Lysosome Escape

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Pre-occupation of Empty Carriers Decreases Endo/Lysosome Escape and Reduces the Protein Delivery Efficiency of Mesoporous Silica Nanoparticles Wenqing Li, Liping Sun, Yiqiu Xia, Si-Jie Hao, Gong Cheng, Zhigang Wang, Yuan Wan, Chuandong Zhu, Hongzhang He, and Si-Yang Zheng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18577 • Publication Date (Web): 18 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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ACS Applied Materials & Interfaces

Pre-occupation of Empty Carriers Decreases Endo/Lysosome Escape and Reduces

the

Protein

Delivery

Efficiency

of

Mesoporous

Silica

Nanoparticles

Wen-Qing Li†,‡, Li-Ping Sun†,‡, Yiqiu Xia†, Sijie Hao†, Gong Cheng†, Zhigang Wang†, Yuan Wan†, Chuandong Zhu†,§, Hongzhang He†, and Si-Yang Zheng†,*

†

Department of Biomedical Engineering, Material Research Institute, The Pennsylvania State

University, University Park, PA 16802, United States §

The Second Hospital of Nanjing, Affiliated to Medical School of Southeast University,

Nanjing, 210003, China

KEYWORDS: mesoporous silica nanoparticles (MSN), endo/lysosome escape, saporin, protein delivery, pre-occupation

ACS Paragon Plus Environment

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ABSTRACT

Endo/lysosome escape is a major challenge in nanoparticles-based protein delivery for cancer therapy. To enhance endo/lysosomal escape and increase the efficacy of proteins delivery, current strategies mainly focuses on destroying endo/lysosomes by employing modified nanoparticles, such as pH-sensitive polyplexes, cell-penetrating peptides, and photosensitive molecules. Herein, we hypothesize that pre-treatment with empty nanocarriers might make endo/lysosomes occupied and affect the endo/lysosomal escape of subsequent protein delivery by nanocarriers. We first treated breast carcinoma MDA-MB-231 cells with a high concentration of empty nanocarriers mesoporous silica nanoparticles (MSN), to occupy endo/lysosome. After 2 hours, we treated the cells with a lower concentration of fluorescein isothiocyanate-labeled MSN (MSN-FITC) and investigated the intracellular spatial and temporal distribution of MSN-FITC and its colocalization with endo/lysosomes. We discovered the pre-occupation of endo/lysosomes by the empty nanocarriers did exist, mainly through changing the spatial distribution of the subsequently introduced nanocarriers. Furthermore, for the protein delivery, we observed reduced MSN-saporin delivery after endo/lysosome pre-occupation by MSN empty carriers. Similar result is observed for delivery of Cytochrome C by MSN, but not for small molecule anti-cancer drug doxorubicin. The results show that the empty nanocarriers inhibit the endo/lysosome intracellular trafficking process and decrease the endo/lysosome escape of proteins subsequently delivered by the nanocarriers. This new discovered phenomenon of declined endo/lysosome escape after endo/lysosome pre-occupation indicates that repeated treatment by nanomaterials with low protein loading capacity may not yield good cancer therapeutic effect. Therefore, it


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provides a new insightful perspective on the role of nanomaterial carriers in intracellular protein delivery.


