Site-Selective in Situ Growth-Induced Self-Assembly of Protein

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Site-Selective in situ Growth-Induced Self-Assembly of Protein-Polymer Conjugates into pH-Responsive Micelles for Tumor Microenvironment Triggered Fluorescence Imaging Pengyong Li, Mengmeng Sun, Zhikun Xu, Xinyu Liu, Wenguo Zhao, and Weiping Gao Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01368 • Publication Date (Web): 23 Oct 2018 Downloaded from http://pubs.acs.org on October 24, 2018

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Site-Selective in situ Growth-Induced SelfAssembly of Protein-Polymer Conjugates into pHResponsive Micelles for Tumor Microenvironment Triggered Fluorescence Imaging Pengyong Li, Mengmeng Sun, Zhikun Xu, Xinyu Liu, Wenguo Zhao, and Weiping Gao* Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, China *Corresponding author: [email protected] KEYWORDS: protein-polymer conjugate; controlled polymerization; self-assembly; pHresponsive; tumor imaging

ABSTRACT ：

Self-assembly of site-selective protein-polymer conjugates into stimuli-

responsive micelles is interesting owing to their potential biomedical applications ranging from molecular imaging to drug delivery, but remains a significant challenge. Herein we report a method of site-selective in situ growth-induced self-assembly (SIGS) to synthesize site-specific human serum albumin-poly(2-(diisopropylamino) ethyl methacrylate) (HSA-PDPA) conjugates that can in situ self-assemble into pH-responsive micelles with tunable morphologies.

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Indocyanine green (ICG) was selectively loaded into the core of sphere-like HSA-PDPA micelles to form pH-responsive fluorescence nanoprobes. The nanoprobes rapidly dissociated into protonated individual unimers at a transition pH of around 6.5 that is the extracellular pH of tumors, which resulted in a sharp fluorescence increase and markedly enhanced cellular uptake. In a tumor-bearing mouse model, they exhibited greatly enhanced tumor fluorescence imaging as compared to ICG alone and pH-nonresponsive nanoprobes. These findings suggest that pHresponsive and site-selective protein-polymer conjugate micelles synthesized by SIGS are promising as a new class of tumor microenvironment-responsive nanocarriers for enhanced tumor imaging and therapy.

1. INTRODUCTION Self-assembly of proteins into nanostructured biomaterials with complex functions is the unique phenomenon in biological systems. Inspired by this phenomenon, engineered proteins with self-assembly behaviors have been designed as artificial building blocks for the construction of self-assembled protein biomaterials.1-5 Recently, amphiphilic protein-polymer conjugates synthesized by conjugating proteins to hydrophobic polymers has emerged as new and versatile building blocks to form self-assembled protein-polymer conjugate micelles with controlled morphologies and new functions offered by the attached polymers.6-10 Protein-polymer conjugate micelles are potentially interesting as a new class of nanocarriers of imaging agents and drugs for biomedical applications;11-13 however, the potential has not been well exploited. Notably, it remains a considerable challenge to synthesize stimuli-responsive protein-polymer conjugate micelles in a site-selective and controllable manner,14-17 which may limit their potential applications in biomedicine. For instance, pH-responsive polymers were randomly conjugated to


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a protein to form pH-responsive and site-unselective protein-polymer conjugate micelles by the conventional “grafting to” method, resulting in a product mixture composed of positional isomers with reduced protein activity.16 Therefore, it is valuable to develop new strategies to synthesize pH-responsive and site-selective protein-polymer conjugate micelles for molecular imaging and drug delivery. In this proof-of-concept study, we propose a general method of site-selective in situ growthinduced self-assembly (SIGS) to synthesize pH-responsive and site-selective human serum albumin-poly(2-(diisopropylamino) ethyl methacrylate) (HSA-PDPA) conjugate micelles for tumor microenvironment triggered fluorescence imaging (Scheme 1). HSA is the most abundant plasma protein that is soluble and stable. Particularly, it has a very long circulatory half-life of 19 days due to its large size and FcRn (neonatal Fc receptor) mediated recycling.18-19 These attributes make it interesting as a carrier for drug half-life extension.20 The only free cysteine-34 position of HSA is site-selectively attached with a maleimide-functionalized atom transfer radical polymerization (ATRP) initiator to yield a site-selective and stoichiometric (1:1) HSA macro-initiator (HSA-Br) due to the high selectivity of maleimides to the thiol (Scheme 1a).21,22 PDPA is a pH-responsive polymer with a pKa of around 6.5 that is close to the extracellular pH of tumors.23-25 It can change from hydrophobic to hydrophilic when the solution pH decreases to be below its pKa. This is because the tertiary amino groups of PDPA are protonated and hydrated. PDPA is directly grown from HSA-Br by atom transfer radical polymerization (ATRP) to generate stoichiometric (1:1) and site-selective HSA-PDPA conjugates. During the ATRP reactions, the as-formed HSA-PDPA conjugates in situ self-assembled into micelles with different morphologies in aqueous solution at pH 7.4. Due to the hydrophobicity of PDPA at pH 7.4, it can aggregate to form the core of the micelles, while HSA, the hydrophilic block, can form


