In Situ Growth of Self-Assembled ProteinâPolymer Nanovesicles for

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Letter

In Situ Growth of Self-Assembled Protein-Polymer Nanovesicles for Enhanced Intracellular Protein Delivery Xinyu Liu, and Weiping Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14132 • Publication Date (Web): 05 Jan 2017 Downloaded from http://pubs.acs.org on January 6, 2017

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ACS Applied Materials & Interfaces

In Situ Growth of Self-Assembled Protein-Polymer Nanovesicles for Enhanced Intracellular Protein Delivery Xinyu Liu，and Weiping Gao*

Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, P. R. China. KEYWORDS: self-assembly, protein-polymer conjugates, nanovesicles, protein delivery,atom transfer radical polymerization

ABSTRACT:We report a new and general method, in situ growth, for designing self-assembled protein-polymer nanovesicles for intracellular protein delivery.In situ polymerization of a watersoluble monomer from a protein attached with a polymerization initiator yields amphiphilic protein conjugates of a water-insoluble polymer. These conjugates can in situ self-assemble into nanostructures with tunable morphologies from spheres to vesicles. Interestingly, an exogenous protein can be in situ encapsulated inside protein-polymer nanovesicles for enhanced intracellular protein delivery. The in situ growth method may open up new opportunities for designing a variety of self-assembled protein-polymer nanostructures tailored to specific applications.

ACS Paragon Plus Environment

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TEXT: Most proteins are incapable of spontaneously entering cells due to their membrane impermeability. As a result, protein therapeutics have proven effective for extracellular targets but not for intracellular ones. To explore the potential of proteins for intracellular targets, it is of significance to develop strategies for translocation of proteins inside cells.1 Liposomes have been used to wrap proteins for transferring them into the cytoplasm, but with low efficiency.2Cellpenetrating peptides3 or superpositively charged proteins4 have been fused or conjugated with proteins to facilitate endocytosis in cell culture, but they cannot improve the stability of the proteins against protease digestion and are hard to show success in vivo due to their non-specific cell targeting and poor pharmacokinetics and biodistribution. Recently, polymeric nanocapsules have been employed to encapsulate proteins for intracellular protein delivery,5 but the interfacial polymerization for protein encapsulation is out of control in the conjugation site, polymer length and conjugate stoichiometry, which may hamper its practical use. Hence it is highly desirable to develop novel strategies for intracellular protein delivery. A hallmark of biological systems is the self-assembly of proteins into highly ordered nanostructures that exhibit highly sophisticated functions. The unique functional opportunities provided by protein self-assembly have inspired the development of strategies to engineer selfassembled protein nanostructures. Recently, conjugating hydrophobic polymers to proteins to form amphiphilic protein-polymer conjugates has emerged as a promising approach to design self-assembled protein nanostructures.6-11 For instance, hydrophobic polymers like polystyrene are conjugated to hydrophilic proteins, by covalent bonding between amino acid residues of the proteins and the end groups of polymers,7 or non-covalent interactions of enzyme-cofactor recognition like hemin-horseradish peroxidase6,8 and bioaffinity coupling like biotinstreptavidin,9 to form amphiphilic protein-polymer conjugates. These giant amphiphiles can self-


