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Nanoscale Proteinosomes Fabricated by Self-Assembly of a Supra-molecular Protein-Polymer Conjugate Guangda Han, Jin-Tao Wang, Xiaotian Ji, Li Liu, and Hanying Zhao Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00704 • Publication Date (Web): 30 Dec 2016 Downloaded from http://pubs.acs.org on January 4, 2017

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Bioconjugate Chemistry

Nanoscale Proteinosomes Fabricated by Self-Assembly of a Supramolecular Protein-Polymer Conjugate Guangda Han, Jin-Tao Wang, Xiaotian Ji, Li Liu*, and Hanying Zhao* Key Laboratory of Functional Polymer Materials, Ministry of Education, College of Chemistry, Nankai University; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, China. ABSTRACT: Proteinosomes are a type of protein-based spherical capsules, which have potential applications in drug delivery, cell imaging, gene expression and biocatalysis. In this research, a novel approach to the fabrication of proteinosomes entirely composed of protein molecules based on self-assembly of a supramolecular protein-polymer conjugate, is proposed. A supramolecular protein-polymer conjugate was prepared by mixing βCD-modified bovine serum albumin (BSA) and adamantane-terminated poly(Nisopropylamide) (Ad-PNIPAM) in aqueous solution. The BSA-PNIPAM bioconjugate self-assembled into micelles with PNIPAM cores and BSA coronae at a temperature above lower critical solution temperature (LCST) of PNIPAM. After cross-linking of BSA in the coronae, and followed by addition of excess βCD, PNIPAM chains were cleaved from the micellar structures, and nanoscale proteinosomes were prepared. The dual-responsive proteinosomes dissociated in the presence of trypsin or glutathione.

Inspired by nature, the fabrication of synthetic ensembles to mimic biological systems has attracted considerable interest in the chemistry and materials science research communities.1 Among these studies, one of the most active research areas is the study of capsules, i.e. nano- or microscale hollow structures composed of solid shells that surround core-forming voids.2,3 To date, a variety of capsules, including liposomes,4,5 polymersomes,6-10 and inorganic colloidosomes,11,12 have been synthesized, and these hollow structures have found applications in drug delivery,13,14 cell imaging,15 multicompartmentalization,16,17 gene expression and enzyme catalysis.18 In order to improve the biocompatibility of the capsules and construct artificial chemical cells, proteinosomes, a type of capsules composed of protein walls and hydrophilic voids, have been synthesized in these years.19-24 In general, proteinosomes can be synthesized by self-assembly of amphiphilic proteins in aqueous solutions or by interfacial self-assembly of protein-polymer conjugates in emulsions. For example, Hammer and coworkers demonstrated that engineered recombinant surfactant proteins made self-assembly into proteinosomes at both the nano- and micro- scales, and the membrane thickness was controlled by the ratio of the length of the hydrophilic arms to the length of the central hydrophobic block of the protein.19 Thordarson and coworkers fabricated proteinosomes by self-assembly of a protein-polymer bioconjugate composed of poly(N-isopropylamide) (PNIPAM) and a green-fluorescent protein variant.20 Mann, Huang and coworkers fabricated proteinosomes based on the interfacial assembly of protein − polymer conjugates at oil-water interface.21-23 Very recently, Landfester, Wurm and coworkers prepared proteinosomes entirely composed of albumin proteins by using an inverse miniemulsion protocol and interfacial cross-linking reaction.24 In this paper, a novel approach to the synthesis of nanoscale proteinosomes based on self-assembly of a supramolecular protein-polymer conjugate, is reported. Over the past decade, synthesis of covalently-connected protein-polymer conjugates

