Differentiation of Intracellular Hyaluronidase Isoform by Degradable

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Differentiation of Intracellular Hyaluronidase Isoform by Degradable Nanoassembly Coupled with RNA-Binding Fluorescence Amplification Yuan Li, Sheng Yang, Lei Guo, Yue Xiao, Jinqiu Luo, Yinhui Li, Man Shing Wong, and Ronghua Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01242 • Publication Date (Web): 16 Apr 2019 Downloaded from http://pubs.acs.org on April 16, 2019

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Analytical Chemistry

Differentiation of Intracellular Hyaluronidase Isoform by Degradable Nanoassembly Coupled with RNA-Binding Fluorescence Amplification Yuan Li,† Sheng Yang,‡,* Lei Guo,§ Yue Xiao,† Jinqiu Luo,‡ Yinhui Li,† Man Shing Wong,§ and Ronghua Yang†,‡,* †

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and

Chemical Engineering, Hunan University, Changsha, 410082, P. R. China ‡

School of Chemistry and Food Engineering, Changsha University of Science and Technology,

Changsha, 410114, P. R. China. § Department

of Chemistry and Institute of Molecular Functional Materials, Hong Kong Baptist

University, Hong Kong SAR, P. R. China.

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Analytical Chemistry 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

ABSTRACT: Hyaluronidase has two cruical isoforms, hyaluronidase-1 (Hyal-1) and hyaluronidase-2 (Hyal-2), which are essential for cellular hyaluronic acid (HA) catabolism to generate different-sized oligosaccharide fragments for performing different physiological functions. In particular, Hyal-1 is the major tumor-derived hyaluronidase. Thus, specific detection of one hyaluronidase isoform, especially Hyal-1, in live cells is of scientific significance but remains challenging. Herein, by using of differentiated tolerance capability of an amphiphilic HA-based nanoassembly to Hyal-1 and Hyal-2, we rationally design a Hyal-1 specific nanosensor, consist of cholesterylamine-modified HA nanoassembly (CHA) and RNAbinding fluorophores (RBF). The RBF molecules were entrapped in CHA to switch off their fluorescence via aggregation cause quenching. However, CHA can be disassembled by Hyal-1 to release RBF, resulting in fluorescence activation. Moreover, the fluorescence of the released RBF is further enhanced by cytoplasm RNA. Owing to this cascade signal amplification, this nanosensor RBF@CHA displays significant change of signal to background ratio (120-fold) toward 16 μg/mL Hyal-1 in cellular lysates. In contrast, it is resistant to Hyal-2. By virtue of its selective and sensitive characteristics under complicated matrix, RBF@CHA had been successfully applied for specifically visualizing Hyal-1 over Hyal-2 inside live cells for the first time, detecting low level of intracellular Hyal-1, and distinguishing normal and cancer cells with different expressions of Hyal-1. This approach would be useful to better understand biological functions and related diseases of intracellular Hyal-1.


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INTRODUCTION Hyaluronic acid (HA), composed of repeating D-glucuronic acid and N-acetyl-Dglucosamine disaccharide units, is a linear and anionic glycosaminoglycan and widely distributed in the extracellular matrix.1 It can be degraded into oligosaccharide fragments by hyaluronidases, a family of endoglycosidases, to maintain diverse physiological functions such as angiogenesis, fertilization, embryogenesis, and cell motility.2 In human, two homologous hyaluronidase isoforms, hyaluronidase-1 (Hyal-1) and hyaluronidase-2 (Hyal-2), are predominately involved in the cellular pathway of HA catabolism.3 Hyal-2, mainly locating in the external surface of cell membrane, primarily cleaves high-molecular-weight HA to 20 kDa intermediate fragments of approximately 50 disaccharide units that are highly angiogenic, immuno-stimulatory, and inflammatory.4 The products of Hyal-2 digestion can be transported intracellularly and further enzymatically degraded by the action of Hyal-1 in lysosomes to generate tetrasaccharides, which are demonstrated to be anti-apoptotic and inducers of heat shock proteins.5 In addition to its normal functions in metabolic process, Hyal-1 is identified as the major tumor-derived hyaluronidases expressed in bladder, prostate, and breast cancer tissues.6-8 Thereby, the specific detection of one hyaluronidase isoform, especially Hyal-1, in living cells is of great significance to better elucidate its role in physiological and pathological events. Among various cellular biology tools, fluorescence probes possess the advantage of noninvasiveness, high resolution, and real-time monitoring.9-12 To date, several enzymeactivatable fluorogenic probes had been proposed for hyaluronidase assay.13-24 Generally, HAbearing fluorophore/upconversion luminescence material was covalently linked with another fluorophore13,14 or nanoquencher15-17 including gold nanoparticle(AuNP) and poly(mphenylenediamine) nanosphere to form an energy transfer donor−acceptor pair, which can 3 ACS Paragon Plus Environment