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INTRODUCTION

Proteins and peptides have been developed as potent therapeutic agents in many medical applications including cancer therapy, vaccination, and regenerative medicine.1-4 Successful protein and peptide delivery of the therapeutics relies on the development of highly effective and safe vectors to overcome their instability and side effects.2, 5 Nanoparticles as carriers for protein and peptide delivery have been extensively studied.6-9 However, the intracellular trafficking of proteins from endo/lysosomes into the cytoplasm represents a major ratelimiting step for many nanomedicine delivery approaches.10-11 The delivery pathway of protein-loaded nanoparticles into the cells normally includes the endocytic vesicles, early endosomes, late endosomes, and lysosomes.12-13 Late endosomal vesicles are acidified (pH 5~6) by membrane-bound proton-pump ATPase. The endosomal content is then relocated to lysosomes. Lysosomes are membrane-enclosed organelles (pH ~4.5) that contain an array of enzymes capable of breaking down all types of biological polymers such as proteins, nucleic acids, carbohydrates, and lipids. When delivering proteins into cells, the transport through endo/lysosome is an important process, in which the delivered proteins are degraded and inactivated.13 Therefore, endo/lysosomal escape is a major barrier for efficient intracellular protein delivery. Currently, there are various nanoparticle modification strategies to promote endo/lysosomal escape, such as pH-sensitive polyplexes,14-15 cell-penetrating peptides (e.g. TAT, VP22)16-18 and photosensitive molecules.19 These approaches enhance endo/lysosomal escape and increase the efficacy of proteins delivery by destroying endo/lysosome. However, besides destroying the endo/lysosomal, we reason that there are other methods that may affect endo/lysosomal


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escape, and thereby influencing protein delivery efficiency. Herein, we hypothesize that pretreatment with empty nanocarriers would occupy the endo/lysosomes and affect its endo/lysosomal escape. We first treated breast carcinoma MDA-MB-231 cells with a high concentration of mesoporous silica nanoparticles (MSN) to occupy endo/lysosomes. After 2 hours, we treated the cells with a lower concentration of MSN labeled with fluorescein isothiocyanate (MSN-FITC), and investigated the spatial and temporal intracellular distribution of MSN-FITC and the colocalization of MSN-FITC with endo/lysosomes. The empty nanocarriers occupied the endo/lysosomes, decreased the endo/lysosome escape, and thus reduced the subsequent protein delivery efficiency of drug-loaded nanocarriers. Our results suggest a potential new intrinsic drug resistance mechanism, which is dependent on the intracellular drug delivery pathway through endocytosis/endosome/lysosome and the treatment regimens including drug dosage and treatment cycles.

RESULTS AND DISCUSSION Synthesis and Characterizations of MSN. Here, we used MSN as a carrier to deliver proteins. As a drug delivery system, MSN has attracted more and more attention for their potential biomedical applications in nanobiotechnology and nanomedicine.20-26 First, to increases cell uptake, we synthesized the MCM-41 type MSN modified with (3Aminopropyl)triethoxysilane (APTES) to obtain positive zeta potential.27 MSN-FITC was prepared using the reported co-condensation method to trace the locations of nanoparticles in vitro.28 As shown in Figure 1A, the transmission electron microscopy (TEM) images show both the MSN and the MSN-FITC nanoparticles have a spherical morphology with an


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average diameter of 100 nm. The zeta-potential of MSN and MSN-FITC are 35.0 mV and 29.9 mV, respectively (Figure 1B). The average diameter of the MSN-FITC in water is similar to that of the MSN at about 145 nm (Figure 1C). The nitrogen adsorption–desorption isotherm of the MSN is type-IV (Figure S1A), and the average pore size is 3.9 nm (Figure S1B and Table S1). The Brunauer–Emmett–Teller (BET) surface area and the total pore volume of MSN are 379.3 m²/g and 0.3735 cm³/g, respectively (Table S1). Intracellular transport of MSN-FITC after MSN pretreatment and the cytotoxicity of MSN. One reason for the low efficiency of protein delivery is the protein loading capacity is normally very low. When the cells are treated with the protein-loaded nanoparticles, a relatively large number of nanoparticles is needed, including some empty nanoparticles due to inefficient protein loading. These empty nanoparticles are delivered to the cells and they may affect the therapeutic efficacy of the protein. To investigate the effects of these empty nanoparticles on protein delivery, we used the empty nanoparticles to pretreat the cells to magnify this process, and studied the subsequent protein delivery by the protein-loaded nanoparticles. We hypothesize that an initial cell treatment with high concentration of empty MSN would result in the occupation of endosomes and lysosomes. After a short time (e.g. 2 hours), if we treat the cells again with a lower concentration of protein-loaded MSN, the endo/lysosome pre-occupation due to the treatment of empty MSN might affect the endo/lysosome escape of MSN-protein from the second treatment. Before testing the hypothesis, we first investigated the cytotoxicity of the MSN and MSNFITC. MDA-MB-231 cells were treated either with 50 µg/mL MSN-FITC for 24 hours, or with 300 µg/mL MSN for 2 hours first and then washed and treated with 50 µg/mL MSNFITC for 24 hours. The cells without MSN pretreatment were served as a control. The MSN