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assemble into nanostructures like spheres, worms and vesicles in aqueous solutions. However, the hydrophobic nature of these polymers or monomers makes it inevitable to employ organic solvents7-9 or emulsification10,11 for the protein-polymer conjugation, which often leads to denaturation of the proteins and thus loss of their biological activity. Moreover, it is difficult to control protein self-assembly by the post-polymerization conjugation and emulsion polymerization methods. These problems limit further investigations and potential applications of these protein nanostructures. Hence novel strategies that can circumvent these limitations are of considerable interest. Herein we describe a new and general method, called in situ growth, to construct proteinpolymer nanostructures with tunable morphology for enhanced intracellular protein delivery. In this proof-of-concept study, we illustrate the in situ growth method in Scheme 1 using human serum albumin (HSA) as a model protein. The method consists of two steps: (i) site-specific HSA modification with an atom transfer radical polymerization (ATRP) initiator to form a macroinitiator (HSA-Br), followed by (ii) in situ ATRP of a water-soluble monomer, 2hydroxypropyl methacrylate (HPMA),12 from HSA-Br in aqueous solution without any organic solvent to yield HSA conjugates of a hydrophobic polymer, poly(2-hydroxypropyl methacrylate) (PHPMA).13,14 Due to the amphiphilic nature of the HSA-PHPMA conjugates in which HSA and PHPMA are the hydrophilic and hydrophobic blocks, respectively, they insitu self-assemble into HSA-PHPMA nanostructures with tunablemorphologies from spheres to worms and then to vesicles. Interestingly, green fluorescence protein (GFP) as a model protein can be in situ encapsulated inside HSA-PHPMA nanovesicles for enhanced intracellular GFP delivery. We chose HSA as a model protein in this proof-of-principle study since it is not only an important protein in our body15 but also widely used for treating hypoalbuminemia, surgery,


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burns, shock, cardiopulmonary bypass, trauma, hemodialysis, acute respiratory distress16 and for drug delivery.17 We utilized the accessible and single free cysteine group (Cys 34) in HSA for site-specific modification as it can allow us to synthesize site-specific and stoichiometric (1:1) HSA conjugates.18,19 It should be pointed out that ca. 45% Cys 34 in HSA remains intact and thus is available for site-specific modification.20 A maleimide-functionalized ATRP initiator of 2-(2(2-(2-(2,5-dioxo-2H-pyrrol-1(5H)yl)ethoxy)ethoxy)ethoxy) ethyl 2-bromo-2-methylpropanoate (DBMP) was attached to the Cys 34 residue of HSA, through the bioorthogonal Michael addition reaction between the maleimide group of DBMP and the thiol group of Cys 34, to produce a stoichiometric and site-specific HSA-initiator conjugate (HSA-Br). The site-specificity of the attachment was confirmed by electrospray ionization mass spectrometry (ESI-MS) (Figure 1a). The molecular weights of HSA and HSA-Br were determined to be 66765 and 67188 Da, respectively, which matched with their theoretical values of 66765 and 67186 Da. These data showed that the initiator was successfully attached to HSA to form the HSA-Br, as expected. Subsequently, in situ ATRP of HPMA in phosphate buffered saline (PBS) in an ice-water bath was performed to graft PHPMA from HSA-Br at a given protein concentration of 50 µM. A series of HSA-PHPMA conjugates with different molecular weights of the PHPMA blocks were prepared by adjusting the feeding molar ratio of HPMA to HSA-Br (HPMA/HSA-Br). The ATRP solutions were directly analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 1b). After in situ ATRP, new bands with higher molecular weights than that of HSA were observed, depending on HPMA/HSA-Br, indicating the successful in situ growth of PHPMA from HSA-Br. In a control experiment in which in situ ATRP was carried out by using HSA instead of HSA-Br as an initiator, we did not observe a new band with a higher molecular weight than that for HSA, indicating that in situ ATRP did not take place as HSA was


