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An approach to deliver two antioxidant enzymes with Mesoporous Silica Nanoparticles into cells Yu-Hsuan Lin, Yi-Ping Chen, Tsang-Pai Liu, Fan-Ching Chien, Chih-Ming Chou, Chien-Tsu Chen, and Chung-Yuan Mou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05834 • Publication Date (Web): 29 Jun 2016 Downloaded from http://pubs.acs.org on July 4, 2016

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ACS Applied Materials & Interfaces

An approach to deliver two antioxidant enzymes with Mesoporous Silica Nanoparticles into cells Yu-Hsuan Lin , Yi-Ping Chen*, , Tsang-Pai Liu , Fan-Ching Chien , Chih-Ming Chou⏊, Chien

§

‖

Tsu Chen⏊, and Chung-Yuan Mou*,

Department of Chemistry, National Taiwan University, Taipei 106, Taiwan

Graduate Institute of Nanomedicine and Medical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei 110, Taiwan §Mackay Junior College of Medicine, Nursing and Management, Taipei 112, Taiwan; Department of Surgery, Mackay Memorial Hospital, Taipei 104, Taiwan ‖Department of Optics and Photonics, National Central University, Taoyuan City 320, Taiwan

⏊Department of Biochemistry and Molecular Cell Biology, College of Medicine, Taipei Medical University, Taipei 110, Taiwan. KEYWORDS: mesoporous silica nanoparticle, antioxidant enzyme, cascade reaction, codelivery, reactive oxygen species, superoxide dismutase, glutathione peroxidase

ACS Paragon Plus Environment

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ABSTRACT

Reactive oxygen species (ROS) are important factors in many clinical diseases. However, direct delivery of antioxidant enzymes into cells is difficult due to poor cell uptakes. A proper design of delivery of enzymes by nanoparticles is very desirable for therapeutic purpose. To overcome the cell barrier problem, a designed mesoporous silica nanoparticle (MSN) system with attached TAT-fusion denatured enzyme for enhancing cell membrane penetration have been developed. Simultaneous delivery of two up-down stream antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase(GPx), reveals synergistic efficiency of ROS scavenging, compared to single antioxidant enzyme delivery. TAT peptide conjugation provided a facile nonendocytosis cell-uptake and escape from endosome while moving and aggregating along the cytoskeleton that would allow them to be close to each other at the same time, resulting in the cellular anti-oxidation cascade reaction. The two-enzyme delivery shows a significant synergistic effect for protecting cells against ROS-induced cell damage and cell cycle arrest. The nanocarrier strategy for enzyme delivery demonstrates that intracellular anti-ROS cascade reactions could be regulated by multifunctional MSNs carrying image fluorophore and relevant anti-oxidation enzymes.


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1. Introduction Targeted delivery of antioxidant enzymes, such as glutathione peroxidase (GPx) and superoxide dismutase (SOD), is a promising therapeutic approach for protecting tissues and organs from inflammation and oxidative stress.1 Antioxidant capacities of cells may be compromised in various disease states such as inflammation, vascular oxidative stress, ischemiareperfusion, and cancer.2, 3 A delivery of antioxidant enzymes may thus restore the balance in ROS production and annihilation.4 For example, it has been reported that suppression of the malignant phenotype of pancreatic cancer can be achieved by overexpressing glutathione peroxidase.5 The therapeutic applications of antioxidant enzymes are however hindered by several problems including inadequate delivery, stability, and immune response. Intracellular delivery of non-functionalized enzyme is a difficult problem because enzymes are not permeable to cell membrane. Delivery of cascade enzymes is even more difficulty. The delivered enzyme as part of inclusion body might not be active in the biological cell due to incorrect folding. Welldesigned nanomaterials as an enzyme carrier may overcome these problems with enhanced cell targeting/uptake mechanism and protection against protease digestion.6 There is a critical need for nanocarriers that will improve the intracellular accessibility of enzymatic therapeutics. Although stabilization of enzyme on solid supports has been of interest for many years in enzyme biotechnology,7-10 medical delivery of enzyme using nanocarrier for cellular delivery is not well-developed. The main problem is the selection of suitable nanocarrier for the enzyme immobilization and cell-uptake while keeping the enzyme active.11-13 Mesoporous silica nanoparticles (MSNs) possess many advantages as nanocarriers. It is known MSN is biocompatible. Secondly, it is easier to build multifunctionality in MSN due to its much higher surface area and uniform internal pores. In this study, internal pores of MSNs are functionalized