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the soluble shell to reduce the surface tension of the PDPA aggregates. Herein, we call this method SIGS. Indocyanine green (ICG) is a Food and Drug Administration (FDA) approved near infrared (NIR) fluorophore, which can form a complex with HSA for in vivo imaging and photothermal therapy.26 We chose to load ICG into the core of sphere-like HSA-PDPA micelles to form HSAPDPA/ICG (HDI) nanoprobes (Scheme 1a). We reasoned that above their transition pH, the nanoprobes would be silent due to the aggregation-caused quenching (ACQ) effect,27,28 but they could be activated to recover the fluorescence below their transition pH because of micelle dissociation into protonated individual unimers (Scheme 1b). Indeed, the nanoprobes dramatically dissociated into positively-charged unimers in a narrow pH range from 6.6 to 6.4, which resulted in a steep fluorescence increase and significantly enhanced cellular uptake. Furthermore, we hypothesized that the nanoprobes would be relatively “silent” in blood, but could be activated in the acidic tumor microenvironment when accumulating into a tumor by the enhanced permeability and retention (EPR) effect,29 which is essential for inhibition of blood fluorescence and for fluorescence amplification in the tumor (Scheme 1c). Indeed, in a tumorbearing mouse model, the nanoprobes were much more effective in tumor fluorescence imaging than ICG alone and non-pH-responsive nanoprobes.


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Scheme 1. Schematic illustration of synthesis of HSA-PDPA/ICG (HDI) nanoprobes (a) with pH-responsiveness (b) for acidic tumor microenvironment-triggered fluorescence imaging (c).

2. EXPERIMENTAL SECTION 2.1. Synthesis of HSA-Br. HSA-Br were synthesized using a previously reported method.13 Briefly, 425 μL 2-(2-(2-(2-(2,5-dioxo-2H-pyrrol-1(5H)-yl)ethoxy)ethoxy)ethoxy)ethyl 2-bromo2-methylpropanoate (DBMP) in DMF (50 mM) was added into 280 mg HSA dissolved in 40 mL Tris-HCl (50 mM, pH 7.4). After 1 h incubation at room temperature, the reaction mixture was purified with a desalting column to produce the initiator HSA-Br. The conjugation efficiency was determined by Ellman’s reagent.30 2.2. Synthesis of HSA-PDPA Micelles. HSA-PDPA micelles were synthesized by ATRP with different feeding molar ratio. One typical synthetic procedure of HSA-PDPA micelles with a feeding molar ratio of 500/1 (DPA/HSA-Br) is depicted as follows: A solution of HSA-Br in phosphate buffered solution (PBS, 2 mL, 10.5 mg/mL) and 2-(diisopropylamino) ethyl


