Controlled release of dexamethasone from peptide nanofiber gels to modulate inflammatory response
Introduction
One major challenge in the field of biomaterials is to develop strategies to combat the innate host inflammatory response [1], [2]. Many commonly used biomaterials, including collagen and alginate, elicit some cell-mediated immune response at the implantation site [3], [4], [5], [6], [7]. Though a mild innate immune response is part of the normal healing process following implantation of a foreign material, such a response could prove detrimental when combined with the delivery and transplantation of fragile cells, islets, or tissue [8], [9], [10], [11]. Using biomaterials to deliver therapeutic cells adds additional factors that could influence the inflammatory response, including issues of cell source or transplant location. For example, strategies delivering autologous cells as therapy for cardiovascular disease have demonstrated limited efficacy, resulting in part from the enhanced inflammatory environment of ischemic tissue [12]. Islet transplantation to combat type 1 diabetes mellitus, meanwhile, relies on allogeneic (or even xenogeneic) tissue sources, which makes managing the inflammatory response paramount for effective therapy [11]. Strategies to combat immune-destruction of transplanted islets have included immunoisolation through encapsulation in biomaterials, but such strategies have demonstrated only limited success thus far as the construct size is often too big for injection and the encapsulation results in limitations to nutrient and oxygen transport [13], [14], [15]. The development of new materials that actively mitigate the local host immune response and facilitate improved efficacy in cell or tissue transplantation therefore remains an important objective.
One approach that has been used to modulate the host immune response is to design biomaterials with controlled release of anti-inflammatory drugs such as dexamethasone (Dex), a potent steroidal anti-inflammatory and immune response-suppressing drug [16]. In addition, Dex is known to be an important signaling molecule to promote differentiation in bone marrow-derived stromal cells and mesenchymal stem cells [17], [18], [19], [20], [21]. In order to harness its anti-inflammatory function or to stimulate stem cell differentiation, non-covalent Dex incorporation and release has been included in the design of a number of biomaterials [22], [23], [24], [25], [26], [27]. Several constructs incorporating this drug have been evaluated in vivo, demonstrating a significant reduction in the post-implant inflammatory response to a material [28] and also promoting osteogenesis in transplanted stem cells when its release is combined into the delivery scaffold [29], [30]. In a few instances, biomaterials have been designed to control the release of Dex using covalent attachment via hydrolyzable linkages, including hydrazones and lactide esters, which are labile in aqueous conditions to release the free drug [31], [32], [33], [34], [35].
Over the last decade, our group and others have investigated self-assembling peptide-based materials for applications in regenerative medicine, drug delivery, and cancer [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46]. In particular, our group has focused on a class of molecules, termed peptide amphiphiles (PAs), which contain a short peptide sequence covalently attached to a saturated alkyl tail and self-assemble into fiber-like nanostructures [47], [48]. The assembly of these molecules occurs through hydrophobic collapse of their alkyl tails and hydrogen bonding leading to the formation of β-sheet structures near the core of the nanofiber [49], [50]. On the end opposite the hydrophobic tail, PAs can be synthesized to contain a bioactive peptide designed to interact with biological targets such as cell receptors [51], [52], biopolymers [53], [54], or proteins [55]. In other systems, PAs can be integrated with commonly used biomaterials such as collagen or poly(l-lactic acid) [56], [57]. The conserved amphiphilic molecular design enables the modular construction of PA nanofibers that combine different PA molecules within the same assembled nanofiber as a way to multiplex different bioactivities or optimize the spacing of bioactive ligands [55], [58].
We report here the design, synthesis, and function of a PA that could allow controlled release of Dex through the use of a hydrazone linkage. While previous attempts to combine drug release with PA technology have focused on solubilizing hydrophobic drugs in the lipid core of the assembled fiber [59], [60], [61], this approach using covalent attachment via a hydrazone could facilitate enhanced control over the kinetics of drug release [62], [63]. A Dex-releasing PA (Dex-PA) could potentially be easily integrated with other bioactive PAs to therapeutically reduce or eliminate the immune response. In this work, a PA conjugated with Dex was synthesized and PA nanofiber gels were prepared that could release the soluble drug over a period of several weeks. Using in vitro methods, we evaluated the anti-inflammatory and cytoprotective activity of released Dex. Finally, an in vivo model was used to evaluate the ability of the Dex-PA to mitigate the inflammatory response to cell surrogate microparticles delivered within a Dex-PA nanofiber gel.
Section snippets
Dex-PA synthesis
Rink Amide MBHA resin and Fmoc-protected amino acids were purchased from EMD. Dex was purchased from Spectrum and used as received. All solvents were ACS reagent grade and purchased from Mallinckrodt. Other peptide synthesis reagents were purchased from commercial sources and used as received. 1-[N, N, N′-Tris((tert-butyloxycarbonyl))hydrazide]-adipic acid (HAA(Boc)3) was synthesized as previously described and used to synthesize a PA containing a free hydrazide, as also described previously,
PA design and characterization
The Dex-PA was designed to release free Dex over time in physiological conditions through the use of a hydrolyzable hydrazone linkage. Briefly, tri-Boc-hydrazido adipic acid (HAA(Boc)3) was synthesized as previously described [62] and added to a PA with the sequence C16–V2A2E2K via the ε-amine of the lysine residue on resin after selective removal of an Mtt protecting group on the lysine. Following cleavage in 95% TFA, a stabile hydrazide was generated. After purification by HPLC, the
Conclusions
We have described a peptide amphiphile conjugated covalently through a labile hydrazone bond to the steroidal anti-inflammatory drug dexamethasone. This material, along with similar drug-conjugated peptide amphiphiles, could be useful as gel networks to provide prolonged and localized drug delivery at the site of injection. In this work, we showed through in vitro studies the cytoprotective and anti-inflammatory properties of this material. Additionally, in vivo studies in mice demonstrated the
Acknowledgments
This work was supported by NIH grant number 5R01DE015920. JBM was supported by an NIH postdoctoral fellowship (1F32AR061955-01) and MJW was supported by the NIH through the Northwestern University Regenerative Medicine Training Program (5T90-DA022881). Additionally, support was provided through a Northwestern Feinberg School of Medicine Dixon Translational Research Stewardship Award. The authors also gratefully acknowledge helpful discussions with Prof. Dong Wang (University of Nebraska), Prof.
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MJW and JBM contributed equally to this work.