D. Channe Gowda

Biocompatibility of the Bioelastic Materials, Poly(GVGVP) and Its γ-Irradiation Cross-Linked Matrix: Summary of Generic Biological Test Results

The complete series of the recommended generic biological tests for materials and devices in contact with tissues and tissue fluids and blood have been carried out by an independent testing laboratory on the elastic protein-based (bioelastic) polymer, Poly(L-Val1-L-Pro2-Gly3-L-Val 4-Gly5) with a degree of polymerization greater than 120, and its 20 Mrad γ-irradiation cross linked elastic matrix, X20-poly(VPGVG). The specific tests and the summarized results given in parentheses are: (1) the Ames mutagenicity test (non- mutagenic), (2) cytotoxicity-agarose overlay (non-toxic), (3) acute systemic tox icity (non-toxic), (4) intracutaneous toxicity (non-toxic), (5) muscle implantation (favorable), (6) acute intraperitoneal toxicity (non-toxic), (7) systemic antigenic ity (non-antigenic), (8) dermal sensitization—the Magnusson and Kligman maximization method (non-sensitizing), (9) pyrogenicity (non-pyrogenic), (10) Lee White clotting study (normal clotting time), and (11) in vitro hemolysis test (non-hemolytic). Thus, this new elastomeric polypeptide biomaterial which is based on the most striking repeating sequence in the mammalian elastic fiber exhibits an extraordinary biocompatibility. This parent bioelastic material and a wide range of component peptide variations are under development for an equally wide range of potential medical applications such as prevention of adhesions, drug delivery, and synthetic arteries.

Comparison of electrostatic-and hydrophobic-induced pKa shifts in polypentapeptides. The lysine residue

Abstract Acid—base titrations at 37°C determined the p K a values in water and saline of twelve synthesized polypentapeptides poly( f K (GVGIP), f v (GKGIP), where f v + f K = 1 with f K ranging from 1 to 0.06 and where K is the lysine (NH + 3 /NH 2 ) residue. In water the p K a was 9.60 near f K = 0.9 decreasing to 9.20 at f K = 1 and to 8.18 at f K = 0.06. At 37°C in the NH 2 state, the polymers form a viscoelastic phase of 50% water by volume where for f K = 0 previous dielectric permittivity data place the dielectric constant near 65. For f K K a could not be explained by the usual electrostatic self-energy argument of decreased dielectric constant as Lys (K) is replaced by Val (V). Instead, an apolar—polar repulsive free energy of hydration is discussed which provides a basis for hydrophobic-induced p K a shifts.

Synthetic polypeptide sleeve for strabismus surgery.

A synthetic polypentapeptide sleeve was placed around the superior rectus muscle of five New Zealand white rabbits in hopes of preventing postoperative fibrous scarring. Two forms of the polypentapeptide were used. No significant inflammation or scarring occurred with either form of the polypentapeptide when compared to controls. One form elicited a fibrous membrane surrounding the sleeve within 2 weeks. The other elicited no such reaction after 2 months. The latter form of the polypentapeptide may be useful in preventing scarring following strabismus surgery.

Cell Adhesive Properties of Bioelastic Materials Containing Cell Attachment Sequences

The biocompatibility, conformational and inverse temperature transition properties of poly(Vall-Pro2-Gly3-Va14- Gly5), i.e., poly(VPGVG), and its 7-irradiation crosslinked matrix and the poly(VPGVG)-derived hydrophobicity scale are noted. Also noted are the capacities of varying the bioactive role of bioelastic materials; that is, the bioelastic materials can be designed (1) to exhibit a range of elastic moduli, (2) to exhibit different rates of degradation, (3) for various modes of drug release, (4) to perform numerous free energy transductions, (5) to contain functional enzyme sites, and (6) to contain functional cell attachment sequences that promote growth to confluence.

Synthesis, Characterizations, and Medical Applications of Bioelastic Materials

Bioelastic materials are elastomeric polypeptides composed of repeating sequences. They are a relatively new class of polymers that may also be called elastic protein-based polymers, having their origins in repeating sequences found in the mammalian elastic protein, elastin. The most striking and longest sequence between cross-links in pig and cow is the polypentapeptide-(PPP), poly(VPGVG) or (Val1-Pro2-Gly3-Val4-Gly5)n, where n is 11 (Sandberg et al., 1985; Yeh et al, 1987). Another repeat first found in porcine elastin is a polytetrapeptide-(PTP), poly(VPGG) or (Val1-Pro2-Gly3-Gly4)n, but this repeat has not been found to occur with n greater than 2 without substitution (Sandberg et al., 1981). The next most common recurring sequence in mammalian elastin is a polyhexapeptide-(PHP), poly(APGVGV) or (Ala1-Pro2-Gly3-Val4-Gly5-Val6)n where, with but a couple of isomorphous hydrophobic residue replacements such as Val by He or Leu, n is 8 in man (Indik et al., 1987). The monomers, oligomers, and high polymers of these repeats have been synthesized and conformationally characterized (Urry and Long, 1976). The high polymers of these repeating sequences have been cross-linked into sheets, rods, and tubes, and the PPP and PTP have been found to be elastomeric with the former being capable of an elastic modulus similar to that of the natural elastic fiber (Urry et al., 1976, 1981, 1982).

