Elsevier

Biomaterials

Volume 31, Issue 15, May 2010, Pages 4268-4277
Biomaterials

Structure-biocompatibility relationship of dendritic polyglycerol derivatives

https://doi.org/10.1016/j.biomaterials.2010.02.001Get rights and content

Abstract

Nanocarriers possess advanced physicochemical properties that improve bioavailability, enhance cellular dynamics, and control targetability in drug delivery. In particular, dendritic polyglycerol is a promising new biocompatible scaffold for drug delivery. The present explores the structure-biocompatibility relationship of dendritic polyglycerol (dPG) derivatives possessing neutral, cationic, and anionic charges. The effect of solution pH on the surface charge was studied in buffered aqueous solution between pH 4.8 and 7.4. Surface charge properties of dPG derivatives are discussed in terms of surface functionalities and compared with amine and hydroxyl terminated polyamidoamine (PAMAM) dendrimers. Zeta potential measurements and fluorescence quenching studies address the binding interactions of dPGs to bovine serum albumin in order to explore the applicability of dPG derivatives for systemic delivery. Cellular entry of dPG-dye conjugate was evaluated using A549 lung epithelial cells, while in vitro toxicity was studied for various dPGs and compared to PAMAM dendrimers, polyethyleneimine (PEI), dextran, and linear polyethylene glycol (PEG) using human hematopoietic cell line U-937. Cellular uptake studies of dye labelled dPGs inferred that the charged derivatives (dPG-sulfate and dPG-amine) are more rapidly internalized primarily inside the cytosol of A549 cells compared to the neutral dPG. The cell compatibility results show that the dendritic polyglycerols are as safe as linear PEG polymer or dextran, which indicates the suitability of dPG derivatives in delivering therapeutic agents systemically.

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.

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