Sphere-Like ProteinâGlycopolymer Nanostructures Tailored by

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Sphere-like Protein-Glycopolymer Nanostructures Tailored by Polyassociation Franka Ennen, Philipp Fenner, Susanne Boye, Albena Lederer, Hartmut Komber, Brigitte Voit, and Dietmar Appelhans Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00975 • Publication Date (Web): 01 Dec 2015 Downloaded from http://pubs.acs.org on December 10, 2015

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Sphere-like Protein-Glycopolymer Nanostructures Tailored by Polyassociation Franka Ennen,*,§,# Philipp Fenner,§,# Susanne Boye,§ Albena Lederer,§,# Hartmut Komber,§ Brigitte Voit,§,# and Dietmar Appelhans*,§ §

Leibniz-Institut für Polymerforschunng Dresden e.V., Hohe Straße 6, D-01069 Dresden,

Germany #

Technische Universität Dresden, 01062 Dresden, Germany

*Corresponding author: [email protected]; Tel.: +49 (0)351 4658 353; Fax: +49 (0)351 4658 565. [email protected]

KEYWORDS: Dendritic glycopolymers, Nanoparticles, Avidin-biotin conjugation, Polyassociation, Ligand-Receptor stoichiometry

ABSTRACT. Key parameters allow a reproducible polyassociation between avidin and biotinylated glycopolymers in order to fabricate defined supramolecular nanostructures for future (bio)medical and biotechnological applications. Thus the polymerization efficiency of

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biotinylated glycopolymers in the fabrication of biohybrid structures (BHS) was investigated with regard to the influence of (i) the degree of biotinylation of the dendritic glycoarchitectures, (ii) two biotin linkers, (iii) the dendritic scaffold (perfectly branched vs. hyperbranched) and (iv) the ligand-receptor stoichiometry. The adjustment of all these parameters opens the way to fabricate defined sizes of the final biohybrid structures as multi-functional platform ready for their use in different applications. Various analytical techniques, including purification of BHS, were used to gain fundamental insights into the structural properties of the resulting proteinglycopolymer BHS. Finally, the elucidation of pivotal conformational properties of isolated BHS with defined sizes by asymmetrical flow field-flow fractionation study revealed that they mainly possess spherical-/star-like properties. From this study, the fundamental knowledge can be likely transferred to other assemblies formed by molecular recognition processes (e.g. adamantane-βcyclodextrin).

Abbreviation Used abbreviation - AF4 = Asymmetrical flow field flow fractionation, BHS = biohybrid structure(s); CB = six carbon atoms containing biotin ligand attached to the dendritic scaffold of hyperbranched poly(ethylene imine) or poly(propylene imine) dendrimer; Dh = hydrodynamic diameter determined by three independent experiments based on the volume distribution unless otherwise stated; Dh, max = maximum hydrodynamic diameter obtained within the probed ligandreceptor stoichiometry of avidin/biotinylated glycopoymer (based on three independent experiments); Dh,

min

= minimum hydrodynamic diameter obtained within the probed ligand-

receptor stoichiometry of avidin/biotinylated glycopoymer (based on three independent experiments); DS = dense shell; EA = elemental analysis; eq = equivalent(s); bGP = the term


2

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refers to both PPI- and PEI based biotinylated glycopolymer(s); G4 = 4th generation; HABA = 4'hydroxyazobenzene-2-carboxylic acid; HFF = hollow fiber filtration; LILBID MS = laser induced liquid beam/bead ion desorption mass spectrometry; n.d. = not determinable; mPES = modified polyethersulfone; PEG = poly(ethylene glycol); PB = biotin ligand, attached to the dendritic scaffold of hyperbranched poly(ethylene imine) or poly(propylene imine) dendrimer with a spacer of 12 poly(ethylene glycol) units; PEI = hyperbranched poly(ethyleneimine) with a Mw = 25000 g/mol or 25 kDa; PEI-BHS = BHS formed by avidin and biotinylated poly(ethyleneimine) glycopolymers; PEI-bGP = biotinylated hyperbranched poly(ethyleneimine) glycopolymer(s); PPI = poly(propyleneimine) dendrimer of the 4th generation; PPI-BHS = BHS formed by avidin and biotinylated poly(propylene imine) glycodendrimers; PPI-bGP = biotinylated poly(propylene imine) glycodendrimer(s); Rg = radius of gyration; Rh = hydrodynamic radius; LS = static light scattering; RI = refractive index.

1.

INTRODUCTION

The design of novel tailor-made, multifunctional and bioactive nanostructured materials has revealed itself as a promising avenue towards biomedical applications in today’s life sciences. Hence, the formation of higher ordered molecularly organized nanostructures has been explored tremendously during the past decades mainly against the background of (bio-)nanotechnology. Here, non-covalent interactions have been widely applied to a variety of soft or hard nanoparticles in order to expand the scope of their applicability through a so called “value added” bottom up strategy. This results in abilities, which are beyond the sum of their single components.1-3 Although a variety of non-covalent interactions (e.g. avidin-biotin, NiIINTA-His


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or adamantane-β-cyclodextrin) can be adopted to such nanoparticles through the introduction of functional ligands (e.g. biotin, NiIINTA, His-tags, adamantane or β-cyclodextrin), one of the most frequently utilized non-covalent interaction is that of avidin-biotin. This is mainly caused by the avidin´s tremendously high avidity toward biotin (KD = 10-15 M)4 facilitating stable conjugations of various biological and/or artificial compounds to avidin or its analogs. Moreover avidin offers the opportunity for effective drug delivery through both active and passive targeting. Firstly this is mediated through the enhanced permeability and retention effect due to increased molecular weights of the avidin´s associates5-8 with soft and hard nanoparticles. Secondly active targeting is facilitated through its ability to bind different lectins expressed on several cancer cell surfaces.9-14 In addition it possesses no harmful immunogenicity.15 Among the soft nanoparticles, due to their unique and perfect structure, dendrimers represent a promising platform for nanocarriers. These three-dimensional dendritic polymers are characterized by a central core, branching units and high numbers of terminal functional groups. This makes them suitable candidates for complex formation, conjugating or encapsulating therapeutic drugs and imaging moieties or the stabilization of nanoparticles. Their terminal functional groups can be used in order to tailor their solubility and chemical behavior.16-19 Moreover, their high number of reactive surface groups facilitates the introduction of a variety of functional groups that can (i) work as biocompatibility mediators, (ii) enhance the blood circulation times in drug or gene delivery vesicles (e.g. PEG terminated or OH group terminated dendrimers),20, 21 or (iii) work as recognition moieties.22,

