Diffusion-controlled release of the theranostic protein-photosensitizer Azulitox from composite of Fmoc-Phenylalanine Fibrils encapsulated with BSA hydrogels
Introduction
Hydrogel systems are applied in several diverse pharmaceutical and medical research areas. In recent years, the research on hydrogels has been widely intensified due to the rising awareness for their potential and the tremendously increased amount of possible precursor materials over the last decade (Calo and Khutoryanskiy, 2015, Kopeček, 2007, Lee and Konst, 2014). Apart from natural materials like collagen, elastin, lysozyme fibrin, peptides, polysaccharides, or DNA (Jiyuan Yang, 2010; Liu and Chan-Park, 2010; Mithieux et al., 2004; Oh et al., 2012; Skopinska-Wisniewska et al., 2016; Xing et al., 2011; Ziv et al., 2014), artificial precursors like polyethylene glycol (PEG) (Jiyuan Yang, 2010; Lin and Anseth, 2009; Missirlis and Spatz, 2014) or polyvinyl alcohol (PVA) (Hayes and Kennedy, 2016) have been utilized vastly to obtain a variety of hydrogels with different properties. One major advantage is the high biocompatibility of hydrogel systems: the high water content along with the biocompatibility of most natural hydrogel precursors make them an excellent replacement for natural structures within living organisms (Hoffman, 2012), a promising template for 3D cell culture systems (Sung et al., 2013, Thoma et al., 2014) and material with smart or responsive properties (Gonçalves et al., 2013, Sasaki et al., 2010) for the application in a variety of field in modern technology, e.g., as actuators and valves (Ebara et al., 2013, Techawanitchai et al., 2012), to catch enzymes and cells (Dong and Hoffman, 1986, Kim et al., 2012) and as sensors (Miyata et al., 2002). Up to date, hydrogels in biotechnology are mostly found as wound dressings (Sun et al., 2011), as membranes (Yang et al., 2011), as contact lenses (Calo and Khutoryanskiy, 2015), as coatings for medical devices (Butruk-Raszeja et al., 2015) or as cosmetics (Mitura et al., 2020).
In drug delivery application, it is crucial to find drug formulations that do not interfere with the active pharmaceutical ingredients and do not alter any properties of the encapsulated compound. To find novel drugs, nature is exploited, and more complex biomolecules with highly promising features are discovered (Cragg and Newman, 2013). As many of those novel biomolecules suffer from rather low stabilities, different galenic formulations must be developed to maintain the proteins or peptides’ specific features (Hoare and Kohane, 2008). In this context, hydrogels possess huge potential: their high-water content makes them an excellent material for encapsulating specific drugs as hydrogels can be tailored closely to the natural compartments. Another essential and promising feature is the selective and time-controlled release from a specifically designed matrix (Li et al., 2015), e.g., materials that release a substance when in contact with harmful reactive oxygen species (Saravanakumar et al., 2016) (Scheme 1).
The huge variety of hydrogels - ranging from soft natural to highly resilient, artificial materials - and the possibility to alter most features opens new possibilities for their application in drug delivery. The drug delivery process can be categorized into different release systems: diffusion-controlled, swelling controlled, chemically controlled, or environmental responsive systems (Hoare and Kohane, 2008). The great potential of hydrogel systems lies in the adjustability of their properties like elasticity, chemical and thermal resistance, charge, pore sizes, and water content (Zhu and Marchant, 2011). Many studies aim towards the understanding of fundamental properties of one component hydrogel systems and their manipulation, which often leads to all-purpose materials with promising features, e.g., responsiveness towards specific factors such as a pH shift (Wang et al., 2020), temperature shift (Tao et al., 2020, Yang et al., 2020), or light-responsive materials which can serve as molecular valves for microfluidic (Ter Schiphorst et al., 2015). This is often done by changing precursor molecules, ratios of components, or alteration of 3D architectures in polymerized matrices to optimize the hydrogel’s performance. However, sometimes it might be beneficial to combine different hydrogels to achieve the advantages of both systems.
In this study, we aim to combine the stiffness and stability of a protein-based material with the gentle encapsulation procedure of a fibrillary peptide hydrogel. Bovine serum albumin (BSA) is a 66 kDa protein derived from cow serum and is used frequently as a tool in many laboratories due to its biocompatibility and low costs (Bujacz, 2012). BSA has already been shown to have a vast potential for drug delivery applications (Benko et al., 2015, Iemma et al., 2006, Yu et al., 2014, Zhao et al., 2010). Many described protein-based hydrogels are produced by partial heating of the protein or covalent crosslinking of reactive groups in the matrix backbone, e.g., amine to hydroxy groups. While those approaches can result in tough, highly resistant biocompatible materials, they cannot be used for protein encapsulation as the protein would denature or get inactivated during the production process, limiting the possible applications of the material.
