PEG-Immobilized Keratin for Protein Drug Sequestration and pH-Mediated Delivery

Protein drugs like growth factors are promising therapeutics for damaged-tissue repair. Their local delivery often requires biomaterial carriers for achieving the therapeutic dose range while extending efficacy. In this study, polyethylene glycol (PEG) and keratin were crosslinked and used as sponge-like scaffolds (KTN-PEG) to absorb test proteins with different isoelectric points (pI): albumin (~5), hemoglobin (~7), and lysozyme (~11). The protein release kinetics was influenced by charge at physiological pH 7.4. The keratin network, with pI 5.3, electrostatically attracted lysozyme and repulsed albumin generating the release rate profile: albumin > hemoglobin > lysozyme. However, under acidic conditions (pH 4), all proteins including keratins were positively charged and consequently intermolecular repulsion altered the release hierarchy, now determined by size (MW) diffusion: lysozyme (14 kDa) > hemoglobin (64 kDa) > albumin (66 kDa). Vascular endothelial growth factor C (VEGF-C), with properties comparable to lysozyme, was absorbed into the KTN-PEG scaffold. Endothelial cells cultured on this substrate had significantly larger numbers than on scaffolds without VEGF-C suggesting that the ionically bound and retained growth factor at neutral pH indirectly increased acute cell attachment and viability. PEG and keratin based sequestrations of proteins with basic pIs are therefore a feasible strategy with potential applications for selective biologics delivery.


Introduction
Protein drugs, also called protein therapeutics and protein biologics, are proteins that provide healing, repair, and regenerative functionalities to injured and damaged cells and tissues. These include cell-secreted extracellular growth factors (GFs) and signaling proteins for induction of cell growth, cell division (proliferation), movement, changes in shape, survival and inhibition of apoptosis, differentiation, and tissue morphogenesis [1][2][3][4]. GFs bind to target enzymelinked cell-surface receptors and activate intracellular signaling pathways leading to expression of genes involved in macromolecular synthesis, metabolism, and alteration of cellular behaviors. Effective levels of GFs are usually in the pico-to nanomolar ranges, acting in the order of hours [5]. To extend this range and regulate the spatiotemporal dose release for treatment applications, drug-delivery systems or biomaterial carriers are utilized [6][7][8]. A charge-based sequestration strategy can be employed wherein the protein drug load and the carrier matrix have opposing electrical charges to provide electrostatic or coulombic attraction. Proteins and peptides are zwitterions; that is, they can alter their net charges depending on the aqueous environmental pH; those with isoelectric points (pIs) lower than the pH of the medium will have negative molecular electrical charges, while those with higher pIs will be positively charged [9,10]. Accordingly, proteins with acidic and basic pIs in phosphatebuffered saline (PBS, pH 7.4) carry negative and positive net charges, respectively, and can potentially associate together.
Polyethylene glycol (PEG) and hair keratin (KTN) biomaterials have been shown to be safe, biocompatible (with minimal fibrous encapsulation), and appropriate drug-delivery vehicles for tissue engineering purposes [11][12][13][14][15][16][17][18][19]. Diacrylates of linear PEG (PEGDA) can be photopolymerized for PEG chain growth to form scaffolds [20,21]. Reduced KTN proteins containing free thiols (-SH) can be gelled by reforming of disulfide bonds (-S-S-) [22,23]. PEGDA and KTN can also be combined and crosslinked via a photopolymerization  thiol-ene reaction [24,25]. We capitalized on these reactions ( Figure 1) to form stable KTN-PEG scaffolds. KTN, having a pI of 5.3, is negatively charged in PBS, pH 7.4 (Table 1) [22]. Moreover, the reactions do not target any of the ionizable amino acid groups [9] and should not significantly alter the crosslinked keratin pI. The KTN network of KTN-PEG, hence, can theoretically hold onto positively charged proteins with basic pIs. Several GFs, including vascular endothelial growth factor C (VEGF-C), exhibit pIs greater than 7.4 [26,27]. As a result, they are likely to ionically associate with the KTN-PEG bulk material. In this study, we investigated the diffusion release profiles of KTN-PEG scaffold-absorbed soluble proteins with varying pIs (charges) and sizes in physiological pH and, additionally, in acidic PBS, pH 4 (Table 1). At a pH level lower than the KTN's pI, the biomaterial scaffold is expected to gain positive charges, thereby inducing repulsion and quick release of sequestered positively charged proteins. The functional bioactivity of a bound-protein, represented by VEGF-C, was tested through endothelial cell culture onto the KTN-PEG substrate with absorbed VEGF-C.