23

Additionally, peripheral functional groups (e.g. alkylic, aromatic or

host entities) can act in the outer shell of dendrimers to initiate uncontrolled but also controlled self-assembly processes.24,

25

In this context certain studies in solution revealed a significant

dependence of the biotinylated materials´ properties on the final structure of the self-assembled


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nanostructures,26-32 also determining the final sizes, molar masses and dispersities of those nanostructures. These parameters have a huge impact on the final fate of such nanoparticular formulations. For instance, they influence key factors for drug delivery such as blood circulation time, distribution in the body or excretion.33-37 In 1971 Green et al. showed the polymerization of avidin by bisbiotinyl compounds, whereas structure, shape and size were critically depending on the length of the bisbiotinyl compounds.38 However, in combination with biotinylated polymers avidin is used mainly as a central unit and so far no comparable fundamental study has been conducted that evaluates the polymerization ability of biotinylated perfectly branched and hyperbranched polymers. The reaction of avidin with highly valent biotinylated glycopolymers can be categorized as a polyassociation reaction in analogy to the so called polymer chain reaction. The adjustment of conjugation parameters for the design and fabrication of biohybrid structures is

one

of

the

most

crucial

parameter

when

considering

the

potential

use

of

controlled/uncontrolled (spherical) nanoparticles´ assembly in life sciences and material science.39-57 To achieve those different biological supramolecular and biohybrid structures the most frequently approaches are directed to use non-covalently driven41-45 (e.g. electrostatic, hydrophobic and/or H-bonds) and protein-ligand40, the

conjugation

of

different

biotinylated

49-53, 58

mediated interactions. For example,

nanoparticles

(polymeric,

enzymatic

or

metallic/magnetic nanoparticles) with (strept-)avidin generally results in larger aggregates with different size dimensions and shapes,49-52, 58 while the bottom up approach tailored by avidinbiotin conjugation on planar substrates produces stable layered enzymatically-active nanoparticle structures.53 Thus, there is a strong demand on the establishment of defined avidin-biotin conjugation processes for the nanoparticle assembly between enzymes, avidin and other


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spherical nanoparticles, but also to further integrate them in even more complex biohybrid structures with versatile and advancing properties. To aim such a new class of hierarchically and biologically-active sophisticated supramolecular nanoparticles architectures controlled (selflimiting) assembly is already available, for example, between concanavalin A and glucose oxidase,40 gold nanoparticles and virus particle,55 dendrons and virus particles.43 Moreover, further efforts are directed to program supramolecular biohybrids as precision therapeutics.39 Establishing such new class of biohybrid nanoparticle structures it is also essential to use light/temperature-sensitive,59-62 cleavable,43 reversible/dynamic59,

60, 62-64

linking units between

different nanoparticles for the logical fabrication composition and the modular rearrangement of various nanoparticles´ assembly. Because of these scientific challenges we are strongly interested to establish a toolbox of biotinylated spherical nanoparticles (e.g. enzymes, dendritic glycopolymers and other proteins) with nanometer-sized diameters (4-15 nm) for the fabrication of controlled nanoparticles´ assembly triggered by protein-ligand mediated conjugation.40, 49-53, 58 Residual binding pockets of proteins (e.g. avidin or concanavalin A) in the BHS can be further used, for example, to introduce cell-recognition ligands. Basic concepts for the design and fabrication of complex biohybrid structures are adopted from the promoted work of D. Tomalia.65-67 Moreover recent progresses are directed to develop (bio-)hybrid structures attributed by non-spherical shapes that allow enhanced cellular uptake against spherically shaped (bio-)hybrid structures.68 In our recent publication32 we have shown the successful fabrication of biohybrid structures (BHS) composed of avidin and mono-, bi- and tetravalent biotinylated poly(propylene imine) glycodendrimers (PPI-bGP) with sizes ranging from 12 nm to 100 nm in diameter depending on the degree of biotinylation and ligand-receptor stoichiometry. Furthermore, we also found that


6

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the conjugation of avidin with biotinylated glycodendrimers leads to the formation of conjugation adducts of avidin and the glycodendrimers next to high molecular weight associates (denoted as nanostructures) with significantly increased molar masses and larger diameters compared to those of the conjugation adducts. This is exemplarily illustrated for a bivalent glycodendrimer in Figure 1A.

A Composition of non-purified avidin / bGP biohydrid structure C Topic of the study avidin

Dynamic biological conjugation between avidin and spherical biotinylated glycopolymer

bivalent bGP conjugation adducts

nanostructures

B Simplified molecular architecture of bGP based on PPI dendrimer and PEI core and a dense maltose shell (DS)

cis position trans position

Biotin ligand (R1) with spacer connected to PPI dendrimer or PEI scaffold via amidation

Association and dissociation processes between avidin and bGP due to sterical demand [32]

monovalent

bivalent

tetravalent

octavalent

PPIDS-CB1*

PPIDS-CB2*

PPIDS-CB4*

PPIDS-PB1* PEIDS-CB1 PEIDS-PB1



PPIDS-CB8 PPIDS-PB8

1. Formation process in dependence of (i) kind of dendritic scaffold, (ii) length of biotin ligand and

O

R1 = 2

N H

S H HN

C6-linked biotin (CB)

Still open questions before the use of BHS in biological experiments

O

H NH

O

R1 =

O 12

N H

H HN

PEG12-linked biotin (PB)

(iii) concentration

S H NH

2. Reproducibility and stability of BHS in consideration of association/dissociation processes during formation

O

3. Reproducibility and dispersity of BHS after purification

* Evaluated principle key parameters (ligand-receptor stoichiometry and degree of biotinylation) for the design and fabrication of biohydrid structures in solution

under shear forces [32]

