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. 2021 May 22;13(6):779.
doi: 10.3390/pharmaceutics13060779.

Translational Studies on the Potential of a VEGF Nanoparticle-Loaded Hyaluronic Acid Hydrogel

Joanne O'Dwyer  1   2   3 , Robert Murphy  4 , Arlyng González-Vázquez  2   3 , Lenka Kovarova  5   6 , Martin Pravda  5 , Vladimir Velebny  5 , Andreas Heise  4   7 , Garry P Duffy  2   3   7   8   9 , Sally Ann Cryan  1   2   3   7   8
Affiliations

Translational Studies on the Potential of a VEGF Nanoparticle-Loaded Hyaluronic Acid Hydrogel

Joanne O'Dwyer et al. Pharmaceutics. .

Abstract

Heart failure has a five-year mortality rate approaching 50%. Inducing angiogenesis following a myocardial infarction is hypothesized to reduce cardiomyocyte death and tissue damage, thereby preventing heart failure. Herein, a novel nano-in-gel delivery system for vascular endothelial growth factor (VEGF), composed of star-shaped polyglutamic acid-VEGF nanoparticles in a tyramine-modified hyaluronic acid hydrogel (nano-VEGF-HA-TA), is investigated. The ability of the nano-VEGF-HA-TA system to induce angiogenesis is assessed in vivo using a chick chorioallantoic membrane model (CAM). The formulation is then integrated with a custom-made, clinically relevant catheter suitable for minimally invasive endocardial delivery and the effect of injection on hydrogel properties is examined. Nano-VEGF-HA-TA is biocompatible on a CAM assay and significantly improves blood vessel branching (p < 0.05) and number (p < 0.05) compared to a HA-TA hydrogel without VEGF. Nano-VEGF-HA-TA is successfully injected through a 1.2 m catheter, without blocking or breaking the catheter and releases VEGF for 42 days following injection in vitro. The released VEGF retains its bioactivity, significantly improving total tubule length on a Matrigel® assay and human umbilical vein endothelial cell migration on a Transwell® migration assay. This VEGF-nano in a HA-TA hydrogel delivery system is successfully integrated with an appropriate device for clinical use, demonstrates promising angiogenic properties in vivo and is suitable for further clinical translation.

Keywords: angiogenic growth factor; catheter delivery; chick chorioallantoic membrane model; hyaluronic acid hydrogel; nanoparticle-loaded hydrogel; protein delivery; sustained release; vascular endothelial growth factor nanoparticles.

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Conflict of interest statement