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with fluorescence molecules for particle tracking. Moreover, mesopores could be loaded with other drug molecules as another agent for future development. Recently, mesoporous silica nanoparticles (MSN) has been explored as enzyme delivery agent for SOD and catalase.14, 15 The MSN-bound enzymes have been shown to be active while protected from denaturation by trypsin or denature agents. Furthermore, the well-documented possibilities in building multifunctionality (including targeting, sensing and imaging capacity) with MSN are advantageous. Recently, we developed a unique delivery system for denatured protein which combined a MSN nanocarrier and 6xHis-TAT-SOD fusion protein which is produced by genetically engineered E. Coli.14 The six histidine groups linked to the protein provide the selectivity and oriented conjugation to the Ni-complex surface functional groups of MSN. Also, the designed tether in 6XHis-TAT-SOD provides a flexibility space for protein folding. The combination of Ni-Histagbioconjugation, nanomaterial, and genetic engineering provide a simple method for protein purification and delivery with the same nanomaterials. The 6xHis-TAT-SOD fusion protein, after E. coli overexpression, was conjugated, purified and isolated by using MSN-NTA-Ni via a one-step metal affinity binding at the same time. The combination of protein engineering and immobilization of enzymes could be a powerful tool for improving protein delivery and largescale preparation. The bio-conjugation method should be generally applicable to other engineered enzyme. Thus, we would like to further develop the conjugation method with the following goals: (i) it should be simpler, with one-step isolation, conjugation and protein denaturation at the same time; (ii) it should be demonstrated for other protein of different expressing type; (iii) it can be mass produced; and (vi) it should be capable of two or multiple enzymes delivery to regulate cascade reactions. To address the challenge, we chose to deliver


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two antioxidant proteins with cascade function for free radicals scavenging, e.g. superoxide dismutase (SOD) and glutathione peroxidase (GPx). In prokaryotic protein expression system, genetic proteins by molecular cloning are often expressed in the supernatants (supernatant’s type) and pellets (pellet’s type) of E. coli crude lysates based on their property. For example, SOD is a metalloprotein expressed in the supernatants (mainly) and pellet (partly).16 GPx, a special selenium-protein, is mainly expressed and precipitated in the E. coli pellets. The pellet type overexpression protein is more difficult to prepare; N-Lauroylsarcosine (sarkosyl) is commonly used to lyse the E. coli pellets and stabilize the expression protein activity, according to standard purification procedure of prokaryotic overexpressed protein system.17 One of the goals of the proposed study was to establish a method to verify that two kinds of genetic engineering protein containing supernatant or pellet’s types could be applied simultaneously for easy and quick preparation, without having to separate the types of protein overexpressed in E. coli. Then, the TAT-SOD or TAT-GPx protein functionalized MSN, named as MSN-TAT-SOD and MSN-TAT-GPx, would be denatured, conjugated and isolated at the same time by adding MSN-NTA-Ni nanoparticles. Hence, this protein conjugated nanocarrier system is simpler and easier than our previous method.14 The enzyme-nanoparticle conjugation method is general and applicable to different types of overexpressed protein by genetic engineering. Delivering two or more enzyme-nanoparticles at the same time would enable a sequential steps reaction.18 Simultaneous delivering of two cascade enzymes into biological cells have been recently reported using co-encapsulating two enzymes in a nanocarrier. Tanner et al. reported a coupled superoxide dismutase/ lactoperoxidase enzyme system working within triblock copolymers vesicle that could be serve as artificial peroxisomes to protect cells from ROS


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stress.19, 20 Chang et al used hollow silica nanospheres to co-encapsulate SOD and catalase for protecting against ROS.15 Successfully bringing about two enzymes playing cascade roles in catalytic reactions under a special confinement would represent a further achievement, but as of right now, the process is not well-understood. For example, we do not have detailed knowledge about the spatial distribution of the enzymes involved, nor the factors influencing the total catalytic activities. 21-24 Taking advantages of our system of recombinant protein production by genetic engineering, in this study we fabricated two differently conjugated MSNs with two different types of proteins and then delivered them into cells to investigate synergetic effects on the cascade reaction in cells.(see Scheme 1) We rationalize that these nanoparticles, when they are moving and aggregating along the cytoskeleton of the microtubule-organizing center (MTOC) after the delivery, could move close to each other and result in cellular cascade reaction at the short distance. The method of combining genetic engineering and simple bio-conjugation would allow its applications to other cascade enzyme reaction systems.

2. Experimental 2.1 Synthesis of MSN-NTA-Ni-TAT-Proteins. Details of synthesis of MSN-NTA-Ni were reported in our previous study.14, 25 To immobilize 6xHis-TAT-Proteins on MSN-NTA-Ni, the pellet of E. coli crude lysates which containing 6xHis-TAT-Protein were dissolved in 8 M urea and then mixed with nanoparticles at 4 ℃ for 2 h. The protein-conjugated particles were isolated by centrifugation and washed with ethanol. The protein-functionalized particles are denoted as MSN-TAT-Protein (MSN-TAT-SOD and MSN-TAT-GPx). 2.2 Characterization of mesoporous silica nanoparticles. Transmission electron microscopy (TEM) images were taken on a JEOL JSM-1200 EX II operating at 120 kV, and the samples