by “grafting to” and “grafting-from” method have been extensively studied.25-28 In the bioconjugates, the polymer chains are covalently bonded to the protein molecules. Supramolecular chemistry provides an efficient and versatile method to prepare protein-polymer conjugates.29-32 Supramolecular proteinpolymer conjugates are formed by bridging polymer chains and protein molecules via highly directional and reversible noncovalent interactions such as π-π interaction, metal-ligand interaction, hydrogen bonding, hydrophobic interaction and so on. Compared to the covalently-connected protein-polymer conjugates, one of the significant advantages of the supramolecular bioconjugates is their reversible feature, i.e. the noncovalent bonds can be easily cleaved under the external environmental stimuli, such as pH, temperature, metal ions, organic solvents and excess host or guest molecules.33,34 Herein, nanoscale proteinosomes are fabricated based on selfassembly of a supramolecular protein-polymer conjugate. Bovine serum albumin (BSA) is used as a model protein in this research, but other proteins are also available for the fabrication of proteinosomes by using this approach. As shown in Scheme 1, β-cyclodextrin (βCD) modified BSA (BSA-βCD) was obtained after thiol-disulfide exchange reaction between thiols on BSA and pyridyldisulfide βcyclodextrin (βCD-s-s-Py). Adamantane-terminated PNIPAM (Ad-PNIPAM) was synthesized by reversible additionfragmentation chain transfer (RAFT) polymerization. Due to the strong inclusion interaction between βCD and Ad, a supramolecular BSA-PNIPAM conjugate was formed upon mixing of BSA-βCD and Ad-PNIPAM in aqueous solution. The protein-polymer conjugate self-assembled into micelles with PNIPAM in the cores and BSA in the coronae at a temperature above lower critical solution temperature (LCST) of PNIPAM. The hybrid micelles were used as templates for the fabrication of nanoscale proteinosomes.35 After cross-linking of BSA coronae, excess βCD was added to the micellar solution. The native βCD molecules competed with βCD groups in the hostguest inclusion complexes, resulting in the cleavage of the

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bridges between PNIPAM and BSA. Upon removal of the cleaved PNIPAM, nanoscale proteinosomes were prepared. Comparing with previous researches, this method has following features: (1) the fabrication of the proteinosomes is conducted in aqueous solution and no organic solvent is necessary, (2) the proteinosomes are entirely composed of BSA protein, which is biocompatible and biodegradable,36,37 (3) the sizes of the proteinosomes are in the nanoscale range.

Scheme 1. Schematic depiction for the fabrication of proteinosomes based on self-assembly of a supramolecular bioconjugate. Adamantane-containing RAFT agent was synthesized by reaction of S-1-dodecyl-S’-(α,α’-dimethyl-α’’-acetic acid) trithiocarbonate and amantadine. 1H NMR spectrum and ESI-mass spectrum of the RAFT agent are shown in Figure S1. AdPNIPAM was synthesized by RAFT polymerization of NIPAM. Gel permeation chromatography (GPC) traces of AdPNIPAM obtained by using right angle laser light scattering (RALS), low angle laser light scattering (LALS) and refractive index (RI) detector are shown in Figure S2a, which show symmetric and monomodal elution peaks. The apparent number-average molecular weight (Mn) and the dispersity of the polymer are 20.6 kDa and 1.18, respectively. The absolute molecular weight (Mw) and dispersity of the polymer, determined by GPC equipped with light scattering detector, is 28.5 kDa and 1.13. The 1H NMR spectrum and signal assignments for the polymer are presented in Figure S2b. Based on 1H NMR result, the average repeating unit number of AdPNIPAM is 192. A BSA molecule contains only one free cysteine (Cys-34) which is usually partially oxidized resulting in about 52% cysteine available for the further conjugation.37 In order to prepare BSA with multiple thiols on the surface, native BSA was treated with Traut’s reagent (2-iminothiolane), a cyclic thioimidate compound for sulfhydryl addition. Traut’s reagent