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methacrylate (DPA, 34 mg, 160 μmol) were added into a Schlenk tube. Meanwhile, a catalyst solution of CuCl (12.8 mmol) and 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA) (32 mmol) in 1 mL PBS was prepared. After deoxygenated by bubbling with nitrogen, the catalyst solution was transferred into the deoxygenated HSA-Br and DPA solution. The polymerization was allowed to proceed overnight in an ice-water bath and then stopped by exposure to air. After dialysis (dialysis bag: MWCO 8-10 kDa) against PBS to remove the unreacted DPA monomer and catalyst, the HSA-PDPA micelles were further purified by Hiload 26/600 Superdex 75 pg (GE Healthcare) performed on AKTA purifier. HSA-PDPA micelles with different feed ratio (500/1, 3000/1, 5000/1, 10000/1) were named as HSA-PDPA 1, HSA-PDPA 2, HSA-PDPA 3 and HSA-PDPA 4, respectively. Meanwhile, the non-pH-responsive HSA-poly(2-hydroxypropyl methacrylate) (HSA-PHPMA) micelles as a positive control was synthesized by the same method13. 2.3. Synthesis of HDI Nanoprobes. To investigate the drug loading capacity, ICG was mixed with HSA-PDPA 1 micelles at different molar ratios of ICG/HSA (0.5/1, 1/1, 2/1, 5/1 and 10/1). One typical synthetic procedure of HDI nanoprobes at the feeding ratio of 5:1 is shown below: ICG (0.3 mg, 0.38 μmol) in 100 μL dimethyl sulfoxide (DMSO) was added into an HSA-PDPA 1 solution (1 mL, 5 mg HSA-equivalent/mL). The mixture was allowed to stand overnight at 25 °C

in the dark. The product was further purified by dialysis (dialysis bag: MWCO 810 kDa) to

remove DMSO and unbound ICG. HSA-PHPMA/ICG (HHI) was synthesized as the control by the same way. 2.4. Cleavage of the Grafted Polymer from the Conjugates. The grafted polymers were cleaved from the conjugates by hydrochloric acid hydrolysis31,32. 12 mg HSA-PDPA and 3 mL HCl (6 M) were added into a hydrolysis tube. The hydrolysis was allowed to proceed at 120 °C


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for 12 h in vacuum. After that, the cleaved polymers were purified by dialysis against water (dialysis bag: MWCO 3500 Da). The molecular weights of the polymers were analyzed by gel permeation chromatography (GPC). Chemical structure of the cleaved PDPA was confirmed by proton nuclear magnetic resonance (1H NMR). 2.5. Evaluations of Entrapment Efficiency and Drug Loading. The ICG concentrations of samples were determined by comparisons of the absorbances of the samples to the standard curve of ICG absorbance versus concentration. Briefly, a series of ICG solutions with different concentrations in PBS containing 1% HSA were prepared. The absorbances of the above solutions at 805 nm were recorded with a SpectraMax M3 Microplate Reader (Molecular Devices) to obtain a linear standard curve for further determining the concentrations of ICG in the samples. The HSA concentration of HSA-PDPA 1 micelles was determined by bicinchoninic acid (BCA) assay according to the directions of BCA kit (Beyotime Biotech). The entrapment efficiency was determined by the weight ratio of the loaded ICG to the initially added ICG, and the drug loading was determined by the weight ratio of the loaded ICG to the ICG-loaded micelles. 2.6. Measurements of Absorption and Emission Spectra. The absorption and emission spectra of HDI, HHI, HSA-ICG, PDPA+ICG and ICG at different pH values were recorded with a SpectraMax M3 Microplate Reader. The fluorescence emission intensity at 810 nm was recorded at an excitation wavelength of 740 nm. All the samples contained 10 μg/mL ICG. 2.7. Measurement of Critical Micelle Concentration (CMC). The CMC of HSA-PDPA 1 micelles was measured using Nile Red (NR) solution. First, a micelle stock solution (HSA concentration: 2.6 mg/mL) was diluted to different concentrations with PBS. 4 μL ethanol solution of NR (0.31 mM) was added into 1 mL of each micelle solution to produce a final NR


