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10.6AFFINITY-BINDING ALGINATE BIOMATERIAL FOR MULTIPLE GROWTH FACTOR DELIVERY

Bio-inspired by ECM interactions with heparin-binding proteins, our group has developed an affinity-binding alginate biomaterial to enable precise control over factor release and to allow the release of combinations of growth factors.

10.6.1SULFATION OF ALGINATE HYDROGELS AND ANALYSIS OF BINDING

Alginate biomaterial with affinity binding sites for heparin-binding proteins was synthesized by sulfation of the uronic acid monomers in alginate, using carbodiimide chemistry (Fig. 10.3) [48].

The infrared (IR) spectrum of the product alginate-sulfate confirmed the appearance of a new major peak at 1250 cm−1 (assigned to S=O symmetric stretching) and a minor peak at 800 cm−1 (assigned to S–O–C stretching). According to nuclear magnetic resonance spectroscopy (C13-NMR) spectra, the sulfate groups are added to either C-2 or C-3 or both, in an identical manner. The percentage sulfation by the Sheniger method was 8% (wt. sulfur per wt. alginate).

Surface Plasmon Resonance (SPR) analysis revealed the specific and strong binding of various heparin-binding proteins to alginate-sulfate, with equilibrium binding constants at the same order of magnitude as their binding to heparin [48, 49] (Fig. 10.3D and Table 10.2). No such interactions were recorded with pristine alginate. Thus, it appears that the binding to alginate-sulfate mimics in large the interactions of growth factors, chemokines, and cell adhesion molecules, collectively known as heparin-binding proteins (Fig. 10.3E). These molecules bind the proteoglycans heparin and heparan sulfate via high affinity, specific electrostatic interactions with the lowand high-sulfated sequences in these glycosaminoglycans (GAG) [50]. In this aspect, heparan sulfate GAGs play an important role in sequestering and storage of the proteins, and also participate in the formation of active signaling complex with a respective cell surface receptor.

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Figure 10.3: Alginate sulfation for attaining affinity-binding biomaterial. A. O-sulfation of the uronic acid on alginate. Reaction scheme of the sulfation of uronic acids in alginate involves the formation of protonated DCC–H2SO4 intermediate, followed by a hydroxyl nucleophilic attack to produce sulfated alginate and dicyclohexylurea.The latter is removed by extensive dialysis. B. 13C NMR spectra of alginatesulfate and raw sodium alginate, showing that sulfation occurs on C2 and C3. C. FTIR spectra of alginatesulfate and raw sodium alginate. The product, alginate-sulfate, has a new major peak at 1250 cm −1, assigned to S=O symmetric stretching (arrow). Reprinted with permission from [48].

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Figure 10.3: Alginate sulfation for attaining affinity-binding biomaterial. D. A representative SPR sensogram of bFGF binding to unmodified alginate, heparin, and alginate-sulfate, showing strong and specific binding of bFGF to heparin and alginate-sulfate, and not to unmodified alginate. E. The model of reversible binding. Reprinted with permission from [48].

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Table 10.2: Equilibrium binding constants (KA) calculated from the interactions of alginate-sulfate with proteins (SPR analysis) [48, 49]

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10.6.2BIOCONJUGATION WITH ALGINATE-SULFATE AND PROTEIN PROTECTION FROM ENZYMATIC PROTEOLYSIS

The bioconjugation of the growth factors with alginate-sulfate was found to shield the proteins from enzymatic proteolysis. Exposure of the bioconjugates to trypsin and analysis for degradation products by Matrix-Assisted Laser Desorption/Ionization – Time of Flight (MALDI-TOF) mass spectroscopy, revealed the presence of intact protein and much fewer digestion fragments in the spectrum compared to unprotected protein samples where no intact protein was detected [50, 51] (Fig. 10.4).

The shielding effect is likely due to bioconjugation leading to nanoparticle formation, wherein the alginate-sulfate found on the surface protects the core protein. The formation of nanoparticles has been established by high-resolution microscopy techniques, such as Atomic Force Microscopy (AFM),Transmission Electron Microscopy (TEM), and cryogenic-TEM, while zeta potential studies validated the presence of alginate-sulfate on the surface of the nanoparticles (Ruvinov et al, paper in preparation).

The effect of protein protection from proteolysis is of great importance,if the delivered proteins are to remain active for prolonged periods of time in harsh environments, where extensive protein

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Figure 10.4: Bioconjugation with alginate-sulfate protects IGF-1 and HGF from enzymatic proteolysis. A. MALDI-TOF spectra of IGF-1, soluble or in the bioconjugate form. Reprinted with permission from [50, 51].

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Figure 10.4: Bioconjugation with alginate-sulfate protects IGF-1 and HGF from enzymatic proteolysis. B. MALDI-TOF spectra of HGF, soluble or in the bioconjugate form. Reprinted with permission from [50, 51].

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degradation takes place, i.e., the infarct area during the first few weeks after the initial ischemic event.

