(84a) Synchronized Delivery of Vascular Endothelial Growth Factor With Protease Cues Sustains Activation Toward Vessel Formation

Synchronized delivery of vascular endothelial growth
factor with protease cues

sustains activation toward vessel formation

Suwei Zhu, Yunfeng
Lu, Tatiana Segura

Chemical
& Biomolecular Engineering Department, University of California Los Angeles

Over 6.5 million patients annually in the United States are
affected by wound-related health problems resulting from impeded healing
processes. Following cutaneous wounding, dermal angiogenesis -- the
formation of new blood vessels to bring in nutritive substances and oxygen -- is important for the reconstruction of the normal aspects
of skin physiology and yet to be improved in tissue regeneration. More
specifically, the signal protein of vascular endothelial growth factor (VEGF), which
enhances the survival, proliferation and migration behaviors of endothelial
cells, needs to be presented at the wound site in a cell-responsive and
sustainable fashion. We have designed a nanocapsule delivery system of VEGF
(nVEGF) allowing cargo to be liberated only in the presence of
cell-secreted proteases.  Unlike conventional protein therapeutic delivery
approaches that resort to multi-dose administrations or matrix-assisted
retention of growth factors, nVEGF act as a reservoir to sustain modulated
release of active VEGF, based on a nanocapsule platform that can be applied
alone or within versatile scaffolds.

In our strategy, a nanogel shell comprised of acrylamide
based monomers and plamin-sensitive peptide (KNRVK) crosslinker was weaved
around VEGF to synthesize protein nanocapsules (nVEGF), which were characterized
by transmission electron microscopy (TEM) to be 15~30 nm in diameter, dynamic
light scattering (DLS) and enzyme-linked immunosorbent assay (ELISA) for
targeted release (Fig. 1). In the aspect of molecular
biology, nVEGF altered the phosphorylation profile of VEGF receptor-2 on
human umbilical endothelial cells (HUVECs) and downstream signalings into a
prolonged activation state significantly different than naked VEGF did (Fig. 2). A
three-dimensional in vitro assay of HUVEC sprouting and lumen formation showed
that one dose of plasmin-degradable nVEGF supported such behaviors of HUVECs
while naked VEGF was soon depleted and supplemental additions were required (Fig. 3). Ex vivo application of nVEGF
on the chicken chorioallantoic membrane stimulated the formation of capillaries
(Fig. 4). In mouse skin wound healing model, the application of nVEGF within
fibrin matrices promoted a faster wound closure with a significantly higher
density of infiltrated endothelial cells and capillaries (Fig. 5).

Our results demonstrated plasmin-degradable VEGF nanocapsules
sustained the activity of VEGF (preventing clearage and degradation) and
achieved the same degree of angiogenesis as re-dosing with exogenous VEGF. Here
enzymatically-triggered release of VEGF from the nanocapsule reservoir
synchronized the dosage with tissue endogenous protease levels. By reducing the
frequency of therapeutic administration, such applications of growth factor
nanocapsules would be clinically relevant and practically feasible to
continuous deliver active growth factors in regions with proteases and at times
of demand.

Figure 1 Characterization of the size of nVEGF and the
degradability of its nanogel shell to release cargo VEGF. TEM image of purified
nVEGF showing spherical particle morphology (left) and measured distribution of
nanocapsule diameters (N = 322) (upper center). Hydrodynamic sizes of different
batches of nVEGFs and naked VEGF (lower center). Release of VEGF from
nVEGF upon protease degradation of nanogel shells (n = 3; mean ± SEM)
(right).

Figure 2 nVEGF prolongs the phosphorylation of VEGFR-2 (Tyr
1175) due to VEGF release by cell-produced proteases. Western blot analysis
from HUVECs exposed to native VEGF and nVEGF for different lengths of time (left)
and normalized phosphorylation profiles (n = 3; mean ± SEM) (upper
center). Phospho-ELISA analysis of HUVECs exposed for 30 min to consecutively
reapplied VEGF and nVEGF (right) and the prolonged phosphorylation profile
reproduced by additions of a small amount of VEGF in the last 5 min of each
incubation period (lower center) (n = 3; mean ± SEM).

Figure 3 Single dose plasmin-degradable nVEGF supports the
sprouting of HUVECs in fibrin matrices over 7 days in the bead assay.

Figure 4 Ex vivo implantation of fibrin matrices with VEGF
or nVEGF induces capillaries in chicken chorioallantoic membrane assay.

Figure 5 Mouse wound healing model and angiogenesis.