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were deposited on carbon-coated copper grids. The nickel amount of sample was determined by inductively coupled plasma mass spectrometry (ICP-MS) using Agilent 7700e instrument. Small fragments of the samples (1-2 mg) were dissolved in a mixture of HF and HCl solution for one day. These solutions were then diluted to 10 mL with 2 wt% HNO3 and filtered with 0.22 µm Millipore membrane before analysis. Size measurements were performed using dynamic light scattering (DLS) on a Malvern Zetasizer Nano ZS (Malvern, UK). Zeta potential was determined by the electrophoretic mobility and then applying the Henry equation on Malven Zetasizer Nano ZS (Malvern, UK). 2.3 Cell culture. HeLa cells, a human epithelial cervical cancer cell line obtained from the American Type Culture Collection (Manassas, VA), were maintained in Dulbecco's modified eagles medium supplemented (DMEM; GIBCO) with 10% fetal bovine serum (FBS; GIBCO), 100 U mL−1 penicillin, and 100 µg mL−1 streptomycin (GIBCO) at 37 °C in a humidified 5 % CO2 atmosphere. When adherent cells reached about 60–70 % confluence, they were detached with 0.25 % trypsin-EDTA growth medium to allow for continued passaging. 2.4 Western blotting analysis. Collected cell lysates were separated by 10 % SDS-PAGE and then transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane which was blocked in blocking buffer [1X Tris-buffered saline (TBS)- 0.1 % (v/v) Tween20, 5 % (w/v) non-fat milk] for 1 h. The membrane was incubated with primary antibodies against SOD (GenScript; 1:12000 dilute), GPx-1 (Cell Signaling Technology; 1:100 dilution), p-p38 (Cell Signaling Technology; 1:500), COX II (Cayman; 1:1000), α-tubulin (Oncogene Science; 1:24000) overnight at 4℃. The PVDF membranes were extensively washed and incubated with secondary immunoglobulin G antibody (1:2000 dilution, Santa Cruz) for 1.5 h at room temperature. Immunoreactive bands were visualized with the enhanced chemiluminescence


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substrate kit (Amersham Pharmacia Biotech, GE Healthcare UK Ltd, Bucks, UK) according to the manufacturer’s protocol. 2.5 Cell viability assay. 3×104 cells per well were seeded in 24-well plates for proliferation assays. After incubation with different amounts of nanoparticles suspended in serum-free medium for 4 h, N, N’-dimethyl-4, 4’-bipyridinium dichloride (paraquat) of 500 µM was added to the culture medium for 24 h. Particle-treated cells were then washed twice with phosphatebuffered saline (PBS) and incubated with 200 µL WST-1 (10%) in DMEM for 4 h. Cell viability was estimated by a formazan dye generated by the live cells and the absorbance at 450 nm was measured using a microplate reader (Bio-Rad, model 680). Cell numbers were determined from a standard plot of known cell numbers versus the corresponding optical density. Enhanced cell viability formulation was calculated as

A: cell viability (Abs. value) of adding nanoparticles P: cell viability (Abs. value) of treating with PQ only 2.6 Superoxide detection. The production of superoxide anion was fluorometrically estimated using a fluorescent probe, dihydroethidium (DHE) which is oxidized to a fluorescent intercalator-ethidium, by cellular oxidants which are superoxide radicals especially. Therefore, to measure superoxide anion generation, dihydroethidium (DHE; 2 µM; Invitrogen) was used. The increase in DHE fluorescence upon paraquat (superoxide anion generator) stimulation indicated an increase in superoxide anion levels. After various experimental treatments, cells were incubated with DHE for 20 min and then trypsinized, followed by cytofluorimetric analysis with a FACS Calibur flow cytometry (BD Biosciences).


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2.7 Cell cycle analysis. HeLa cells were seeded in 6 well plates (2×105 cells per well) for 24 h incubation at 37 ℃. Then, the medium was removed and cells washed in serum-free medium. HeLa cells were incubated with nanoparticles in serum-free medium for 4 h. Then, washed cells were exposed to 500 µM PQ in serum medium for 24 h. After washing and re-suspension, HeLa cells were analyzed by flow cytometry using propidium iodide (PI) staining. Statistical analyses were performed using ModFit LT software (Verity Software House, Topsham, ME). 2.8 Flow Cytometry Analysis. The uptake efficiency of nanoparticles by HeLa cells was determined by a FACS Calibur flow cytometry (BD Biosciences). The green emitting fluorescein dye (FITC) conjugated onto the MSNs serves as a marker to quantitatively determine their cellular uptake. 2×105 HeLa cells per well were seeded in 6-well plates and allowed to attach for 24 h. Cells were incubated with 25-250 µg mL-1 of nanoparticles in serum-free medium for 4 h and then washed twice with PBS and harvested by trypsinization. After centrifugation, the cells were resuspended in trypan blue solution to quench the FITC fluorescence of nanoparticles adsorbed on the cell surface and flow cytometry analysis was carried out. 2.9 Cellular uptake mechanism. For non-endocytosis pathway, HeLa cells were pre-treated with 4 ℃ condition or ATP-depletion solution (10 mM sodium azide and 6 mM 2-deoxyglucose) at 37 ℃ for 1 h. Then 50 µg mL-1 MSN-TAT-SOD was added and the incubation was continued for another 4 h. Finally, cells were analyzed by flow cytometry. To identify endocytosis pathway, we chose three inhibitors, 5 µg mL-1 filipin III, 30 µM chlorpromazine and 10 µM amiloride. After an hour pre-incubation with inhibitor in serum-free medium, the cells were treated with 50 µg mL-1 MSN-TAT-SOD and various inhibitor for 4 h respectively. After washing with PBS, the cells were trypsinized and were observed with flow cytometry.


