Combining autophagy-inducing peptides and Brefeldin A delivered by perinuclear-localized mesoporous silica nanoparticles: a manipulation strategy for ER-phagy †

Yimin Wang, a Zhao Zhao, a Fujing Wei, a Zewei Luo, a and Yixiang Duan*a

Autophagic degradation of endoplasmic reticulum (ER-phagy) has been found to play a critical role in human sensory neuropathy. So far, however, specific and efficient intervention means for ER-phagy remains unexplored. Herein, Brefeldin A (BFA), a blocking agent on protein transport between ER and Golgi, was screened from ER stress inducers. BFA was then delivered to the perinuclear area co-localized with ER by mesoporous silica nanoparticles based drug-carrier functionalized with autophagy-inducing peptides of TAT-Beclin 1 (MSNs-BFA), to evoke a perturbation of ER-phagy. The molecular mechanism of ER-phagy regulated by BFA was explored by biochemical evaluation including time-lapse live-cell fluorescence imaging. We found that MSNs-BFA treatment caused a lower mRNA/protein expression level of FAM134b even under a compensation of autophagic flux in U2OS cells, and resulted in ER-expansion. The fragmentation of ER was blocked as a response to ER stress mediated by inactivation of AKT/TSC/mTOR pathway. Our work developed an efficient external manipulation strategy to regulate ER-phagy and may contribute to therapeutic application of autophagy-related major human diseases.

1 Introduction

Autophagy, also known as macroautophagy, is a lysosome- dependent catalysis, degradation and recycling process that maintains the homeostasis of organelles and macromolecules in eukaryote cells.1 Pathologic autophagy is responsible for a number of major human diseases including neurodegenerative diseases, cancers, cardiomyopathy, diabetes, and infectious diseases.2 Therefore, the coming of post-Nobel Prize era in autophagy pushes researchers to focus on the drug discovery field concerning these diseases.3, 4 In the last few years, the pathophysiological roles of autophagic degradation of ER membrane, which is called ER-phagy (or reticulophagy), have been uncovered both in mammalian cells/animal model and yeasts.5, 6 In these studies, ER-phagy was identified to play a significant role in ER size and quality control-related human sensory neuropathy. In pathological conditions, accumulation of a large number of misfolded proteins at ER may disturb the functional balance of ER and induce ER stress response.7, 8 As an abnormal phenotype, uncontrolled fragmentation of ER may also destroy the ER function and is responsible for nervous system related diseases, such as neuritic degeneration.9 However, little attention, so far, has been paid to develop an efficient modulating tool that can specifically regulate the activity of ER-phagy, thus making it out of reach for ER-phagy regulation based therapeutic applications. It has been known that ER stress may deactivate the mTOR pathway and induce autophagy, which is also closely linked to the ER expansion and ER-phagy.10, 11 Therefore, some small chemical molecules known as ER stress inducers including Brefeldin A (BFA, a blocking agent of protein transport between ER and Golgi), Thapsigargin (Tha, an ER Ca2+ ATPase inhibitor), Tunicamycin (Tun, blocking the N-linked glycosylation), and 1,4-Dithiothreitol (DTT, inhibiting disulfide bond formation and disturbing protein folding),12 are potential candidates for ER- phagy regulators.

Evidences in vivo concerning nanomaterial-associated autophagy modulation for medical use have been growing rapidly recently.13 Chen and his co-workers developed a nanocarrier system for sinomenium delivery based on chitosan microspheres and methacrylate hydrogel for treatment of osteoarthritis through inducing autophagy in mice.14 Wang et al. demonstrated an efficient interference method for inhibiting tumor development of breast cancer in vivo, in which a high level of autophagy was induced by micelle-like nanoparticles functionalized with autophagy-inducing peptide and pH-response polymers.15 As successfully and frequently used drug carriers with controllable mesoporous structures, high surface area, and easy surface functionalization, mesoporous silica nanoparticles (MSNs) have been attracted widespread interest during the past 10 years, especially in the cancer-targeted therapy field.16, 17 MSNs with diameter sizes below 50 nm have been synthesized by Pan L. and its co- workers for nuclear-targeted drug delivery, which is based on the fact that MSNs of 50 nm size exactly match the diameter of nuclear pore complexes and are able to pass through the nuclear membrane.18 Additionally, to facilitate the intracellular delivery of nanoparticles, a cell-penetrating peptide, such as the HIV-1 TAT peptide, has been used to conjugate with the MSNs.19 Based on those advantages as nanocarriers of drug delivery system, the biomedical applications of MSNs in previous work mainly focused on the therapeutic drugcarriers, intracellular/intravital imaging, and regeneration of artificial tissues.20-22 The applications of MSNs related nanocarrier as an autophagy-interfering tool, whereas have rarely been addressed.