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concentration of 1.25 μM. The fluorescence intensity values of the samples at 630 nm were recorded at an excitation wavelength of 550 nm on a SpectraMax M3 Microplate Reader and were plotted as a function of HSA concentration. The CMC was determined from the curve. 2.8. Evaluation of Esterase-like Activity of HSA. 195 µL of HSA, HSA-Br or HSA-PDPA 1 (0.1 mg/mL of HSA) in PBS (10 mM) was added into wells of a 96-well plate, followed by the addition of 5 µL of p-nitrophenyl acetate (8 mM) in PBS containing 20% (v/v) acetonitrile. The reaction at 25 °C was monitored with a SpectraMax M3 Microplate Reader at 400 nm absorbance to calculate the amount of the product p-nitrophenol. The reaction rate was calculated from the plot of absorbance against time, as described in the literature33. Each sample was tested in triplicate. 2.9. Confocal Laser-Scanning Microscopy Analysis of Cellular Uptake. C8161 melanoma cells expressing green fluorescent protein (GFP) were seeded at 5 × 104 cells per well in a 35 mm glass bottom culture dish (NEST) and were cultured overnight. The cultured cells per well were incubated with 20 μg ICG equivalent/mL of HDI at pH 7.4 or pH 6.4 for 30 min, and then washed with PBS to remove the unbound samples and fixed with 4% (w/v) cold paraformaldehyde for 10 min. Then, the cell nucleus was stained with 2.5 µg/mL Hoechst 33342 (Sigma) for 10 min. The cells were then washed with PBS three times and imaged with an LSM710 confocal laser scanning microscope (Carl Zeiss). The excitation and emission wavelengths of Hoechst, GFP and ICG were 346/460 nm, 488/544 nm, and 633/664 nm, respectively. Images were analyzed by Zen_2012 (blue edition) software. 2.10. Biocompatibility Evaluation. C8161 cells, L929 cells, MCF-10A cells, or HMECs were firstly seeded at 5000 cells per well in a 96-well plate (Corning) overnight, and then different concentrations (concentrations of HSA: 12.5, 25, 50, 100, 200, 300 µg/mL) of HSA-


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PDPA 1 micelles in medium were added. Wells filled with medium and medium-treated cells only were defined as 0% (background) and 100% cell viability, respectively. After incubation for 48 h, the viability of cells was determined by MTS assay according to Cell Proliferation Assay kit (Promega). 2.11. Flow Cytometry Analysis of Cellular Uptake. For flow cytometry analysis, C8161 cells were seeded in a 6-well plate at 5 × 104 cells per well and cultured overnight, and incubated with 20 µg ICG equivalent/mL of HDI at pH 7.4 or pH 6.4 for 30 min. Then, the cells were washed with PBS for 3 times and harvested using 0.25% Trypsin-EDTA for BD FACS Aria III flow cytometer analysis. 2.12. In vivo Tumor Imaging. Female BALB/c nude mice of 8 weeks’ old were inoculated subcutaneously in the right flank with 1 × 107 C8161 cells. When the tumor volume reached 150200 mm3, the mice were randomly assigned to four groups (n = 3) and intravenously injected with 200 µL PBS, 200 µL HDI (100 µg/mL ICG), 200 µL HHI (100 µg/mL ICG), and 200 µL ICG (100 µg/mL). Images were taken at 1, 8, 24 and 32 h after injection using an IVIS Lumina II in vivo imaging system (PerkinElmer). The mice were sacrificed 32 h post injection. Then the organs and tissues including the heart, liver, spleen, lung, kidney and the tumor were collected and imaged. 2.13. Statistical Analysis. All data are shown in the form of mean ± standard deviation. A two-tailed, unpaired Student’s t test was used to analyze the differences between the two groups. One (*), two (**), and three stars (***) mean statistical significance at P < 0.05, 0.01, and 0.001, respectively.

3. RESULTS AND DISCUSSION


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3.1. Preparation and Characterization of HSA-PDPA Micelles. HSA-PDPA micelles were synthesized via two-step reactions (Scheme 1a). First, an ATRP initiator was solely attached to the only free cysteine-34 position of HSA to form a site-selective HSA-initiator conjugate (HSABr) according to our previous work.13 Ellman’s assays indicated that HSA had approximately 0.6 thiols per albumin and almost all thiols were conjugated with the initiator, which means that about 60% HSA was modified with the initiator (Figure S1). Second, PDPA was grown from HSA-Br by ATRP of DPA in PBS to yield stoichiometric and site-selective HSA-PDPA micelles. The molecular weight of PDPA was controlled by changing the feeding molar ratio of DPA to HSA-Br to tune the morphology of HSA-PDPA micelles (Table 1). For instance, after the polymerization at DPA/HSA-Br = 500 to yield HSA-PDPA1, the ATRP solution was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A smear band with a high molecular weight was observed for the HSA-PDPA conjugate after the ATRP reaction (Figure S2). The HSA-PDPA conjugate was further purified by size exclusion chromatography. SDS-PAGE showed that the unreacted HSA residue was successfully removed from the HSA-PDPA conjugate. To determine the molecular weights of the polymer chains, HSA-PDPA micelles were incubated in 6 M HCl aqueous solution at 110 °C to degrade the protein, and the PDPA residues were analyzed by 1H-NMR and GPC (Figures S3 and S4). The morphologies of HSA-PDPA micelles were observed by transmission electron microscopy (TEM) (Figure S5a), and their hydrodynamic diameters (Dh) were mearsured by dynamic light scattering (DLS) (Figure S5b). Notably, the conversion of monomer was low, typically less than 10%, indicating that the polymerization was not well controlled and thus needs to be optimized in the future. HSA-PDPA conjugates could self-assemble into micelles with different morphologies from sphere, to worm, to aggregate, and then to vesicle with increasing the molecular weights of