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not attached with the ATRP initiator. After purification, the band for unreacted HSA disappeared and only the bands for HSA-PHPMA conjugates were observed (Figure 1c), indicating the successful removal of unreacted HSA. Proton nuclear magnetic resonance (1H NMR) confirmed the in situ growth of PHMPA from HSA-Br (Figure S1). To determine the molecular weights of the PHPMA blocks, we treated the purified products with sodium hydroxide to remove the protein from the PHPMA blocks (Figure S2) and convert the water-insoluble PHPMA blocks to water-soluble hydrolyzed PHPMA blocks by alkaline hydrolysis,21 and thenmeasured the molecular weights of the hydrolyzed PHPMA blocks by gel permeation chromatography (GPC) (Figure 1d). During the reaction processes, the solutions turned from transparent to opaque (Figure S3), suggesting the in situ formation of HSA-PHPMA nanostructures. This was confirmed by dynamic and static light scattering (DLS and SLS) (Figure 2a and Figure S4) and transmission electron microscopy (TEM) (Figure 2b). Based on the GPC, DLS, SLS and TEM data, representative parameters of the HSA-PHPMA conjugates are summarized in Table 1. Interestingly, the morphology of HSA-PHPMA nanostructures could be tuned continuously by changing the molecular weight of the PHPMA block. A monomodal distribution with an intensity-average hydrodynamic radius (Rh) of 39.8 nm (Dispersity, Đ = 0.052) was detected for HSA-PHPMA-1, suggesting the formation of monodisperse spherical micelles. Indeed, spheres with an average diameter of 62 nm were observed by TEM. In contrast, a bimodal distribution with Rh values of 45.6 nm and 229.3 nm (Đ = 0.29) was recorded for HSA-PHPMA-2, implying a possible change in conjugate morphology due to the greater volume fraction of the hydrophobic block of PHMPA. TEM disclosed the coexistence of spheres with an average diameter of 63 nm and short worms with an average length of 182 nm. For HSA-PHPMA-3,


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worms were observed by TEM, with a broad monomodal distribution (Rh = 412.5 nm, Đ = 0.24). For HSA-PHPMA-4, there was a bimodal distribution with Rh values of 198 nm and 741.9 nm (Đ = 0.24), suggesting a possible morphology change due to the increased volume fraction of the PHMPA block. A mixture of worms and vesicles was revealed by TEM. When the molecular weight of the PHMPA block further increased, vesicles were observed for HSA-PHPMA-5, with a Rh of 212.4 nm (Đ = 0.16). The ratios of gyration radius/hydrodynamic radius (Rg/Rh) were determined by combining SLSwithDLS, which provided another supportive evidence to confirm the formation and evolvement of the assembly structures.Taken together, these results revealed that the morphology of HSA-PHPMA nanostructures could evolve from spheres to worms and then to vesicles when the core-forming PHPMA block grew from the shell-forming HSA block. We ascribe the phase transition behaviour to the gradual molecular curvature reduction of the conjugate chains as the PHPMA block grows from the HSA block. In this regard, the HSAPHPMA conjugates behave like small molecule surfactants22 and amphiphilic diblock copolymers23,24 for self-assembly is dominated by the molecular curvature,25,26 which is dependent on the relative volume fractions of the hydrophilic and hydrophobic blocks. The conjugates are more inclined to shape kinetically frozen morphologies such as “sphere & worm” and “worm & vesicle” mixed phases than surfactants. This is also observed for block copolymers, which is attributed to much slower exchange between the individual chains and the aggregates than that for surfactants.27 It is well-known that HSA has esterase-like activity towards many kinds of small-molecule esters, and among them, p-nitrophenyl acetate is most frequently used as a substrate in measurement.28 Hence we further investigated the esterase-like activity of HSA-Br and the representative HSA-PHPMA nanostructures of spheres, worms and vesicles by monitoring the


