Amphiphilic degradable polymers for immobilization and sustained delivery of sphingosine 1-phosphate

Abstract

Controlled delivery of the angiogenic factor sphingosine 1-phosphate (S1P) represents a promising strategy for promoting vascularization during tissue repair and regeneration. In this study, we developed an amphiphilic biodegradable polymer platform for the stable encapsulation and sustained release of S1P. Mimicking the interaction between amphiphilic S1P and its binding proteins, a series of polymers with hydrophilic poly(ethylene glycol) core and lipophilic flanking segments of polylactide and/or poly(alkylated lactide) with different alkyl chain lengths were synthesized. These polymers were electrospun into fibrous meshes, and loaded with S1P in generally high loading efficiencies (>90%). Sustained S1P release from these scaffolds could be tuned by adjusting the alkyl chain length, blockiness and lipophilic block length, achieving 35-55% and 45-80% accumulative releases in the first 8h and by 7 days, respectively. Furthermore, using endothelial cell tube formation assay and chicken chorioallantoic membrane assay, we showed that the different S1P loading doses and release kinetics translated into distinct pro-angiogenic outcomes. These results suggest that these amphiphilic polymers are effective delivery vehicles for S1P and may be explored as tissue engineering scaffolds where the delivery of lipophilic or amphiphilic bioactive factors is desired.

Keywords: Amphiphilic copolymer; Angiogenesis; Drug delivery; Sphingosine-1-phosphate; Tissue engineering.

Figure 1

(A) Schematic illustration of possible interactions between S1P and the amphiphilic polymers. (B) Synthetic schemes for the alkylated lactides and amphiphilic polymers. Reagents and conditions: (a) 2-bromopropionyl bromide (1.05 eq.), Et₃N (2.0 eq.), acetone, rt for 0.5 h, then filtered; Et₃N (2.0 eq.), 65 °C for 2 h. (b) PEG20K, Sn(Oct)₂, 150 °C, 30 min. (c) D,L-lactide, 150 °C, 60 min. (d) PEG20K, D,L-lactide, Sn(Oct)₂, 150 °C, 60 min.

Figure 2

(A) GPC chromatograms of triblock copolymer intermediate P(C₁₄LA)₁₅-b-PEG₄₅₄-b-P(C₁₄LA)₁₅ (M_n = 34,653, PDI = 1.13) and crude pentablock copolymer PLA₃₁₂-b-P(C₁₄LA)₁₅-b-PEG₄₅₄-b-P(C₁₄LA)₁₅-b-PLA₃₁₂ (C14-L, M_n = 113,463, PDI = 1.47). (B) ¹H NMR spectra of the pentablock and random copolymers.

Figure 3

DSC spectra of PELA, pentablock and random copolymers.

Figure 4

(A) SEM micrographs, (B) calculated mesh porosity (by methods described in section 2.10; n = 3) and (C) water contact angles (n = 7) of electrospun fibrous meshes. Scale bar = 50 μm.

Figure 5

(A) S1P loading efficiencies (n = 3) on polymeric fibrous meshes and (B) their cumulative releases over time (n = 3) in PBS with 0.2% FAF-BSA.

Figure 6

Representative micrographs and total tube length quantifications (n = 3-4) of HUVEC-Matrigel cultures after 17 h exposure to free S1P solutions of varying concentrations (A & C) or polymer meshes preloaded with varying doses of S1P (B & D). Scale bar = 100 μm.

Figure 7

Ex-ovo angiogenic effects of amphiphilic polymer meshes preloaded with 0.5-μg S1P examined by CAM assay. (A) Representative photographs of the CAM surrounding the meshes with/without S1P (16× mag.) at day 0 and day 3, and the photographs of the flipped side of the harvested CAM on day 3 (25× mag.) of the boxed area. (B) Quantification of microvessel numbers surrounding each scaffold (n = 4).