Elsevier

Biomaterials

Volume 28, Issue 31, November 2007, Pages 4581-4590
Biomaterials

In vitro biological evaluation of high molecular weight hyperbranched polyglycerols

https://doi.org/10.1016/j.biomaterials.2007.07.011Get rights and content

Abstract

Low molecular weight hyperbranched polyglycerols are highly water soluble and biocompatible polyether polyols, which can be synthesized in a controlled manner with narrow polydispersity. Recently we reported the synthesis and characterization of very high molecular weight (Mn up to 700,000) and narrowly polydispersed polyglycerols which could be potentially used as alternatives to high generation dendrimers which are difficult to make. A detailed biocompatibility testing of these polymers conducted in vitro is reported here. The in vitro studies include hemocompatibility testing for effects on coagulation (prothrombin time (PT), activated partial thromboplastin time (APTT), plasma recalcification time (PRT), thrombelastograph parameters (TEG)), complement activation, platelet activation, red blood cell aggregation and cytotoxicity. Results from these studies show that these high molecular weight polyglycerols are highly biocompatible and are potential candidates for various applications in nanobiotechnology and in nanomedicine. Moreover these polymers are thermally and oxidatively stable.

Introduction

Hyperbranched polymers that can be prepared in a convenient single step make them potential alternatives to dendrimers in applications where a precise structure is not necessary [1]. These applications include viscosity modification and cross linking where the polymer characteristics such as globular structure, low viscosity and the large number of functional groups are exploited [2]. Initially polydispersities were large but more recent theoretical and experimental work has shown that controlled polymerization of AB2 monomers is possible by slow monomer addition [3]. In particular, synthesis of hyperbranched polyglycerols (HPGs) with low polydispersities and predetermined molecular weights were reported using anionic ring opening multibranching polymerization of glycidol with slow monomer addition and partial deprotonation of the initiator [4]. The structure of the polymers is shown in Fig. 1. Recently we reported the synthesis of very high molecular weight HPGs with low polydispersities using high monomer to initiator ratios in the presence and absence of emulsifying solvents [5]. Narrowly polydisperse HPGs (polydispersity index (PDI)=Mw/Mn=1.1–1.4, where Mw and Mn are the weight and number average molecular weights, respectively) were synthesized using dioxane as emulsifying agent. Broader molecular weight distributions with low molecular weight fractions were obtained when diglyme was used although the low molecular weight fractions could be removed by dialysis. We prepared a series of polymers with various molecular weights with a relatively low PDI=1.1–1.8 and the Mw range up to 1,475,000. As the number of functional hydroxyl groups approximately equals the degree of polymerization, synthesis of higher molecular weight HPGs is of significant interest as such materials have numerous potential applications and have properties likely unattainable in a practical sense by other approaches, given the complexity inherent in synthesizing higher generation dendrimers. Such polymers, with low intrinsic viscosity and a very large number of derivatizable hydroxyl groups, would seem to be excellent candidates for applications in nanotechnology and in nanomedicine.

Due to their structural similarities to poly(ethylene glycol) (PEG) and polysaccharides, polyglycerols (PGs) are also expected to be biocompatible [6]. The availability of multiple functional groups in the HPG class of polyether polyols is an advantage in pharmaceutical and medical applications compared to linear PEG. An additional advantage is that HPG is thermally and oxidatively more stable than PEG [7] and of much lower viscosity for comparable molecular weights, potentially opening up the concentration range over which these polyethers can be used. Recently we reported the biocompatibility of low molecular weight polymers (Mn∼3000) using several in vitro techniques and animal toxicity studies where HPGs were found to be as biocompatible as PEG, suggesting that these robust polymers are promising biomaterials [8]. The biocompatibility of polymers is a function of molecular weight, however, as volume exclusion, cell aggregation, polymer adsorption and solution viscosity all increase strongly with molecular weight. In addition, in the case of HPGs, the large number of functional end groups could potentially affect interactions with biological macromolecules or cells. We therefore selected two high molecular weight HPGs and studied their biocompatibility properties in detail using a variety of in vitro techniques. The results are reported in this article.

Tests of coagulation (prothrombin time (PT), activated partial thromboplastin time (APTT), and plasma recalcification time (PRT), thrombelastograph parameters (TEG)), platelet activation (PAC-1, fibrinogen and CD62 expression), complement activation (C3a levels), red blood cell aggregation, plasma protein precipitation as well as cytotoxicity experiments were conducted.

Section snippets

Materials

All chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON) and used without further purification except glycidol (96%) which was purified by vacuum distillation and stored over molecular sieves in a refrigerator (2–4 °C). 1,1,1-tris(hydroxymethyl)propane (Fluka) and potassium methylate solution (25 wt% in methanol, Fluka) were used as supplied. Anhydrous diglyme and dioxane were also obtained from Aldrich and used without further drying. For the in vitro biocompatibility testing

Results and discussion

HPGs are thought to be a promising candidate for biotechnology applications [6]. Detailed descriptions of their synthesis, biocompatibility and applications have been reported [4], [7], [8]. However the data available to date were limited to low molecular weight fractions (Mn⩽20,000). Very recently we reported the synthesis and solution properties of several HPGs with molecular weights as high as 700,000 (Mn) [5]. We have found that narrowly polydispersed polymers can be synthesized using

Conclusions

High molecular weight hyperbranched PGs appear to have very little effect on a variety of measures of biocompatibility in vitro. Although they are of high molecular weight, the inherently compact hyperbranched structure removes many of the disadvantages associated with the exposure of high molecular weight linear polymers to blood and cells. In fact, the HPGs behave in solution more like proteins than linear polymers. The high molecular weight means there are a large number of reactive sites

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