(50c) Solid-Phase Synthesis of Functionalized Peptides as Enzymatically Degradable Crosslinkers for Fabrication of Tissue Engineering Scaffolds

Biomaterials seeded with
cells and coupled with minimally invasive techniques are an attractive
alternative for treating irregularly shaped defects with minimum tissue
dissection and retraction. After hardening in-situ, these three dimensional
matrices can guide the organization, differentiation, proliferation, and
development of seeded cells into the desired tissue. Biodegradable scaffolds
provide the opportunity to form a completely new tissue that can adapt to
natural tissue functions as the matrix degrades. Degradable scaffolds
fabricated from polymers such as poly (lactic-co-glycolic acid) that degrades
by hydrolysis cannot provide the degradation characteristics required for
tissue remodeling. The ability to degrade extracellular proteins is essential for
any individual cell to interact properly with its immediate surroundings and
for multicellular organisms to develop and function normally.

The matrix
metaloproteinases (MMPs) constitute a family of secreted and cell surface
enzymes that degrade numerous structural extracellular matrix proteins such as
collagen. It is well established that MMPs act on and degrade specific
sequences of amino acids on the collagen fibrils. For example, osteoblasts
secret MMP-13 that cleave the amino acid sequence Pro-Gln-Gly-Leu-Ala-NH2 of
human type II collagen. This peptide sequence has been synthesized by
solid-phase peptide synthesis methodology and functionalized with acrylate
end-groups in solution for use as a crosslinker in fabrication of biologically
degradable hydrogel scaffolds. Normally, these peptide sequences, after
synthesis and cleavage from the solid support, are functionalized in solution
requiring extensive purification by dialysis. In this report, we present a
novel method for synthesis of functionalized peptide cross-linkers.

Rink amide resin, HoBt
and Fmoc-protected amino acids were purchased from Novabiochem (San Diego, CA).
Peptide synthesis grade reagents 4-Dimethylaminopyridine (DMAP),
1,3-diisopropylcarbodiimide (DIC), trifluoroacetic acid (TFA), triisopropylsilane
(TIPS), dichloromethane (DCM), dimethyl formamide (DMF), and acetonitrile
(MeCN) were purchased from Acros. All reagents were used as received without
any further purification. RGD peptide and MMP-3 degradable peptide with
unsaturated reactive end groups were synthesized manually using a novel method
on the Rink Amide NovaGelTM resin (NovaGel^TM, 0.62 mmol/g). Resin
(100 mg) was swelled in DMF (3 ml) for 1h and washed with DMF (3 ml). The
Fmoc-protected amino acid derivative (6 eq), HOBt (12 eq), and DIC (6.6 eq) in
dry DMF (3 ml) were mixed and added to the resin. 0.2 ml of 0.05 M DMAP was
added to this mixture and agitated with an orbital shaker for 4-6 h at 30°C. A small volume of the resin was
removed and tested for the presence of unreacted amine groups using Kaiser
reagents. If the test result was positive, the resin was washed with 3 ml of
DMF and the coupling reaction was repeated until a negative result was
obtained. Then, the resin was treated with 20% piperidine in DMF for 15 min, washed
with DMF, and other amino acids were coupled successively using the same
procedure. After coupling the last amino acid of the peptide chain, the resin
was washed with 5ml of DMF and DCM. The Mtt protecting group was selectively
deprotected by treating the peptidyl resin with 3 ml of TFA/DCM (1:99 v/v) for
2 min. The mixture was filtered and treated with the same volume and ratio of
TFA/DCM 7 times. The resin was washed
with 3x3 ml of DCM and DMF and the Fmoc protecting group was removed by
treatment with 20% piperidine in DMF for 2x15 min. The resin was washed
thoroughly with 5x3ml of DMF.

Bifunctional peptide
with unsaturated acrylate end groups was synthesized on resin by coupling
acrylic acid to the amine groups of glutamine and lysine residues at the two
ends of the peptide sequence. Briefly, acrylic acid (12 eq), HoBt (24 eq), and
DIC (13.2 eq) were mixed in 3 ml of DMF, added to the peptidyl resin, and mixed
in an incubator shaker at 30°C
for 6 h. The Kaiser reagents were used to test for the completion of the
coupling reaction. If the test result was positive the coupling reaction was
repeated until a negative test result was obtained. The resin was washed with
3x3 ml of DMF and DCM and treated with a mixture of 95% TFA, 2.5% TIPS, and
2.5% water for 2 h to cleave the peptide from the resin and deprotect the side
chains. The combined cleavage solution was added to cold ether at -20°C for 24 h to precipitate the crude
peptide cross-linker. The suspension was centrifuged, and the supernatant was
decanted. The pellet was dried for 2 h under vacuum. The crude product was
purified by preparative HPLC on a 250x10 mm, 10 mm Xterra® Prep RP18 column (Waters, Milford, MA) at a flow
rate 2 ml/min using a gradient 5% MeCN and 95% 0.1% aqueous TFA solvent mixture.
A photodiode array detector (model 996, Waters) was used for detection at a
wavelength of 214 nm. The fractionated and purified peptide was freeze-dried
and stored at -80°C until used.

Hydrogels as a cell
carrier were prepared using a novel poly (lactide-ethylene oxide-fumarate)
terpolymer as the macromer, peptide crosslinker degradable by MMP-13, and a
neutral redox initiation system. The redox system consisted of ammonium
persulfate (APS) and tetramethylethylenediamine (TMEDA), respectively. In a typical
procedure, 0.63g of PLEOF and 0.3 g of crosslinker were added to 1.65 ml of PBS
and vortexed. To this mixture, 0.21 ml of 0.3 M APS (Aldrich) and 0.21 ml of
0.3 M TMEDA (Aldrich) were added and vortexed. The mixture was degassed,
injected between two glass plates separated by a 0.5 mm gap, and fastened with
clips. The assembly was placed in a convection oven at 37°C for 15 min to crosslink. The
concentrations of APS and TMEDA in the final solution were each 0.03 M. After
crosslinking, the gel was removed from the glass plate and disks were cut from
the gel using a 15 mm cork-borer. The disk-shaped samples were used for
swelling, cell viability, cell function, and degradation studies. The results
of swelling studies, cell adhesion, and degradation by collagenase will be
presented.