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the grafted PDPA chains (Figure S5 and Table 1). The phase transition behavior could be ascribed to the gradual molecular curvature reduction of the conjugate chains as the PDPA block grows from the HSA block.13 Unlike other micelles, sphere-like HSA-PDPA micelles (HSAPDPA 1) were much stable in solution and chosen for further study. The CMC of the micelles was determined to be as low as 0.46 μM (Figure 1a). Circular dichroism (CD) spectra showed that HSA exhibited two negative minima at 209 and 221 nm characteristic of an α-helix structure of protein, the CD shape and the band intensity of HSA-Br and HSA-PDPA were identical to those of HSA (Figure 1b), indicating that the secondary structure of HSA did not change during the whole process. The esterase-like activity of the micelles was measured to be 108% of that of HSA (Figure 1c), which indicated that SIGS did not damage the activity of HSA. Overall, these results demonstrated that SIGS was a biocompatible and enabling method to synthesize pHresponsive and site-selective protein-polymer conjugate micelles. Table 1. Parameters for HSA-PDPA micelles with different molecular weights Sample name

HSA-PDPA 1

Feed ratio

500/1

a)

C% b)

Mthc) (kDa)

Mn d) (kDa)

Mw/Mn

6%

10.6

10.1

1.68

d)

Dh (nm) e)

Morphology f) (size, nm)

42

Sphere (34)

HSA-PDPA 2

3000/1

3%

32.0

29.3

1.61

103, 1113

Sphere, worm, aggregate

HSA-PDPA 3

5000/1

3%

53.2

57.7

1.42

1035, 4040

Aggregate

HSA-PDPA 4

10000/1

4%

142.0

119.6

1.21

267

Vesicle (234)

a)Feed

ratio is the molar ratio of DPA to HSA-Br; b)C% (the conversion of monomer) = ((weight of HSA-PDPA conjugates)-(weight of HSA-Br)) / (weight of initially added monomer); b)Mth represents the theoretical molecular weight, Mth = feed ratio/60% × molecular weight of monomer × C%; d)Mn and Mw/Mn were estimated by GPC; e)Dh was determined by DLS; f)Morphology was observed by TEM.


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Figure 1. Stability and activity of HSA-PDPA 1 micelles. a) CMC of HSA-PDPA 1 micelles. b) CD spectra of HSA, HSA-Br, and HSA-PDPA 1. c) Normalized esterase-like activities of HSA, HSA-Br, and HSA-PDPA 1 (mean ± SD, n=3). The esterase-like activity of HSA was defined as 100%.

3.2. Preparation and pH-Responsive Properties of HDI Nanoprobes. Subsequently, ICG was added into the HSA-PDPA 1 micelle solution to form HSA-PDPA/ICG (HDI) nanoprobes. The optimum feeding molar ratio of ICG to HSA was screened out to be 5:1 (Table S1). The ICG entrapment efficiency and ICG loading in the nanoprobes were measured to be 60% and 3.6%, respectively. The Dh of the nanoprobes was 44 nm with a Ð of 0.26 (Figure 2a). The morphology was observed by TEM to be spherical with an average diameter of 36.7 ± 6.5 nm (Figure 2b), which was consistent with the morphology observed by Cryo-TEM (Figure S6). Additionally, according to our previous work,13 a hydrophobic and non-pH-responsive polymer PHPMA was directly grown from HSA-Br to generate non-pH-responsive HSA-PHPMA micelles as a positive control (Figure S7 and Table S2). ICG was also loaded into the HSAPHPMA micelles to form HHI nanoprobes as a positive control (Figure S8 and Table S3). The