Nanoscale

Scheme 1 Function model of BFA loaded drug-carrier system in regulating ER-phagy. The as prepared MSNs-BFA interacts with cells and is engulfed by cell membrane through endocytosis. BFA is delivered to the perinuclear ER by MSNs and then released between the nuclear membrane and ER, resulting in blocking of FAM134b-mediated degradation of ER fragments, which are sequestered into the phagophore, i.e., ER-phagy.

Considering the research status and application requirements mentioned above, in the present work, we successfully screened and identified a small chemical molecule, BFA, from several ER stress inducers. The nonselective autophagy-inducing peptide, TAT-Beclin 1 (TAT-B), a functional domain fragment derived from the autophagy protein of beclin 1, was synthesized to modify the surface of MSNs. Then BFA was loaded in the drug-carrier system of TAT-B@MSNs to obtain the perinuclear-targeted drug delivery complexes, namely MSNs-BFA, which was subsequently applied for manipulating the FAM134b-mediated ER-phagy in human cancer cells (Scheme 1).

2 Experimental
2.1 Materials

Chemicals. Rapamycin (Rap, MW=914.18, 99.30%), MG132 (MW=457.62, >97%), and Brefeldin A (BFA, MW=280.36, purity=99.01%) were obtained from Selleck Chemicals. Thapsigargin (Tha, MW=650.75, ≥98%) and (3-Aminopropyl) trimethoxysilane (APTMS, MW=179.29, 97%) were from Sigma-Aldrich. Tunicamycin (Tun, MW=840.00, ≥98%) Hexadecyltrimethylammonium chloride (CTAC, MW=320.00, ≥97%), Triethanolamine (TEA, MW=149.19, ≥99.0%) were all purchased from Aladdin Chemistry Co., Ltd (Shanghai, China). Bafilomycin A1 (BafA1, MW=622.83, ≥95%). 1,4-Dithiothreitol (DTT, MW=154.25, ≥97%). Chloroquine phosphate solution was obtained from Genechem Co., Ltd (Shanghai, China). The TAT-Beclin 1 (YGRKKRRQRRR-GG-TNVFNATFEIWHDGEFGT,
FITC-labeled and not) peptides referred from literature24 were customized by Genechem Co., Ltd and dissolved in phosphate- buffered saline (PBS, pH=7.4). Tetraethyl orthosilicate (TEOS, MW=208.33) was obtained from Kelong Chemicals Co., Ltd (Chengdu, China), all chemicals were used directly as received. Antibodies. Anti-FAM134b rabbit monoclonal antibody (ab151755), anti-SQSTM1/p62 mouse monoclonal antibody (ab56416), anti-Sec61A rabbit monoclonal antibody (ab183046) and Alexa488-labeled anti-rabbit IgG polyclonal secondary antibody (ab150077) were from Abcam. Anti-LC3B rabbit polyclonal antibody 1 was from Novus (#NB100-2220). Anti- LC3B rabbit polyclonal antibody 2 (#2775) and anti-phospho- AKT (Ser473) mouse antibody (#9271) were from Cell Signaling Technology. Anti-GAPDH mouse monoclonal antibody (#200306-7E4), FITC-conjugated goat anti-mouse IgG (#511101), and HRP-conjugated secondary antibodies of anti- mouse and anti-rabbit IgG were purchased from Zen BioScience (Chengdu, China).

Cell lines. Human osteogenic sarcoma cells U2OS, human lung carcinoma cells A549 and human hepatoma cells Huh-7 were purchased from Shanghai Zhongqiaoxinzhou Biotech. Co., Ltd. (Shanghai, China). All cell lines were maintained in DMEM medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin pre-mixed solution and 2 mM L- glutamine at 37 ℃ in a humidified atmosphere.

2.2 Synthesis of MSNs-NH2

MSNs-NH2 were synthesized by a classical method modified from reported work.18 Briefly, 4.0 g CTAC and 0.2 g TEA were added in 40 mL ddH2O in a 100-mL three-necked flask. The mixtures were heated to 95 ℃ and refluxed for 60 min under vigorous stirring with a Teflon rod. After that, 3 mL TEOS was added drop by drop and the system was stirred at 95 ℃ for another 60 min. The resulting product was washed with ethanol and water in turn by centrifugation at 14,000 g×15 min. Finally, the solid precipitate was extracted in 40 mL methanol solution consisting of 1% NaCl and 4 mL concentrated hydrochloric acid (v/v) at 75 ℃ under water reflux for 24 h to remove the excess CTAC. The pure MSNs powder was obtained via vacuum freeze-drying. To functionalize the amino group, 50 mL of ethanol solution containing 1 mg/mL MSNs and 100 μL APTMS was refluxed for 4 h at 78 ℃. The resulting products of MSNs-NH2 were centrifuged three times at 14,000 g×15 min in water and dried for the next use.

