Self-assembling peptide hydrogels for the stabilization and sustained release of active Chondroitinase ABC in vitro and in spinal cord injuries
Graphical abstract
Introduction
The neural extracellular matrix (ECM) is a glycoprotein meshwork influencing multiple biological processes, from tissue growth and cellular homeostasis to brain and spinal cord protection [1,2]. It is an acellular structure composed of water, proteins, and polysaccharides, filling the area between neurons and glia with defined organization and characteristics [3]. At the microscopic level, the ECM is a 3D-dynamic molecular system guiding cell proliferation, cytoskeletal structure, cellular differentiation, and receptor signaling through its mechanical and biochemical properties [4]. Among others, ECM remodeling is affected by the composition of native protein chains, types of polysaccharide units, and the number of sulfate substitutions [5,6]. The ECM comprises considerable quantities of glycosaminoglycans (GAGs), which are linear polysaccharides consisting of hexose and uronic acid units linked by β-1,3-glycosidic bond [7]. GAGs are mainly distributed in ECM and on cell surfaces. They guide multiple biological processes, such as neural stem cells (NSCs) proliferation, neuronal patterning, synaptogenesis, and tuning of inflammatory response [8].
In central nervous system (CNS) injuries such as SCI and traumatic brain injury (TBI), the formation of a glial scar is considered as one of the main physical and chemical hurdles impairing axonal regeneration [9]. It contains a dense GAGs meshwork, having the ability to bind small signaling molecules like growth factors, chemokines, morphogens, and interacting with semaphorins, members of the LAR subfamily, PTPsigma [10], LAR1 [11], and Nogo receptors 1/3 (NgR1/3) [12]. They interact with the GAG chains of CSPGs through an Ig-like domain rich in positive-charged amino acids and are responsible for transducing CSPGs repulsive signals to the growing axons. LAR-CSPGs interaction, in particular, triggers a downstream pathway that results in the reduction of Akt-kinase, regulating phosphorylation, and activity of RhoA GTPase [13]. The signaling pathways downstream of PTPsigma share the same effectors and Ras-Raf-MEK-ERK pathways [14]: interestingly, PTPsigma exposure to the excessive amount of CSPGs at the site of injury causes the collapse of axonal growth-cones through the establishment of an over-adhered axonal phenotype [8]. Hence, the degradation of CSPGs in TBI and SCI exerts a positive role in axonal regeneration and subsequent functional recovery [[15], [16], [17]].
Chondroitinase ABC (ChABC) is a depolymerizing lyase that cleaves a broad range of Galactosaminoglycan substrates, including chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronic acid [18]. It received particular attention in the field of SCI regeneration [19] because of its degradation effect over the gliotic scar: however, its thermal instability hampers main its usage in clinics, since it requires multiple spinal injections over time, thus leading to potential infections as well as patient distress [20].
Some biotechnological techniques were adopted to overcome this hurdle, such as point mutation of the original protein sequence [21]. We recently managed to prolong the in vitro Chondroitinase ABC enzymatic activity from a few hours to a couple of weeks: [22] in chronic SCI, we showed that single intraspinal injections of “treated” ChABC stimulated the degradation of CSPGs, generating a permissive environment for nervous tissue restoration.
Sustained local Chondroitinase ABC release from injectable biomaterials may further improve the enzyme's durability and translational impact of ChABC-based therapies. Ni and colleagues employed poly(propylene carbonate)-chitosan microfibers to achieve extended-release of ChABC in SCI in rats [23]. In vitro tests revealed that most of the Chondroitinase ABC was delivered in ten days, resulting in an increased number of regenerating neurons, but with a modest reduction of CSPGs and with no behavioral improvement [23].
In this context, hydrogels based on self-assembling peptides (SAPs), which spontaneously self-organize into braided nanofibers, were demonstrated to release bioactive substances at both short and medium timeframes [24]. Nanostructured SAPs are synthetic (but naturally inspired) biomaterials with excellent tunability of both mechanical and biomimetic properties [25], making them promising candidates for next-generation translational researches in tissue engineering. Interesting data demonstrated slow and sustained release of active cytokines from SAPs, with release kinetics related to SAP sequences and type of functionalization [[26], [27], [28]]. Moreover, it became clear that different protocols of SAP hydrogel preparation significantly affect their kinetics of assembly, nano-topography, porosity, and mechanical properties [26]: as such, they may also affect the release kinetics of the loaded drugs.
In this work, we used different SAP hydrogels for sustained release of active Chondroitinase ABC to enhance nervous tissue repair in chronic SCI. We assessed the influence of SAP concentration, chemical composition, the protocol of dissolution, and the ChABC loading strategy on Chondroitinase ABC release and stability over 42 days.
The neuro-regenerative potential of ChABC-loaded injectable hydrogels was evaluated in a chronic SCI model resulting in the locomotor recovery improvement of the injured rats, degradation of their gliotic scars, and enhanced nervous tissue repair. The tested hydrogel systems may also be useful tools for the delivery of Chondroitinase ABC in the peripheral nervous system (PNS) injuries [29], stroke [30], myocardial infarction [31], skin scarring [32], or as anti-cancers [33,34], novel treatments for multiple sclerosis [35], Alzheimer's [36] and Parkinson's [37] diseases.
Section snippets
Self-assembling peptide synthesis
The NH2-FAQRVPPGGGLDLKLDLKLDLK-CONH2 (named FAQ) and Ac-CGGLKLKLKLKLKLKGGC-CONH2 (named CK) peptides, were synthesized by solid-phase Fmoc-based chemistry on Rink amide 4-methyl-benzhydrylamine resin (0.5 mmol g−1 substitution) by using the Liberty-Discovery (CEM) microwave automated synthesizer, as previously described [38,39]. All the experiments were carried out at concentrations above the critical aggregation concentration (CAC) at which the peptides show a self-assembly (CAC (FAQ) = 0.3% (w
Results and discussion
Different bioactive soluble molecules such as growth and angiogenic factors, cytokines, DNA, and siRNA have been used for the restoration of lost or injured tissues [52]. Nevertheless, reduced bioactivity and solubility, high toxicity due to unnecessary concentration, and uncontrolled distribution of released drugs are some of the primary limitations of standard delivery methods [53,54]. One possible approach is to load bioactive factors in bioresorbable SAP scaffolds for their delivery [55].
Conclusions
In today's health care systems, controlled drug release has become a key factor in pharmaceutical product development. Drug delivery to hard-to-reach areas, such as spinal cord or brain, is particularly challenging, especially in the case of recurring injections. In situ sustained release of active drugs may significantly lower the injection frequency, thus increasing the feasibility of novel approaches in a real clinical scenario. In this work, biodegradable and bioresorbable SAPs were loaded
Authors' contributions
A.R., F.G. designed research; A.R., L.C. performed all in vitro and in vivo experiments; F.F. performed MD simulations and wrote the section dedicated to in silico studies; R.P. conducted the rheological studies and revised the manuscript; A.R., L.C. analyzed data; F.G. provided funding and supervision; A.R. and F.G. co-wrote the manuscript.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
Work was sponsored by the “Ricerca Corrente 2018-2020” funding conferred by the Italian Ministry of Health; by the”5 × 1,000” voluntary donations; and by Revert Onlus.
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