Liquid Absorption and
where is equilibrium volume swelling ratio, swollen is volume of absorbate and scaffold, and dry is volume of scaffold. Consider where ] e is effective crosslink density (mol/mL), is compressive modulus of elasticity (MPa), is gas constant = 8.314 mL⋅MPa⋅K −1 ⋅mol −1 , and is temperature (K). values were obtained from the slope of the linear elastic region of true stress versus true stain curve of equilibrated PBSsoaked scaffolds compressed at 0.1 mm/s using the Instron 3345 mechanical tester (Norwood, MA). Consider , where is molecular weight between crosslinks (g/mol) and polymer is polymer density (g/mL).     Table 2.   Table 3 summarizes the results obtained from measurement of scaffold network properties. It was found that the molecular weight between crosslinks ( ) in KTN-PEG scaffolds immersed in PBS was 25.9 kDa. A 5% KTN gel only was determined to have = 183 kDa. Since was reduced in the presence of PEG additive, this suggests successful photocrosslinking of keratin-to-PEG via thiol-ene reaction. The average molecular weight (MW) of the KTN biomaterial was reported to be 98 kDa [22]. Accordingly, there were about 3.8 (=98 kDa/25.9 kDa) crosslinks per KTN molecule in the fabricated KTN-PEG scaffold. In the KTN-PEG scaffold, any of the possible reactions illustrated in Figure 1 can occur: PEG-PEG chain growth, KTN-KTN disulfide oxidation, and KTN-PEG thiol-ene reaction [33].

Release of Unbound Keratins.
Despite statistical similarities in the absorbate / scaffold ratio among KTN-PEG time groups, there was a downward plateauing trend over time (Figure 3), suggesting that some of the water-absorbing keratins were initially being released into the PBS medium. Total protein assay showed that uncrosslinked keratin proteins were indeed leaving the bulk of the scaffolds (Figure 4) in where is time (d), is release concentration ratio, max is maximum release, and is time at half max . max was found to be 25.5%, implying that 74.5% of keratins were stably integrated into the KTN-PEG scaffold network. At 0.48 days or about 11.5 hours ( ), half of the unbound keratins were released in a static PBS environment at 37 ∘ C. Overall, washing and rinsing of the scaffolds in PBS on a shaker accelerated the removal of "free" or nonnetwork keratins.

