Sequential Self-Assembly Using Tannic Acid and Phenylboronic Acid-Modified Copolymers for Potential Protein Delivery

Abstract

Tannic acid (TA) can form stable complexes with proteins, attracting significant attention as protein delivery systems. However, its systemic application has been limited due to nonspecific interaction. Here, we report a simple technique to prepare systemically applicable protein delivery systems using sequential self-assembly of a protein, TA, and phenylboronic acid-conjugated PEG-poly(amino acid) block copolymers in aqueous solution. Mixing the protein and TA in aqueous solution led to covering of the protein with TA, and subsequent addition of the copolymer resulted in the formation of boronate esters between TA and copolymers, constructing the core−shell-type ternary complex. The ternary complex covered with PEG exhibited a small hydrodynamic diameter of ∼10−20 nm and prevented an unfavorable interaction with serum components, thereby accomplishing significantly prolonged blood circulation and enhanced tumor accumulation in a subcutaneous tumor model. The technique utilizing supramolecular self-assembly may serve as a novel approach for designing protein delivery systems.

Sequentially Self-Assembled Nanoreactor Comprising Tannic Acid and Phenylboronic Acid-Conjugated Polymers Inducing Tumor-Selective Enzymatic Activity

“…17 In addition, our previous study revealed that a 50% introduction of FPBA maintained water solubility of the polymer and permitted the formation of boronate esters multivalently with the galloyl groups of TA, resulting in the formation of the β-Gal/TA/PEG-P[Lys/Lys(FPBA)] ternary complexes. 13 In the present study, PEG-P[Lys/Lys(FPBA)] was added to the β-Gal/TA complex in aqueous solution, and the complex formation was confirmed by electrophoresis (Figure 2a). Compared with β-Gal alone, β-Gal/TA exhibited a longer migration distance toward the anode due to the anionic property of TA, while the β-Gal ternary complex migrated to the cathode, indicating the interaction of the cationic PEG-P[Lys/Lys(FPBA)] with β-Gal/TA.…”

“…The cellular uptake of β-Gal/TA was ∼80% lower than that of free β-Gal, because serum components in the medium may interact with β-Gal/TA, compromising interaction with the cell membrane as previously reported. 13 The ternary complex also exhibited a cellular uptake lower than that of free β-Gal, but it was higher than that of β-Gal/TA. The PEG shell of the ternary complex might inhibit the aforementioned nonspecific interaction of TA, which might lead to the moderate cellular uptake.…”

“…Moreover, under physiological conditions, biological substances such as albumin can interact with TA and TA complexes. 13 Indeed, the hydrodynamic diameter of β-Gal/TA was gradually increased as the concentration of FBS increased due to nonspecific interaction (Figure S6). In contrast, the size of the ternary complex was maintained even in the presence of 50% FBS because the PEG shell covered the ternary complex, inhibiting interaction with biological components.…”

“…Given that the complex formation of β-Gal with TA diminished its enzymatic activity (Table 1), β-Gal/TA might be dissociated in the cell probably due to the diluted condition and endo/lysosomal pH, as we previously reported that GFP/ TA complex formation depended on the concentration and that the high concentration led to complex/aggregate formation even at low molar ratios of TA to GFP. 13 In addition, to demonstrate the pH-responsive release behavior of β-Gal/TA, the recovery of Alexa647-β-Gal fluorescence intensity ([β-Gal/TA]/[β-Gal] fluorescence intensity ratio) was measured at various pH values, in which the increase in fluorescence intensity reflects the release of β-Gal. 21 The relative fluorescence intensity recovered from 45% at pH 7.4 to 100% at pH 5.5, suggesting that TA had dissociated from the complex due to dissociation of the hydrogen bond between TA and the protein in response to the acidic pH.…”

“…In this regard, we recently reported a sequential selfassembled ternary complex constructed from a green fluorescent protein (GFP), tannic acid (TA, Figure 1a), and phenylboronic acid-conjugated polymers (Figure 1b). 13 TA is structured by the conjugation of 10 galloyl groups to the central glucose via ester bonds and has been widely applied as a biomaterial due its biocompatible, biodegradable, and assembly properties. 14 Its aromatic moieties and hydroxy groups can interact with GFP by hydrophobic interactions and hydrogen bonds to form a GFP/TA complex.…”