4. Molecular shape and solution properties of BHS

Figure 1. Various aspects (A and B) described in our previous report32 and open questions addressed in this study (C). A – Composition of raw conjugation solution B – Besides the investigation of PPI-based biotinylated glycopolymer (marked with *) further biotinylated glycopolymer based on PEI core are under investigation. C -


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Favored coupling of two biotinylated glycopolymers,69 and steric shielding of the coupled biotinylated glycopolymer due to similar or even larger sizes of avidin and the biotinylated glycopolymer.32

The present study addresses further pivotal questions (Figure 1C) still open from our previous study32 with regard to potential future biomedical applications of such BHS as a multi-functional platform with short- and long-term stability properties.. As well as the reported avidin-biotin conjugation process of a singly biotinylated polymeric architectures to other (bio-)polymers via avidin,26,

70

also the couplings of multiple biotinylated (bio-)macromolecules through avidin-

biotin interactions have to be considered as dynamically-driven processes (Figure 1C) at which simultaneously, spontaneously and/or sequentially intra- and intermolecular associations and dissociations between biotin ligands of the biotinylated glycopolymer and avidin take place (Figure 1B). Furthermore, the affinity of avidin toward biotinylated sterically demanding derivatives is lower compared to the affinity of avidin toward pure biotin.27, 29, 30 Since the latter consequently increases the number of associations/dissociation of avidin and biotinylated compounds and has a strong impact on the resulting architectures,69, 71 we were highly motivated to investigate further vital key parameters (Figure 1C) for the formation of stable BHS in order to find proper conjugation protocols, before those biohybrid nanoparticles can be used as multifunctional platform, for example, for various biological, enzymatic, sensing and therapeutic applications. In this report the following key parameters are considered in order to get deeper insight in the structure-property/activity relationship between biotinylated glycopolymer and avidin (Figure 1C):


8

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•

The formation process of BHS in dependence of (i) the kind of dendritic scaffold (poly(propylene imine) dendrimers (PPI) or hyperbranched poly(ethylene imine) polymers (PEI)), (ii) impact of the length and structure of the biotin ligands and (iii) the ligand-receptor stoichiometry (LRS = navidin/nbGP, whereas n represents the amount of the substance);

•

Reproducibility and stability of those BHS over time evaluated by the size development within 1 day and 14 days and concentration dependence;

•

Stability and dispersity of BHS after the purification under shear forces by hollow fiber filtration;

•

Analysis of purified BHS by dynamic and static light scattering coupled to asymmetrical flow field flow fractionation (AF4) in order to gain fundamental knowledge about the physical and conformational properties of the formed BHS;

•

Design and fabrication of preferably monodisperse spherical-like polyassociates formed by avidin-biotin interaction, instead of amorphous hydrogels which are generally formed by such non-covalent interactions (e.g. avidin-biotin,72, 73 adamantane-β-cyclodextrin,73-75 α-CD-PEG inclusion complexes75, 76).

Consequently, we are strongly interested in the establishment of a toolbox for the fabrication of defined assemblies of avidin and biotinylated glycopolymers (polyassociates) as a new class of BHS as multi-functional platform for potential biomedical applications. For example, this multifunctional platform can be attributed additionally to integrate the degradable binding of enzymes and hemocompatible proteins at which the cleavage of such biomolecules can be induced by pH,


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light or redox processes. Moreover, the degradability of those platforms can be further strengthened, for example, by the introduction of photo- and pH-cleavable units in the spacer of biotinylated glycopolymers. The influencing factors for reproducible polyassociation may not only account for BHS composed of avidin and biotinylated glycopolymer, thus, the gained fundamental knowledge can be likely transferred to other assemblies formed by molecular recognition processes (e.g. adamantane-β-cyclodextrin) in order to fabricate defined nanoparticles.

2.

EXPERIMENTAL PART AND MATERIAL DETAILS

Materials Sodium tetraborate decahydrate, benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP), dimethylsulfoxide (DMSO), 6-(N-biotinylamino)caproic acid (CB), tris(hydroxymethyl)aminomethane (TRIS), and sodium chloride (NaCl) were purchased from Sigma Aldrich. Hydrochloric acid (Tritisol®) was purchased from Merck KGaA. Alpha-Biotinomega-(propionic acid)-dodecae(ethylene glycol) (PB) was obtained from Iris Biotech GmbH. Triethylamine (NEt3), D-(+)-maltose monohydrate, borane-pyridine complex (8 M in THF) (BH3·Pyr) were purchased from Fluka. 4th generation poly(propylene imine) (PPI) dendrimer was supplied by SyMO-Chem (Eindhoven, Netherlands) as DAB-Am64†. Avidin was purchased from Life Technologies (Darmstadt, Germany). All chemicals were used as received. All photometric measurements were performed in 70 – 850 µL micro UV cuvettes (PLASTIBRAND) from Brand GmbH & Co. KG with the UV/Vis spectrophotometer DU 800


10

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from Beckman Coulter GmbH. A 100 mM TRIS/HCl/0.1 M NaCl solution with pH 7.5 was prepared by solving 6.72 g TRIS, 44 mL 1 N HCl and 5.84 g NaCl in 1 L MilliQ water.

Syntheses. Details about the syntheses of the biotinylated dendritic polymers and glycopolymers can be found in the Supporting Information (SI).

†

Footnote:

Following

suggestion

from

D.A.

Tomalia

and

M.

Rookmaker

for

“Poly(propyleneimine) Dendrimers” in Polymer Data Handbook,78 the nomenclature for Tomalia-type PAMAM dendrimers can be adopted to PPI dendrimers. Hence, the commercially available 5th generation PPI dendrimer (DAB Am 64) is a 4th generation.

Methods Hollow fiber filtration: Purification of conjugation solutions of different ligand-receptor stoichiometry of avidin and biotinylated glycopolymer. 4 mL of an equimolar solution of biotinylated glycopolymer and avidin (2.19 × 10-5 M; 66 kDa) in 0.1 M TRIS/HCl/NaCl was equilibrated for 24 h. The resulting solutions with a total mass concentration of about 2.5 mg/mL were diluted up to approximately 35 mL and subsequently filtrated with different mPES membrane with MWCO of 100, 300, 500 kDa or a pore size of 0.2 µm for up to 100 mL with an approximately pressure of 200 mbar and a volume flow of 15 mL/s. During the filtration samples


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were taken for DLS measurments. (SpectrumLabs, USA), equipped with a polysulfone-based separation module (MWCO: 300 kDa, SpectrumLabs, USA).