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pH-responsive dissociation behaviors of HSA-PDPA 1 micelles and HDI nanoprobes were studied by DLS and zeta potential measurement. A sharp decrease in Dh from 45 nm to 14 nm was observed for HSA-PDPA 1 micelles when the pH value decreased from 6.6 to 6.4 (Figure 2c). A similar phenomenon was also found for HDI nanoprobes, where the Dh suddenly decreased from 53 nm to 12.2 nm with the pH transition from 6.6 to 6.4. These data indicated the speedy dissociation of these assemblies into individual unimers in response to an extremely small pH change of 0.2. The phenomenon was also confirmed by zeta potential measurement. The zeta potentials of both the assemblies suddenly changed from negative to positive values when the pH value changed from 6.6 to 6.4 (Figure 2d), indicating the fast protonation of the tertiary amino groups of the PDPA blocks in response to the tiny pH change of 0.2. In contrast, both HSA-PHPMA micelles and HHI nanoprobes did not show pH-responsive properties as expected (Figures S9). Taken together, these results indicated that HSA-PDPA 1 micelles and HDI nanoprobes were pH-responsive with a transition pH of 6.5.


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Figure 2. Physicochemical characterization of HSA-PDPA 1 micelles and HDI nanoprobes. a) DLS analysis of HDI nanoprobes. b) TEM image of HDI nanoprobes (scale bar = 50 nm). The samples were negatively stained with 2% phosphotungstic acid. c) Hydrodynamic diameter (Dh) of HSA-PDPA 1 micelles and HDI nanoprobes as a function of pH. d) Zeta potentials of HSA, HSA-PDPA 1 micelles and HDI nanoprobes at different pH values. The data are shown as mean ± SD (n = 3). 3.3. Optical Properties of HDI Nanoprobes. We further studied the ol properties of HDI nanoprobes. The UV-vis-NIR spectrum of HDI nanoprobes at pH 7.4 was different fropticam those of ICG and its complex with HSA (HSA-ICG) at pH 7.4 (Figure 3a). Notably, the wavelength of the major absorption peak of HDI nanoprobes (807 nm) was longer than those of HSA-ICG (798 nm) and ICG (774 nm), which suggested that ICG was largely loaded into the


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PDPA core of the nanoprobes (Figure 3b). ICG with a pKa of 3.3 is negative at pH 7.4, so that it could get into the PDPA core of HSA-PDPA 1 micelles, which was driven by the hydrophobic interaction34,35 and electrostatic attraction between ICG and PDPA. As a result, the phenomenon of ACQ was observed at pH 7.4, as indicated by the remarkably reduced fluorescence intensity of HDI nanoprobes at 740 nm excitation compared to HSA/ICG and ICG (Figure 3c). Interestingly, a sudden increase in fluorescence intensity was observed for HDI nanoprobes when the pH value changed from 6.6 to 6.4 (Figures 3d), indicating that HDI nanoprobes dissociated into unimers to release ICG. The UV-vis-NIR spectrum of HDI nanoprobes at pH 6.4 more resembled that of HSA-ICG than ICG and ICG+PDPA (Figures 3a and S10), suggesting that most of released ICG were entrapped into the HSA block of the HSA-PDPA 1 unimers at pH 6.4 (Scheme 1b). In contrast, the wavelength of the major absorption peak of HHI nanoprobes (795 nm) at pH 7.4 was the same as that of HSA-ICG (Figure S11a). Moreover, less fluorescence quenching was observed for HHI nanoprobes than for HDI nanoprobes at pH 7.4 (Figure 3c). These results suggested that ICG was mainly loaded into the HSA shell of HSAPHPMA micelles (Figure S11b). In contrast to HDI nanoprobes, no obvious changes in fluorescence intensity were observed for HHI nanoprobes and ICG alone in the pH range of 5.57.4 (Figure 3e), which confirmed that HHI nanoprobes and ICG alone were not pH-responsive. Collectively, these results indicated that HDI nanoprobes were pH-responsive fluorescence nanoprobes with a transition pH of 6.5.