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hydrolysis process of p-nitrophenyl acetate with a UV-visible spectrophotometer at 400 nm (Figure 3a). HSA-Br showed 98% of esterase-like activity of HSA, suggesting that the attachment of the ATRP initiator to HSA did not reduce its activity, as expected. HSA-PHPMA nanostructures of spheres, worms and vesicles showed 102%, 106% and 96% of esterase-like activity of HSA, respectively, indicating that in situ growth of these HSA-PHPMA nanostructures did not influence the activity of HSA. This was confirmed by circular dichroism (CD) (Figure 3b). The CD scans of them displayed a doublet at 209/222 nm similar to that of HSA, suggesting that the site-specific in situATRP did not impair the secondary structure of HSA.Taken together, the CD and activity assays showed that in situ growth of PHPMA from HSA to yield HSA-PHPMA nanostructures did not change the structure and activity of HSA, due to the excellent biocompatibility of in situ ATRP of HPMA. Motivated by the encouraging results of tunable morphology and high activity retention of HSA-PHPMA nanostructures, we attempted to know whether exogenous proteins could be in situ encapsulated in HSA-PHPMA vesicles(HPMA/HSA-Br= 20000) for intracellular delivery. We chose GFP as a model protein and conducted the in situ growth of HSA-PHPMA vesicles (HPMA/HSA-Br= 20000)in the presence of GFP. Their size and zeta potential were characterizedTable S1. TEM showed that the presence of GFP did not affect the formation of HSA-PHPMA vesicles (Figure 2b and 4a).Confocal fluorescence microscopy (CFM) showed the successful in situ encapsulation of GFP in the vesicles, as indicated by the overlap between green fluorescence of the GFP and red fluorescence of the Rhodamine-labelled vesicles (Figure 4b). In a control experiment, mixing the empty vesicles with GFP could hardly load GFP into the vesicles (Figure S6). The in situ encapsulation efficiency and capacity of GFP in the vesicles were determined to be 11.7% and 4.67%, respectively (Figure S7).These results show that the in


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situ growth method is capable of encapsulating exogenous proteins inside HSA-PHPMA vesicles. Next, we examined the intracellular delivery of GFP-encapsulated vesicles by confocal laser-scanning microscopy (CLSM) (Figure 4c). GFP itself could not enter MCF-7 cells at all, as indicated by the absence of green fluorescence in the cells. HSA itself could be translocated into MCF-7 cells by receptor-mediated endocytosis,29 as showed by the presence of green fluorescence of FITC labelled HSA in the cells. Interestingly, the FITC labelled vesicles showed stronger green fluorescence than the FITC labelled HSA in the cells. As a result, GFPcould be deliveredinto MCF-7 cells by the vesicles, as demonstrated by the green fluorescence in the cells. These results were confirmed by flow cytometry (Figure 4d). Notably, the GFP-loaded vesicles showed a 73-fold increase in cellular fluorescence intensity relative to the free GFP (Figure 4e). We further found that the GFP-loaded vesicles were internalized by cells through a macropinocytosis pathway (Figure S8). The HSA-PHPMA vesicles showed no cytotoxicity toward both tumor cells (MCF-7) and normal cells (MCF-10A) (Figure S9).These results showed that HSA-PHPMA vesicles could be used as biocompatible carriers for intracellular protein delivery. The release of GFP from the vesicles and the escape of the release GFP from lysosomes might be triggered by the lysosomal degradation of the vesicles (Figure S10), but need to be further elucidated in future studies. In conclusion, we have developed a new and general methodology of in situ growth to build selfassembled protein-polymer nanostructures for intracellular protein delivery. The in situ growth method leads to three important findings that have not been achieved by current methods as follows: (i) the morphology of protein-polymer nanostructures can be continuously tuned from spheres to worms and then to vesicles by increasing the molecular weight of the water-insoluble


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polymer block due to the gradual molecular curvature reduction of the conjugate chains as the core-forming polymer block grows from the shell-forming protein block; (ii) the protein structure and activity of self-assembled protein-polymer nanostructures are highly retained as compared to those of the native protein due to the excellent biocompatibility of our in situgrowth method; and (iii) exogenous proteins can be in situ encapsulated inside protein-polymer vesicles for enhanced intracellular protein delivery. These attributes make the in situ growth methodology interesting as a new and general platform for the development of self-assembled protein nanostructures with tunable morphology for enhanced intracellular protein delivery. These findings may open up new possibilities in molecular imaging, drug delivery and other applications that can take advantage of the controlled self-assembly of proteins into well-defined nanostructures enabled by the in situ growth method.