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reacted spontaneously and efficiently with primary amines on BSA at pH 7–9, producing thiol groups.38 BSA-βCD was obtained after an exchange reaction between thiols on BSA and βCD-s-s-Py. The analysis of βCD-s-s-Py can be found in our previous paper.34 In the thiol-disulfide exchange reaction, the amount of βCD grafted to the BSA molecules is equal to the amount of 2-mercaptopyridine produced in the exchange reaction. Based on UV absorption and a standard curve of 2mercaptopyridine made at 343 nm (Figure S3), the amount of mercaptopyridine as well as the average number of βCD grafted to a BSA molecule can be calculated. Our calculation result indicated that on average there were about 2.3 βCD groups on a BSA-βCD molecule. BSA-βCD was characterized with matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, which revealed m/z peaks at 67903, 69082 and 70245 for BSA with one, two and three βCD groups respectively (Figure S4). It is noted that the m/z peak at 66734 corresponding to the Traut’s reagent-modified BSA can also be observed. The protein molecules without βCD groups were removed after the formation of selfassembled structures. The intensity of the peak in the MALDITOF mass analysis cannot be used for quantitative analysis purpose because the ionization of bioconjugate is more difficult than native protein.20 Cross-peaks in 2D 1H NOESY NMR spectrum identify protons of host and guest molecules undergoing “through space” dipolar interactions and the formation of inclusion complexes.39 2D 1H NOESY NMR spectrum of BSA-βCD/AdPNIPAM mixture prepared at an equal molar ratio of βCD to Ad confirms the interaction between Ad and βCD. As shown in Figure 1a, the observed cross-peaks indicate strong interaction between the H3 protons of βCD and the Ha, Hb and Hc protons of Ad in an inclusion complex, demonstrating a deep insertion of Ad end groups into the βCD rings on BSA. The host-guest interaction between BSA-βCD and Ad-PNIPAM was also demonstrated by isothermal titration calorimetry (ITC). Raw and integrated data of ITC measurements for titration of BSA-βCD with Ad-PNIPAM solution are shown in Figure 1b. The binding constant (K) between BSA-βCD and Ad-PNIPAM was measured to be around 2.17±0.10×105 M-1 by fitting the ITC results with One Sites Model.40 As estimated by ITC, the formation of inclusion complexes was also demonstrated by a large negative entropy and enthalpy change (∆S=-70 cal mol-1 K-1, ∆H=-2.767×104 cal mol-1). The complexation showed an experimental n value of 0.902±0.006, which was close to the theory binding ratio of 1:1.


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Figure 1. (a) 2D 1H NOESY NMR spectrum of a 1.7 wt% mixture of Ad-PNIPAM and BSA-βCD at a βCD/Ad molar ratio of 1:1, (b) raw and integrated data of isothermal titration calorimetry (ITC) measurement for titration of BSA-βCD with Ad-PNIPAM solution.

PNIPAM is a temperature-responsive polymer, and exhibits a transition from dehydrated state to hydrated state at LCST in water. LCSTs of Ad-PNIPAM and the bioconjugate composed of BSA-βCD and Ad-PNIPAM (BSA-βCD-Ad-PNIPAM) in neutral water, were measured and compared. In order to determine the LCSTs, the changes of the transmittances of the two aqueous solutions at 600 nm with temperature were recorded (Figure 2a). The transmittances decreased rapidly with temperature, and LCSTs of Ad-PNIPAM and BSA-βCD-AdPNIPAM were determined at 29.3 and 30.7 °C, respectively. The binding of BSA molecules to the ends of PNIPAM chains through host-guest interaction leads to an increase in the LCST of the polymer. The introduction of the hydrophilic BSA to the polymer chains is responsible for the shift of the LCST of PNIPAM to a higher temperature.41,42 It is worthwhile to note that in turbidity measurements BSA-βCD-AdPNIPAM exhibits a broader transition than that of AdPNIPAM, due to the different number of PNIPAM chains grafted to the BSA molecules through host-guest interaction. In previous researches, self-assemblies of “doublehydrophilic” block and graft copolymers with PNIPAM chains in aqueous solutions were investigated.43,44 Generally, these polymers self-assemble into micelles with dehydrated PNIPAM in the cores and the other hydrophilic blocks in the coronae at temperatures above LCST of PNIPAM. It is expected that BSA-βCD-Ad-PNIPAM bioconjugate composed of hydrophilic BSA and the thermal sensitive PNIPAM, is also able to form self-assembled structures in aqueous solution at a temperature above LCST. Figure 2b shows a transmission electron microscopy (TEM) image of micelles formed by BSA-βCD-Ad-PNIPAM bioconjugate in water at 40 °C. The inset in the figure is a magnified TEM image of the micelles. At a temperature above LCST of PNIPAM, the bioconjugate self-assembles into spherical micelles with sizes in the range of 23-47 nm. The PNIPAM chains dehydrate and collapse into the cores of the micelles and the compact hydrophobic interiors are stabilized by protein molecules. In the inset, dark dots on the aggregates representing BSA molecules can be observed, demonstrating the formation of hybrid core–corona structures. Figure 2c shows dynamic light scattering (DLS) curves of BSA-βCD-Ad-PNIPAM bioconjugate measured in water at 20 and 40 °C. The Z-average sizes (Dh,z) of the bio-

