) are of a particular interest since they are the most abundant soluble protein constituents in plasma with a mass of 65 kDa [11]. Additionally, they have many physiological functions, such as maintenance of colloidal osmotic blood pressure, blood pH, and support in transport, distribution, and metabolism of many endogenous and exogenous ligands [12]. Furthermore, they easily transfer many ligands across organ circulatory interfaces in the liver, intestine, kidney, and brain [13]. Most importantly, albumins can reversibly bind to ligands and may change the fate of therapeutically active agents. If the binding is irreversible and significantly high enough to cause a conformational change in native albumin, however, it is likely that there is an associated toxicity. More profoundly, high affinity and irreversible binding between albumin and carrier may lead to excess organelle accumulation, e.g. in the liver, spleen, kidney, etc. Therefore, detailed biophysical evaluation of interactions between albumin and polymeric carriers must be addressed because they may change the pharmacokinetic, pharmacodynamic, and toxicology pattern of the bioactive.
In general, the quantum of interactions between small ligand molecules and SA is evaluated by Stern-Volmer constants, because of the tryptophan fluorescence quenching abilities of the ligands. Furthermore, these interactions are measured in terms of binding constants or binding associations. Currently, fluorescence quenching mechanisms for hyperbranched polymers and nanocarriers are not well evaluated. In the past, there have only being a few studies on the interactions of polyamidoamine (PAMAM) dendrimers (fatty acid free and loaded with fatty acids) with serum albumins (SA), mostly dealing with their physiological significance for developing new nanocarrier systemic delivery systems [14]. Currently, many groups have been using dendritic polyglycerols to complex or conjugate bioactives, but only the pure PG-structure has been evaluated and little is still known about the biocompatibility of functional derivatives which is relevant for the prodrug conjugation [15], [16], [17], [18], [19], [20], [21]. Furthermore, the physical interactions of nanocarriers with or without bioactives in plasma proteins need to be evaluated to demonstrate in vitro safety, before being considered for in vivo delivery. Currently, dPG candidates are considered as delivery enhancers for many bioactives which could substantially increase the internalization of active components, specifically into targeted cells to enhance the ‘specific activity’ of the combined drug delivery system and decrease the adverse side effects [5], [17]. The ‘specific activity’ (e.g. in anticancer drug delivery prodrug systems) is expected to improve, mainly by enhanced permeation and retention (EPR) effect due to the nature of tumor vasculature and the accumulation of high molecular weight polymeric prodrug conjugate [8]. In addition, polyglycerols and hydrophobically derivatized hyperbranched polyglycerols have been shown to increase the plasma half-life of 32 hours in mice for lower molecular weight polymer, whereas it can be as high as ∼57 hours for high molecular weight PGs [21]. In ideal circumstances a nanocarrier could be considered as a suitable delivery candidate if it possesses a combination of the following properties: a) unique chemical surface tunability, b) significantly tolerable surface charge for cell uptake, c) moderate affinity and low interactions with plasma proteins, d) cellular internalization, concentration equilibrium with achieving EPR effect, and e) cellular targetability.
As a result, we report here on the structure-biocompatibility relationship of dPG derivatives with different surface functionalities (Fig. 1). The main objective of the present work is to evaluate the surface charge properties of dPGs of varying polymeric architectures. Special attention has been addressed to zeta potential (ZP) measurements in order to explore the changes of surface charge with pH. In addition, the interactions of dendritic polyglycerol candidates are examined with bovine serum albumin (BSA) using fluorescence quenching analysis in buffered aqueous solutions. Furthermore, the cellular uptake and delivery ability of dPG and dendritic polyglycerol sulfate (dPGS)-labeled with indocarbocyanine-carboxylic acid NHS ester (ICC) dye have been evaluated using fluorescence microscopy with A549 lung epithelial cells. In addition, in vitro cytotoxicity profiles of various dPGs are compared with PAMAM dendrimers, polyethylene imine (PEI), dextran, and polyethylene glycol (PEG) using human hematopoietic cell line U-937 to establish and compare the structure biocompatibility relationship of dPGs for systemic delivery.