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2.10 Method of super-resolution localization microscopy. For the super-resolution localization imaging by direct stochastic optical reconstruction microscopy (dSTORM), a set of fluorescence images was captured by the inverted fluorescence microscope (Olympus) with an excited configuration of total internal reflection fluorescence (TIRF), a dual view imaging system, an emission filter wheel, and a scientific complementary metal-oxide-semiconductor (sCMOS) camera (Andor). The magnification and numerical aperture of oil-immersion TIRF objective (Olympus) were 100X and 1.49. Before capturing the fluorescence images, the specimens were added a phosphate-buffered saline (PBS) solution with 30 mM of β-mercaptoethanol (BME) to make the fluorescent switching behavior of fluorophores in the dSTORM imaging. Three solid state lasers (CNI) with the wavelength of 473, 561, and 637 nm were used to excite the fluorescence signals of FITC, RITC, and Alexa fluor 647. The fluorescence signals were transmitted the dichroic beamsplitter (Di01-R405/488/561/635, Semrock) and split two channels by the dichroic beamsplitter (FF560-FDi01, Semrock) of dual view imaging system. One was filtered by the emission filter (FF01-525/45-25, Semrock) and the other was filtered by a filter wheel with the emission filters (FF01-593/46-25, and FF01-692/40-25, Semrock). After filtered the emission filters, the fluorescence images were recorded using the sCMOS camera with a sequential imaging procedure from Alexa Fluor 647 image, RITC image, to FITC image. Finally, the multicolor dSTORM images were achieved by the localization and reconstructed algorithm. 2.11 Statistical analysis. The data are given as mean ± standard deviation (SD). Statistical analysis was tested by the Student’s t-test. *p < 0.05 was considered statistically significant, and extreme significance was set at **p < 0.01.

3. Results and discussion


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3.1 Characterization of nanoparticles and TAT-fusion proteins. For TAT-SOD and TAT-GPX proteins conjugation, we constructed and overexpressed the His-tag human Cu, Zn-superoxide dismutase (SOD) and human glutathione peroxidase (GPx) which also contain a human immunodeficiency virus (HIV) transducing domain (TAT, 49-57). The genes of TAT-SOD and TAT-GPx were cloned and inserted into prokaryotic protein expression vector of pQE-30 to form pQE-TAT-SOD and pQE-TAT-GPx (Fig. 1a). The vectors were transfected into JM109 E. coli by culturing in a LB broth with IPTG protein induction for 1 and 3 h. The TAT-SOD and TAT-GPx with high protein overexpression were displayed in accordance with increasing induction time in 10 % SDS-PAGE electrophoresis (Fig. S1). To identify the over-expressed protein distribution, we separated E. coli crude lysates in the supernatants (supernatant’s type) and pellets (pellet’s type) by centrifugation after sonicating the lysate. Then, the pellets of E. coli crude lysates were treated with 8 M urea to dissolve the pellets and collected its supernatants after centrifugation. Electrophoresis revealed TAT-SOD was overexpressed in the E. coil supernatants and pellets. However, TAT-GPx shows the significant distribution in the pellets (mainly) of overexpressed protein (Fig. 1b). We then show these His-tagged proteins can be easily separated through bio-conjugation with MSN-NTA-Ni. FITC-conjugated MSN (MSN) was synthesized according to our previously reported methods.14 The NTA-Ni tethers were conjugated onto MSN to form MSN-NTA-Ni with an average loading of Ni at 0.6 wt % by ICP-MS analysis. The strong affinity between the Ni (II) and His-tag protein offered a tight linkage. To attach denatured proteins, the MSN-NTA-Ni was directly mixed with the pellets of E. coli crude lysates, which were dissolved in 8 M urea solution, including the over-expressed TAT-SOD or TAT-GPx proteins. The data about the zeta potential, size distribution by dynamic light scattering (DLS), nitrogen adsorption, and enzyme