conjugate at 20 and 40 °C are 5 and 117 nm, respectively, which indicates that the bioconjugate is molecularly dissolved in water at 20 °C, and self-assemble into micelles at 40 °C. The average size measured by TEM reflects conformations of the hybrid micelles in the dry state, whereas, Dh,z determined by DLS represents the average hydrodynamic diameter of the micelles in water, so the sizes measured by TEM are usually smaller than those measured by DLS. The BSA molecules in the coronae of the micelles were cross-linked with bis(2,5-dioxopyrrolidin-1-yl) 3,3'disulfanediyldipropanoate, a disulfide-containing compound (Scheme 1). The corona crosslinking was confirmed by DLS. Upon cooling from 40 to 20 °C, the average hydrodynamic diameter of the micelles increased from 136 to 164 nm (curve 3 in Figure 2c). PNIPAM chains hydrate at a temperature below LCST, and the extended polymer chains make the crosslinked particles bigger.



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Figure 2. (a) Temperature dependence of the transmittances for aqueous solutions of adamantane-terminated PNIPAM (AdPNIPAM), and bioconjugate of Ad-PNIPAM and BSA-βCD, (b) transmission electron microscopy (TEM) image of micelles formed by BSA-βCD-Ad-PNIPAM bioconjugate in water at 40 °C, (c) dynamic light scattering (DLS) curves of BSA-βCD-AdPNIPAM bioconjugate measured in water at 20 (1) and 40 °C (2), cross-linked hybrid micelles (3), and proteinosome (4) measured at 20 °C.

Native β-CD molecules added to the micellar solutions, compete with the β-CD substituent in host-guest inclusion complexes, resulting in dissociation of the supramolecular complexes and cleavage of the linkages between PNIPAM and BSA. After ultrafiltration, the cleaved PNIPAM chains were removed and proteinosomes entirely composed of BSA molecules were fabricated. 1H NMR spectra of the coronacrosslinked micelles and the proteinosomes using trace of DMF as an internal standard in deuterated water at 25 °C are shown in Figure 3a. In the spectrum of the micelles, the peaks corresponding to the protons on PNIPAM can be observed; however, after addition of β-CD and followed by ultrafiltration, all the signals representing PNIPAM disappear, which demonstrates the cleavage and removal of PNIPAM chains from the micellar structures. In the meanwhile, hydrophilic voids are produced on the structures after removal of PNIPAM. Figure 3b shows TEM images of the proteinosomes. The inset in the figure shows magnified TEM image of typical particles. The TEM specimen was stained with OsO4 and BSA was stained. In the TEM images, spherical hollow structures with sizes in the range from 20 to 40 nm can be observed. Based on TEM measurement, the thickness of the proteinosome membrane was determined to be about 3.5 nm (inset in Figure 3b). Our TEM result indicated that the average size of BSA molecules was around 3 nm (Figure S5). The size similarity between BSA and the membrane thickness suggested that the membrane was composed of single BSA layer. The hollow architecture was also characterized by atomic force microscopy (AFM). The AFM sample was prepared by casting aqueous solution of proteinosomes onto mica and drying in air at room temperature. Figure S6 shows a taping-mode AFM image and height profile of the proteinosomes. A height profile measured along the line marked in the AFM image indicates that the height of a typical proteinosome is about 7 nm, much smaller than the average lateral dimension (65 nm). After the evaporation of water in the voids, the proteinosomes form collapsed structures, and the height of the structures is twice as high as the membrane thickness as measured by TEM.