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and MSN-FITC show no significant toxicity to the MDA-MB-231 cells compared with the control (Figure S2) at 50 µg/mL nanocarrier concentration. Even pretreatment with high concentration of 300 µg/mL MSN for 2 hours does not have a negative effect on cell viability. Colocalization of MSN-FITC and early endosomes. Next, to investigate the endosome escape and trace the location of MSN in vitro, we stained the endosomes with FM 4-64 (a red fluorescent endosome probe) and monitored MSN-FITC (green fluorescence) in MDA-MB231 cells at different time points after the cells being exposed to FM 4-64 and MSN-FITC. As shown in the merged images of Figure 2 and Figure S3, the green fluorescence signal represents MSN-FITC escaped from endosomes, whereas the yellow fluorescence signal is due to co-stain of FM 4-64 and MSN-FITC and shows MSN-FITC trapped inside the endosomes. We found that amount of yellow fluorescent dots in the MSN pretreatment group increased slower than that of the control group without MSN pretreatment from 2 to 24 hours. Additionally, the Pearson’s correlation coefficient calculated by WCIF ImageJ showed that the overlap coefficient between MSN-FITC and endosomes in the MSN pretreatment group was 10%~20% lower than that of the group without MSN pretreatment at each time point, and significant at 2, 4, and 8 hours (Figure 4A). Colocalization of MSN-FITC and lysosomes. We next investigated the lysosome escape. MDA-MB-231 cells were treated with red fluorescent lysosome probe LysoTracker Red DND-99 and green fluorescent MSN-FITC, and then monitored at different time points (2, 4, 8, 12 and 24 h). The green fluorescence signal represents the MSN-FITC outside of the lysosomes, while the yellow fluorescence signal indicates that MSN-FITC is trapped inside the lysosomes (Figure 3 and Figure S4). The amount of yellow fluorescent dots in the group without MSN pretreatment increased from 2 to 12 hours, and decreased from 12 to 24 hours,


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while the amount of yellow fluorescent dots increased continuously from 2 to 24 hours in MSN pretreatment group. We further calculated the colocalization efficiency of MSN-FITC with lysosomes by using Pearson’s correlation coefficient. As shown in Figure 4B, the colocalization efficiency in MSN pretreatment group was lower (10%~20%) than that of non-MSN pretreatment group at 2, 4, 8 and 12 hours post-treatment time points, while it was higher than that of non-MSN pretreatment group at 24 hours. As showed in Figure 4, for most of the time after the start of the MSN-FITC treatment of the cells, the colocalization of MSN-FITC with both early endosomes and lysosomes was lower (10% ~ 20%) in the MSN pretreatment group than the group without MSN pretreatment. We speculate that this can be caused by inhibition of cellular uptake of MSNFITC due to MSN pretreatment. Therefore, we further investigated the uptake efficiency of MSN-FITC for the MSN pretreatment group and the group without MSN pretreatment. However, judged by the MSN-FITC fluorescence intensity per cell using flow cytometry (Figure S5), we found that the uptake of MSN-FITC for MSN pretreatment group was initially decreased at 2 and 4 hours, but then increased at 8, 12, 24 hours in comparison with that of the group without MSN pretreatment. Therefore, the low colocalization of the MSNFITC with both early endosomes and lysosomes in the MSN pretreatment group cannot be contributed to the reduced uptake of MSN-FITC by the cells in the MSN pretreatment group. Intracellular spatial distribution of MSN-FITC after MSN pretreatment. We observed that in the MSN pretreatment group, MSN-FITC mainly located in the periphery of the cells at each time point, while MSN-FITC in the group without MSN pretreatment gradually entered from the periphery to the center of the cells during the 24-hour time period (Figure 2, 3 and Figure S3, S4). In order to quantitatively study the intracellular spatial