Absorption and Then Release of Proteins in Neutral
and Acidic pH. KTN-PEG scaffolds absorbed 70.7 ± 1.4% liquid ( liquid /( liquid + scaffold )) containing 200 g/mL of test globular proteins (Alb, Hem, or Lys). This value can also be interpreted as the gel water content, determined to be less than reported 82% in Table 2. The discrepancy can be accounted for by the loss of uncrosslinked keratins, thereby slightly decreasing the capacity to hold water into the scaffold matrix. No significant difference ( = 0.9627) was observed between the liquid absorption behavior in neutral (pH 7.4) and in acidic (pH 4) PBS ( Figure 5; 70.6 ± 0.7% versus 70.7 ± 2.2%, resp.).
Keratins covalently linked into the scaffold network degraded and released proteinaceous materials into the surrounding liquid medium at a relatively slow pace, 8.46 ± 0.05% in neutral PBS versus 6.64 ± 1.56% in acidic PBS for 6 days (Figure 6), although there was no significant difference between them ( = 0.1139). The keratin degradation profile was relatively slower with higher variability (standard deviation) at the pH of 4, suggesting that the scaffold has more stability in acidic pH. This was consistent with our previous findings in experiments of keratin gel degradation at varying pH [22], possibly because disulfide bonds were more protected during hydrolysis in acidic pH levels [34].
Proteins absorbed into the bulk gel matrix were released into the surrounding PBS through the processes of diffusion and osmosis: movement of mobile masses from higher to lower concentration gradients. The experimental setup allowed for high volume of PBS to scaffold ratio; hence the movement of molecules was not hindered by the saturation effect of the supernatant. In PBS pH 7.4, albumin was released the fastest, followed by hemoglobin and then lysozyme (Figure 7(a)). At this pH level, scaffolding keratin matrix proteins (with pI = 5.3) [22] are negatively charged (pI < pH; Table 1), dictating the overall charge density of the KTN-PEG sponge-like material. While albumin (pI ∼ 5) is also negatively charged, hemoglobin (pI ∼ 7) is almost neutral (though slightly negative) and lysozyme (pI ∼ 11) is positively charged. The observed protein release rates can be explained by electrostatic interactions between charged species; that is, negative-negative repulsion drove albumin diffusion out the fastest. At day six, 28.5% of the original albumin load had been released into the medium. Conversely, the positively charged lysozyme electrostatically bound to the negative keratin backbone which consequently registered minimal release kinetics, at about the same rate as keratin degradation (4.9% for 6 days).
In an acidic environment, the amine groups of amino acids in proteins are more conducive to protonation (-NH 3 + ), inducing net positive charges. Specifically at pH 4, the keratin matrix charge shifts to positive ( Table 1). All loaded proteins, with pIs > 4, also now carry positive charges. Accordingly, repulsive forces dominate between the scaffold matrix and the absorbed globular proteins, thereby inducing even faster release of mobile proteins and the order dictated by size diffusion (the smaller the faster the release). The porous nature of the scaffold enables repulsion to force like-charged molecules out of the bulk material [35,36]. Experimental results (Figure 7(b)) concurred with the projected kinetics wherein the smallest protein, lysozyme (14 kDa), had the highest rate of 48.4% in 6 days. Next in the order of release was hemoglobin (64 kDa) at 29.6% and finally albumin (66 kDa) at 9.8% in the 6-day period. Both lysozyme and hemoglobin were released at a higher rate in pH 4 than in physiological pH (Figure 7  Another possible explanation is that the low pH condition enabled the exposure of hydrophobic regions in both keratin [37] and albumin [38] resulting in hydrophobic attraction and slower albumin release. Growth factors (GFs) are proteins secreted by cells targeting recipient cells via binding and interaction to cellsurface growth factor receptors leading to a variety of downstream effects [2][3][4] including local recruitment of repair stem cells for tissue engineering and regenerative medicine applications [39]. They can be held into the scaffold matrix through affinity-based methods [6] for sustained delivery and extension of efficacy [40]. Lysozyme can act as an inexpensive model for studying the kinetics of GF release since its size and isoelectric point (charge) properties are close to clinically relevant GFs [3] such as bone morphogenetic protein 2 (BMP-2; MW = 18 kDa (monomer); pI = 9) [41,42] and brain-derived neurotrophic factor (BDNF; MW = 14 kDa (monomer); pI = 9) [43]. At neutral pH, lysozyme was sequestered tightly within the scaffold but was extensively released in an acidic condition (Figure 7) when the KTN charge flipped from negative to positive. GFs loaded into the KTN-PEG construct are thus anticipated to behave similarly to lysozyme. For evaluation, vascular endothelial growth factor C (VEGF-C; MW ∼ 15 kDa (monomer); pI = 8.3) [26,27] solution was utilized as an absorbent and its retention and activity were assessed indirectly on the survival of endothelial cells. on KTN-PEG scaffold with electrostatically bound VEGF-C showed statistically more ( < 0.05) cell attachment and survival relative to the other three groups: KTN-PEG without VEGF-C, PEG with VEGF-C, and PEG without VEGF-C ( Figure 8). The bar graph trend indicated that the presence of keratin as a network material generally increased cell growth, likely because of cell-interaction with the inherent cell-binding domains of keratins [44]. Addition of the growth factor VEGF-C further increased the number of cells on the scaffold surface suggesting that VEGF-C bound to the immobilized keratin network allowed cells to interact via their growth factor receptors facilitating attachment and promoted viability. Despite the outcome, the number of cells that anchored onto the substrate material was still relatively low. Application of cyclic sinusoidal pressure may induce endothelial cell proliferation with VEGF-C exposure [45]. Cultured lymphatic endothelial cells may respond better to the sequestered VEGF-C due to VEGF-C's importance 8 Journal of Drug Delivery in lymphangiogenesis or the formation of lymphatic vessels [46][47][48]. Future experiments may also require addition of surface-incorporated integrin-binding molecules such as laminin and its cell-binding peptides, RGD, YIGSR, and IKVAV, to improve the initial endothelial cell attachment [49].

Conclusions
Keratin (KTN) proteins in the recent years are gaining interest as appealing biocompatible biomaterial carriers of potent therapeutics which include growth factors. We have integrated and immobilized KTN with polyethylene glycol (PEG) diacrylate (PEGDA) through photoactivated linkage of KTN free thiols with PEGDA acrylate ends forming a stable KTN-PEG scaffold. KTN in the scaffold matrix enabled increased water uptake along with solubilized proteins. Its water-absorption capacity was found to be similar in physiological phosphate-buffered saline (PBS) pH 7.4 and in PBS pH 4. This network-bound KTN provided low hydrolytic degradation rate over the 7-day period with trend suggesting an even slower KTN release in acidic pH. Scaffold-absorbed proteins interacted with the KTN bulk following the First Law of Electrostatics: like charges repel and opposite charges attract. At pH 7.4, the negatively charged KTN tightly held onto proteins with basic isoelectric points (pIs) while preserving bioactivity. Lowering the pH to 4, below KTN's pI of 5.3, induced the fast release of a sequestered protein. The fabricated KTN-PEG construct can potentially be used as a slowly degradable sponge-like material for burst-release of high quantities of growth factors in acidic environment but also attract endogenous positively charged growth factors in neutral to basic pH states.