All other analytical methods and assays (1H NMR spectroscopy, DLS, UV-vis spectroscopy, HABA displacement assay, and asymmetrical field flow field fractionation (AF4)) used for the characterization of biotinylated glycopolymer and their BHS are presented in the SI. These methods have been also described in our previous publications.32

3.

RESULTS AND DISCUSSION

3.1

Biotinylated dendritic glycopolymers

Figure 1B presents the used mono-, bi-, tetra- and octavalent biotinylated glycopolymers to investigate the polymerization efficiency of biotinylated glycopolymers based on PPI dendrimer and PEI cores against avidin. Biotinylated glycopolymers possess either C6-alkyl (CB) or PEG12 (PB) biotin ligands. Further details for the synthesis and characterization of the various biotinylated glycopolymers, including the determination of the degree of biotinylation, are presented in the Supporting Information (SI). Non-biotinylated dendritic glycopolymers have been used as a control.

3.2

Biohybrid structures determined by HABA displacement assay and dynamic light scattering


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3.2.1 Amount of biotinylated dendritic glycopolymers bound to avidin - HABA displacement assay For the DLS investigations an [avidin.HABA] complex was mixed with certain amounts of the biotinylated glycopolymer taking a specific activity of avidin of 13.6 units/mg into account. Along with the DLS investigations the amount of bound biotinylated glycopolymer to avidin was determined through the displacement of the azo-dye HABA by the biotinylated glycopolymer. The determined values of the amount of bound biotinylated glycopolymer compared to the applied ligand-receptor stoichiometry (summarized in Table SI-2) are summarized in Table SI10. In general, it was found that on average only one half of the biotinylated glycopolymer are bound to avidin, even though avidin-biotin conjugation is characterized by a very low dissociation constant of 10-15 M.4 There could be several reasons for this behavior. One of them is the size of the dendritic glycopolymers, e.g. steric reasons (cf. Table SI-9). This results in potential steric shielding32,71 even though a hampering effect of the azo dye HABA cannot be ruled out as found in another study (Figure 1C).38 Secondly, this effect is also a result of the non-monodisperse distribution of biotin functions in the glycopolymer population, especially for biotinylated glycopolymers with ≥ 2 biotin ligands, at which biotin ligands are randomly distributed over the dendritic glycopolymer surface to keep maximal distance to each other. Thus, lower amounts of biotinylated glycopolymers in the glycopoylmer population hamper the formation of larger BHS as identified later with smaller hydrodynamic diameters in the DLS study. In this context, the molecular flexibility of biotin ligands can be also suppressed by the partly/completely embedding ligands (Figure 1B) in the dense shell of the biotinylated glycopolymer. This inhibits further and desired binding events with the binding pockets of avidin. Future theoretical calculation and modelling experiments are needed to get further


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insights in this complex conjugation process. Overall, this leads to a distribution in the probability of a contact between a biotin unit and an avidin molecule, which is most probably shifted to the maximum of the functionalization distribution of the dendrimers, and corresponds to half of their amount. 3.2.2

Dynamic light scattering (DLS)

The obtained DLS data of the batch measurements were analyzed with the focus on the following issues: (i) range of hydrodynamic diameters (Dh, min – Dh, max) and (ii) maximum diameter (Dh, max) with respect to ligand-receptor stoichiometry and degree of biotinylation and (iii) size stability of the BHS. DLS data were evaluated by three independent experiments. The probed ligand-receptor stoichiometry is summarized in Table SI-2. Figure 2 shows the BHS average Dh formed by avidin with mono-, bi- and tetravalent biotinylated glycopolymer after one day of equilibration. In general, the size distributions of the avidin/PEI-bGP BHS are not broader than those of the avidin/PPI-bGP BHS (cf. Figures SI-13-18). However, it has to be considered that DLS size curves always present averaged sizes. If there are populations present that differ only in a few nanometers in their sizes, one will measure the overlap of the sizes of these populations e.g. avidin and/or bGD and formed associates.32 Standard deviation and polydispersity of DLS data obtained from three different batch measurements are summarized in Table SI-9.

Monovalent biotinylated dendritic glycopolymer – The Dh of the BHS of avidin and monovalent biotinylated glycopolymer are depicted in Figure 2a – d. The mixing of different ligand-receptor stoichiometry of avidin (∅ 7 nm) and the monovalent PEI-bGP (∅ 11 nm) (cf. Table SI-9)


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resulted in PEI-BHS with a Dh, max of more than 20 nm for the 1/3 ligand-receptor stoichiometry of avidin/PEIDS-CB1 (Figure 2c) as well as for the ligand-receptor stoichiometry 1/3 and 1/4 of avidin/PEIDS-PB1 (Figure 2d). The Dh of the PEI-BHS are in general higher than the Dh of the analogous PPI-BHS at the same ligand-receptor stoichiometry of avidin/biotinylated dendritic glycopolymer. The conversion of avidin with PPIDS-CB1 showed Dh, max of about 12 nm at both the 1/2 and 1/3 ligand-receptor stoichiometry (Figure 2a), whereas the Dh of the single PPI-bGP is of about 6 nm (cf. Table SI9). This indicates that there are non-bound particles present, since DLS size curves present the average size distribution of the sample. If there are populations present that differ only a few nanometers in their Dh, one will measure the overlap of the Dh of these populations e.g. avidin, biotinylated glycopolymer or formed BHS. Thus, the higher the amount of non-conjugated biotinylated glycopolymer or avidin is, the lower the measured average Dh of the avidin/biotinylated glycopolymer mixtures is. In general, the standard deviations (Table SI-9) do not exceed 3 nm and are rather low for almost all ligand-receptor stoichiometry of avidin and the monovalent biotinylated glycopolymer. Bivalent biotinylated dendritic glycopolymer – The Dh of the BHS of avidin and bivalent biotinylated glycopolymer are depicted in Figure 2e – h. Dh, max of 23 nm and 29 nm were reached at a ligand-receptor stoichiometry of 1/1.5 in the case of the BHS based on PPI- and PEIDS-CB2, respectively (Figure 2e and g). Furthermore, Dh, max of 21 nm and 32 nm of the BHS were obtained at even lower ligand-receptor stoichiometry of 1/2 in the case of the PPIDSand PEIDS-PB2, respectively (Figure 2f and h). All standard deviations of the Dh of the formed BHS are rather low and indicate a good reproducibility (Figure SI-9).