Section snippets
Materials
All chemicals were of analytical grade and purchased from Fluka, Aldrich, and Merck, respectively. Dendritic polyglycerols (dPGs) with various architectures and functionalities were prepared according to our previously published procedures [20] (see Supporting Information). Polyamidoamine (PAMAM) G4-NH2 and G4-OH dendrimers in methanol (10 wt.%) were purchased from Aldrich. Bovine serum albumin was obtained from Sigma–Aldrich. Water of Millipore quality (resistivity∼18 MΩ cm−1, pH = 5.6 ± 0.2)
Dendritic polyglycerols
dPGs with various architectures and functionalities were synthesized in high yield and controllable degree of conversions according to the published procedures reported by our group [20]. In case of polyglycerolamines different degrees of functionality (7%, 18%, 23%, 45%, and 100%) have been evaluated for surface charge, binding interactions with SA, and in vitro biocompatibility [4], [21]. In addition, precursors in the form of dPG-azide and dPG-phosphate have been evaluated for fluorescence
Discussion
The fact that the dendritic polymers can deliver therapeutic agents more efficiently makes them an attractive vehicle for vascular, extracellular, cytoplasmic and nuclear delivery. However, there are many factors that determine the delivering ability of these polymers for e.g. molecular size, molecular weight, polymeric architecture, surface charge, and chemical functionality [34]. In addition, the safety of these nanocarriers is largely dependent on these physicochemical characteristics which
Conclusions
Dendritic polyglycerols have been designed in regard to control of molecular weight, size, surface charge, and chemical diversity with amine, hydroxyl, sulfate, and phosphate functionalities. The extensive studies on interactions and insights with plasma protein highlight the associated changes by fluorescence quenching. Cellular uptake studies of dye labelled dPGs inferred that the charged derivatives (i.e. dPG-sulfate and dPG-amine) are primarily rapidly internalized inside the cytosol of
Acknowledgements
The authors would like to acknowledge Prof. Dr. Rainer H. Müller, Institute of Pharmacy, Freie Universität Berlin, for his inputs and discussions on zeta potential measurements. The authors also would like thank DFG, IBB, BMBF (NanoFutur Award), and Alexander von Humboldt Experienced Researcher Fellowship for the financial support.
References (46)
- et al.
Polymer–drug conjugates: progress in polymeric prodrugs
Prog Polym Sci
(2006) - et al.
Dendrimers: relationship between structure and biocompatibility in vitro, and preliminary studies on the biodistribution of 125I-labelled polyamidoamine dendrimers in vivo
J Contr Rel
(2000) - et al.
Interactions between PAMAM dendrimers and bovine serum albumin
Biochim Biophys Acta
(2003) - et al.
Effects of ethylene glycol-based graft, star-shaped, and dendritic polymers on solubilization and controlled release of paclitaxel
J Control Release
(2003) - et al.
Development of pH-responsive core-shell nanocarriers for delivery of therapeutic and diagnostic agents
Bioorg Med Chem Lett
(2009) - et al.
Nanoparticles for skin penetration enhancement-a comparison of a dendritic core-multishell-nanotransporter and solid lipid nanoparticles
Eur J Pharm Biopharm
(2009) - et al.
High-performance anion-exchange chromatography of sugar and glycerol phosphates on quaternary ammonium resins
Carbohydr Res
(1999) - et al.
Structure of serum albumin
Adv Protein Chem
(1994) - et al.
Therapeutic and diagnostic applications of dendrimers for cancer treatment
Adv Drug Deliv Rev
(2008) - et al.
Polyamidoamine starburst dendrimers as solubility enhancers
Int J Pharm
(2000)
A distinct intermediate of RNase A is induced by sodium dodecyl sulfate at its pKa
Colloids Surf B
Impact of PAMAM G2 and G6 dendrimers on bovine serum albumin (faty acids free and loaded with different fatty acids)
Colloids Surf B: Biointerfaces
Dendrimer-protein interactions by tryptophan room temperature phosphorescence
Biophys Acta
Serum albumins have five sites for binding of cationic dendrimers
Biochim Biophys Acta
Starburst dendrimers: molecular-level control of size, shape, surface chemistry, topology, and flexibility from atoms to macroscopic matter
Angew Chem Int Ed
Effective suicide gene therapy in vivo by EBV-based plasmid vector coupled with polyamidoamine dendrimer
Gene Ther
Controlled synthesis of hyperbranched polyglycerols by ring-opening multibranching polymerization
Macromolecules
An approach to glycerol dendrimers and pseudo-dendritic polyglycerols
J Am Chem Soc
Dendritic polyglycerol for biomedical application
Adv Mater
Dendritische Polymere für medizinische Anwendungen: auf dem Weg zum Einsatz in Diagnostik und Therapie
Angew Chem
Supramolecular drug-delivery systems based on polymeric core-shell architectures
Angew Chem
Polymer conjugates as anticancer nanomedicines
Nat Rev Cancer
All about albumin: biochemistry, genetics and medical applications
Cited by (0)
- 1
Authors contributed equally to this work.