Sustained Release of Vascular Endothelial Growth Factor from Poly(ε-caprolactone-PEG-ε-caprolactone)-b-Poly(l-lactide) Multiblock Copolymer Microspheres

Abstract

Vascular endothelial growth factor (VEGF) is the major regulating factor for the formation of new blood vessels, also known as angiogenesis. VEGF is often incorporated in synthetic scaffolds to promote vascularization and to enhance the survival of cells that have been seeded in these devices. Such applications require sustained local delivery of VEGF of around 4 weeks for stable blood vessel formation. Most delivery systems for VEGF only provide short-term release for a couple of days, followed by a release phase with very low VEGF release. We now have developed VEGF-loaded polymeric microspheres that provide sustained release of bioactive VEGF for 4 weeks. Blends of two swellable poly(ε-caprolactone)–poly(ethylene glycol)–poly(ε-caprolactone)- b -poly( l -lactide) ([PCL–PEG–PCL]- b -[PLLA])-based multiblock copolymers with different PEG content and PEG molecular weight were used to prepare the microspheres. Loading of the microspheres was established by a solvent evaporation-based membrane emulsification method. The resulting VEGF-loaded microspheres had average sizes of 40–50 μm and a narrow size distribution. Optimized formulations of a 50:50 blend of the two multiblock copolymers had an average VEGF loading of 0.79 ± 0.09%, representing a high average VEGF loading efficiency of 78 ± 16%. These microspheres released VEGF continuously over 4 weeks in phosphate-buffered saline pH 7.4 at 37 °C. This release profile was preserved after repeated and long-term storage at −20 °C for up to 9 months, thereby demonstrating excellent storage stability. VEGF release was governed by diffusion through the water-filled polymer matrix, depending on PEG molecular weight and PEG content of the polymers. The bioactivity of the released VEGF was retained within the experimental error in the 4-week release window, as demonstrated using a human umbilical vein endothelial cells proliferation assay. Thus, the microspheres prepared in this study are suitable for embedment in polymeric scaffolds with the aim of promoting their functional vascularization.

Vascular Endothelial Growth Factor–Releasing Microspheres Based on Poly(ε-Caprolactone-PEG-ε-Caprolactone)-b-Poly(L-Lactide) Multiblock Copolymers Incorporated in a Three-Dimensional Printed Poly(Dimethylsiloxane) Cell Macroencapsulation Device

“…VEGF-loaded and placebo microspheres were prepared with Symbiosys multiblock copolymers using a membrane emulsification-based double emulsion method and characterized as reported previously. 35 Microspheres were prepared with a 50:50 blend of Symbiosys multiblock copolymers A and B. Polymer A (~54 kDa) consisted of 30 wt% of an amorphous, hydrophilic poly(ε-caprolactone)-PEG 3000 -poly(ε-caprolactone) (PCL-PEG 3000 -PCL) block with an M w of 4000 g/mol and 70 wt% of a semi-crystalline poly(L-lactide) block with an M w of 4000 g/mol. The total PEG weight fraction of polymer A is 22.5%.…”

“…The obtained parameters of fitted VEGF release from microspheres filled in PDMS devices were compared to the parameters of the fit of the VEGF release curves from microspheres in IVR buffer, which have been reported previously. 35…”

“…The bioactivity of released VEGF from the implants was analyzed by Alamar Blue proliferation assay with HUVECs, as described previously. 35 In short, a 12 mg/mL microsphere dispersion of VEGF-loaded microspheres in 1% HA carrier liquid was prepared as described in the section Release of VEGF From Microspheres Filled in PDMS Devices and filled in PDMS devices (see section Filling of PDMS Devices). Filled PDMS devices were incubated in 10 mL cell-compatible IVR buffer ("bioactivity IVR buffer"; Dulbecco's PBS pH 7.4, 0.2 mm filtered, 0.5% bovine serum albumin, 30 mg/mL gentamicin, and 15 ng/mL amphotericin) at 37 C under mild agitation.…”

“…These multiblock copolymers form amorphous domains [primarily consisting of poly(ε-caprolactone)poly(ethylene glycol)-poly(ε-caprolactone) blocks] and semicrystalline domains [mainly composed of poly(L-lactic acid) blocks], and are attractive for protein delivery due to their wellcontrolled swelling properties which allow continuous release through diffusion with low burst release. [32][33][34][35] The release of proteins can be tailored by the weight fraction and molecular weight (M w ) of poly(ethylene glycol) (PEG), 33,36 which was also demonstrated for microspheres loaded with VEGF. 35 In this study, we incorporated VEGF-loaded microspheres in poly(dimethylsiloxane) (PDMS)-based devices 37 and studied the release of VEGF and its bioactivity.…”

“…[32][33][34][35] The release of proteins can be tailored by the weight fraction and molecular weight (M w ) of poly(ethylene glycol) (PEG), 33,36 which was also demonstrated for microspheres loaded with VEGF. 35 In this study, we incorporated VEGF-loaded microspheres in poly(dimethylsiloxane) (PDMS)-based devices 37 and studied the release of VEGF and its bioactivity. Hyaluronic acid (HA) solution served as carrier liquid for VEGF-loaded microspheres.…”