Highly Bioactive VEGF Release from Liquid Injectable Poly(trimethylene carbonate-co-5-hydroxyl trimethylene carbonate)

Mohajeri, Sara (Department of Chemical Engineering, Queen’s University, Kingston, Canada)
Burke-Kleinman, Jonah (Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Canada)
Maurice, Donald H. (Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Canada)
Amsden, Brian (Department of Chemical Engineering, Queen’s University, Kingston, Canada)

Introduction

Peripheral arterial disease is a chronic condition caused by the formation and hardening of fatty plaque within arteries and arterioles, resulting in narrowing or blocking of the vessels carrying blood to the limbs.  Gradually, the flow of blood becomes restricted leading to critical limb ischemia in the tissues fed by the diseased artery.(1) A potential treatment approach for this condition consists of administering angiogenic growth factors, such as vascular endothelial growth factor (VEGF), directly to the ischemic site via a biocompatible, and biodegradable delivery vehicle.(2,3) Viscous liquid injectable polymers possess certain attributes that make them an attractive delivery vehicle for such delivery. They allow minimally invasive administration through standard gauge needles and elicit minimal irritation when implanted in soft tissue. In this approach, the growth factors are co-lyophilized with a non-reducing cryoprotectant sugar, such as trehalose, and incorporated into the polymer by simple mixing.(3–6) However, the aliphatic polyesters commonly used to prepare such delivery vehicles degrade to acidic products that have been demonstrated to denature incorporated protein therapeutics.(7)(8)

We have recently demonstrated that poly(5-hydroxy trimethylene carbonate) is rapidly degraded in mild alkaline environments, producing glycerol and carbon dioxide as the degradation products.(9)  We reasoned that by co-polymerizing 5-hydroxyl trimethylene carbonate (HTMC) with trimethylene carbonate, a gradually degradable viscous liquid copolymer, P(TMC-co-HTMC), could be formed that degrades to cytocompatible products. It was further reasoned that this copolymer would be a suitable VEGF delivery vehicle. Herein we report the influence of P(TMC-co-HTMC) molecular weight and composition on its in vitro degradation rate and on the in vitro release and bioactivity of VEGF from a P(TMC-co-HTMC) delivery vehicle.

Materials and Methods

P(TMC-co-HTMC)s of 30 and 50 mol% of HTMC and molecular weight (Mn) range of 600-2000 Da were prepared via ring opening polymerization using either butanol (BU) or octanol (OCT) as an initiator. Mass loss of the resulting copolymers was determined at 37 ºC in pH 7.4 PBS. VEGF particles were prepared by dissolving bovine serum albumin (BSA), VEGF and trehalose in pH 7.4 PBS (Formulation A) or phosphate buffer (PB) (Formulation B) at a VEGF:BSA:trehalose weight ratio of 0.2:97.8:2 and a total concentration of 5 wt% at room temperature. The solutions were frozen in liquid N2, and lyophilized. The lyophilized particles were then mixed into the copolymer. In vitro protein release was determined from 100 mg of the formulation placed in a glass vial at 37 ºC under horizontal shaking in 1 mL pH 7.4 PBS containing 0.02% (v/v) Tween 20, 0.02% (w/v) Na azide. The VEGF concentration in the release medium was measured using a human VEGF-A ELISA kit. For bioactivity assessment the release medium was 1% BSA and 1% antibiotics in pH 7.4 PBS at 37 ºC.(5) The bioactivity of the VEGF released into the latter medium was assessed through its ability to stimulate the proliferation of telomerase-immortalized human aortic endothelial cells (TeloHAEC) with cell number measured using a WST-1 assay kit.

Results

Poly(TMC-co-HTMC) was successfully synthesized with compositions and Mns close to target values. The copolymers exhibited moderate to fast mass loss, with the rate of mass loss depending on the initial copolymer HTMC content and Mn, and the hydrophilicity of the initiator (Fig. 1). Importantly, the buffer pH remained close to neutral throughout the degradation period.

Roughly 90% of the VEGF was released within 9 days from Formulation A, but over 42 days from Formulation B, which contained 9 times less salt (Fig. 2A). The released VEGF from both formulations retained greater than 80% bioactivity throughout the release period (Fig. 2B).

Discussion and Conclusion

The HTMC content of the copolymer was the most influential parameter affecting its mass loss. Hydrolysis of the copolymer occurs through intramolecular nucleophilic attack of the pendant hydroxyl on the HTMC on the carbonate bond.(9) The polymer chain length decreases until polymer fragments are formed that dissolve in water. Thus, incorporation of a higher content of the hydrophilic HTMC facilitated both water diffusion into the copolymer bulk and provided more cleavage sites leading to faster generation of water-soluble products. The rate of VEGF release was strongly influenced by the salt content in the incorporated particles, suggesting that release was affected by the osmotic activity of the solution formed within the polymer by the dissolved particles.(10) The lower osmotic pressure produced by the particles in Formulation B reduced the extent of water penetration into the copolymer which subsequently retarded the dissolution and release of the VEGF, leading to a slower release rate. This study introduced P(TMC-co-HTMC) as a potential vehicle to deliver acid sensitive therapeutic proteins such as VEGF without loss of their bioactivity.


Figure 2. A) Influence of formulation parameters on the release profiles of VEGF, n=3. B) Bioactivity of the released VEGF.

Figure 1. Mass loss of P(TMC-co-HTMC)s initiated with BU or OCT at different number of repeating units (P6, P10 and P18) and HTMC mol% (50H or 30H) in PBS at pH 7.4, n=3.

Acknowledgements

Funding for this work was provided by a CIHR Open Operating Grant.

References

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