15

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Tetravalent biotinylated dendritic glycopolymer – The Dh, max that was found for BHS consisting of avidin and PPIDS-CB4 or -PB4 are 46 nm and 50 nm, respectively. The Dh, max of the BHS composed of avidin and PEIDS-CB4 or -PB4 are even higher with 53 nm and 57 nm, respectively (cf. Figures 2i - l). Dh, max was found to be at the 1/1 ligand-receptor stoichiometry in the most cases. The BHS composed of avidin and tetravalent biotinylated glycopolymer possess a higher standard deviation compared to the standard deviation of BHS composed of avidin and mono- or bivalent biotinylated glycopolymer (Table SI-9).

Dh [nm]

40

a)

b)

c)

d)

30 20

*

*

*

*

*

Dh, max

Dh,PEI-bGP

10

Dh,avidin; Dh,PPI-bGP

Dh [nm]

PPIDS-CB1 40

e)

PPIDS-PB1

f)

PEIDS-CB1

g)

PEIDS-PB1

h)

*

*

30

*

*

*

20

Dh, max

Dh,PEI-bGP

10


PPIDS-CB2 70 60

i)

PPIDS-PB2

j)

*

*

PEIDS-CB2

k)

PEIDS-PB2

l)

*

*

50

Dh [nm]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 47

40 30

*

20

Dh, max

Dh,PEI-bGP

10


PPIDS-CB4

PPIDS-PB4

PEIDS-CB4

PEIDS-PB4

Figure 2. Average Dh and their corresponding standard deviations (further details presented in Table SI-9) of different BHS composed of [avidin.HABA] and biotinylated glycopolymer after 1 d of equilibration in 0.1 M TRIS/HCl/NaCl at pH 7.5 determined by DLS batch measurements of three independent experiments.


16

Page 17 of 47

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Biomacromolecules

Octavalent PPI-bGP – Figure 3a shows the Dh of the single components [avidin.HABA], PPIDSCB8 and PPIDS-PB8 as well as the Dh of the BHS formed by different ligand-receptor stoichiometry of avidin and PPIDS-CB8 or PPIDS-PB8 (Figure 3b - e). The ligand-receptor stoichiometry of 1/0.25 of avidin and the octavalent PPI-bGP resulted in a broad and partly bimodal size distribution after one day, which was still present after 14 days for both avidin/ PPIDS-CB8 and avidin/ PPIDS-PB8 BHS (Figures 3b – e – black lines, cf. also Figure SI-19be). Largest particles with a Dh, max of about 860 nm for PPIDS-CB8 based BHS and 1740 nm for PPIDS-PB8 based BHS were obtained at a ligand-receptor stoichiometry of 1/0.5 after one day (Figure 3b and d – red lines). However, after 14 days they showed either a bimodal size distribution (avidin/ PPIDS-CB8 1/0.5, Figure 3c, red lines) or instability in size (avidin/ PPIDSPB8 1/0.5, Figure 3e, red lines). The 1/1 ligand-receptor stoichiometry resulted in Dh of about 20 – 40 nm, which were bimodal and rather stable for up to 14 days (Figure 3 - blue lines). The 1/1.5 and 1/2 ligand-receptor stoichiometry resulted in even smaller particles (Figures 3b – d – pink and green lines). The shoulder of the 1/2 ligand-receptor stoichiometry of avidin/ PPIDSCB8 after 14 days indicates the presence of single macromolecules (Figure 3c, green line). The observed conjugation results for the octavalent biotinylated dendritic glycopolymers are in agreement with a former study by Kisak et al.47 It was found that an increase in the amount of biotin moieties incorporated in lipid vesicles - meaning an increase of the degree of biotinylation of the vesicles - increased the critical ratio of [streptavidin/exposed biotin] at which the transition from complete to limited aggregation occurred. This means for the structure-activity relationship


17

Biomacromolecules

between avidin and octavalent biotinylated PPI glycodendrimers that unequal molar ratios against 0.5/1 ligand-receptor stoichiometry will result in smaller particle formation. Only in the case of 1/0.5 a molar saturation of all binding pockets of avidin is given. Similar observations were obtained by the less biotinylated dendritic glycopolymers.

a)

20

[avidin.HABA] PPIDS-CB8 PPIDS-PB8

Vol %

15 10 5 0 1

10

[avidin.HABA]/PPIDS-CB8

25 20

Vol %

1/0.25 1/0.5 1/1 1/1.5 1/2

*

15 10

*D

5

h, max

c)

1000

10000 after 14 d [avidin.HABA]/PPIDS-CB8

1/0.25 1/0.5 1/1 1/1.5 1/2

25

*

20 Vol %

b)

100 Dh [nm]

after 1 d

15 10

*D

h, max

5 0

0 1

10

100

1000

1

10000

10

1/0.25 1/0.5 1/1 1/1.5 1/2

20 15 10 5

e)

1000

10000

[avidin.HABA]/PPIDS-PB8

1/0.25 1/0.5 1/1 1/1.5 1/2

25 20

Vol%

[avidin.HABA]/PPIDS-PB8

d) 25

100 Dh [nm]

Dh [nm]

Vol %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 47

15 10 5

0

0 1

10

100 Dh [nm]

1000

10000

1

10

100

1000

10000

Dh [nm]

Figure 3. Dh of a) avidin, PPIDS-CB8 and PPIDS-PB8; different ligand-receptor stoichiometry of avidin/PPIDS-CB8 after b) 1 d and c) 14 d and different ligand-receptor stoichiometry of avidin/PPIDS-PB8 after d) 1 d and e) 14 d in 0.1 M TRIS/HCl/NaCl at pH 7.5 measured by DLS in batch (cf. Figure SI-19, Table SI-7 and SI-8).


