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Coil-like Enzymatic Biohybrid Structures Fabricated by Rational Design: Controlling Size and Enzyme Activity over Sequential Nanoparticle Bioconjugation and Filtration Steps Franka Ennen,*,§,# Philipp Fenner,# Georgi Stoychev,§,# Susanne Boye,§ Albena Lederer,§,# Brigitte Voit,§,# and Dietmar Appelhans*,§ §
Leibniz-Institut für Polymerforschung Dresden e.V., Hohe Strasse 6, 01069 Dresden, Germany Organische Chemie der Polymere, Technische Universität Dresden, 01062 Dresden, Germany
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S Supporting Information *
ABSTRACT: Well-defined enzymatic biohybrid structures (BHS) composed of avidin, biotinylated poly(propyleneimine) glycodendrimers, and biotinylated horseradish peroxidase were fabricated by a sequential polyassociation reaction to adopt directed enzyme prodrug therapy to protein−glycopolymer BHS for potential biomedical applications. To tailor and gain fundamental insight into pivotal properties such as size and molar mass of these BHS, the dependence on the fabrication sequence was probed and thoroughly investigated by several complementary methods (e.g., UV/vis, DLS, cryoTEM, AF4-LS). Subsequent purification by hollow fiber filtration allowed us to obtain highly pure and well-defined BHS. Overall, by rational design and control of preparation parameters, e.g., fabrication sequence, ligand−receptor stoichiometry, and degree of biotinylation, well-defined BHS with stable and even strongly enhanced enzymatic activities can be achieved. Open coil-like structures of BHS with few branches are available by the sequential bioconjugation approach between synthetic and biological macromolecules possessing similar size dimensions. KEYWORDS: avidin−biotin, glycodendrimer, enzyme, sequential bioconjugation, coil-like structure
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INTRODUCTION Nature shows us tremendous examples of highly efficient complexes and machines attributed to specific biological functions.1−6 The design and fabrication of those highly sophisticated supramolecular biostructures are exclusively tailored by a balanced self-assembly, complexation, or aggregation process. This also includes the use of unidirectional and sequential formation processes. Various biological functional units are now widely available for the preparation of smart nanocontainers7 and artificial organelles,7 for mimicking cell functions8 or for realizing biohybrid structures (BHS) suitable for biomedical applications, diagnosis, and analysis.9−16 Especially, the use of various biological building blocks such as (strept)avidin,17−19 concanavalin A,20 and other (ligandrecognizing) proteins21 gives the possibility to realize versatile functional materials in life and material science.2,7−16 Recent developments are directed not only to establish complex BHS with desired biofunctions but also to consider the fabrication of advantageous molecular shapes (e.g., nanorods or tubular assemblies) compared to spherelike structures for better uptake processes in biomedical applications.22 It would be desirable to integrate the well-known biological building blocks (strept-)avidin or concanavalin A in a highly efficient conjugation process for the fabrication of BHS with preferential © XXXX American Chemical Society
nonspherical molecular shapes. Most conjugation processes between avidin and biotinylated nanoparticles result in the formation of spherelike BHS17,18 or in uncontrolled aggregation processes.23 In this context, the avidin−biotin conjugation has evolved a frequently used fabrication strategy in the formation of functional conjugates with biotinylated bioactive compounds for their preferential use in drug delivery systems and in biosensing. Due to the low avidin−biotin dissociation constant (10−15 M),24 the potential of such larger bioconjugates25−28 for targeted delivery is facilitated by the enhanced permeability and retention effect in the case of cancer treatment. On the other hand, the intrinsic properties of avidin allow for the binding to certain lectins on cancer cell surfaces.29−32 Furthermore, the applicability of polyassociates composed of avidin and biotinylated dendritic glycopolymers (bGP) to biomedical fields is favored not only by avidin’s lack of harmful immunogenicity33 but also by the high biocompatibility of oligosaccharide-modified dendritic polymers.34−38 Furthermore, control over the size of the polyassociates is further Received: October 27, 2015 Accepted: February 12, 2016
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DOI: 10.1021/acsami.5b07305 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
targeted drug delivery. Other ligand-modified and sugardecorated dendrimers can also be considered as a drug carrier units for BHS consisting of avidin, bHRP, and GD-B4 in future DEPT. Overall, the use of two different biotinylated biomacromolecules opens the potential for sequential conjugation approaches with avidin to realize any kind of nonspherical and uniform biohybrid structure. Initial sequential self-assembly and/or complexation of different biological building blocks have been successfully established for which templates such as larger metallic and natural particles and wormlike structures have been further modified with avidin, antibodies, enzymes, etc.51−54 So far, no multiple use of avidin has been applied to the sequential conjugation process to fabricate larger BHS (>30 nm) in solution where avidin is used as a bridge or linear unit. Furthermore, the nontemplated self-assembly of three different nanoparticles has still not been reported compared with previously described approaches for sequential conjugation and/or complexation.51−54 Our study also exclusively includes the use of similar nanometer-sized nanoparticles in the sequential polyassociation process of avidin (Scheme 1).