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loading analysis are given in Table 1 and Supporting information (Fig. S2). We note there are small degrees of aggregations after loading of proteins(due to a decrease of zeta potential). But the aggregation is not too serious to keep them from cell-uptake. From TEM images (Fig. 1c, low-magnification images are shown in Fig. S3), the size of MSNs are general ~60 nm. DLS determination gives somewhat bigger size, indicating slight aggregation of MSNs in the medium. However, the MSNs are still well-suspended for good cell-uptake. The loadings of proteins on MSN-NTA-Ni, as dissociated from the linkage between tether and proteins by the action of 500 mM imidazole solution, was determined by Bradford assay. We found the weight percent of TAT-SOD and TAT-GPx on MSN was 1.19 wt% and 1.22 wt%, respectively. To identify the proteins associated with nanoparticles, we utilized Western blotting assay to examine the dissociated proteins (Fig. 1d) and confirmed the TAT-SOD and TAT-GPx were attached to MSN through the coordinated covalent bond between Ni (II) and 6xHis-tag. In brief, this method takes the advantage of one-step NTA-Ni conjugation and direct purification; it also could be applied to other insoluble proteins in protein expression system. 3.2 Deliver TAT-fusion denatured enzymes into HeLa cells. For delivering the MSM-TATSOD or MSN-TAT-GPx into HeLa cells, 25-250 µg mL-1 of MSM-TAT-SOD or MSN-TATGPx was added to a DMEM medium and incubated with HeLa cells for 4 h. Afterward, harvested cells were lysed with RIPA buffer containing 500 mM imidazole for dissociating proteins from MSN. Moreover, specific proteins in cell lysates were identified by Western blotting, and the results indicate that both MSN-TAT-SOD and MSN-TAT-GPx have been efficiently transduced into the HeLa cells in a dose-dependent manner (Fig. 2a). To further quantify cell uptake efficiency of MSNs, we incubated HeLa cells in culture medium containing different concentrations of nanoparticles (25-250 µg mL-1) for 4 h, and then using trypan blue


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staining method for quenching the autofluorescence of HeLa cells26. Not surprisingly, flow cytometry assay revealed that the cell uptake percentages increased with increasing concentrations of nanoparticles. (Fig. 2b) More specifically, the uptake of MSN-TAT-SOD and MSN-TAT-GPx reached a saturation level at 100 µg mL-1 and 50 µg mL-1 and the values are about 80% and 60%, respectively. Under the same condition, we also studied the cytotoxicity of MSN-TAT-Proteins using HeLa cells by the WST-1 method. Fig. S4 shows that no cytotoxicity was detected in the presences of MSN-TAT-SOD or MSN-TAT-GPx, indicating protein functionalized MSN have good cell biocompatibility. According to our previous report,14 we demonstrated that TAT-SOD on MSN could be successfully uptaken by HeLa cells giving good cellular SOD specific activity in a dosedependent manner. In this study, we anticipate that delivery of both TAT-SOD and TAT-GPx not only have catalytic activity but also may give a synergetic effect because the cascade updownstream reactions involved in anti-oxidation. To address this, MSN-TAT-SOD and MSN-TAT-GPx were delivered at the same time into HeLa cells and the specific activity was carried out individually (Fig. 2c). As expected, MSNTAT-SOD exhibited enzyme activity in a dose-dependent manner; however, we found the activity of MSN-TAT-GPx achieved saturation level at 50 µg mL-1. We suggested that the excess GPx might form inclusion body resulting in limitation of the total activity. As described above, both of the TAT-SOD and TAT-GPx proteins could be delivered into HeLa cells and restore their anti-oxidation activity. These enzymes are well-known antioxidant enzyme which are involved in scavenging free radicles in cells for protecting cell against ROSinduced stress. To evaluate the biological function of SOD and GPx as ROS scavenger, paraquat (N, N’-dimethyl-4, 4’-bipyridinium dichloride, PQ), a specific superoxide anion generator, was


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used to induce superoxide anion (O2-•) production. First, we treated cells with different concentrations of MSN-TAT-SOD or MSN-TAT-GPx for 4 h, then added 500 µM of PQ for another 24 h. The cell viability determined the cell protection ability. Fig. 2d shows that the treatment with MSN-TAT-SOD or MSN-TAT-GPx significantly increased the cell viability compared with the control MSN-NTA-Ni. Although both MSN-TAT-SOD and MSN-TAT-GPx enhanced cell viability in a dose-dependent manner, MSN-TAT-GPx did show better ability to protect PQ-induced ROS damage than MSN-TAT-SOD. One of the reasons is that MSN-TATGPx plays a role in quickly removed the hydrogen peroxide to avoid the Fenton and HaberWeiss reaction to generate the extremely hydroxyl radical (•OH-).27 Besides, the MSN-NTA-Ni control exhibited a slight anti-oxidation effect due to the mesoporous structure might adsorb some superoxide anion or paraquat directly in cells. 3.3 Co-delivering two MSN-TAT-Proteins and its synergetic effect. It is well-known that SOD and GPx are powerful up- and down-stream antioxidant enzymes and play critical roles in cellular defense against oxidative stress by cascade reaction.28 Therefore, the two enzymes are suitable to study intracellular cascade reactions. We delivered MSN-TAT-SOD and MSN-TAT-GPx into HeLa cells with a fixed total particles weight (50 µg mL-1) and chose the ratio of 0:1, 1:1 and 1:0 of SOD and GPx (e.g. for 1:1 case, MSN-TATSOD, 25 µg mL-1; MSN-TAT-GPx 25 µg mL-1) to investigate whether the combination can present the cascade reaction and enhance the cell viability after treating with PQ. Fig. 3a shows the comparison of beneficial efficiency for scavenging ROS and reducing cell damage of single enzyme delivery (25 or 50 µg mL-1) with simultaneous delivery of MSN-TAT-SOD and MSNTAT-GPx. To be specific, for single enzyme delivery, both MSN-TAT-SOD and MSN-TATGPx did enhance the cell viability to some extent. However, simultaneous delivery of MSN-