respond hyaluronidase to realize ratiometric/turn on fluorescence change. Nevertheless, this covalent attachment strategy was often synthesis-complicated and time-consuming. As well as, these AuNP-based ones may suffer from activity decline due to steric hindrance and poor stability because of glutathione (GSH) competition and being apt to precipitation in physiological salt environments. Alternatively, lots of assemblies were developed via electrostatic adsorption of negatively charged HA with cationic fluorophores and positively charged luminescence nanomaterials.18-24 Despite such simplicity and flexibility, unavoidable interference by other charged components in living cells hampers label-free approach further application to tackle biological issues. Moreover, the aforementioned ones were mainly designed for all hyaluronidases, to the best of our knowledge, none is capable of differentiating one isoform from another. The main reason is that they usually use single HA chain as the recognition and signal transduction moiety. In such case, both Hyal-1 and Hyal-2 can cleave HA chain indiscriminately to generate fluorescence signal, indicating that HA itself is not very selective for Hyal-1 over Hyal-2. For in-situ imaging of intracellular Hyal-1, the inevitable challenge is how to overcome the false signals by nonspecific cleavage of Hyal-2. Meanwhile, several difficulties including exceedingly low concentration and unstable activity are present.25 Therefore, available strategy for developing fluorogenic probes with biological compatibility, high sensitivity, and in particular specificity to monitor trace Hyal-1 in living cells is urgently required. To address this issue, we turn our attention to amphiphilic HA conjugate by chemical modification of hydrophobic moiety to hydrophilic HA chain. The hydrophobically modified HA derivatives with proper chain length and degree of substitution could be capable of being selfassembled to form stable nanoparticles in an aqueous environment.26 Since the HA degradation


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fragments by Hyal-1 and Hyal-2 are different-sized, we hypothesized that amphiphilic HA-based nanoassembly would display differentiated tolerance toward them, Hyal-1 might be more liable to dissociate such nanoassembly than Hyal-2. Inspired by this and responsive fluorescence sensing systems based on structurally-sensitive nanocarriers,27 we herein present our first attempt to construct a novel Hyal-1 activatable nanosensor based on self-assembly of cholesterylaminemodified HA (CHA) containing RNA-binding fluorophores (RBF), namely RBF@CHA, as depicted in Scheme 1. Owing to the self-quenching effect of RBFs entrapped in self-assembled CHA,28 RBF@CHA displays low fluorescence signal. While internalization into cells via CD44mediated endocytosis,29 RBF@CHA is resistant to Hyal-2, whereas intracellular Hyal-1 triggers disassembly of CHA to release the quenched RBF molecules, resulting in fluorescence activation. Moreover, the escaped PBF can immediately bind with cytoplasmic RNA to exhibit a second fluorescence gain. Due to its specificity and signal amplification characteristics, RBF@CHA was successfully applied to visualize Hyal-1 over Hyal-2 in live cells and to sensitively differentiate between normal and cancer cells with different Hyal-1 expressions.

EXPERIMENTAL SECTION Synthesis of Nanosensor (RBF@CHA). According to the synthetic procedure as depicted in Scheme S1, cholesteryl chloroformate (Chol) was firstly aminated by 2,2’-(ethylenedioxy)-bisethylamine (EDEA) to give the intermediate amino-Chol (Chol-NH2), which was successively linked into HA to synthesize amphiphilic HA conjugate Chol-HA. Their detailed syntheses are attached to the Supporting Information. Subsequently, Chol-HA (5 mg) was incubated without or with RBF (2 μM), an indole-based cyanine previously prepared, in bath sonicator for 30 min at 25 °C. After dialyzed in semipermeable tubes (MWCO 12-14 kDa) against water three times at