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distribution of the MSN-FITC over time, the distances to the membrane of all green fluorescence pixels inside the cell are calculated using Matlab software. First, the outline of each cell is detected in its bright field image by the contrast difference between the cell and background by applying the Sobel operator for edge detection of the cell (Figure S6A, S6B). Then, according to the histogram of fluorescence intensity inside the cell (Figure S6C), all pixels with fluorescence intensity lower than a determined threshold are considered as the noise. After removing all fluorescence spots outside the cell and coming from the noise, the outline of the cell and fluorescence image of the cell are combined (Figure S6D, S6E). Finally, the shortest distance of each fluorescence pixel inside the cell to the cell contour is calculated. To calculate the distance, a circle is drawn in the image with the chosen fluorescence pixel as the center. The radius of the circle increases by 1 each iteration until the circle contacts the cell contour. Then, this radius is defined as the distance between that fluorescence spot and the cell membrane. The intensity of the chosen fluorescence spot and its distance to the cell membrane are recorded. The distributions of normalized sum of fluorescence (FITC) intensity as a function of distance to cell membrane are plotted in Figure 5. Clearly, in the group without MSN pretreatment, the fluorescence signal propagates from the periphery of the cell membrane into the cell center as the time proceeds, which indicates that MSN-FITC is transported to the inner areas of the cells after hours (Figure 5A). However, the fluorescent signal stays close to the membrane area in the MSN pretreatment group (Figure 5B), suggesting the intracellular transport of MSN-FITC is inhibited by the MSN pretreatment. Model of endo/lysosome pre-occupation of cells. Previous studies showed that, in the normal process without MSN pretreatment, MSN-FITC would be endocytosed into endocytic


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vesicles, carried into early endosome, late endosome, and lysosome, then be exocytosed (Figure 6A).29-30 The MSN-FITC would escape the endo/lysosome pathway in the stages such as early endosome, later endosome, and lysosome. If only considering cellular accumulation of MSN-FITC in lysosomes from 2 to 12 hours in Figure 4B, one may think the observed decrease in colocalization efficiency of MSN-FITC with endosome/lysosomes in the MSN pretreatment group might be due to the endo/lysosome escape. However, as the quantitative image processing of the time-dependent fluorescence images, we found most of the MSN-FITC signals came from the periphery of the cells in MSN pretreatment group from 4 h to 24 hours, which is very different from the group without MSN pretreatment (Figure 5). Taken together, the periphery intracellular spatial distribution, the decreased colocalization with early endosomes and lysosomes, and steady overall uptake, suggest that more MSNFITC are in the endocytic vesicles in the MSN pretreatment group (Figure 6B). Thus, pretreatment of cells with high concentration of MSN leads to the endosome and lysosome pre-occupation. The pre-occupation prevents the intracellular transport of the MSN-FITC, which keeps more MSN-FITC in the endocytic vesicles. Therefore, the escape of MSN-FITC from endo/lysosomes is decreased by the MSN pretreatment. The above model indicates that the endo/lysosome pre-occupation induced by empty nanoparticles reduces protein delivery efficiency due to inhibition of the intracellular transport and reduction of endo/lysosome escape. Validation of the pre-occupation model in protein delivery by MSN. To validate the model of endo/lysosome pre-occupation and inhibited escape from endo/lysosomes after empty MSN pretreatment in intracellular protein delivery, we synthesized MSN with large pore size for saporin delivery using mesitylene as pore-expanding agent.24 Saporin is a plant