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TAT-SOD and MSN-TAT-GPx (ratio: 1:1) revealed a synergistic effect and helped cells against the oxidative stress significantly. To further verify the hypothesis of synergistic effect for ROS protection, ROS was stained with dihydroethidium (DHE) after the PQ treatment following detection by flow cytometry.29 In Fig. 3b, we found the same synergistic effect that the codelivery of MSN-TAT-SOD and MSN-TAT-GPx had the best ability for scavenging ROS than delivery of single type MSN-TAT-Proteins. Then, we also imaged the ROS production by fluorescence microscopy after the co-delivery two MSN-TAT-Proteins. The upper panel of Fig. 3c shows the intracellular distribution of MSN, and bottom panel displays ROS production level. HeLa cell as control had a weak background fluorescence resulting from cell metabolism,30 while treatment with 500 µM PQ (positive control) dramatically increased red fluorescence intensities, which indicate a large amount of intracellular ROS. In contrast, ROS fluorescence decline when HeLa cells were incubated in the presence of MSN-TAT-SOD and MSN-TATGPx (ratio: 1:1), implying the excellent protection ability of co-delivery strategy. Oxidative stress may lead to DNA damage31 and contribute to cell death, and the early effect can be evidenced in cell cycle progression. The influence of co-delivery of MSN-TAT-SOD and MSN-TAT-GPx on the change of cell cycle phase (G1,S andG2/M) was analyzed and quantified by flow cytometry. Under the oxidative stress condition, the accumulation of cells in S phase reflect that slow replication fork progression because of DNA damage which leads by ROS attacking.32, 33 As show in Fig. 3d-e, both control cells and MSN-TAT-Ni delivered cells have the same tendency on three phases after treating with PQ, displaying PQ induced S phase arrest that indicated cells cannot process from mitosis into G2/M phase. Nevertheless, co-delivery improved the accumulation phenomenon in the S phase and all the phase were progressed. Thus, the promoting proliferation property of co-delivery of MSN-TAT-SOD and MSN-TAT-GPx


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could be attributed to increase cell cycle progression (Figure 3a). With the MSN-based denatured protein delivery strategy, we have demonstrated co-delivery of two up-downstream enzymes has a beneficial ability for scavenging intracellular ROS. Because intracellular ROS could induce cell damage and affect a variety of signal pathways,3437

we examine if p-p38 and COX II protein expression levels could be up-regulated in HeLa

cells. As a stress-activated mitogen-activated protein kinase (MAPK) pathway, p-p38 protein is an important biomarker to detect and respond to intracellular oxidative stress.38-40 Additionally, ROS may trigger the pro-inflammatory responses and then induce cyclooxygenase II (COX II) generation which is an important inflammation biomarker.41 From the western blotting results of Fig. 3f, the expression levels of p-p38 and COX II in HeLa cell were enhanced after treating with PQ. Also, the negative control, MSN-NTA-Ni, did not inhibit the expressions of COX II and pp38 under PQ treatment. However, simultaneous delivery of MSN-TAT-SOD and MSN-TATGPx (ratio: 1:1) did inhibit COX II and p-p38 significantly, implying good protection ability under oxidative stress. Briefly, our results revealed that delivery of the two anti-oxidative enzymes enhanced the cascade reaction and protected cells from ROS attack. 3.4 The cell internalization mechanism and transport pathway of nanoparticles. Cellular uptake of nanoparticles can be classified into energy-independent (non-endocytosis) and energy-dependent (endocytosis) routes. Endocytosis often is the primary cell- uptake mechanism of nanoparticles which may involve different pathway such as caveolae-mediated, clathrin-mediated and macropinocytosis.42, 43 It has been reported previously that conjugation of TAT peptide can provide a non-endocytosis pathway and an endosome escape mechanism for nanoparticles.14, 44 In order to investigate the internalization mechanism of MSN-TAT-Proteins by HeLa cell, we delivered nanoparticles to inhibitor-treated cells (Fig. 4). The control means the