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Figure 3. pH-responsive optical properties of HDI nanoprobes. a) UV–vis–NIR spectra of ICG, HSA-ICG, HHI and HDI at pH 7.4 and 6.4. b) Schematic illustration of the structure of HDI nanoprobes. c) Emission spectra of ICG, HDI, and HHI at pH 7.4 when excited at 740 nm. d) Emission spectra of HDI nanoprobes at different pH when excited at 740 nm. e) Emission intensity at 810 nm of ICG, HDI and HHI as a function of pH when excited at 740 nm. The data are shown as mean ± SD (n = 3). 3.4. pH-Responsive Cellular Uptake. The pH-responsive cellular uptake of HDI nanoprobes was evaluated by confocal laser-scanning microscopy (CLSM) (Figure 4a). After incubation of the nanoprobes with C8161 melanoma cells for 30 min at pH 7.4 and 6.4, respectively, the intracellular fluorescence intensity at pH 6.4 was remarkably stronger than at pH 7.4. These data implied that the activated nanoprobes at pH 6.4 were much easier to be taken up by the cells than the unactivated nanoprobes at pH 7.4. This result could be attributed to the enhanced electrostatic interactions36 between the protonated HDI unimers and the cells at pH 6.4. In contrast, no


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significant difference in intracellular fluorescence intensity was observed after incubation of HHI nanoprobes with the cells for 30 min at pH 7.4 and 6.4, respectively. These results were confirmed by flow cytometry (Figures 4b and 4c). The intracellular fluorescence intensity of HDI nanoprobes at pH 6.4 was 5.8-fold higher than at pH 7.4. On the contrary, the intracellular fluorescence intensity of HHI nanoprobes at pH 6.4 was similar to that at pH 7.4, as expected. Notably, the intracellular fluorescence intensity of HDI nanoprobes at pH 6.4 was 7.1-fold higher than that of HHI nanoprobes at pH 6.4. Additionally, both of the nanoprobes were not cytotoxic to C8161 melanoma cells and normal cells such as MCF-10A cells, L929 cells and HMECs (Figure S12). Overall, these results indicated that the activation of HDI nanoprobes at pH 6.4 could significantly enhance the cellular uptake, suggesting that HDI nanoprobes would be activated and taken up by tumor cells in vivo for enhanced tumor fluorescence imaging since the extracellular pH in tumors is close to the transition pH (6.5) of the nanoprobes.

Figure 4. pH-responsive cellular uptake of HDI nanoprobes. a) CLSM analysis of pH-responsive endocytosis of HHI and HDI into C8161 cells. C8161 cells expressing green fluorescence protein (GFP) were incubated with HHI or HDI at pH 7.4 or pH 6.4 for 30 min (Scale bar = 60 μm). The


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cell nuclei were stained in blue. GFP and ICG are shown in green and red, respectively. b) Fluorescence-activated cell sorting (FACS) analysis of cellular uptake of HHI or HDI at pH 7.4 or 6.4. c) Quantitative fluorescence (FL) intensity comparisons based on the FACS analysis.

3.5. In Vivo Tumor Imaging. To prove the hypothesis, we evaluated the in vivo tumor imaging of HDI nanoprobes in a mouse model of C8161 melanoma (Figure 5). After an intravenous injection of HDI nanoprobes via tail vein, the NIR fluorescence images of the mice were obtained at different time points (Figure 5a). At 1 h post injection, the fluorescence was observed all over the body, and mainly focused in the liver. The strong fluorescence was observed in the tumor at 8 h, indicating that the nanoprobes accumulated into the tumor presumably owing to the EPR effect.29 Interestingly, at 24 h and 32 h, the fluorescence was observed only in the tumor. Moreover, the fluorescence intensity at 32 h was stronger than at 24 h. In contrast, at 24 h and 32 h, the fluorescence was almost not observed in the tumors for the mice treated with HHI nanoprobes and ICG alone, respectively. Due to the acidity of tumor microenvironment, HDI could be protonated and become positively-charged when accumulating into the tumor by the EPR effect, so that HDI could electrostatically interact with tumor cell membranes that are negatively charged, which could significantly increase the tumor accumulation of HDI. Simultaneously, ICG could be released from HDI, which could restore the fluorescence of ICG encapsulated in HDI that had been quenched by the homo-FRET effect. In contrast, although HHI could accumulate into the tumor by the EPR effect, the fluorescence of ICG in HHI could not be restored in the tumor. As a result, the fluorescence of HHI in the tumor was very weak, in striking contrast to HDI. As expected, the tumor accumulation of free ICG was very low, as indicated by the very weak fluorescence in the tumor, presumably due to its