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toxin belonging to the type 1 ribosome-inactivating protein (RIP) family, a class of toxic enzymes that is widely distributed among plant genera, causing apoptosis activation (both caspase-dependent or caspase-independent apoptosis), autophagy, necrosis, oxidative stress and inhibition of protein synthesis.31 The cellular transduction of saporin poses a great challenge due to its susceptibility and fragility. Therefore, efficient delivery of saporin into the cell is a unmet demand.32 TEM showed that MSN had an average diameter of 100 nm (Figure S7A) after pore-expanding. And the average diameter of MSN in water is 150 nm (Figure S7B). Nitrogen adsorption–desorption plot of the MSN manifests it as a type-IV isotherm, and the average pore size is 4.9 nm (Figure S8 and Table S1). The Brunauer– Emmett–Teller (BET) surface area and the total pore volume of the MSN are determined to be 539.9 m²/g and 0.6641 cm³/g, respectively (Table S1). We pretreated MDA-MB-231 cells with 300 µg/mL MSN of large pores, as well as with PBS as a control. After 2 hour pretreatment, we then washed and treated cells with 1 µg/mL MSN-saporin (saporin concentration) and 10 µg/mL MSN-saporin (saporin concentration), respectively. Figure 7 showed that the cell viability was 87% and 84% under 1 and 10 µg/mL free saporin, respectively. In the group without MSN pretreatment, the cell viability was 67% and 50% with 1 and 10 µg/mL MSN-saporin, respectively. However, the cell viability only increased to 76% and 63% under 1 and 10 µg/mL MSN-saporin in the MSN pretreatment group, respectively. The results showed that empty MSN pretreatment decreased saporin delivery efficacy significantly at each dose compared with the group without non-MSN pretreatment. To investigate whether this phenomenon holds for other proteins, we used MSN to deliver cytochrome C (Cyto C), which has been identified as an important mediator in apoptotic pathways.33 The release of mitochondrial Cyto C into the cytoplasm stimulates apoptosis and


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is commonly used as an indicator of the apoptotic process in the cell.34 We pretreated MDAMB-231 cells with 300 µg/mL MSN of large pores. After 2-hour pretreatment, we washed cells and then treated them with 2 µg/mL MSN-Cyto C (Cyto C concentration). Again, PBS and MSN were served as a negative control and 2 µg/mL MSN-Cyto C delivery without MNS pretreatment was used as a positive control. Figure S9 showed that the cell viability was 83 % and 97 % in the group without MSN pretreatment and with MSN pretreatment, respectively, while negative controls had cell viability of 100% or above. This result is similar to that of the MSN-Saporin, showing MSN pretreatment mitigates the therapeutic effect of protein drugs delivered by MSN. In previous reports, MSN was often used as a delivery carrier of the small molecules for cancer chemotherapy. We investigated if this pre-occupation by MSN would impact the subsequent MSN-based deliver of small molecule drugs, such as doxorubicin hydrochloride (DOX.). We first pre-treated MDA-MB-231 cells with PBS or 300 µg/mL MSN for 2 hours, then measured cell viability after treatment with MSN-DOX at different concentrations for 24 h. As shown in Figure S10, there are no significant differences between the PBS and MSN pretreatment group. This result indicated that pre-treatment of empty MSN nanocarriers does not affect the cytotoxicity of DOX subsequently delivered by MSN. This small molecule drug is permeable to plasma and internal membranes. It doesn’t depend on endo/lysosome escape. Therefore, the pre-treatment and pre-occupation of endo/lysosomes by empty MSN does not affect its therapeutic effect. Nanomedicine has been proposed to combat drug resistance in cancer.35-36 Our results suggest that nanoparticle-based drug carriers may induce intrinsic drug resistance by saturating the endo/lysosomes and inhibit the intracellular transport of the drug-loaded