tunable by adjusting the degree of biotinylation of the bGP and the molar ratio of them to avidin.17,18,39 The coupling of an enzyme to those avidin/bGP polyassociates allows their use in directed enzyme prodrug cancer therapies (DEPT).39,40 The development of those structures for DEPT requires a proper choice of enzyme. In this context, human and nonhuman enzymes can be used for this therapeutic treatment. A huge disadvantage of human enzymes is that they cause prodrug transformation wherever they are in the human body. In opposition to this, nonhuman enzymes provide a high specificity when arriving at the targeted tissue. A disadvantage of them is that they increase toxicity and activate an immune response.40 A promising enzyme−prodrug combination suggested for cancer treatment due to its apoptotic effect toward human cancer cells41−44 is horseradish peroxidase (HRP) and indole-3-acetic acid (IAA).41,42 It was found that neither HRP45 nor IAA46 harms healthy cells. This combination is also very attractive because only trace amounts of H2O2 are required to form the cytotoxic products.46 Moreover, fixation of enzymes is a feasible way to maintain or increase their enzymatic activity.47 For example, through the integration of bHRP within avidin layers, its enzymatic activity was maintained or was even increased.48 Similar findings may also be expected for the established enzymatic biohybrid structures in this study (Scheme 1).
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EXPERIMENTAL PROCEDURES
Materials. Biotinylated glycodendrimers (bGD) were synthesized as reported previously.18,39 Nonbiotinylated horseradish peroxidase (HRP) and biotinylated horseradish peroxidase (bHRP) and avidin were purchased from Life Technologies (Darmstadt, Germany). All chemicals were used as received. All photometric measurements were performed in 1.5−3.0 mL PMMA cuvettes (Plastibrand) from Brand GmbH & Co. KG with a UV/vis spectrophotometer DU 800 from Beckman Coulter GmbH. A 100 mM Tris/HCl/0.1 M NaCl solution at pH 7.5 was prepared by dissolving 6.72 g of Tris, 44 mL of 1 N HCl, and 5.84 g of NaCl in 1 L of Milli-Q water. Methods. Further details of the methods used (dynamic light scattering (CLS), UV−vis spectroscopy, asymmetric flow field flow fractionation, transmission electron microscopy) for the characterization of BHS are presented in the Supporting Information and were previously described.39 Synthesis of GD-B4. Synthesis and characterization of fourthgeneration poly(propyleneimine) glycodendrimer functionalized with a C6-linked biotin ligand (GD-B4) was performed according to the literature.18,39 See also Figure S1, Supporting Information. The experimental descriptions for the preparation of BHS based on avidin, bHRP, and GD-B4, the determination of bound GD-B4 and bHRP to avidin, the purification of BHS by hollow fiber filtration, and the horseradish peroxidase activity assay are presented in the Supporting Information and were previously described.39
Scheme 1. Sequential Conjugation Approaches for (A) Avidin/bHRP/GD-B4 and (B) Avidin/GD-B4/bHRP BHS39
The main aim of this work was to fabricate rather large enzymatically active BHS constituted by avidin, a tetravalent biotinylated glycodendrimer (GD-B4)17,18,39,49 and bHRP. These polyassociates were fabricated by two different stepwise approaches (Scheme 1). Hollow fiber filtration (HFF) was used to isolate the nanostructures, which are a major fraction in the raw conjugation solution.17,18,39,49 Complementary analytical techniques such as DLS, TEM, and cryoTEM were applied to specify their attributes such as shape, size, or enzymatic activity.39 The HABA displacement assay was used to investigate the content of biotinylated macromolecules conjugated to avidin.39 Asymmetric flow field flow fractionation (AF4) coupled to static and dynamic light scattering (AF4-LS) was also performed to analyze the molar mass, dispersity, the radius of gyration (Rg), and hydrodynamic radius (Rh) of these complex structures.39 The maltose attached to the outer sphere of the dendrimer not only increases the biocompatibility of those dendrimers36,38 but also represents a model sugar for potential interaction with lectins such as the model lectin concanavalin A.50 Even though these interactions are weak and can be easily destroyed by lower blood concentrations of glucose,50 it clearly shows the potential of oligosaccharide modification of those dendrimers in