2.3 Synthesis of MSNs-BFA

Firstly, MSNs-NH2 was functionalized with nonselective autophagy-inducing peptides (TAT-B). 5 mg FITC-TAT-B, 38 mg EDC and 57.5 mg NHS were dispersed in 10 mL PBS (pH 7.4) and stirred for 15 min to activate the –COOH of the peptides. Subsequently, 10 mg MSNs-NH2 powder were added. And the above system was shaken at 120 rpm under room temperature for 24 h. Residual EDC, NHS and FITC-labeled peptides in the supernatant were removed from the FITC-TAT-B@MSNs by centrifugation and characterized by fluorescence spectra. For preparation of unlabeled MSNs, FITC-labeled TAT-B peptides were replaced by nude TAT-B peptides. At last, the small molecule drug of BFA was loaded onto the functionalized MSNs. Briefly, 5 mg BFA and 5 mg FITC-TAT-B@MSNs were co- incubated in PBS and shaken for 24 h at room temperature to obtain the drug-carrier complex.

2.4 Characterizations

Morphological analysis of MSNs and MSNs-BFA was obtained by transmission electron microscopy (TEM, Tecnai G2 F20STWIN, FEI, USA) and scanning electron microscopy (SEM, JSM-7500F, JEOL, Japan). The particle size distributions were measured by dynamic light scattering (DLS, ZEN3600, Malvern instrument, UK). The functional groups of MSNs and MSNs-BFA were characterized by Fourier transform infrared spectroscopy (FTIR) (Frontier FT-IR/NIR, PerkinElmer, USA) and fluorescence spectra (LS-55, PerkinElmer, USA). Thermogravimetric analysis (TGA) of MSNs, TAT-B@MSNs and MSNs-BFA was performed on a Mettler Toledo system (TGA/DSC2) at a heating rate of 10 ℃ /min. Surface area and pore size distribution of TAT-B@MSNs and MSNs-BFA were measured through N2 adsorption- desorption (Gemini VII 2390, Micrometrics, USA) and calculated by Brunauer-Emmett-Teller (BET) and Barrett- Joyner-Halenda (BJH) methods, respectively. For in vitro cytotoxicity evaluation of MSNs, TAT-B, TAT-B@MSNs and MSNs-BFA, the CCK-8 kit (Dojindo, Japan) was used following the recommended method of the manufacturer described in our previous work,25 and then performed on a microplate reader (Multiskan MK3, Thermo, USA).

2.5 Immunofluorescence

Target protein expression levels of U2OS and A549 were analyzed by immunofluorescence imaging. U2OS cells were treated with the indicated chemicals and drug-carrier system, fixed with ice-cold paraformaldehyde (PFA, 4%) for 10 min, permeabilized with 0.05% Triton X-100, and then blocked in 5% goat serum for 30 min. Primary antibodies including anti-LC3B (rabbit, 1:400), anti-p62 (mouse, 1:400) and anti-Sec61A (rabbit, 1:500) were used to probe the corresponding proteins for 60 min at room temperature. Then cells were labeled with FITC-conjugated goat-anti-mouse (1:500, probing p62) or Alexa488-conjugated goat-anti-rabbit (1:500, probing LC3B and Sec61A). For fluorescence co-localization of ER, the same cells were incubated alive with CellLight® ER-RFP molecular probe (BacMam 2.0, Life Technologies) for 16 h before immunofluorescence. After that, cell nuclei were stained with 10 μg/mL of Hoechst 33342 at 37 ℃ for 10 min. Images were acquired on a confocal/super-resolution microscope (N-SIM, Nikon, Japan).

2.6 Western blot

U2OS and A549 cells were treated with chemicals as indicated in the experimental results, and harvested by PBS washing and centrifugation at 200 g. Cells were lysed with RIPA (containing 50 mM Tris at pH 7.4，150 mM NaCl，1% Triton X-100，0.1% SDS, and 1% sodium deoxycholate ) and 1 mM protease inhibitor cocktail (ThermoFisher, USA). Total proteins were quantified by the BCA kit (ThermoFisher, USA) The cell lysis samples were denatured at 95 ℃ for 8 min and separated by SDS–PAGE. The proteins in the gel were transferred to the PVDF membrane. Immunoblot was carried out by incubating the membrane with the primary antibodies above (1:2,000 dilution) and the HRP–conjugated secondary antibodies (against mouse and rabbit, 1:5,000). Anti-GAPDH primary antibody (1:5,000) was stained as the loading control.