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instability and rapid renal clearance.37 The major organs and tissues were collected from the mice at 32 h post injection for ex vivo imaging (Figure 5b). The fluorescence of each organ or tissue was further semi-quantified for comparison (Figure 5c). Notably, the tumor fluorescence intensity of HDI nanoprobes was 3- and 6-fold higher than those of HHI nanoprobes and ICG alone, respectively. These results indicated that HDI nanoprobes could significantly enhance tumor fluorescence imaging due to their activation in response to the acidic tumor microenvironment.

Figure 5. In vivo and ex vivo fluorescence imaging of C8161 melanoma after intravenous injection of HDI nanoprobes. a) Time-lapse NIR fluorescence images of C8161melanomabearing mice at different time points after intravenous injection of ICG, HHI or HDI at the same dose of 10 µg equivalent ICG/kg body weight. b) NIR fluorescence images of major organs and tumors collected at 32 h after intravenous injection of ICG, HHI or HDI. c) The averaged fluorescence intensities of major organs and tumors collected at 32 h after intravenous injection


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of ICG, HHI or HDI. Note: Backgrounds of organs and tumors collected from untreated mice were correspondingly deducted from the measured fluorescence intensity values. The data are shown as mean ± SD (n = 3). (*) P < 0.05 for HDI versus HHI, (**) P < 0.01 for HDI versus ICG.

4. CONCLUSIONS In summary, we have reported a new method of SIGS to synthesize pH-responsive and siteselective protein-polymer conjugate micelles with controlled morphologies. The potential of them as a new class of pH-responsive nanocarriers has, for the first time, been demonstrated by selectively loading ICG into the core of sphere-like HSA-PDPA micelles to form pH-responsive fluorescence nanoprobes. The nanoprobes possess highly desirable attributes for enhanced tumor fluorescence imaging. First, they are highly sensitive to the change of pH. Especially, they can suddenly dissociate into protonated unimers in a very narrow pH range from 6.6 to 6.4, resulting in a steep increase in fluorescence due to the pH-responsive switch from an ACQ state to activated state. Second, they can easily be taken up by tumor cells at the extracellular pH of tumors to show enhanced intracellular fluorescence imaging, due to the strong electrostatic interaction between the protonated PDPA block of HSA-PDPA/ICG unimers and the negatively charged cell membrane of tumor cells. Third, in a tumor-bearing mouse model, they can selectively accumulate into tumors for considerably enhanced tumor fluorescence imaging due to the pH-responsive activation in response to the acidic tumor microenvironment. These unique attributes suggest that these pH-responsive fluorescent nanoprobes might be highly valuable to precise excision of tumors, which would be pursued in the future. Based on these findings, we believe that SIGS is promising as a new and general method to synthesize a variety of pH-


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responsive and site-selective protein-polymer conjugate micelles for biomedical applications, especially as a new class of tumor microenvironment-responsive nanocarriers of imaging agents and drugs for enhanced tumor imaging and therapy.

ASSOCIATED CONTENT The Supporting Information is available free of charge. Materials, cells and mice, and measurements are included in Supplementary Experimental Section. Physicochemical characterization for HSA-PDPA, HDI, HSA-PHPMA and HHI, and biocompatibility evaluation are shown in Supplementary Figures and Tables 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. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT

This study was financially supported by Grants from National Natural Science Foundation of China (Grant No. 21534006). The Laboratory Animal Facility at the Tsinghua University is accredited by the AAALAC (Association for Assessment and Accreditation of Laboratory


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Animal Care International), and all animal protocols used in this study are approved by the Institutional Animal Care and Use Committee (IACUC).

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For Table of Contents Use Only Site-Selective in situ Growth-Induced Self-Assembly of Protein-Polymer Conjugates into pH-Responsive Micelles for Tumor Microenvironment Triggered Fluorescence Imaging Pengyong Li, Mengmeng Sun, Zhikun Xu, Xinyu Liu, Wenguo Zhao, and Weiping Gao*


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Site-Selective in Situ Growth-Induced Self-Assembly of Protein

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