On the other hand, peptide hydrogels are attractive candidates for the direct encapsulation of particles, proteins, or biological structures due to their gentle self-assembly (Huang et al., 2011, Ischakov et al., 2013, Koutsopoulos et al., 2009). Many peptide-based hydrogels are formed via pi-pi interaction or hydrophobic interactions, resulting in soft, stable materials (Roy and Banerjee, 2011). Peptides can be produced via the solid-state synthesis in which it is simple to introduce specific modifications that might be useful for several setups, e.g., cell adhesive moieties for cell culture applications. They are attractive candidates for hydrogelation and can often self-assemble into hydrogels via pi-pi or hydrophobic interactions (Singh et al., 2015). Different amino acids have already been successfully investigated for their potential to form hydrogels, e.g. tryptophan, tyrosine, methionine, glycine, and isoleucine (Draper et al., 2015). Those types of hydrogels have been used to immobilize and grow cells and to encapsulate and deliver drugs like doxorubicin, vitamins, or antibodies (Huang et al., 2011, Ischakov et al., 2013, Koutsopoulos et al., 2009, Malinen et al., 2012, Wang et al., 2008). However, most systems use specifically designed peptides (Gungormus et al., 2010; Jiyuan Yang, 2010; Pochan et al., 2003; Zhu and Marchant, 2011), whereas the number of examples in literature for hydrogels made of pure, individual, and amino acids modified with a fmoc-protection group are rather limited. The phenylalanine-based system has been shown to produce transparent hydrogels, to be biocompatible and well-diffusible by even bigger molecules and nanoparticles (Singh et al., 2015). The transparent material polymerizes at concentrations as low as 0.1% (w/v) (Roy and Banerjee, 2011) and is easy to handle, making it an attractive system for the encapsulation of proteins. Furthermore, pure fmoc protected peptides can be bought for several euros per gram, while the synthesis of specifically designed peptides requires more financial outlay for less yield/outcome. Despite those advantages, the low mechanical properties of fmoc-protected amino acids and their susceptibility towards external stimuli e.g., changing pH, temperature, or shear stress significantly limit their possible use. The Fmoc-phenylalanine fibril hydrogel used in this study is inexpensive (100 g for 80 euro is enough to polymerize 100 liters) and easy to handle, but also lacks mechanical strength and resistance towards environmental changes, e.g., temperature resistance. We could stabilize Fmoc-phenylalanine fibrils in two different approaches by either surrounding them with a layer of covalently crosslinked protein matrix or by stabilizing it within a freeze-dried, macroporous BSA-based matrix, improving the mechanical properties of the material as well as influencing the release pattern of an encapsulated protein.As a model protein, the novel, potential anticancer fusion protein Azulitox (EcFbFP-P28) was used (Raber et al., 2020). This theranostic protein consists of two domains as functional components: The first part of the fusion protein codes for a 28 amino acid peptide, which is derived from the 14 kDa protein azurin and has cell-penetrating activity as well as specificity for cancer cells (Raber et al., 2020). Non-cancer cells are barely targeted by azurin, while cancer cells internalize azurin – as well as the truncated P28 peptide (Jia et al., 2011, Lulla et al., 2016, Taylor et al., 2009, Yamada et al., 2005). The second part of the fusion protein, namely the Escherichia coli Flavin mononucleotide binding fluorescent protein (EcFbFP) is derived from Bacillus subtilis (Drepper et al., 2007). It was originally intended for use as a marker that maintains its fluorescence under anaerobic conditions. This protein-photosensitizer can produce reactive oxygen species when excited with blue light and then can kill cells, while this effect is increased significantly when the fusion protein is internalized by cancer cells (Raber et al., 2020). In combination with the P28 fragment, it can serve as a potent anti-cancer drug.
Section snippets
Materials and methods
BSA, Fmoc-Phe-OH, Na2HPO4, NaH2PO4, trypsin-EDTA (0.05% (w/v)), Tetrakis (methyl hydroxy) Phosphonium Chloride (THPC), and 2-propanol were purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and appropriate stocks were created prior to use. Dimethylsulfoxide (DMSO), Phosphate Buffer Saline (PBS), Dulbecco’s Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), Penicillin-Streptomycin (1000 U/mL), and non-essential amino acid solution (MEM NEAA) were purchased from life technologies
Material properties
In recent years, the focus on hydrogel materials has been intensified due to their versatile application possibilities (Butruk-Raszeja et al., 2015, Calo and Khutoryanskiy, 2015, Mitura et al., 2020). Therefore, hydrogels should have the appropriate properties to meet the requirements for the desired applications e.g., chemical, or physical stability. Fig. 1 shows the degradation profile of the Fmoc-phenylalanine fibril hydrogel. The phenylalanine rings stack with each other via pi-pi
Conclusions
We have used Fmoc-phenylalanine fibril hydrogels for the encapsulation and delivery of a potential anticancer drug with cell-penetrating and fluorescent properties. The substance could be encapsulated in a single step during the gel-to-sol transition and showed an even distribution in the material. By either coating the fibrillary hydrogel with layers of a BSA-based protein hydrogel or stabilizing it with a macroporous system, the mechanical integrity of the fibrillary hydrogel could be
Funding
This work was supported by the Ministry of Science, Research and Arts of the State of Baden-Württemberg, Germany in the framework of the Ph.D. program: pharmaceutical biotechnology, the Baden-Württemberg Stiftung in the framework of “Bioinspired material synthesis” and “Biofunktionelle Materialien und Oberflachen” (BiofMO_005, Nano-Mem-to-Tech), and the European Union project “Horizon 2020″ (no. 686271) in the framework “AD-gut”. This work was also supported by the German Federal Ministry of
CRediT authorship contribution statement
Patrizia Favella: Investigation, Data curation, Writing − original draft. Ann-Kathrin Kissmann: Investigation, Data curation, Writing − original draft. Heinz Fabian Raber: Investigation, Data curation, Writing − original draft. Dennis Horst Kubiczek: Investigation, Validation. Patrick Bodenberger: Investigation. Nicholas Emil Bodenberger: Investigation, Validation. Frank Rosenau: Conceptualization, Supervision, Funding acquisition, Resources, Writing − review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We thank Vittoria Rimola for assistance in the data acquisition and Paul Walther and Thomas Heerde (Central Facility for Electron Microscopy, Ulm University, 89081 Ulm, Germany) for their help with Cryo-electron Microscopy.
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These authors contributed equally to this work.