25 °C for 24 h, the solution was then concentrated in vacuo to obtain uniform nano-sized assembly CHA and RBF@CHA. Spectrophotometric Experiments. First, RNA sensing properties of RBF (200 nM) were examined in 10 mM phosphate buffer (PB, pH 7.4) containing different concentrations of RNA extract from HeLa cell. The reaction solutions were kept at room temperature for 1 h in dark, and the emission spectra were recorded in the range from 500 to 650 nm with excitation wavelength at 458 nm. Next, for Hyal-1 in vitro assay, RBF@CHA (2 μg/mL) was incubated with different concentrations (0 to 16 μg/mL) of Hyal-1 in PB solution (10 mM, pH 5.0) containing RNA extract (5 mg/mL) from HeLa cell for 100 min (37 °C). Then the emission spectra were recorded in the range from 500 to 650 nm with excitation wavelength at 458 nm. Fluorescence Imaging in Living Cells. For cellular imaging experiments, HeLa cells were pretreated with or without RNase (5 U/mL), and then stained with RBF (1.0 μM) and cell nucleus tracker 4',6-diamidino-2-phenylindole (DAPI, 0.5 μg/mL) for 30min. For imaging of intracellular Hyal-1, HeLa cells were incubated without or with RBF@CHA (10 μg/mL) and FITC-labeled hyaluronidase antibody (1:200 dilution) in the absence or presence of indomethacin (20 μg/mL) for 4 h. In order to verify intracellular visualization of Hyal-1 over Hyal-2, HeLa cells were incubated in the fresh culture medium containing RBF@CHA and FITC-labeled hyaluronidase antibody under different conditions: control, mineralization treatment, UV-B irradiation, and treatment of 10 ng/mL interleukin-1 (IL-1). To determine different Hyal-1 expression between cancer and normal cell, HepG2 and L02 cell lines were treated with RBF@CHA and FITC-labeled hyaluronidase antibody in the absence or presence of indomethacin for 4 h. After washing with PBS, these treated cells were subjected to imaging analysis using an Olympus FV1000 laser confocal microscope. Relative fluorescence intensity


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values in the fluorescence images were analyzed by ImageJ software. (nanosensor channel: λex=458 nm; λem=540-640 nm; FITC-labeled hyaluronidase antibody channel: λex=488 nm; λem=505-530 nm). A 40× objective lens was used.

RESULTS AND DISCUSSION Synthesis and Characteristic of CHA. To obtain an amphiphilic HA-based nanoassembly, water-soluble HA backbone was firstly conjugated with hydrophobic cholesterylamine (Chol) through two-step amidation reactions to synthesize amphiphilic HA conjugate Chol-HA (Scheme S1).30 The intermediate Chol-NH2 was structurally confirmed by MS peak at m/z 560.1 (Figure S1). The synthetic process of Chol-HA was monitored by Fourier transform infrared spectroscopy (FTIR) and Zeta potential measurement (Figure S2). In comparison with bare HA chain, Chol-modified HA present increased absorption bands at 1560 cm-1 (C-N vibration of acylamide groups) and disappeared absorption bands at 1700 cm-1 (C=O stretching of carboxyl groups), consistent with the amidation reaction. Moreover, the zeta potential of Chol-HA is less negatively charged than HA due to the modification of Chol-NH2 (-25.5 mV vs -18.9 mV). The chemical structure of Chol-HA was further characterized with 1H NMR (Figure S3). Successful conjugation of Chol-NH2 with HA was confirmed by the characteristic peaks appearing from the ethylene group at 5.75 ppm, and the degree of substitution (DS, the number of Chol molecules per 100 sugar residues of HA) was calculated to be 8.5 % by the integration ratio between the characteristic peak of the N-acetyl group in HA and that of the methyl group in Chol-NH2 (1.97 ppm vs 0.65 ppm). After successful synthesis, Chol-HA spontaneously self-assembled to form the nanoassembly CHA by a simple sonication method.31 Transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements (Figure 1A) show that CHA is a



