Synthetic Morphogens and Pro-morphogens for Aided Tissue Regeneration - Biologically Responsive Biomaterials for Tissue Engineering - page 57

Table 3.3 Typical protocol for the solid-phase synthesis and characterisation of a growth factor
analogue (relevant amino acid sequences are reported in Table 3.1 )
Growth factor mimic synthesis
• Peptides were synthesised by a solid-phase synthesis method on a microwave system (Biotage)

at mg scale

• Amino acids were loaded sequentially into the reaction vessel and coupled to 0.5 g resin

(0.1 mmol -NH 2 ) as follows:

- Rink Amide linker and HBTU/DIPEA

- Fmoc-amino acid -OH and HBTU/DIPEA

- Sequential addition of ×4 molar excess of each amino acid (0.4 mmol) in turn

• Deprotection step 20 % piperidine in DMF was added to remove Fmoc groups

• Peptides cleaved from resin using Trifluoroacetic acid:H 2 O:Triisopropyl silane (95:2.5:2.5)

and precipitated in cold diethyl ether. A series of centrifugation and diethyl ether washing
steps are used to collect peptide

• Peptides were purified by preparative HPLC

• Peptides were characterised by

- Analytical HPLC

- Mass spectrometry (microTOF)

- Gravimetric analysis

and the native conformation. Protein scaffolds have been engineered through
different biotechnological approaches and include [ 66 ] :

• Cystine knot miniproteins derived from plant cyclotides able to present bioactive

peptides
Tetracorticopeptide modules presenting a variable paratope based on a natural •

protein fold
Human I-set domains encompassing cell adhesion molecules

•

Peptide-based dendrimers have been proposed as scaffolds capable of enhancing
epitope presentations and of mimicking protein secondary structures.
Dendrimers are highly and 3D ordered, hyperbranched polymers forming
nanostructures with tuneable physico-chemical properties. Dendrimers are obtained
from a core molecule to which synthetic (e.g. polyamido amine, PAMAM), amino
acid (e.g. lysine) and carbohydrate monomers are bonded [ 67 ]. There are two main
methods to synthesise dendrimers [ 68 ] :

1. The divergent synthesis where a core molecule with multiple reactive sites is
used to form chemical bonding with a reactant and where the formed complex is
later reacted with a molecule capable of generating another branching point
2. The convergent synthesis where fragments of dendrimers are added to the core
molecules and thus assembled

When the synthesis is performed in liquid phase, although the shape and symmetry
of the dendrimer depend on the physico-chemical properties of the molecules used
for its synthesis, the polymer branching generally leads to an open ball, spherical
structure [ 69 ] (Fig. 3.3 ).