2.7 Autophagic flux and time-lapse live-cell microscopy

Autophagic flux was assessed by a tandem fluorescence construct RFP-GFP-LC3 by lentiviral transfection, which is described in our previous work.26 Cell lines transfected with RFP-GFP-LC3 probe by a lentiviral vector were treated with the indicated chemicals or drug delivery system for 8 h or specially indicated, and imaged on an inverted fluorescence microscope (IX83, Olympus, Japan) equipped on a spinning-disk confocal system. As an absolutely negative control in theory, a mutation at LC3 of RFP-GFP-LC3 construction (RFP-GFP-LC3-mut, data is shown in Fig. S1) was used. For live-cell imaging experiments, cells were cultured in a mini 5% CO2 incubator containing humidified atmosphere at 37 ℃, and observed on the same microscope above (60×oil objective lens, NA=1.4). An EMCCD camera (Andor iXon Ultra) was used to record the time-lapse images. And the real-time videos were acquired using the Olympus cellSens software.

2.8 SiRNA transfection

For FAM134b gene silencing, an anti-FAM134b small interfering RNA (Forward, 5’-GGACUGAUAAUGGGACCUUTT-3’ Reverse, 5’-AAGGUCCCAUUAUCAGUCCTT-3’) was screened from three synthesized target siRNA candidates and a scrambled control siRNA (Table S1) and transfected in U2OS cells with Lipo6000 (Beyotime Biotech., China) following the manufacturer’s instructions.

2.9 Real-time quantitative PCR (qPCR)
SYBR green based qPCR was carried out as the procedures modified from previously described methods on the LightCycler® 96 instrument (Roche, Shanghai, China).27 RNA preparation, reverse transcription (RT–PCR) and qPCR experiments were performed using the Roche RNA isolation kit, cDNA synthesis kit and qPCR mix, respectively. The mRNA expression levels of FAM134b was quantified as relative values to that of control gene (GAPDH) based on the double-standard curves method. Primers for PCR are as following: GAPDH (Forward, 5’-TGATGACATCAAGAAGGTGGTGAAG-3’; Reverse, 5’- TCCTTGGAGGCCATGTAGGCCAT-3’), FAM134b (Forward, 5’- TGACCGACCCAGTGAGGA-3’; Reverse, 5’-GGGCAAACCAAGGCTTAA-3’). Reaction conditions of q-PCR are as following: initial denaturation (95 ℃ for 600 s), 45 cycles of 3 steps amplification (95 ℃ for 10 s, 55 ℃ for 10 s and 72 ℃ for 10 s), and melting (95 ℃ for 10 s, 65 ℃ for 60 s, and 97 ℃ for 1 s). Reaction for each group was repeated three times to achieve a mean ±SD.

2.10 TEM analysis of ER expansion

U2OS cells treated with DMSO or MSNs-BFA were collected and centrifuged to pellets. Then cells were fixed with 2.5% glutaraldehyde, embedded in epoxy resin and sectioned into a series of ultrathin slices. Ultrastructural structures were observed under TEM.

2.11 Statistical analysis

Images of immunoblot were processed by the AzureSpot software. For fluorescence imaging, pictures were acquired from three random areas under the scanning field. Unless indicated, quantitative experiments were repeated independently for three times to reach the reproducibility (mean±SD). P<0.05 was defined as a statistically significant difference.

3 Results

3.1 Screening and Identification of BFA from ER stress-inducers

At the beginning of this research, four commonly used compounds which are known for inducing ER stress were used as candidates of potential pharmacological manipulation tools for ER-phagy. Immunofluorescence imaging shows that cells treated with these compounds exhibit an increase in proteins expression level of LC3B (Fig. 1a). In immunoblot experiments (Fig. 1b), 10 μg/mL of BFA induces a higher rate of LC3II/LC3I than the untreated control, which indicates a promotion of autophagy intensity. On the other hand, cells treated with BFA exhibited a lower expression level of FAM134b, which is a receptor mediating degradation of ER membrane, indicating a deactivation of ER-phagy. Fig. 1c shows the expression changes of autophagy-related proteins in U2OS cells treated with TAT-B peptides. In addition, the LC3II/LC3I ratio increased from 0.29 to 0.49 under the incubation of peptides. And then co- treatment of Chloroquine (Chl), a compound that inhibits the fusion of autophagosome and lysosome, further increases the ratio of LC3II/LC3I, suggesting that TAT-B peptides promote the autophagic flux. Above all, the mRNA expression level changes were also verified by qPCR. As shown in Fig. 1d, the treatment of BFA led to the most evident downregulation of FAM134b genetic transcription comparing with the other groups, which is consistent with the results in FAM134b proteins expression. Cells stably expressing a tandem fluorescence protein construct, RFP-GFP-LC3, exhibited an evident accumulation of RFP+/GFP--LC3 spots (Red) under the treatment of BFA (Fig. 2a), indicating an increasing amounts of autolysosome. A live-cell imaging based time-lapse video confirmed this result (Fig. S2 and Movie S1). We also compare the ER-phagy regulation effects between Tun and BFA treatments and found that BFA resulted in higher LC3 and p62 levels than that of Tun, which is in accordance with the qPCR results explained above (Fig. 2b). Additionally, we compare the effects of TAT-B peptides and BFA treatments on protein expression levels of FAM134b. As shown in Fig. 2c, after treating TAT-B peptides alone or with BafA1 (an inhibitor of acid hydrolase in lysosomes that disables the fusion of lysosome and autophagosome), no significant change was found in FAM134b expression, suggesting that peptides alone have little effects on ER-phagy. However, for cells treated with different concentrations of BFA, the FAM134b levels decreased in all groups (Fig. 2d).