homogeneous spherical nanostructure with hydrodynamic particle size at 187 ± 10.1 nm, indicating that the hydrophobic interaction among Chol groups help to form HA nanoassembly. Moreover, Figure S4 revealed that this nanoassembly was found to be dispersed stably for several days under physiological conditions (PBS, pH = 7.4, 37 ℃), indicating the possibility of its application in biological systems. In contrast, upon treatment of CHA with Hyal-1, the assembly form disappeared and the hydrodynamic particle size is deeply reduced to 8.0±0.9 nm, revealing the full dissociation of CHA proceeded by fracturing HA chains with Hyal-1 (Figure 1B). Whereas decrease in size of CHA (hydrodynamic particle size at 149.0±9.5 nm) can be observed in the presence of Hyal-2, but it still maintains the nanosphere form, indicating that CHA is relatively tolerant to Hyal-2. Furthermore, gel-filtration chromatography confirmed that amounts of low-molecular weight fragments were generated from Hyal-1-treated HA but Hyal-2-treated HA mainly give relatively large degradation product with 50 disaccharide units (Figure S5). Above experiments really demonstrate that CHA can distinguishably response to Hyal-1 over Hyal-2. Encouraged by the fully-dissociated feature of CHA by Hyal-1, we envision that a novel fluorescence probe specific to Hyal-1 may be developed based on the nanoassembly of amphiphilic HA. Design of RBF@CHA. Owing to its overexpression in body fluids such as urine and serum, hyaluronidase had been considered as potential prognostic marker and therapeutic target of malignant tumors.32 However, the role of intracellular Hyal-1 in tumor progression is concentration dependent, and it may function as a tumor suppressor at low concentration.33 On the basis of its biocompatibility, susceptibility to hyaluronidase, and loading capability, amphiphilic HA-based nanoassembly has been served as stimuli-responsive reservoir for tumor


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imaging and theranostic recently.34-37 Regrettably, these fluorophore-labeled imaging systems exhibit insufficient “off/on” behavior of fluorescence signal, which are not suitable for fully elucidating the function of intracellular Hyal-1 in cancer biology. Obviously, more effective signal transduction approach is a requisite for constructing amphiphilic HA-based fluorogenic probe for Hyal-1 to achieve high sensitivity and large signal to noise ratio. Inspired by the drug release strategy of amphiphilic HA-based delivery platform, we proposed an amplified nanosensor specific to Hyal-1 by utilizing CHA as switchable scaffold to regulate the behavior of fluorophore with cytomatrix-enhanced property. Different from the previous design plan that signal molecules were conjugated to the HA chain, the cytomatrixenhanced fluorophores are anticipated to be entrapped in the hydrophobic inner core of CHA, in which they undergo self-quenching due to their aggregation behaviors, but intracellular Hyal-1 can specifically disassemble CHA to release the quenched fluorophores, accompanying the fluorescence activation and further amplification assisted by abundant cytomatrix. To this end, it is prerequisite to acquire ideal signal molecules. We had previously synthesized an ethyl-substituted indole-based cyanine derivate, which exhibits weak fluorescence emission in aqueous medium.38 Interestingly, this cyanine dye fluoresces brighter in cell lysates than that in buffer solution (Figure S6). To reveal the puzzle of fluorescence enhancement, various cellular components including metal ions, anions, nucleic acids, proteins, glucose, phospholipids, and amino acids were considered. Among of them, only DNA and RNA dramatically increased the fluorescence (Figure S7-S10), suggesting nucleic acid binding could be the main reason for signal enhancement. Nonetheless, the non-overlapping of cell nucleus tracker DAPI and dye in cellular imaging pattern revealed that this dye owns robust staining ability in cytosol rather than cell nucleus (Figure 2A), and the fluorescence signal would