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cellular uptake of MSN-TAT-SOD (50 µg mL-1) without pathway inhibitors treatment at 37 ℃, indicating the result contains the endo and non-endocytosis uptake. Endocytosis is an energydependent uptake process; thus, we decreased endocytosis energy by lowering temperature to 4 ℃ 45 or by treatment with sodium azide and 2-deoxyglucose (ATP depletion).46 The results revealed about 60 % reduciotn in the cellular uptake, indiciating the majority of energydependent endocytosis was hindered effectively. We suggested the rest of MSN-TAT-SOD might go through another uptake pathway; e.g. by direct penetration into the cytosol. To further distinguish the three endocytosis pathways, e.g. caveolae-mediated, clathrin-mediated, and macropinocytosis, HeLa cells were treated with different inhibitors. The uptake of MSN-TATSOD was reduced by about 40% under the Filipin III treatment which has been reported to block speciﬁcally caveolae-mediated uptake.47 No signiﬁcant effect was observed on the uptake of MSN-TAT-SOD with the treatment of chlorpromazine, an inhibitor of clathrin-mediated pathway.48 However, while the treatment with amiloride, a well-known macropinocytosis inhibitor,49 the uptake of MSN-TAT-SOD was reduced approximately by 30%. Taken together, our data revealed that MSN-TAT-SOD was internalized by cells via both energy-independent (direct)

and

energy-dependent

(caveolae-mediated

endocytosis

and

macropinocytosis)

processes.50 It is interesting that clathrin-mediated endocytosis plays little role in the uptake of MSN-TAT-SOD. Previous reports indicated the different level of cellular uptake and routes of endocytic pathways may explain the difference in gene transfection efficiency by nanocarriers.51, 52

It has been reported that only caveolae-dependent uptake is highlighted with gene delivery and

subsequent gene expression, resulting from avoiding the endo/lysosomal degradation. By contrast, clathrin-dependent uptake would target to lysosomal compartment for degradation.51, 52 These findings offer the possibility of MSN-TAT-Proteins for increased enzyme activity due to


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Page 18 of 38

highly efficient uptake through a caveolae-mediated endocytosis pathway which avoids lysosomal fate which may be due to efficient endosomal escape to the cytosol. In an effort to explore why the synergistic effect in antioxidation could occur as shown in Fig. 3, the intracellular spatial distribution of MSN-TAT-Proteins was observed by using confocal microscopy images. HeLa cells were co-delivered with FITC labeled MSN-TAT-SOD and MSN-TAT-GPx (ratio: 1:1) for 4 h and stained with SOD (Alexa-568, blue) and GPx (Alexa-647, red) primary antibodies. Confocal microscopic images show both nanoparticles could enter HeLa cells with good uptake (Fig. 5 a-c). As shown in Fig. 5, the merged images (cyan) indicated TAT-SOD (blue) was co-localized with MSN (green). The merged images (yellow) displayed the overlapping of MSN (green) and TAT-GPx (blue) in Fig. 5e. The results of Fig. 5d-e revealed both proteins stayed on the MSN in HeLa cells. It is worth noting that many of MSN-TAT-SOD and MSN-TAT-GPx were co-localized in the cytoplasm from the colocalization images of MSN (green), TAT-SOD (blue) and TAT-GPx (red) (as indicated by the white arrow in Fig. 5f). The co-localization of the two enzymes could explain why the cascade reactions could occur efficiently. To confirm more the synergistic phenomenon, TAT-SOD and TAT-GPx were separately conjugated with different fluorescence nanoparticle (FITC-MSN-SOD and RITC-MSN-GPx) to trace their spatial distribution. Super-resolution (SR) localization microscopy was employed for imaging the system. Thus, after the co-delivery into HeLa cell, direct stochastic optical reconstruction microscopy (dSTORM), a kind of super-resolution localization microscopy, was utilized to observe the distribution of FMSN-TAT-SOD (FITC-labeled, green), RMSN-TATGPx (RITC-labeled, red) and α-tubulin (Alexa fluor 647-labeled, blue) with a nanoscale imaging resolution of 25 nm.53 In general, most nanoparticles internalized with to form vesicles in the


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initial endocytosis process. Nanoparticles in the vesicles then moved along the cytoskeleton such as microtubule (MT) into the microtubule-organizing center (MTOC) by kinesin and dynesin.5457

As shown in Fig. 6, we observed many of nanoparticles were moving and lined up along the α-

tubulin; the images revealed yellow dots on the α-tubulin, which indicate co-localization of FMSN-TAT-SOD and RMSN-TAT-GPx. Fig. 6a displays an example of co-localization of FMSN-TAT-SOD and RMSN-TAT-GPx not sitting on the α-tubulin, indicating TAT mediated non-endocytic or endosome escape mechanism could promote cytosolic co-localization where the cascade reaction will take place. Many more co-localizations of the nanoparticles could be observed on the α-tubulin (Fig. 6b-d). It has been long reported that there are strong interactions between TAT peptide and cytoskeleton; the resulting TAT peptide interaction with actin can remodel the cytoskeleton.58, 59 It could happen that TAT on the nanoparticles help them moving on the α-tubulin and the one-dimensional confinement along the tubule promotes further assist the co-localization. In brief, via TAT-mediated non-endocytic mechanism and caveolaedependent uptake, we demonstrated that TAT-MSN-protein could co-localize closely, and the cascade reactions are thus favored by co-delivery of two up-downstream anti-oxidant enzymes. Finally, we would like to discuss the relative merits of two different approaches to dual enzymes delivery: co-conjugation and simultaneous delivery. Co-conjugating two cascading enzymes on the same nanoparticle in principle could provide benefits of close proximity more. However, there are several drawbacks to using the co-conjugation approach. First of all, excess enzyme binding on the nanocarrier precludes the possibility of an increase in stabilization.60 Further, direct contact of multiple enzymes on a nanoparticle often leads to undesirable interactions and aggregations. Controlling the ratio of enzymes or the percentage of each enzyme on the nanocarrier is not easy for different batches and different protein sizes. Moreover, in our