Fig. 1 Drug screening from ER stress inducers and autophagy-inducing peptides. (a) immunofluorescence staining of LC3 protein expression of U2OS cells treated with DMSO (negative control), Rap (positive control, 500 nM), Tha (1 μM), Tun (2 μg/mL),U2OS cells treated with four ER stress inducers (b) and TAT-beclin 1 peptides (10 μg/mL) (c). Chl (50 μM) was used as control. (d) qPCR analysis of relative mRNA expression levels. Data represent mean ± SD. n=3. Statistical significance comparisons were analyzed by unpaired T-test. (*) P<0.05; (**) P < 0.01.

3.2 Preparation of BFA-loaded drug-carrier system

The morphological characterizations of as-synthesized MSNs were achieved with SEM, DLS, and TEM. As shown in Fig. 3a, the average size of nude nanoparticles is ~70 nm in SEM image. The average diameter size of MSNs-BFA measured by DLS in Fig. 3b is larger than the measurement results of TEM. This is due to the hydrodynamic environment (for example, formation of hydrated layers) around the particles.28 TEM images in Fig. 3c show the high-resolution of particle morphology. The porous-like nanostructures are apparent on the surface of the MSNs and are pointed out by triangles (part C in Fig. 3c). Correspondingly, the BFA loaded particles exhibited much less mesopores and the average diameter of MSNs-BFA is about 72 nm, which is a little larger than nude MSNs, which is due to the loading of BFA and coating of TAT-B peptide chains (arrows in Part D of Fig. 3c).29 Fig. 3d shows the FTIR spectra of BFA, MSNs-NH2 and MSNs-BFA. The absorption BFA (10 μg/mL), and DTT (4 mM) for 8 h. Immunoblot of ER-phagy-related proteins of band at 1542 cm-1 in MSNs-NH is due to the bending vibration.

Based on these results that BFA promoted the accumulation of autolysosomes and may inhibit ER-phagy, and the TAT-B peptides promoted the autophagic flux, BFA and TAT- B peptides were selected as an ER-phagy modulator and an autophagic flux promoter for following uses.

Fig. 2 Regulation of autophagy by BFA and TAT-B. Autophagic flux and ER-phagy affected by BFA treatment (a). Western blot images of U2OS lysates treated with BFA versus Tun (b), BFA versus TAT-B (c) and different concentrations of BFA as indicated (d). BafA1 (100 nM) treatment was used as control. Scale bar, 10 μm.

Fig. 3 Characterizations of MSNs and MSNs-BFA. MSNs were characterized by SEM images (a) and DLS (b). (c) TEM images of MSNs (A) and MSNs-BFA (B). Triangles indicate the pores in the nanoparticles, and arrows indicate the peptides attached on the surface of the nanoparticles, which is also decorated with BFA. (d) FTIR spectra of MSNs, BFA and MSNs-BFA. (e) Fluorescence spectra of FITC-TAT-B, FITC-TAT-B@MSNs- BFA and supernatant over the FITC-TAT-B@MSNs-BFA after centrifugation. Cytotoxicity evaluation for U2OS cells treated with different concentrations of MSNs for 24 h and 48 h (f) and MSNs-BFA for 12 h and 24 h (g). Data were shown as mean ± SD (n=3). Two- way ANOVA with multiple comparisons was used. NS, no significant difference; (*) P < 0.05, (****) P < 0.0001. Scale bars, 100 nm in (a), 50 nm in c(A and B) and 10 nm in c(C and D).