Figure 3. (a) 1H NMR spectra of coronae-crosslinked micelles before (spectrum I) and after (spectrum II) removal of PNIPAM chains, (b) TEM images of the proteinosomes composed of crosslinked BSA at two different magnifications.

The proteinosomes composed of BSA molecules and disulfide-containing crosslinker exhibit dual stimuli responsiveness. BSA molecules are degraded in the presence of protease, and disulfide bonds in the crosslinker are reduced to thiols by a reducing agent. Herein, the dissociation of the proteinosomes with trypsin or glutathione (GSH) was investigated. Part a and b of Figure 4 show TEM images of proteinosomes after treatment with trypsin or reduced GSH, respectively, where no spherical proteinosome particles can be observed, indicating that the proteinosomes dissociate completely in the presence of trypsin or reduced GSH. Trypsin cleaves peptide chains of BSA mainly at the carboxyl side of the amino acids lysine or arginine, resulting in the dissociation of the proteinosomes; GSH cleaves the disulfide bonds of the cross-linker, which leads to the degradation of the crosslinked protein membrane and the dissociation of the proteinosomes. Figure 4c shows DLS curves of proteinosomes after treatment with reduced GSH or trypsin. Because of the dissociation of the proteinosomes and aggregation of BSA molecules, big aggregates with broad size distributions are observed. The application of the proteinosomes for glucose sensing was investigated in this research. Methylene blue (MB) is a frequently used dye. In neutral water, the positively charged MB molecules bind to the negatively charged BSA by electrostatic interaction.45 In the meanwhile, MB can be quenched by


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glucose in alkaline medium, as a result of reduction reaction.46 In this research, MB was added to the proteinosome solution, and after 4 h stirring the excess dye molecules were removed by dialysis. Figure S7 shows the chemical reaction equation and the fluorescence emission spectra of MB recorded at different time. Upon addition of glucose at pH=9.0, the intensity of the fluorescence emissions at 678 nm deceased with time significantly, which demonstrated that the dye molecules adsorbed by the proteinosomes were reduced to colorless reduced forms of MB (MBH) by glucose. It is expected that the proteinosomes prepared in this research will find bioanalysis and biomedical applications.

supramolecular complexes between βCD and Ad. After removal of cleaved PNIPAM from the solution, proteinosomes entirely composed of BSA molecules were prepared. The proteinosomes have potential applications in the bioanalytical, pharmaceutical and biomedical sciences. More researches on the applications of the proteinosomes are being investigated in this laboratory.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org at DOI: Experimental details, 1H NMR spectrum and ESI-mass spectrum of the RAFT agent, GPC curves and 1H NMR spectrum of AdPNIPAM, MALDI-TOF result of βCD modified BSA, TEM, AFM and DLS results of the self-assembled structures.

AUTHOR INFORMATION Corresponding Authors * E-mails: [email protected], [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT ACKNOWLEDGMENT This project was supported by the National Natural Science Foundation of China (NSFC, 51473079 and 21374047), and the National Basic Research Program of China (973 Program, 2012CB821500).

REFERENCES

Figure 4. TEM images (a, b) and DLS curves (c) of proteinosomes after treatment with trypsin or reduced GSH.

In summary, supramolecular protein-polymer conjugate was synthesized based on strong inclusion interaction between βCD modified BSA and Ad-PNIPAM. The bioconjugate selfassembled into micelles with PNIPAM in the cores and BSA in the coronae at a temperature above LCST of PNIPAM. After cross-linking of BSA, hybrid micelles with cross-linked BSA coronae were fabricated. The addition of excess native βCD to the miecllar solution resulted in the dissociation of

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Nanoscale Proteinosomes Fabricated by Self-Assembly of a Supramolecular Protein-Polymer Conjugate Guangda Han, Jin-Tao Wang, Xiaotian Ji, Li Liu, Hanying Zhao


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