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Figure 1. The in situ growth of HSA-PHPMA conjugates. (a) ESI-MS of HSA and HSA-Br. (b) SDS-PAGE of five representative HSA-PHPMA conjugates before purification, lanes 1: HSAPHPMA-5 (HPMA/HSA-Br= 20000), lane 2: HSA-PHPMA-4(HPMA/HSA-Br= 10000), lane 3: HSA-PHPMA-3 (HPMA/HSA-Br= 8000), lane 4: HSA-PHPMA-2 (HPMA/HSA-Br= 5000), lane 5: HSA-PHPMA-1(HPMA/HSA-Br= 2000), lane 6: HSA for ATRP as a control experiment, lane 7: HSA-Br, lane 8: HSA, lane 9: protein marker. Region I: stacking gel (the stacking gel allows the proteins in the loaded sample to be concentrated into a condensed band when they enter the separation gel); region II: separation gel (the separation gel can separate proteins according to their different molecular weights). (c) SDS-PAGE of five representative HSAPHPMA conjugates after purification, lanes 1: purified HSA-PHPMA-5 (HPMA/HSA-Br= 20000), lane 2: purified HSA-PHPMA-4(HPMA/HSA-Br= 10000), lane 3: purified HSAPHPMA-3 (HPMA/HSA-Br= 8000), lane 4: purified HSA-PHPMA-2 (HPMA/HSA-Br=


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5000),lane 5: purified HSA-PHPMA-1(HPMA/HSA-Br= 2000), lane 6: HSA-Br, lane 7: HSA, lane 8: protein marker. (d) GPC traces of the hydrolyzed PHPMA chains for five representative HSA-PHPMA conjugates with different PHPMA molecular weights (Mn). Black line: HPMA/HSA-Br= 2000; green line: HPMA/HSA-Br= 5000; red line: HPMA/HSA-Br= 8000; brown line: HPMA/HSA-Br= 10000; blue line: HPMA/HSA-Br= 20000.

Figure 2. DLS and TEM analyses of HSA-PHPMA nanostructures. (a) DLS analysis of five representative HSA-PHPMA nanostructures, where Rh is hydrodynamic radius. (b) TEM images of five representative HSA-PHPMA nanostructures.


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Figure 3. Characterization of the secondary structure and activity of HSA in HSA-PHPMA nanostructures. (a) CD traces of HSA, HSA-Br and HSA-PHPMA nanostructures of spheres, worms and vesicles. (b) Normalized esterase-like activity of HSA, HSA-Br and the HSAPHPMA nanostructures of spheres, worms and vesicles.

Figure 4. The in situ encapsulation of GFP inside HSA-PHPMA vesicles for the intracellular delivery of GFP. (a) TEM images of GFP-encapsulated HSA-PHPMA vesicles. (b) CFM images of GFP-encapsulated Rhodamine-labelled-HSA-PHPMA vesicles under different excitation wavelengths of GFP and Rhodamine.(c) The intracellular delivery of GFP into MCF-7 cells by HSA-PHPMA vesicles. MCF-7 cells were incubated with GFP, FITC-labelled HSA or vesicles,


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or GFP-encapsulated vesicles for 24 h. The cell membranes were stained in red and the cell nuclei were stained in blue. GFP and FITC are shown in green.(d)Fluorescence-activated cell sorting (FACS) analysis of cell uptakeof free GFP and GFP-loaded vesicles. (e) Quantitative comparisonsbased on the FACS analysis. Scheme 1.Schematic illustration of (a) in situ growth of HSA-PHPMA nanostructures with tunable morphologies and (b) in situ encapsulation of GFP inside HSA-PHPMA nanovesicles(HPMA/HSA-Br= 20000) for the enhanced intracellular delivery of GFP.