10.6.3SCAFFOLD-BASED APPROACH USING AFFINITY-BINDING ALGINATE FOR MULTIPLE AND CONTROLLED GROWTH FACTOR DELIVERY

The combination of alginate-sulfate with pristine alginate in one device represents a unique affinitybinding alginate biomaterial, which is capable of controlling the delivery of multiple proteins, while retaining the supporting and ECM replacing properties of the alginate hydrogel.

Macroporous alginate-sulfate/alginate scaffolds were fabricated by a freeze-dry technique as described in Chapter 4 [52, 53]. The alginate-sulfate was mixed with pristine alginate solution, then the mixture was cross-linked by calcium ions and freeze-dried [49] (Fig. 10.5). As revealed by SEM analysis, incorporation of 10% (dry weight polymer) of alginate-sulfate into the alginate scaffold did not affect scaffold porosity or mechanical stability in culture (Fig. 10.5) [49].

The ability of the affinity-binding alginate scaffolds to control the release of multiple growth factors was tested using the combination of three known angiogenic factors: vascular endothelial growth factor (VEGF), platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-β1 (TGF-β1). Initial loading and binding of the factors was achieved by the addition of protein solutions to the dry scaffolds and subsequent incubation for one hour at 37◦C. In vitro release studies revealed a sequential order of protein release from the scaffold: VEGF was released first, followed by PDGF-BB, and then TGF-β1 [49]. Importantly, the observed release order coincided with the predicted order of the release based on the values of the equilibrium binding constants to alginate-sulfate and initial loading concentration of the factor (Fig. 10.6, also see Table 10.2). By contrast, factor release from the scaffolds lacking alginate-sulfate was rapid and was governed mainly by burst effect.

The sequential delivery of VEGF, PDGF-BB, and TGF-β1 from the affinity-binding scaffold mimics the signal cascade acting in angiogenesis, namely the initiation of the process by VEGF with endothelial cell (EC) assembly, followed by PDGF-BB-mediated smooth muscle cell and pericyte recruitment, and finally, vessel remodeling and stabilization induced by TGF-β1 (Fig. 10.6B) [54, 55].Thus, we tested the efficacy of the system to induce the formation of stable vessels.The scaffolds were implanted subcutaneously in the dorsal area in rats, and the tissues were assessed for blood vessel number and maturation, one and three months after implantation, by immunohistochemistry for lectin (a marker of endothelial cells) and α-smooth muscle actin (SMA, a marker of smooth muscle cells) (Fig. 10.7).

Consistent with the pattern of sequential factor delivery, vessel density increase was observed at one month after implantation, while effects on vessel maturation were observed after three months, as revealed by the density of α-SMA-immunostained vessels, which increased by two-fold compared to the situation after one month. By contrast, the instantaneous delivery of the three factors from pristine alginate scaffolds resulted in two-fold lower blood vessel density, smaller sized vessels and

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Figure 10.5: Affinity-binding alginate scaffolds for the controlled delivery of heparin-binding proteins. A. The concept of affinity-binding alginate scaffolds. The scaffold fabricated from alginatesulfate/alginate can bind multiple heparin-binding proteins (HBP) via specific affinity sites on alginatesulfate. The release rate from such scaffolds is correlated with the equilibrium binding constants (KA) of the factors (Table 10.2). B-C. SEM visualization of internal morphology of affinity-binding (alginatesulfate containing) (B) or unmodified alginate (C) scaffolds. Reprinted with permission from [49].

a similar percentage of mature vessels at one and three months, indicating the short-term effect of the adsorbed factors on scaffold vascularization [49].

In a later study, alginate scaffolds with affinity-bound mixture of pro-survival and angiogenic factors (SDF-1, IGF-1, and VEGF) were also used for the creation of a vascularized cardiac patch by pre-implantation into the omentum for 7 days (Section 8.3) [56]. Such omentum-generated pre-vascularized cardiac patch showed improved structural and electrical integration into host myocardium. Moreover, the vascularized patch induced thicker scars, prevented further dilatation of the chamber and ventricular dysfunction. Interestingly, a similar scaffold, but without seeded cardiac

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Figure 10.7: Sequential delivery of VEGF, PDGF-BB and TGF-β1 induce angiogenesis and vessel maturation in affinity-binding alginate scaffolds after subcutaneous implantation in rats. Quantification of blood vessel densities by counting (A) lectin-positive vessels or (B, next page) SMA-positive vessels in sections from implanted scaffolds retrieved after 1 and 3 months. Scale bar 100 μm. Reprinted with permission from [49].

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Figure 10.7: Sequential delivery of VEGF, PDGF-BB and TGF-β1 induce angiogenesis and vessel maturation in affinity-binding alginate scaffolds after subcutaneous implantation in rats. Quantification of blood vessel densities by counting (A, previous page) lectin-positive vessels or (B) SMA-positive vessels in sections from implanted scaffolds retrieved after 1 and 3 months. Scale bar 100 μm. Reprinted with permission from [49].