3.3 Regulation of autophagic flux by MSNs-BFA

As shown in Fig. 4a, an increase accumulation of total LC3 was observed when U2OS cells were treated with BFA alone or MSNs-BFA. The LC3 level of cells treated with MSNs-BFA is even higher than that of cells treated with BFA alone, which is attributed to accumulation of autolysosomes by BFA induction and the promotion of autophagic flux by TAT-B peptides.24 To further validate these results, MG132, a commonly used inhibitor of proteasomes, was employed to investigate the effects of MSNs-BFA on nonselective autophagy, which is also called bulk autophagy (hereafter named autophagy for short). In Fig. 4b, cells treated with MSNs-BFA alone exhibit a higher expression level of p62, which is an autophagic degradation substrate; and the co-treatment of MG132 or free BFA exhibited a further increase of p62 level, indicating that MSNs- BFA treatment drives the sequestration pace of cellular cargo during the initial stage of autophagy but not inhibits the degradation process in autolysosomes at late stage of autophagy. Additionally, a dynamic and real-time evaluation of autophagic flux was carried out by a long-time live-cell observation. As shown in Fig. 4c, a large number of RFP+/GFP-- LC3 dots (red) that indicate the autolysosomes accumulate in cells treated with MSNs-BFA for 4 h. However, with co- treatment of BafA1, no evident difference of fluorescence intensity between RFP-LC3 and GFP-LC3 was observed in the first 4 h (yellow dots). The total number of fluorescence dots at 4 h is more than that in the untreatment group, suggesting a nonselective autophagic induction at the early stage with MSNs-BFA treatment. The whole dynamic processes of live- cells by time-lapse video microscopy were presented in Movie S2 and Movie S3.

Fig. 4 Regulation of autophagic flux by MSNs-BFA. (a) LC3 proteins expression levels of U2OS cell lysates treated with BFA, MSNs and MSNs-BFA. (b) Co-treatment of MSNs- BFA, MG-132 and BFA. P62 and LC3 proteins were analyzed by immunoblot. (c) Time- lapse observation of live-cell imaging of RFP-GFP-LC3 expressing U2OS cells treated with MSNs-BFA or MSNs-BFA+BafA1. Line region profiles on the right panels show the fluorescence intensity of GFP and RFP channels along the white arrows. See Movie S2 and Movie S3 for more information in online supplementary materials. Scale bars, 5 μm in left three channels and 2 μm in the inset panel.

Fig. 5 Autophagic flux detection with nuclear localization of U2OS cells treated with MSNs-BFA. (a) Autophagic flux detection of RFP-GFP-LC3 and co-localization of nuclei (Hoechst) in U2OS treated with nude MSNs or BFA. (b) Fluorescence co-localization of autophagosome (RFP) and MSNs-BFA (FITC) in U2OS cells treated with FITC labeled MSNs-BFA. Rap treatment was used as control. Scale bar, 5 μm.

3.4 ER co-localization imaging of U2OS cells treated with MSNs- BFA

To identify the subcellular locations of MSNs-BFA in U2OS, a confocal microscope was used. Cells treated with 500 nM Rap for 8 h were employed as the positive control. The RFP+/GFP+- LC3 dots (yellow) increased in cells treated with BFA alone or Rap (Fig. 5a). And most of the LC3 dots localized around the nuclei, which is stained with Hoechst (blue). Cells stably expressing RFP-LC3 were treated with FITC-TAT-B@MSNs-BFA. Compared with the free BFA, larger LC3 dots aggregate at the perinuclear area and co-localized with FITC-TAT-B@MSNs-BFA. In addition, a part of FITC-TAT-B@MSNs-BFA was found entering through the nuclear membrane under co-incubation of Rap, suggesting a more efficient targeting behavior to perinuclear area (Fig. 5b). As is known that the perinuclear ER membrane is usually conjugated with the nuclear membrane, an ER probe of RFP was used and co-stained with LC3 and p62 by immunofluorescence imaging. Compared with control groups, more autophagic compartments were found in cells treated with MSNs-BFA. However, most of these compartments were still not co-localized with ER, demonstrating that a higher nonselective bulk autophagy level, but not selective ER-phagy, exists near the ER in cells incubated with MSNs-BFA (Fig. 6).

Fig. 6 ER co-localization imaging of U2OS cells treated with MSNs-BFA. Immunofluorescence staining of LC3 and p62 co-localized with the ER probe (ER-RFP). Nuclei were stained with Hoechst (blue). Scale bar, 5 μm.

3.5 Regulation mechanism of MSNs-BFA induced ER-phagy

Since the ER membrane expansion is related with ER stress- induced ER-phagy, Sec61A, an ER-resident protein, was used as the marker of cisternal structures of ER.33, 34 Compared with the negative control group treated with DMSO, both BFA and MSNs-BFA treatments caused a strong ER expansion in U2OS cells (Fig. 7a), which indicate an intense stress response at the ER membrane. FAM134b-targeted knockdown experiments mediated by siRNA were used as positive control, confirming that a low expression level of FAM134b leads to ER expansion. Consistent morphologic characteristics were observed in ultrastructures of TEM images, in which cells stimulated with MSNs-BFA exhibited expanded ER in the perinuclear area and plenty of autophagosomes (Fig. 7b).