Table 1. The representative parameters of HSA-PHPMA nanostructures with different PHPMA molecular weights. Sample HSA-PHPMA-1

a

Ratioa Mnb (kDa)b Mwc (kDa) 2000

47.9

65.2

Dispersityd (Đ)

Morphologye

Rhf(nm)

Diameterg (nm)

1.36

S

39.8

62

Rgh (nm) Rg/Rhi 29.2

0.73j

HSA-PHPMA-2

5000

64.1

86.5

1.35

S&W

45.6, 229.3

63

n.a.

n.a.

HSA-PHPMA-3

8000

85.1

115

1.35

W

412.5

65

n.a.

n.a.

HSA-PHPMA-4 10000

97.3

129

1.33

W&V

198, 741.9

66, 250

n.a.

n.a.

HSA-PHPMA-5 20000

130

173

1.32

V

212.4

360

231.3

1.09k

Feeding molar ratio of HPMA to HSA-Br (HPMA/HSA-Br).b Number-average molecular

weight.c Weight-average molecular weight.dDispersity (Đ). Mn, Mw and Đ were determined by GPC calibrated with poly(ethylene glycol) (PEG) standards.e Morphologies of HSA-PHPMA


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nanostructuresidentified using TEM: S = spheres, W = worms, and V = vesicles. Coexisting phases are indicated using two letters.fHydrodynamic radius (Rh) of HSA-PHPMA nanostructures determined by DLS.

g

Diameter determined by TEM, and ‘n.a.’ means that the

diameters cannot be measured since their nanostructures were not regular.hRg values of different samples given by fitting data of static light scattering, ‘n.a.’ means these Rg data cannot be calculated by Zimm model as the sample was a mixture.i The ratio of Rg and Rh.jThe value 0.73 is very close to the theoretical one (0.77) of sphere. k The value 1.09 is close tothe theoretical one (1.0) of vesicle.

ASSOCIATED CONTENT Supporting Information. Materials, full experimental details, including synthesis, purification and 1H-NMR, alkaline hydrolysis, photograph and activity characterization of the HSA-PHPMA, and fluorescence emission spectrum, endocytosis mechanism and cytotoxicity of GFP-loaded vesicles. (Word)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


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Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This study was financially supported by grants from the National Natural Science Foundation of China (Grant No. 21274043, 21534006). REFERENCES (1) Gu, Z.; Biswas, A.; Zhao, M.; Tang, Y. Tailoring Nanocarriers for Intracellular Protein Delivery.Chem. Soc. Rev.2011,40, 3638-3655. (2) Torchilin, V. P. Recent Advances with Liposomes as Pharmaceutical Carriers.Nat. Rev. Drug discovery 2005,4, 145-160. (3) Wadia, J. S.; Stan, R. V.; Dowdy, S. F. Transducible TAT-HA Fusogenic Peptide Enhances Escape of TAT-Fusion Proteins after Lipid Raft Macropinocytosis.Nat. Med.2004,10, 310315. (4) Cronican, J. J.; Thompson, D. B.; Beier, K. T.; McNaughton, B. R.; Cepko, C. L.; Liu, D. R. Potent Delivery of Functional Proteins into Mammalian Cells in vitro and in vivo Using a Supercharged Protein. ACS chem. biol.2010,5, 747-752. (5) Yan, M.; Du, J.; Gu, Z.; Liang, M.; Hu, Y.; Zhang, W.; Priceman, S.; Wu, L.; Zhou, Z. H.; Liu, Z.; Segura, T.; Tang, Y.; Lu, Y. A Novel Intracellular Protein Delivery Platform Based on Single-Protein Nanocapsules. Nat.Nanotechnol.2010,5, 48-53. (6) Reynhout, I. C.; Cornelissen, J. J.; Nolte, R. J. Self-Assembled Architectures from BiohybridTriblock Copolymers. J. Am. Chem. Soc.2007,129, 2327-2332. (7) Velonia, K.; Rowan, A. E.; Nolte, R. J. Lipase Polystyrene Giant Amphiphiles. J. Am. Chem. Soc.2002,124, 4224-4225.


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