18

Page 19 of 47

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Biomacromolecules

The lack of reproducibility of the sizes of the BHS formed by octavalent PPI-bGP can be strongly attributed to a high frequency of association-dissociation processes caused by steric hindrance and the higher amount of competitive biotin ligands present in solution but also unspecific non-covalent interactions cannot be ruled out. However, unspecific non-covalent interactions can be considered as minor events, but not as the main reason for the nonreproducible Dh of the BHS.

3.2.3

Stability of BHS

Another vital point of these BHS is the stability of the Dh over time. The differences of the Dh after one day and 14 days (∆ Dh) of the particular PPI- and PEI-BHS are shown in Figure 4. Monovalent biotinylated dendritic glycopolymer – No significant change in the Dh of the BHS based on monovalent biotinylated glycopolymer within 14 days was found (Figures 4a - d). Thus neither the dendritic scaffold nor the chemical nature of the biotin ligand or its length influences significantly the stability of the BHS, since the changes in the diameter do not exceed 2 nm.


19

b)

PPIDS-CB1

PPIDS-PB1

e)

f)

c)

d)

5 0

10 ∆Dh [nm]

a)

Page 20 of 47

PEIDS-CB1

g)

PEIDS-PB1

h)

5 0

PPIDS-CB2 25

i)

PPIDS-PB2

j)

PEIDS-CB2

k)

PEIDS-PB2

l)

20 15 ∆Dh [nm]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

∆Dh [nm]

Biomacromolecules

10 5 0

PPIDS-CB4

PPIDS-PB4

PEIDS-CB4

PEIDS-PB4

Figure 4. Change of the hydrodynamic diameters ∆Dh with ∆Dh = Dh,14 d -Dh, 1 d of avidin/biotinylated glycopolymer BHS and Dh is the average of three independent experiments; ratios given as [avidin.HABA]/biotinylated glycopolymer.

Bivalent biotinylated dendritic glycopolymer – Comparable low differences in the Dh were found for BHS based on bivalent biotinylated glycopolymer (Figure 4e – h) apart from few exceptions (cf. Figure 4e and g). The BHS based on PEIDS-CB2 showed size growth with up to 5 nm at the ligand-receptor stoichiometry of 1/1 and 1/1.5 (Figure 4g). In contrast, size decrease with up to 5 nm was also observed for avidin/ PPIDS-CB2 at the 1/1 ligand-receptor stoichiometry.


20

Page 21 of 47

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Biomacromolecules

Tetravalent biotinylated dendritic glycopolymer – A significant trend to growth of the BHS can be observed in the case of the tetravalent biotinylated glycopolymer. This is even more distinct for the PEI-BHS (Figure 4k and l). Here, increased Dh of the BHS among almost all ligandreceptor stoichiometry were found. The growth was found to be up to more than 20 nm. In contrast, this trend only occurs at certain ligand-receptor stoichiometry of the PPI-BHS, namely avidin/PPIDS-CB4 1/0.5 and 1/1 ratio as well as for avidin/PPIDS-PB4 1/1. The maximum size growth amounts up to 15 nm. Octavalent biotinylated dendritic glycopolymer – As can be seen in Figure 3 monomodal size distributions along with stability over a period of 14 days is only given for BHS based on octavalent PPI-bGP at the 1/1.5 and 1/2 ligand-receptor stoichiometry. Higher ligand-receptor stoichiometry resulted in instability and/or bimodality of the size distributions (cf. Figure SI19.). A comparison of the PPI based and PEI based BHS is given in Table 1.

Table 1. Overview of diameter ranges, Dh, max, corresponding ligand-receptor stoichiometry (LRS) of Dh, max and differences in Dh, max of the PPI- and PEI-BHS. Complete view on standard deviation for the average Dh, including the polydispersity with standard deviation, is presented in Table SI-9.

BHS based on

size range (Dh, min – Dh, max)

avidin and

1d

14 d

PPIDS-CB1

9 – 12 nm

9 – 12 nm

PEI -CB1

12 – 21 nm

13 – 22 nm

PPIDS-CB2

8 – 23 nm

12 – 28 nm

10 – 29 nm

10 – 34 nm

DS

DS

PEI -CB2

Dh, max corresponding LRS

Differences in Dh, max[a] 1d

14 d

1/3

75 %

83 %

1/1.5

26 %

21 %


21

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

PPIDS-CB4

21 – 46 nm

22 – 59 nm

PEIDS-CB4

35 – 53 nm

40 – 68 nm

PPIDS-PB1

9 – 15 nm

9 – 13 nm

PEI -PB1

13 – 21 nm

12 – 21 nm

PPIDS-PB2

8 – 21 nm

7 – 20 nm

PEIDS-PB2

9 – 32 nm

11 – 42 nm

PPIDS-PB4

43 – 50 nm

45 – 55 nm

32 – 57 nm

45 – 77 nm

DS

DS

PEI -PB4 [a]

Page 22 of 47

1/1

15 %

15 %

1/4

40 %

40 %

1/2

52 %

110 %

1/1

14 %

40 %

ܲ‫ܫܧ‬ differences in Dh, max calculated by ‫ܦ‬ℎ,݉ܽ‫ݔ‬ − ‫ܦ‬ℎܲܲ‫ܫ‬ ,݉ܽ‫ݔ‬ ܲܲ‫ܫ‬ ‫ܦ‬ℎ,݉ܽ‫ݔ‬

3.3

Influences of vital key factors in BHS formation – concluding remarks

Evaluating the previous DLS and HABA displacement assay results and the following points can be concluded: (i)

Influence of ligand-receptor stoichiometry – Self-limiting aggregation77

The self-limiting nature of the conjugation/aggregation as found by Kisak et al.77 of avidin with biotinylated compounds lead to the following phenomenon: the higher the amount of biotinylated glycopolymer is added to avidin or the lower the ligand-receptor stoichiometry of avidin and biotinylated glycopolymer is, the larger the Dh of the BHS are. But importantly, this was found to occur only up to the ligand-receptor stoichiometry of avidin/biotinylated glycopolymer that corresponds to the Dh, max (Table 1), at which, in the most cases, a molar saturation of the 4 binding pockets of the avidin by 4 biotin ligands of the corresponding biotinylated dendritic glycopolymers should be given. Thus, for example, a ligand-receptor stoichiometry of 4 to 1 for monovalent dendritic glycopolymers and of 0.5 to 1 for the octavalent dendritic glycopolymers is