disappear in RNase-treated cells (Figure S11), indicating that this dye is sensitive to cytoplasmic RNA. It is further demonstrated by significant fluorescence enhancement of dye with cytoplasmic RNA extracted from cells (Figure 2B), the largest signal gain of over 10-fold can be achieved for 5 mg/mL cytoplasmic RNA (Figure 2C). In addition, CD spectra showed that in the presence of dye, there was significant change in typical ellipticity (θ) at 168 nm (negative) and 175 nm (positive) of RNA, identifying the interaction between dye and RNA (Figure S12). Therefore, this cyanine dye is an RNA-binding fluorophore (RBF). On account of its cytoplasmic RNA, RBF was chosen as the prototype of cytomatrix-enhanced fluorophore in our design plan. Subsequently, RBF molecules were integrated into CHA to prepare the nanosensor RBF@CHA. Zeta potential and DLS measurements reveal that the RBF@CHA possesses slightly increased size with attenuated zeta potential than CHA (Figure S13), demonstrating RBFs were loaded into the inner core of CHA. As expected, RBF fluorescence exhibit a dramatic decrease after it being wrapped by CHA (Figure S14), which may attribute to aggregation caused quenching (ACQ) effect via π-π stacking and hydrophobic interactions.39 The loading capacity of CHA toward RBF was estimated to be ～142 μmol/g (Figure S15), representing a foundation to achieve excellent sensitivity. This quenching effect combined with enhancement by cytoplasmic RNA would afford a very high sensitivity and large signal-noise ratio for the designed nanosensor. And then the design feasibility was verified by real-time dye release experiment. As shown in Figure S16, dramatic release of RBF could be observed for RBF@CHA in the presence of active Hyal-1 rather than Hyal-2. However, the release efficiency was remarkably decreased if denaturation of Hyal-1 by heat treatment, suggesting that the release quantity of signal reporter is Hyal-1 activity-dependent.


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Sensing Performances of RBF@CHA for Hyal-1 over Hyal-2. After verifying the feasibility, in vitro performances of RBF@CHA response to Hyal-1 over Hyal-2 were then evaluated. Figure S17 depicted that the free RBF@CHA fluoresced weakly and maintains steady under physiological pH range (3.0–8.0). In contrast, gradually incremental fluorescence was observed with increasing concentration of active Hyal-1 (0 to 16 μg/mL) into the buffer solution of RBF@CHA (Figure S18), and the corresponding signal to background ratio (S/B) increased from 1 to 12, where S/B = (F-Fblank)/(F0-Fblank),40 Fblank, F0 and F are fluorescence intensities at 540 nm of the blank, RBF@CHA free, and RBF@CHA treated with Hyal-1, respectively. As well as, its respond toward Hyal-1 is pH dependent (Figure S19), and the best response occurred at pH value of 5.0, consistent with the optimum pH condition for Hyal-1 hydrolysis.41 Compared with the sample in buffer, RBF@CHA display much stronger response to Hyal-1 in cytoplasmic RNA extracted from cells (Figure 3A), eliciting a more dramatic enhancement trend with maximum S/B value approximated to 120 (Figure 3B). This ultrahigh enhancement can be rationalized by cascade amplification process. First, the release of RBF resulted in 12-fold enhancement. Then, the binding of the released RBF with lysate RNA further enhanced 10-fold, reaching a total of 120-fold. There was an excellent linear relationship between the S/B values and low Hyal-1 concentrations ranging from 0 to 0.01 μg/mL (inset of Figure 3B). The detection limit (3σ/slope) was calculated to be 1×10-5 μg/mL. In contrast, Hyal-2 did not produce the striking fluorescence enhancement and large signal to background ratio observed for Hyal-1 (Figure 3C). Moreover, Figure 3D revealed that RBF@CHA was comparatively inert to the potentially competing species including typical reactive oxygen species hydrogen peroxide (H2O2), biothiol glutathione (GSH), and other enzymes (cathepsin, trypsin, thrombin, lysozyme, ribonuclease, galactosidase),



confirming the nanosensor responses to Hyal-1 with excellent specificity. Taken together, all in vitro results strongly manifested that RBF@CHA would be potentially adaptive for ultrasensitive detection of low-expression Hyal-1 over Hyal-2 and other enzymes. Specific Visualization of Hyal-1 in Live Cells. In view of excellent responsive behaviors of RBF@CHA toward Hyal-1 in vitro, we proceeded to explore its potential for fluorescence imaging of Hyal-1 activity in living cells. Cytotoxicity test using standard MTT assay demonstrated that RBF@CHA present minimal toxic effects on cell viability (Figure S20). HeLa cells, which overexpress hyaluronidase,42 were firstly chosen to carry out confocal fluorescence imaging. As shown in Figure 4A, HeLa cells themselves display rather weak background fluorescence. Whereas RBF@CHA-incubated HeLa cells generate strong fluorescence. Moreover, clear fluorescence disappears when dealing with indomethacin (a Hyal-1 inhibitor43). Moreover, flow cytometry analysis is consistent with the imaging results (Figure 4B). In contrast, there is no significant change in immunofluorescence of FITC-labeled hyaluronidase antibody after treatment with inhibitor (Figure 4C, Figure S21). The above results strongly support that internalization of RBF@CHA into cells and its possible reaction with intracellular Hyal-1 to generate fluorescence response. Although Hyal-2 was usually considered as a hyaluronidase isoform located on the surface of cell membrane, it is also present in lysosomes of cells together with Hyal-1.44 To further attest that RBF@CHA indeed responds Hyal-1 rather than Hyal-2 inside living cells, we stimulated HeLa cells with different treatments to regulate intracellular Hyal-1 and Hyal-2 expression. As shown in Figure 5A and 5B, compared to control, a significantly enhanced fluorescence can be observed within HeLa cells treated with UV-B irradiation, which is able to upregulate Hyal-1 and downregulate Hyal-2 simultaneously.45 In sharp contrast, immunofluorescence does not