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Page 20 of 38

MSN-NTA-Ni-Histag approach, we were not able to directly control the relative amount of the two enzymes immobilized on the same particle. For these reasons, co-delivery, or using separate nanoparticles to deliver two or more enzymes into cells at the same time is the preferred alternative. Our results indicate that there are enough spatial overlaps of the two enzyme carriers for effective cascade reactions to take place. Because of the independent fabrication processes of the two enzyme-MSNs, controlling the relative amount of the two delivered enzymes is straightforward. 4. Conclusions In this study, a general approach for multi-protein delivery for cascade enzymatic reactions in cells by using a nanoparticles strategy was reported. We chose E. coli overexpression system as our protein source in this work. We fabricated mesoporous silica nanoparticles (MSN) conjugated with two different expression type of genetic engineering proteins, superoxide dismutase (SOD) and glutathione peroxidase (GPx), respectively and then delivered both of them into HeLa cells. Later on the denaturation in 8 M urea, the linear conformation of enzymes not only increase penetration efficiency due to the highly positive charge of TAT peptide but could be restored native enzymatic activity by using the intracellular refolding mechanism. We demonstrated that the method is easy, efficient, and specific which allows an excellent access of the substrates to the delivered enzymes. The denatured up-downstream antioxidant enzymes could be refolded to restore their activity after cellular delivery, protecting the cell against ROSinduced stress and cell cycle arrest. TAT peptides provide a facile non-endocytosis cell-uptake and help dual enzymesMSNs could able to escape from endosome and move and aggregate along the cytoskeleton into the microtubule-organizing center (MTOC) that would allow them to be close to each other at the same time, resulting in the cellular anti-oxidation cascade reaction at a


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short distance. The protein delivery system using MSNs as nanocarrier could be a versatile platform technology for producing and delivering other intracellular catalytic system for a potential protein therapy approach in the future.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Data of mesoporosity data, nitrogen adsorption, Lowmagnification TEM images, TAT-fusion proteins overexpression gel, and cytotoxicity. Process of

plasmid

construction,

protein

expression

and

purification,

activity

assay,

and

immunocytochemical staining.

AUTHOR INFORMATION Corresponding Author * C. –Y. Mou. E-mail: [email protected] * Y. –P. Chen. E-mail: [email protected] ACKNOWLEDGMENT This research was funded by the National Taiwan University (104R7621) and the Ministry of Science and Technology (MoST) of Taiwan (MOST 103-2113-M-002-021-MY2). We also thank to Ms. Chia-Ying Chien for the assistance in the TEM experiments.


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Table 1. Physical properties of nanoparticles: Dynamic light scattering (DLS) for particle size and zeta potential for surface charge of MSN. Nickel content is determined by ICP-MS and protein amount is quantified by Bradford assay. DLS (nm)

Zeta (mV)

Ni (wt%)

Protein (wt %)

MSN-NTA

277.6(32.3)

19.3(0.2)

-

-

MSN-NTA-Ni

361.4(16.7)

-1.8(0.6)

0.6

-

MSN-TAT-SOD

599.3(49.2)

-8.38(0.3)

1.19 0.6

MSN-TAT-GPx

557.4(1.0)

-7.49(0.1)

1.22


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FIGURES

Figure 1. Characterization of nanoparticles and TAT-fusion proteins. (a) The gene construction of pQE-TAT-vectors. (b) The isolation of crude lysates from E. coli overexpression proteins in 10% SDS-PAGE. S: supernatants; P: pellets. (c) TEM images, low-magnification images are shown in Fig. S3 (d) Protein identified by using Western blotting. After the protein conjugation, TAT-SOD or TAT-GPx proteins were dissociated from MSNs via 500 mM of imidazole digestion.


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Figure 2. Delivery of TAT-fusion enzymes into HeLa cells. HeLa cells were treated with different concentrations of MSN-TAT-SOD and MSN-TAT-GPx (25-250 µg mL-1) for 4h, respectively. (a) Western blotting results for intracellular SOD and GPx levels after the delivery. (b) Cellular uptake efficiency was analyzed by using flow cytometry. (c) Enzymes activity assay. (d) The protection ability of MSN-TAT-SOD and MSN-TAT-GPx under 500 µM of paraquat treatment by using WST-1 assay. (*p

Approach To Deliver Two Antioxidant Enzymes ... - ACS Publications

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