22

Page 23 of 47

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Biomacromolecules

needed. If a ligand-receptor stoichiometry is chosen that is lower than the ligand-receptor stoichiometry, that corresponds to Dh, max,, BHS are formed with Dh, which are smaller than the Dh, max. With respect to the latter BHS sizes can also be directed by the ligand-receptor stoichiometry of avidin/biotinylated glycopolymer (cf. Figures 2 and 3). (ii)

Influence of degree of biotinylation

• Increased BHS sizes were obtained with increased degree of biotinylation at the same ligandreceptor stoichiometry of avidin/biotinylated glycopolymer. Consequently, the final diameters of the BHS can be controlled by the degree of biotinylation (Figures 2 and 3). • The standard deviations of Dh of the BHS formed by tetravalent biotinylated glycopolymer are higher compared to those observed for the BHS formed by mono- or bivalent biotinylated glycopolymer. A rather poor reproducibility was also observed for BHS formed by octavalent PPI-bGP. Here, stable BHS with monomodal size distributions were only obtained at ligandreceptor stoichiometry 1/1.5 and 1/2, respectively. Thus the reproducibility of the sizes of the BHS is clearly depending on the degree of biotinylation and increases with decreased degree of biotinylation (cf. Figure 2). • The higher the degree of biotinylation is the lower the ligand-receptor stoichiometry is that corresponds to the Dh,

max.

In this regard no significant dependence on the dendritic scaffold

(PPIDS vs. PEIDS) or the biotin ligand (CB vs. PB) was observed (cf. Figures 2 and 3 and Table 1). • Even though PEI-BHS are found to be larger than their PPI analogs, this becomes less distinct the higher the degree of biotinylation is. For instance, sizes differences of the Dh, max PPI- versus


23

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 47

PEI-BHS decrease from about 75 % down to 14 % in the case of the CBx-substituted dendritic glycopolymers (cf. Table 1). (iii)

Influence of biotin ligands

• At similar ligand-receptor stoichiometry of avidin/biotinylated glycopolymer BHS composed of avidin and PB-substituted biotinylated glycopolymer showed similar, slightly lower or slightly larger diameters compared to those biohybrid structures formed by avidin and CB-functionalized biotinylated glycopolymer (Figure 2 and Table SI-9), even though the amount of bound biotinylated glycopolymer, determined through the HABA displacement assay, showed rather similar values (Table SI-10). Generally, there is no obvious advantage for the use of PEG spacers in PB biotin ligand to fabricate extreme larger biohybrid structures as only found in the case of avidin/PEIDS-PB4 with 1/1 ligand-receptor stoichiometry after 14 days (Table SI-9). Moreover, one can assume that there are only very minor non-specific interactions of PB ligands with avidin during the formation of biohybrid structures. • Dh, max of biohybrid structures formed by PB-substituted biotinylated glycopolymers were obtained at higher ligand-receptor stoichiometry (Table SI-9: 1/4) of avidin/biotinylated glycopolymer compared to the BHS formed by CB-substituted biotinylated glycopolymer (Table SI-9: 1/3) in the case of mono- and bivalent biotinylated glycopolymers based on PPI dendrimer. Larger or similar ligand-receptor stoichiometry (Table SI-9) is needed to obtain Dh, max of biohybrid structures for PB-containing mono- and bivalent biotinylated glycopolymer based on PEI core macromolecule, when comparing Dh, max of biohybrid structures for bivalent biotinylated glycopolymers with CB units (Table SI-9). It looks likely that it gives no obvious


24

Page 25 of 47

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Biomacromolecules

differences between CB and PB ligands for mono- and bivalent biotinylated glycopolymers during the BHS formation process. In the case of the conjugation of avidin with tetra- and octavalent biotinylated glycopolymers the Dh, max of the biohybrid structures was found to be mainly at the 1/1 ligand-receptor stoichiometry (avidin/biotinylated glycopolymer) for both the tetravalent CB- and PB-substituted biotinylated glycopolymers (Table SI-9 and Figure 2i – l) and at 1/0.5 for BHS based on the octavalent biotinylated glycopolymers (Figure 3b – e).

(iv)

Influence of dendritic scaffold

• Hyperbranched polymers are generally known for their higher dispersities regarding their molar masses compared to their perfectly branched dendrimer relatives. This is barely reflected in the standard deviation of the Dh of the formed BHS or in their size distributions. The size distributions of the PPI-BHS and PEI-BHS showed a similar extent (cf. Table SI-9). • The structural rearrangements over 14 days mainly resulted in a growth of the BHS. PEI-BHS based on PEI-bGP undergo the most distinct growth especially in the case of the BHS formed by tetravalent PEI-bGP (Figure 2, Table 1 and Table SI-9). • Since PEI-bGP are larger building blocks than PPI-bGP they contribute more than the latter ones to the resulting Dh of the BHS. However, a high degree of biotin functionalization of the PPI-bGP results in comparable Dh values of the BHS (Figure 2, and Table SI-9).