To identify the molecular mechanism of MSNs-BFA regulated ER stress and ER-phagy, we analyzed the expression of p-AKT proteins, whose downregulation plays a primary role in ER stress-induced autophagy,35 in A549 cells exposed under MSNs-BFA. We found that cells treated with MSNs-BFA exhibited a significant lower expression level of p-AKT, which is consistent with that of cells treated with Rap, a classic inhibitor targeting mTOR pathway.36 However, treatment of MSNs alone even caused an upregulation of p-AKT (Fig. 7c). These results were verified by the previous work,11 which demonstrated that blocking AKT/TSC/mTOR pathway triggers the ER stress-induced autophagy. Accordingly, to test whether the blocked degradation of ER fragments is recovered in this autophagy-inducing peptide-induced autophagy, we further investigated the relationship between ER-phagy receptor FAM134b (whose degradation represents the induction of ER- phagy5) and the autophagy-related marker proteins of LC3B and p62 under treatments of MSNs-BFA with or without BFA. It was found that cells treated with MSNs-BFA suffered a decrease of FAM134b proteins expression. However, those marker proteins including p62 and LC3 increased under the same treatment, indicating a promotion of autophagic flux, comparing with the negative control treated with DMSO. These results suggest that the downregulation of FAM134b induced by MSNs-BFA exists even under an autophagic compensation caused by TAT-B peptides. Interestingly, co- incubation of MSNs-BFA with free BFA exhibited a further decrease of FAM134b level (Fig. 7d). Finally, we designed a comprehensive control experiment in which co-treatment of drugs including BFA and MG132 was carried out to explore the attenuation effect of MG132 against MSNs-BFA on regulation of ER-phagy. Consistent with our initial hypothesis, MG132 blocked the degradation of FAM134b, even under the addition of MSNs-BFA. Cells co-treated with MG132 and BFA exhibited no downregulation of FAM134b anymore. Similarly, co- treatment of MG132 and MSNs-BFA resulted in a double-level of the FAM134b comparing with the control group treated with DMSO (Fig. 7e). These results demonstrate that the decrease of FAM134b level caused by MSNs-BFA treatment is resulted from inhibition in upstream transcriptional regulation of FAM134b, but not an accelerated degradation of FAM134b proteins in ER-phagy.

4 Discussion

So far, there are few chemical molecules that are able to specifically target a designated kind of selective autophagy, resulting in a challenge in drug development for therapy of autophagy-related human diseases, such as cancer and neurodegenerative diseases.37 Based on the results in this work, we have successfully identified a small chemical molecule, BFA, which negatively drives ER-phagy both in U2OS and A549 cells, from the candidates of ER stress inducers. BFA is a small molecule metabolism product from Penicillium brefeldianum that has been proved to be a useful blocking agent in protein secretion in vesicular trafficking system both in mammalian cells and plant cells.38, 39 A series of biochemical evaluation methods including immunofluorescence imaging, western blot and qPCR have been used to demonstrate that MSNs-BFA promote autophagy from early to late stage, resulting in an accumulation of autolysosomes. However, BFA negatively regulates ER-phagy by inhibiting the expression of FAM134b, which depends on downregulation of p-AKT pathway. On the other hand, the co-treatment of the autophagy-inducing peptides compensates the balance of autophagic flux to maintain cell viability and reinforces the interference effect of MSNs-BFA on ER-phagy (Scheme 2). Consistent with these results, it is worth noting that non- colocalization of fluorescence between ER probe and autophagic marker LC3/p62 was observed in Fig. 6, indicating that nonselective autophagy and selective autophagy could be induced and inhibited respectively via independent mechanisms at the same time in U2OS cells.

Fig. 7 Regulation mechanism of ER-phagy by MSNs-BFA. (a) Immunofluorescence imaging of Sec61A in U2OS cells treated with BFA or MSNs-BFA for 8 h. Cells transfected with FAM134b-targeted siRNA were imaged as positive controls. Scale bar, 5 μm in the middle two panels and 2 μm in inset panels. (b) Ultrastructural analysis of MSNs-BFA induced ER expansion by TEM. White arrows in b1 and red arrows in b2 indicate normal ER and expanded ER, respectively. White arrowheads in b3 and b4 indicate MSNs-BFA particles colocalized with autophagosomes. Red arrowheads in b2 and b3 indicate autophagosomes. Scale bar, 1 μm in b1-b3, 0.5 μm in b4 and enlarged images. (c) Cell lysates treated with MSNs-BFA were probed with p-AKT antibody. DMSO and Rap (500 nM) treatments were set as negative and positive controls, respectively. Ratios show the relative values of p-AKT to the loading control of GAPDH. (d) ER-phagy evaluation in U2OS cells treated with MSNs-BFA. (e) ER-phagy evaluation under co-treatment of MG132, BFA, and MSNs-BFA in A549 cells. Nuclei were stained with Hoechst (blue).