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RESULTS AND DISCUSSION Formation of BHS. In our previous publications we reported the polyassociation reactions of avidin and biotinylated dendritic glycopolymers.17,39,49 In this report we fabricate complex structures consisting of three components: avidin, a tetravalent biotinylated glycodendrimer (GD-B4), and bivalent biotinylated horseradish peroxidase (bHRP). Their characteristics are summarized in Table 1. bHRP carries about 2.4 C6− C6-linked biotin ligands, whereas the GD-B4 carries an average of 3.6 C6-linked biotin moieties and possesses the maximum number of maltose units attached to the outer sphere of the dendrimers (see also Figures S1 and S2).39 To evaluate to what extent the sequential conjugation process or the degree of biotinylation of macromolecules influences the key characteristics of enzyme-containing BHS,39 two fabrication approaches have been evaluated: (i) avidin/ bHRP/GD-B4 (BHS A) and (ii) avidin/GD-B4/bHRP (BHS B
DOI: 10.1021/acsami.5b07305 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
twice smaller intermediate with bHRP (⌀ 14 nm) (Figure 1, left panel) compared to the intermediate with GD-B4 (⌀ 26 nm) (Figure 1, right panel). Also the final BHS A shows a smaller Dh of about 21 nm compared to BHS B with a Dh of 35 nm.39 To understand the significant difference in the diameters for both intermediates and the final BHS, one can exclude the influence of the spacer length in both biotin ligands. The longer spacer (C6−C6) of the biotin ligand (Figure S4) bound to bHRP should facilitate a preferred accessibility for the polyassociation process with avidin. In contrast, the short C6linked biotin ligand of the glycodendrimer (Figure S1) should result in a lower access to the conjugation with avidin caused by its protection of sugar architecture in GD-B4. However, this was not observed, most likely because other circumstances have to be taken into consideration. First, electrostatic repulsion can be considered as one key issue for the receipt of different polyassociation properties between bHRP and glycodendrimer during the two different sequential conjugation processes (Scheme 1 and Figure 1) as discussed above. Both avidin and bHRP are cationic nanoparticles at neutral pH due to the isoelectric point of 10.5 for avidin55 and >9 for bHRP.56 On the other hand, GD-B4 provides a zeta potential of zero at a pH of approximately 8 (Figure S5). Thus, a higher electrostatic repulsion between avidin and bHRP can be expected compared to avidin and GD-B4. This possible key issue may provide a lower number of bHRP conjugated to avidin as found for the glycodendrimer in the intermediates and the final BHS (Table 2), respectively. Overall, the most prominent key issue for the low conversion of bHRP compared to that of GD-B4 emphasizes the degree of biotinylation, as discussed in previous studies,17,18,39,49 that polyassociation takes place to a smaller extent when a biotinylated component bears a low number of biotin ligands. This is clearly the case here because bHRP has about 2.6 biotin ligands, while GD-B4 possesses 3.6 biotin ligands (Table 1). Finally, the combination of both differing degrees of biotinylation of bHRP and GD-B4 and their differing repulsive interaction with avidin renders the sequence of bHRP and GD-B4 addition to avidin a pivotal influencing parameter in the control of the BHS diameter, while the kind of biotin ligand plays a minor role (Scheme 1). Both of these nonpurified enzymatic BHS showed no significant alteration of their enzymatic activities within 2 days compared to the pure enzyme (Figure S6). Hollow Fiber Filtration (HFF). Raw BHS particles of about 16 nm in the case of BHS A and of about 11 nm in the case of BHS B were removed by filtration (Figure S7). Considering the diameters of the spherical nanoparticles (
