Injectable fibroblast growth factor-2 coacervate for persistent angiogenesis

Abstract

Enhancing the maturity of the newly formed blood vessels is critical for the success of therapeutic angiogenesis. The maturation of vasculature relies on active participation of mural cells to stabilize endothelium and a basal level of relevant growth factors. We set out to design and successfully achieved robust angiogenesis using an injectable polyvalent coacervate of a polycation, heparin, and fibroblast growth factor-2 (FGF2). FGF2 was loaded into the coacervate at nearly 100% efficiency. In vitro assays demonstrated that the matrix protected FGF2 from proteolytic degradations. FGF2 released from the coacervate was more effective in the differentiation of endothelial cells and chemotaxis of pericytes than free FGF2. One injection of 500 ng of FGF2 in the coacervate elicited comprehensive angiogenesis in vivo. The number of endothelial and mural cells increased significantly, and the local tissue contained more and larger blood vessels with increased circulation. Mural cells actively participated during the whole angiogenic process: Within 7 d of the injection, pericytes were recruited to close proximity of the endothelial cells. Mature vasculature stabilized by vascular smooth muscle cells persisted till at least 4 wk. On the other hand, bolus injection of an identical amount of free FGF2 induced weak angiogenic responses. These results demonstrate the potential of polyvalent coacervate as a new controlled delivery platform.

Fig. 1.

(A) Design of a coacervate delivery matrix. The crystal structure of [FGF∶heparin∶FGFR] complex indicates that heparin actively participates in the interaction of FGF and its receptor whose heparin-binding domains are labeled yellow and pink, respectively (Top Left). (Top Right) Scheme represents the design of the coacervate where a synthetic polycation replaces the heparin-binding domain of FGFR and forms a complex with heparin and FGF. (B) Chemical structure of poly(ethylene argininylaspartate diglyceride) (PEAD). The backbone of PEAD composed of aspartic acid and ethylene glycol diglyceride is linked by ester bonds (arrows). The conjugation of arginine provides the polymer with two cationic groups per repeating unit: ammonium and guanidinium. (C) The three components dissolve well in water individually as represented here by heparin. Adding FGF2 induces no apparent changes in solubility. Upon addition of PEAD, the solution turns cloudy. Charge neutralization between the polycation and heparin forms the [PEAD∶heparin∶FGF2] coacervate, which is insoluble in the aqueous solution, enabling local delivery of FGF2. Upon standing for 24 h, the coacervate aggregates to the bottom of the tube. (D) SEM micrograph revealed that [PEAD∶heparin∶FGF2] mainly consisted of globular domains that fuse together. The globular nature of the coacervate is more distinguishable at higher magnification. Scale bars: 10 μm (low magnification) and 1 μm (high magnification). (E) The loading efficiency is > 95% for FGF2 (500 μg PEAD, 100 μg heparin, FGF2 range tested: 100–1,000 ng). Western blot demonstrated that the intensity of the coacervate and the loading solution is the same. S: FGF2 in the supernatant after centrifugation. C: FGF2 in the settled coacervates. L: total amount of FGF2 in the loading solution. (F) Protection from proteolysis by the coacervate. FGF2 and trypsin (mass ratio 1∶200) was incubated for 30 min or 2 h at 37 °C. The results indicated that free FGF2 (I) was completely degraded within 0.5 h. On the other hand, heparin (II) and the coacervate (III) protected FGF2 from degradation for at least 2 h. (G) The coacervate localized FGF2 release in fibrin gel. Fibrin gels were prepared with free FGF2 or FGF2-containing coacervate. The amount of FGF2 in the medium was determined by Western blot. Less FGF2 was present in the medium of the coacervate group, suggesting that FGF2 was localized better in the fibrin gel than the free FGF2 group. (H) Endothelial tube formation in fibrin gels. HUVECs mixed with free FGF2 (50, 250, or 500 ng/mL) or the same amount of FGF2 in the coacervate were encapsulated in the fibrin gel. After incubation of 3 d, the coacervate induced extensive tube network formation at 250 and 500 ng/mL of FGF2. On the contrary, FGF2 alone induced sparse tube formation at all growth factor concentrations. Scale bar: 100 μm. (I) Chemotaxis of pericytes by the coacervate. After incubation for 12 h, migrated pericytes were stained by PicoGreen. Quantitative comparison suggested that the coacervate induced significantly higher extent of chemotaxis than free FGF2 (254 ± 44 vs. 108 ± 14 per mm², p < 0.05, Student’s t test). Scale bar: 100 μm.