Owing to the controllable sizes of diameter and mesoporous structures, MSNs were prepared by a strategy modified from the CTAC template based method,40 and employed as a nanocarrier to load BFA, which was screened from ER stress inducers. Unlike the nuclear-targeted property of MSNs (<50 nm) described in previous work,41 we found that MSNs with average size at 72 nm (taking into consideration the peptides modification), which are larger than the nuclear pore complexes, have the advantage of perinuclear-localization. Additionally, it has been discovered in previous reports that a nutritional sufficiency of amino acid signal (for instance, by addition of 2 mM L-glutamine in our work) maintains the perinuclear distribution of mTOR complex.42, 43 Owning to these advantages and the assistance of TAT-B peptides, the nanocarrier was able to “ship” and “spit out” BFA molecules to the perinuclear area, where is co-localized with the membrane of perinuclear ER. This controlled release results in ER expansion and stress, thus modulating ER-phagy specifically (Fig. 8).

Scheme 2 AKT/TSC/mTOR signaling pathway involved in FAM134b-mediated ER-phagy process regulated by MSNs-BFA.

FAM134b, the selective receptor of ER-phagy, maintains morphological feature and physiological function of ER through ER-phagy, thus can be used to characterize the intensity of ER stress-induced ER-phagy.44 As indicated in the biochemical evaluation methods used in this work, the drugcarrier system of MSNs-BFA has been proved to inhibit FAM134b mediated ER-phagy under a promotion of the early stage of autophagic flux and the accumulation of autolysosomes. This perinuclear-targeted drug delivery strategy combining autophagy-inducing peptides and ER stress inducers of BFA could block the fragmentation of ER in autophagic activities, which maintains the physiological function of ER. In other words, the promotion of autophagy flux by TAT-B peptides balances the drugcarrier complex- inhibited ER-phagy, which is essential for cell survival. In fact, the autophagic intensity induced by BFA was concentration- and time-dependent in our work (Fig. 2d and Fig. S2). However, a high concentration (>20 μg/mL) and a long-time (up to 48 h) co-incubation of the nanocarrier resulted in evident cytotoxicity in U2OS cells (Fig. 3f), a phenomena that has been received a widespread attention.45-47 Considering safe application of MSNs in our work, 10 μg/mL of MSNs-BFA was used throughout the biological evaluation. Moreover, previous reports show that autophagy, especially for an overhigh activity of autophagy induced by TAT-B peptides, may cause cell death.48, 49 The biocompatibility evaluation of MSNs-BFA shows that MSNs-BFA causes no evident cytotoxicity at 20 μg/mL (Fig. 3g). However, due to the phototoxicity in live-cell observation, the cytotoxicity appeared after treating for 12 h (Movie S1). Therefore, the time-lapse video of the first 8 h during co-incubation of MSNs-BFA and U2OS cells, which caused no evident cell death and is sufficient for observing autophagy process, was suitable for the subsequent use.

Since basic level of autophagy occurs in U2OS cells, the detection of autophagic flux in drug treated condition becomes knotty. In Fig. 4c, for example, the fluorescence intensity of autophagic dots seems high even in the control group before drug treatment, thus making it puzzling how to distinguish between autophagy induction and autophagy blocking. To overcome this limitation of naked-eye observation, we plotted a line region to profile the substantial fluorescence intensity of RFP and GFP channels by using the CellSens software. Results in the line profiles (right panel of Fig. 4c) verify the fact that the RFP+/GFP- dots significantly increased in cells treated with MSNs-BFA for 4 h, comparing with the control group in which the RFP and GFP exhibited nearly the same fluorescence intensity, which represents the basic autophagic flux. As the GFP fluorescence is quenched in acid environment of autolysosomes, the RFP+/GFP- dots (red) represent the late stage of autophagy, while the RFP+/GFP+ dots (in yellow) indicate the early stage of autophagy. Therefore, the autophagic flux at different stages can be characterized by this line region profiling method above. Additionally, the time-lapse live-cell imaging performed in another commonly used cell line, Huh-7, confirmed the feasibility of this method in evaluating autophagic flux (Fig. S6).

5 Conclusion

In this study, all the results reveal that we have developed an efficient intervention strategy for ER-phagy by using the autophagy- inducing peptide functionalized MSNs nanocarrier system with average size at 72 nm. The diameter-dependent nanoparticles ensure a perinuclear-localized distribution of BFA, and entitle the drug delivery system to a perinuclear ER-targeted ability, which finally facilitates specific manipulation of ER-phagy. Thus the size of ER can be stabilized by blocking the fragmentation of ER. Our work provide a potential therapeutic proposal in designing chemical drug concerning therapy of ER-phagy and ER fragmentation related major human diseases.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was financially supported by the Fundamental Research Funds for the Central Universities (No. 2012017yjsy154), the National Recruitment Program of Global Experts (NRPGE), and the Key Research and Development Project from Department of Science and Technology, Sichuan Province (2017SZ0013), PR China.

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