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reveal such change, since FITC-labeled Hyaluronidase antibody do not possess the ability of distinguishing Hyal-1 from Hyal-2. However, fluorescence disappeared while cells were mineralized to specially downregulate Hyal-1 without influence of Hyal-2.46 Moreover, if cells were pretreated with interleukin-1(IL-1), a known reagent associated with up-regulation of Hyal2 level,47 no obvious fluorescence change can be found comparative to control. These fluorescence imaging results reveal the change of intracellular Hyal-1 expression, entirely consistent with Western blot analysis (Figure 5C, 5D). More importantly, it manifested that RBF@CHA was capable of selectively visualizing Hyal-1 over Hyal-2 in living cells. Recently accumulating evidences manifest that cancer cells, in comparison with normal cells, are associated with higher level of Hyal-1.48 Thus, we further tested HepG2 and L02 cell lines with RBF@CHA to determine differential expression of Hyal-1 between cancer and normal cell. Similar to HeLa cells, RBF@CHA-incubated HepG2 cancer cells exhibited much stronger fluorescence than themselves, as depicted in Figure S22. To our surprise, visible fluorescence signal also appeared in L02 normal cells incubated with RBF@CHA, despite of less than HepG2 cancer cells. In the contrary, immunofluorescence of FITC-labeled Hyaluronidase antibody did not appear in this case due to low Hyal-1 expression. The above experiments confirm that our nanosensor is sensitive enough to detect low level of intracellular Hyal-1, and could potentially be utilized to differentiate between normal and cancer cells based on their different Hyal-1 expressions. CONCLUSIONS In summary, by virtue of the differentiated hyaluronic acid degradation capabilities of Hyal1 and Hyal-2, we have developed a novel nanosensor combining amphiphilic HA-based



nanoassembly with RNA-enhanced dye for the selective detection of hyaluronidase isoform Hyal-1 for the first time. Due to taking the advantage of cytoplasmic RNA-assisted signal amplification, this nanosensor exhibits high fluorescence enhancement and ultrasensitivity with a detection limit of 1×10-5 μg/mL Hyal-1. The specificity of the sensor allows robust visualization of intracellular Hyal-1 over Hyal-2. Moreover, it was successfully applied to differentiate normal and cancer cells with different Hyal-1expressions, which would promote our better understanding of biological functions and related diseases of intracellular Hyal-1. Additionally, our strategy has the potential to be expanded to design specific and sensitive probes for other enzyme isoforms with different-sized catalytic reaction products. ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge on the ACS Publications website at DOI: materials and instruments, synthesis of Chol-NH2 and Chol-HA, examination of HA profile, experimental details on mineralization and UV-B irradiation and cytotoxicity assay, additional spectroscopic data and imaging patterns, and NMR and mass spectra. (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (S.Y.) *E-mail: [email protected] (R.Y.) Notes The authors declare no competing financial interest. 14 ACS Paragon Plus Environment

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ACKNOWLEDGMENT The authors are grateful for the financial support from the National Natural Science Foundation of China (21735001, 21505006, 91853104, 21575018), Hunan Provincial Natural Science Foundation of China (2017JJ3332), the Scientific Research Fund of Hunan Provincial Education Department (16C0033), and the Open Fund of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University (2015011, 2018009).