25

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 47

• The biotin functionalization of the high numbers of reactive surface groups of the hyperbranched polymers results in a loading distribution that is reported to be a normal distribution.78 Hence especially in the case of monofunctionalized biotinylated glycopolymer there are non-biotinylated glycopolymer macromolecules present, which are not available for an avidin-biotin conjugation. • Figure 2 and Table 1 elucidate also the influence of the dendritic scaffold. In general, PPIBHS showed lower Dh and Dh, max compared to the analogues PEI-BHS. This becomes rather conclusive considering that the Dh of PEI-bGP are almost twice as high as the Dh of PPI-bGP (cf. Table SI-9). • Furthermore, a better accessibility of the biotin ligand of the PEI-bGP for the avidin binding sites has to be taken into account as well. A higher flexibility can be attributed to the PEIDS scaffold compared to the rather rigid PPIDS scaffold leading to a higher accessibility of the binding sites and larger BHS. The differences in the Dh, max of the PPI- and PEI-BHS decrease from 75 % to down to 15 % with higher degree of biotinylation of PPIDS-CBx and PEIDS-CBx and after 1 d and 14 d, respectively (Table 1). 3.4

Purification of BHS and characterization of purified nanostructures

The investigation of certain avidin/PPI-bGP BHS by asymmetrical flow field flow fractionation (AF4) coupled to SLS and DLS revealed that the conjugation solutions consist of three different species: (i) non-conjugated components, (ii) conjugation adducts and (iii) high molecular weight associates (denoted as nanostructures).32 In order to isolate the nanostructures from the rest of the components in the solution hollow fiber filtration (HFF) was applied to the raw conjugation solutions. The purification processes were followed by DLS and UV/Vis. The HFF filtration of


26

Page 27 of 47

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Biomacromolecules

avidin/PPIDS-PB2 in a ligand-receptor stoichiometry of 1/1 purified with a HFF membrane with a molecular weight cut off (MWCO) of 300 kDa is shown in Figure 5. The DLS plot in Figure 5a shows increased Dh of the retentate with each filtration step, which indicates the removal of smaller particles. The comparison of the Dh of the initial particle composition (BHS) with the Dh of the final structure (nanostructure) shows a growth of the BHS of up to ca. 7 nm. The DLS measurement of the filtrate shows the removal of particles with a Dh of about 5 - 11 nm (Figure 5b). With regard to the sizes of avidin and PPIDS-PB2 this can be attributed to both of them (Figure 5b). The UV/Vis measurements of the filtrate proved the removal of avidin with every filtration step indicated by the absorbance at λ = 280 nm (A280 nm) (Figure 5c). This is supported by a decreasing A280 nm in the retentate with every filtration step (Figure 5d). Similar findings were mainly found for BHS purifications of avidin with mono- and bivalent PPI-bGP (cf. also Figure SI-20). Thus, the purification process followed by UV/Vis and DLS measurements confirms the success of the particular purifications. Furthermore, the analysis of three independent HFF purifications of avidin/PPIDS-PB2 in 1/1 ligand-receptor stoichiometry showed Dh with low standard deviation of the initial compositions, final structures and the resuspended lyophilisate (Figure SI-21). Along with the purification of BHS composed of avidin and PPIbGP we also proved the successful purification of BHS composed of avidin and PEI-bGP (cf. Figure SI-22 and Figures 6d – f).


27

Biomacromolecules

size↑

20

retentate after 0 mL 20 mL 40 mL 60 mL 80mL

Vol%

15 10 5

b)

20 15

Vol%

a)

initial particle removed composition final particle particles structure

10 5

0

0 10

100

10 Dh [nm]

Dh [nm]

retentate after 0 mL 20 mL 40 mL 60 mL 80 mL

0.30 0.25 0.20 0.15 0.10

d)

100 filtrate after 20 mL 40 mL 60 mL

0.30 Absorbance

c)

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 47

0.25 0.20 0.15 0.10

0.05

0.05

250

300 wavelength [nm]

250

300 wavelength [nm]

Figure 5. HFF purification of avidin/PPIDS-PB2 1/1 with 300 kDa mPES membrane in 0.1 M TRIS/HCl/NaCl at pH 7.5: DLS volume plot shows a) increase of the Dh of the BHS with ongoing filtration steps, b) comparison of the Dh of the initial compositions (∅ 13 nm), removed particles (∅ 8 nm) and the resulting particle composition (∅ 20 nm), c) and d) corresponding UV/Vis spectra of the retentates and filtrates of the filtration steps.

The diameters determined by TEM match the Dh of the purified PPI- and PEI-nanostructures, respectively (purified avidin/PPIDS-PB2: Figures 6a vs. 6b; purified avidin/PEIDS-PB2: Figures 6d vs. 6e) and show rather narrow particle size distribution. The successful purification of avidin/PEIDS-PB2 in a 1/1 ligand-receptor stoichiometry with membranes of a MWCO of 300 kDa and 500 kDa was proven by DLS as well (Figure SI-22). After the purifications size increase of the BHS up to about 44 nm was found in the case of BHS of avidin/ PEIDS-PB2 in a 1/1 ligand-receptor stoichiometry. The corresponding TEM image show spherical particles with a diameter range of about 30 – 40 nm (Figures 2d and f). Applicability of HFF purification was


28

Page 29 of 47

also probed with other BHS in dependence on the MWCO of the HFF membranes. The used membranes, their MWCO and the Dh of the BHS before and after the purification as well as of the removed particles are summarized in Table 2.

purified avidin/PPIDS-PB2

a)

15

35

10

Vol%

40

counts

30

b)

c)

5 0

25

10

20

100 Dh [nm]

15 10 5

100 nm 10

20

30

40

50

60

500 nm

70

d [nm]

purified avidin/PEIDS-PB2

d)

15 Vol%

35 30

e)

f)

10

25 counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

5 0

20

10

100 Dh [nm]

15 10 5

100 nm 10

20

30

40

50

500 nm

60

d [nm]

Figure 6. a) Particle size analysis of purified avidin/PPIDS-PB2 in an initial ligand-receptor stoichiometry of 1/1, b) corresponding DLS volume plot, c) corresponding TEM images and d) particle size analysis of purified avidin/PEIDS-PB2 in an initial ligand-receptor stoichiometry of 1/1, e) corresponding DLS volume plot, f) corresponding TEM images.

Table 2. Overview of Dh, including polydispersity (PDI) and standard deviation (SD), of removed particles as well as BHS before and after HFF purification. [a]

MWCO

Before HFF

After HFF


Removed particles

29

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

mPES Dh SD PDI avidin molar with ratio membranes [nm] [nm]

PPIDSPB1

1/1

PPIDSPB1

½

PPIDSPB2

1/1

PPIDSPB2

½

PEIDSPB2

1/1

PPIDSPB4

[a]

SD

Dh [nm]

SD

PDI

Page 30 of 47

SD

Dh [nm]

SD

PDI

SD

100 kDa

8

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