Pressure effect on inverse temperature transitions: biological implications

Abstract Elastic protein-based polymers of the form poly [ f x (VPGXG), f v (VPGVG)], where f x and f v are mole fractions with f x + f v =1, exhibit inverse temperature transitions in the form of a phase separation in which folding and aggregation of polymer chains into more-ordered states of the condensed (coacervate) phase occurs on raising the temperature. When X = Trp, Phe or Tyr, an increase in pressure causes a substantial increase in the temperature of the transition. The data are interpreted to indicate that water molecules surrounding the aromatic side chains of Trp(W), Phe(F) or Tyr(Y) occupy less volume than water molecules in bulk water. The calculated volume change for poly[0.8(GVGVP), 0.2(GFGVP)], for example, on going from coacervate phase, where hydrophobic associations have largely eliminated waters of hydrophobic hydration, to be dispersed in water where the hydrophobic moieties are surrounded by water is 80 cm 3 /mol of mean pentamers or some 400 cm 3 /mol of (GFGVP). The results provide an understanding of the source of pressure effects in biological systems and for the capacity to design materials capable of exhibiting baromechanical transduction.

Non-linear hydrophobic-induced pKa shifts: Implications for efficiency of conversion to chemical energy

Abstract By using one Asp or one Glu per thirty residues in a polytricosapeptide capable of exhibiting a hydrophobic folding and assembly transition and stepwise converting a set of the five Val residues (most proximal to the Asp or Glu residue) to more-hydrophobic Phe residues, a non-linear hydrophobic-induced p K a shift was observed with a Δ p K a of 0.4 (Asp) and 0.3 (Glu) on addition to 2 Phe residues per 30mer but with a Δ p K a of 4.7 (Asp) and 2.7 (Glu) on going from 4 Phe/30mer to 5 Phe/30mer. As a shift in p K a can be equivalent to the conversion to chemical energy from whatever energy input — mechanical, chemical, electrochemical, pressure or light — which effects a change in hydrophobicity, the non-linear hydrophobic-induced p K a shift means increased efficiency of energy conversion with increased hydrophobicity of the protein-based polymer.

Hierarchical and Modulable Hydrophobic Folding and Self-assembly in Elastic Protein-based Polymers: Implications for Signal Transduction

When the hydrophobic (apolar) and polar moieties of elastomeric polypeptides are properly balanced, the polypeptides are soluble in water at lower temperatures but undergo folding and assembly transitions to increased order on raising the temperature. The temperatures, T t , and heats, ΔH t , of these inverse temperature transitions are determined by differential scanning calorimetry for a series of elastomeric polypentapeptides: poly(VPAVG), poly(IPAVG), poly(VPGVG), poly(IPGVG), poly[0.5(VPGVG),0.5(IPGVG)] and poly[0.82(IPGVG),0.18(IPGEG)] where V = Val, P = Pro, A = Ala, G = Gly, I = lle and E = Glu. On increasing the hydrophobicity as when replacing V(Val) by I(lle) which is the addition of one CH 2 moiety per pentamer, the temperature of the transition is lowered by 15 to 20°C and the heat of the transition is increased by more than one kcal/mole, for the above examples, by more than a factor of two. When differential scanning calorimetry thermograms are obtained on mixtures of poly(VPAVG) plus poly(IPAVG) or of poly(VPGVG) plus poly(IPGVG), it is found that the polypentapeptides self-separate, i.e., they de-mix, even though in the latter case the conformations have been shown to be essentially identical before and after their respective transitions. When the polymer, poly[0.82(IPGVG),0.18(IPGEG)], is studied as a function of pH, increasing the degree of ionization is found to increase the temperature and to decrease the heat of the transition such that, with the correct balance of I with the variable E(GluCOO − ), the values of T t and ΔH t can be made to approach those of poly(VPGVG). Acid-base titration studies indicate that less than one Glu(COO − ) in 200 residues can raise the value of T t by 25°C and decrease ΔH t by 90%. These and additional data are interpreted to mean that there exists an hierarchical hydrophobic folding, that the hierarchical hydrophobic folding can be modulated by changing the degree of ionization or by changes in a number of intensive variables, that changes in these intensive variables can be used to drive folding/unfolding-assembly/disassembly transitions under isothermal conditions, and that these unfolding/folding and disassembly/assembly transitions can be used to achieve signal transduction. This is called the ΔT t mechanism of free energy (signal) transduction.

ΔT t -Mechanism in the Design of Self-Assembling Structures

Protein-based polymers can be designed in which self-assembly occurs as the temperature is raised above the onset temperature, Tt, of an inverse temperature transition for hydrophobic folding and assembly. Instead of changing the temperature, however, by many means the value of Tt can be lowered from above to below an operating temperature to drive hydrophobic folding and assembly. This is the ΔTt- mechanism. Modulation of charges on the polymer provides the most dramatic means of controlling Tt and therefore becomes the most effective means for controlling self-assembly. The formation of charge raises the value of Tt and causes disassembly, whereas neutralization of charge by lowering degree of ionization or by increasing ion-pairing drives self-assembly.