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Scheme 1. Schematic depiction of the response and amplification mechanism of amphiphilic HA-based nanoassembly RBF@CHA toward Hyal-1 over Hyal-2 inside living cells.



Figure 1. The size distribution of CHA (10 μg/mL) in the absence (A) and presence of Hyal-1 (20 μg/mL) (B) or Hyal-2 (20 μg/mL) (C) determined by DLS. Inset: The corresponding TEM images. Scale bars: 100 nm.


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Figure 2. (A) Confocal fluorescence images of HeLa cells incubated with nucleus tracker DAPI (0.5 μg/mL) and RBF (1 μM). Bar scale: 20 μm. (B) Fluorescence spectra of RBF (200 nM) in PB solution treated with different concentration of RNA extracted from HeLa cells (0-5 mg/mL). λex = 458 nm. (C) The corresponding fluorescence enhancement ratio (F/F0) of RBF, where F and F0 are the fluorescence maximum intensities at 540 nm in the presence and absence of RNA, respectively. λex/λem = 458 nm/490 nm.



4

B

Hyal-1(+)

120 90

3

30

2 1

60 0 0.00

4

RNA(-)

0 550

600

0

650

4

8

12

16

Hyal-1(μg/mL)

Wavelength(nm)

C

0.01

30

0

0 500

D 120

Hyal-2(+) 16 μg/mL

90

S/B

3 2 1

60 30

0

0

0 500

550

600

650

Wavelength(nm)

H ya H lC ya 1 at l-2 he p Tr si y n Th ps r in Ly om G so b i n al a zy R cto me ib s on id uc ase le as H e 2O G 2 SH

6

RNA(+)

16 μg/mL

S/B

6

Fluorescence(x10 )

A

Fluorescence(x10 )

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

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Figure 3. Fluorescence spectra of RBF@CHA (2 μg/mL) incubated with a series of concentrations of Hyal-1 (A) or Hyal-2 (C) ranging from 0 to 16 μg/mL in the presence of RNA extracted from HeLa cells (5 mg/mL). λex = 458 nm. (B) The corresponding fluorescence signal to noise ratio (S/B) of RBF@CHA (2 μg/mL) treated with increasing concentrations of Hyal-1 (0-16 μg/mL) in the presence and absence of RNA (5 mg/mL). λex/λem = 458 nm/540 nm. (D) The corresponding fluorescence signal to noise ratio (S/B) of RBF@CHA incubated with Hyal-1 (10 μg/mL), Hyal-2 (10 μg/mL), H2O2 (100 μM), GSH (10 mM), and other enzymes (cathepsin, trypsin, thrombin, lysozyme, galactosidase, ribonuclease, each at 10 μg/mL). Error bars indicate s.d. (n=3). λex/λem = 458 nm/540 nm.


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Figure 4. (A) Fluorescence images of HeLa cells under different conditions: (left) blank; (middle) incubation with nanosensor RBF@CHA; (right) incubation with nanosensor RBF@CHA in the presence of Hyal-1 inhibitor indomethacin. Scale bar: 20 μm. (B) Fluorescence histograms obtained by flow cytometry of (A). (C) Normalized fluorescence intensity of cells staining with nanosensor RBF@CHA (red channel) and FITC-labeled hyaluronidase antibody (green channel) under the same treatments. Error bars indicate s.d. (n=3). (red channel: ***p≤0.001; green channel: n.s.=no significance, #p≤0.05, ##p≤0.01).



Figure 5. (A) Fluorescence images of HeLa cells co-incubated with RBF@CHA (upper row) and FITC-labeled hyaluronidase antibody (lower row) after different conditions. From left to right: control; UV-B irradiation; mineralization treatment; in the presence of IL-1. Scale bar: 20 μm. (B) Normalized fluorescence intensity of (A). Error bars indicate s.d. (n=3). (red channel: *p≤0.05, **p≤0.01;

green channel: n.s.=no significance, #p≤0.05). (C) Western blot analysis of

Hyal-1, Hyal-2 and β-actin (internal reference) within cells after the same treatments as (A). (D) The corresponding Hyal-1 and Hyal-2 expression of (C). Error bars indicate s.d. (n=3). (blue channel: *p≤0.05; purple channel: n.s.=no significance, #p≤0.05).


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Differentiation of Intracellular Hyaluronidase Isoform by Degradable

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