Fig. 2.

(A) Macroscopic observation of subcutaneous tissue showed that the coacervate clearly induced new blood vessel formation at the injection site (2-wk pictures from the same mouse). The injection site was marked by a circle. (B) Hematoxylin and eosin staining of subcutaneous tissues after 4 wk. For the saline, delivery vehicle, and free FGF2 groups, there was no clear growth of vasculature in the subcutaneous region. The coacervate group, on the contrary, revealed the feature of blood vessel that had a closed inner layer of nucleated cells surrounded by smooth muscle bundles (arrow). Scale bar: 50 μm. (C) Hemoglobin quantification compared the extent of angiogenesis between different groups. The result suggested that the coacervate group had a higher amount of hemoglobin 2 wk postinjection, whereas free FGF2 did not have statistical difference between the saline and delivery vehicle groups. This difference lasted at least for 4 wk (mean ± SD, n = 4–8 for each condition). Normalized to the saline group. One-way ANOVA followed by Bonferroni correction was applied for multiple comparisons. *p < 0.05, **p < 0.01. (D) The ratio of hemoglobin at the injection sites and the contralateral sites. For the coacervate, the ratio was significantly higher than that of the free FGF2 group. The result explained that FGF2 was well localized at the injection site by the delivery vehicle. Student’s t test was used as a statistical tool. *p < 0.05, **p < 0.01.

Fig. 3.

[PEAD∶heparin∶FGF2] coacervate induces more potent angiogenesis in vivo. (A) Representative confocal micrographs showed the distribution of blood-vessel associated markers CD31 (endothelial cell, red) and α-SMA (mural cell, green) of each group at three time points. Both the free and coacervate FGF2 groups revealed a higher quantity of endothelial cells than saline control after 1 wk, but only the coacervate induced an increase of α-SMA expression after 2 wk. The circular vessel-like structures were observed in the field. After 4 wk, more endothelial and mural cells were present in the coacervate group demonstrating the long-term efficacy of the FGF2 coacervate. Scale bar: 50 μm. (B) High magnification revealed the maturation of the blood vessels induced by the coacervate. The endothelial tubes were clearly surrounded by mural cells. Scale bar: 50 μm. (C) Comparison of CD31 and α-SMA expression in the four injected groups. The number of endothelial cells in the coacervate group was higher than those of the control groups by 47% to 120%. More significantly, the number of mural cells in the coacervate group was 2.02 folds of that in the free FGF2 group. One-way ANOVA followed by Bonferroni correction, *p < 0.05; **p < 0.01. (D) Comparison of the number of blood vessels in a given size range between free and coacervate FGF2 groups as previously described; the value represents the cumulative number of all the slides examined (24). The coacervate induced more blood vessel formation than free FGF2. Furthermore, the coacervate group contained more large vessels (> 1,000 μm², likely associated with arterioles and venules).

Fig. 4.

Enhanced maturity of the nascent blood vessels by the coacervate. (A) The colocalization of CD31-positive and PDGFR-β-positive cells suggested the coacervate quickly recruited pericytes to interact with endothelial cells in the nascent vessels. This phenomenon was absent in the free FGF2 group. (B) Significant colocalization (green + red = yellow) of VWF- and CD31-positive cells suggested that the endothelial cells in the nascent vessels can potentially participate in hemostasis. (C) Both α-SMA and desmin are markers for mural cells. Their expression pattern revealed that larger vessels coexpressed these markers, whereas smaller vessels were dominated by the expression of desmin (arrows). (D) Calponin, a calmodulin associated with vascular smooth muscle cells contraction, was costained with α-SMA to examine the potential functionality of the new blood vessels. The result indicated that the blood vessels in the coacervate group had abundant expression of calponin. In addition, the blood vessels were much larger in the coacervate group than those in the free FGF2 group. Scale bars indicate 50 μm for both low and high magnification.