The authors declare no conflict of interest. Authors L.K., M.P. and V.V. are employees of Contipro. The funders had no role in the design of the study; in the collection, analysis, or interpretation of the data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Experimental outline for the chick chorioallantoic membrane (CAM) experiment.
Figure 2
Figure 2
(a) The AMCath catheter used for hydrogel injection (Adapted from [35], SAGE Publications, 2018). (b) Syringes containing nano-VEGF-HA-TA attached to the prototype catheter and connected via a 3D printed adaptor to the 50 N load cell of the Zwick for testing the force required for injection. (c) Full length of catheter; tip is set to inject into the 200 µL injection mold (foreground).
Figure 2
Figure 2
(a) The AMCath catheter used for hydrogel injection (Adapted from [35], SAGE Publications, 2018). (b) Syringes containing nano-VEGF-HA-TA attached to the prototype catheter and connected via a 3D printed adaptor to the 50 N load cell of the Zwick for testing the force required for injection. (c) Full length of catheter; tip is set to inject into the 200 µL injection mold (foreground).
Figure 3
Figure 3
Chick chorioallantoic membrane at day twelve, exposed to (a) HA-TA alone, (b) free VEGF-HA-TA and (c) nano-VEGF-HA-TA. No hyperemia is present in any group. VEGF dose in all cases is 500 ng per 200 µL hydrogel portion. One representative image is shown from each group. Circle indicates hydrogel position. (n = 5).
Figure 4
Figure 4
(a) A section of CAM from a membrane exposed to HA-TA alone. Some branch points are evident, but they are not as numerous as those observed in (b) a section of CAM from the free VEGF-HA-TA group or (c) a section of CAM from the nano-VEGF-HA-TA group. (d) Quantification of the number of branch points in a 16 mm region of interest surrounding the hydrogels. Free VEGF-HA-TA and nano-VEGF-HA-TA both contain 500 ng VEGF. * p < 0.05. (n = 5).
Figure 5
Figure 5
Regions of interest around (a) HA-TA alone, (b) free VEGF-HA-TA and (c) nano-VEGF-HA-TA, representative of those used to calculate vessel number, length density and branch points. One representative image is shown from n = 5 in each group. (d) Quantification of the number of vessels in the region of interest around HA-TA alone, free VEGF-HA-TA (500 ng VEGF) and nano-VEGF-HA-TA (500 ng VEGF). (e) Vascular length density in the region of interest surrounding the hydrogels compared to the vascular length density of a distant area of the chorioallantoic membrane. * p < 0.05, ** p < 0.01, *** p < 0.001. (n = 5).
Figure 6
Figure 6
(a) Young’s modulus at day zero and day seven of hydrogels formed either via injection through the Benchtop Hydrogel Mixer (BHM) or via injection through AMCath connected to a syringe pump. (b) % cumulative VEGF release from nano-VEGF-HA-TA hydrogels formed via AMCath injection based on a dose of 50 ng VEGF per hydrogel sample. * p < 0.05. (n = four technical replicates for mechanical testing, n = three technical replicates for release testing).
Figure 7
Figure 7
(a) Metabolic activity of human umbilical vein endothelial cells (HUVECs) exposed to release supernatant from AMCath-injected nano-VEGF-HA-TA, compared to cells fed with normal media without VEGF, or media containing 30 ng fresh free VEGF. (b) Quantified total tubule length on a Matrigel® assay produced by 42-day pooled release supernatant from nano-VEGF-HA-TA formulations formed via injection through AMCath compared to the tubule length produced by untreated cells or those exposed to a similar dose (30 ng) of fresh free VEGF. (c) Quantification of remaining gap width on a scratch assay, where zero indicates complete gap closure. HUVECs were exposed to nano-VEGF-HA-TA release supernatant or fresh free VEGF, each containing 30 ng VEGF, and cells not treated with VEGF were used as a control. (d) Cell migration as determined by number of calcein stained cells per field on a Transwell® migration assay. Quantification of the migration of HUVECs in medium without VEGF (cells alone in serum-free medium) is compared to that achieved by HUVECs treated with 30 ng fresh free VEGF or pooled release medium from AMCath formed nano-VEGF-HA-TA also containing 30 ng VEGF. * p < 0.05. (n = 3 technical replicates).
Figure 7
Figure 7
(a) Metabolic activity of human umbilical vein endothelial cells (HUVECs) exposed to release supernatant from AMCath-injected nano-VEGF-HA-TA, compared to cells fed with normal media without VEGF, or media containing 30 ng fresh free VEGF. (b) Quantified total tubule length on a Matrigel® assay produced by 42-day pooled release supernatant from nano-VEGF-HA-TA formulations formed via injection through AMCath compared to the tubule length produced by untreated cells or those exposed to a similar dose (30 ng) of fresh free VEGF. (c) Quantification of remaining gap width on a scratch assay, where zero indicates complete gap closure. HUVECs were exposed to nano-VEGF-HA-TA release supernatant or fresh free VEGF, each containing 30 ng VEGF, and cells not treated with VEGF were used as a control. (d) Cell migration as determined by number of calcein stained cells per field on a Transwell® migration assay. Quantification of the migration of HUVECs in medium without VEGF (cells alone in serum-free medium) is compared to that achieved by HUVECs treated with 30 ng fresh free VEGF or pooled release medium from AMCath formed nano-VEGF-HA-TA also containing 30 ng VEGF. * p < 0.05. (n = 3 technical replicates).
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