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nanocarriers. This new mechanism depends on the dose and cycles of the treatment regimen, and should be considered for nanomedicine-based intracellular drug delivery, especially for protein drugs. CONCLUSIONS In summary, our data support the existence of endo/lysosome pre-occupation in the nanoparticle-based protein delivery, and its negative impact on the subsequent endo/lysosome escape. By investigating the colocalization of MSN-FITC with endosomes and lysosomes, we found that endo/lysosomes pre-occupation can be induced by high concentration of empty MSN. This endo/lysosome pre-occupation inhibits the subsequent intracellular transport process and decreases the endo/lysosome escape, by keeping more subsequent protein-loaded MSN in endocytic vesicles close to the plasma membrane. The proposed endo/lysosome pre-occupation model was validated with MSN-saporin and MSNCyto C delivery after empty MSN treatment. The finding indicates that prolonged drug delivery by nanoparticles may occupy endo/lysosomes, and lead to inhibition of intracellular transport, reduction in endo/lysosome escape, and decrease of the subsequent protein delivery efficiency. Therefore, short or pulsed protein delivery by nanocarriers and materials and methods of high protein loading capacity are preferred over prolonged use of nanocarriers of low protein loading capacity.


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MATERIALS AND METHODS Synthesis of amino-functionalized mesoporous silica nanoparticles (MSN). The amino functionalized MSN were synthesized by a CTAB-templated, base-catalyzed condensation reaction of silicate.27 Generally, NaOH aqueous solution (2.0 M) was prepared firstly. Then, CTAB (204 mg) was dissolved in a solution with deionized water (96 mL) and 2.0 M NaOH aq (0.7 mL). The solution was heated to 80 °C under vigorous stirring. After 30 min, TEOS (1.0 mL) was added dropwise into the reaction solution under vigorous stirring. The solution was stirred at 80 °C for 2 h. The synthesized nanoparticles were collected by centrifuging the reaction solution at 7,830 rpm for 10 min, and were then thoroughly washed with ethanol and toluene for three times. Amino functionalized mesoporous silica nanoparticles were prepared by grafting APTES (200 µL) to the surface of the obtained MSN in refluxing toluene (50 mL) in 110 °C for 20 h. The resulting materials were centrifuged at 7,830 rpm for 10 min and washed thoroughly with ethanol for three times. To remove the CTAB template, the MSN were resuspended in ethanol (90 mL), and the condensed HCl (37%) solution (10 mL) was added into the suspension. The solution was heated at 80 °C under reflux for 24 h. The resulting nanoparticles were collected by centrifugation at 7,830 rpm for 10 min and washed thoroughly with ethanol and deionized water three times. The collected nanoparticles were dried under vacuum at 80 °C for 24 h, and the MSN in powder MSN was obtained. Synthesis of fluorescein isothiocyanate-labeled amino-functionalized mesoporous silica nanoparticles (MSN-FITC). The MSN-FITC were synthesized according to previously


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reported methods. Briefly, FITC (1 mg) was dissolved in absolute ethanol (0.25 mL), APTES (20 µL) was added to the solution, and the mixture solution was kept in the dark for overnight. Then, the FITC/APTES solution was added into TEOS/CTAB solution, and the following procedures were the same as the aforementioned method. Finally, yellow colored MSN-FITC were obtained. Synthesis of amino-functionalized mesoporous silica nanoparticle (MSN) with large pores. The MSN with large pores were synthesized by adding a pore-expanding agent (mesitylene) to a CTAB-templated, base-catalyzed condensation reaction of silicate. Briefly, CTAB (204 mg) was dissolved in a solution of deionized water (96 mL) and 2.0 M NaOH aq (0.7 mL). Mesitylene (1.4 mL) was then added to the solution. The mixture was stirred vigorously at 80 °C for 30 min. TEOS (1.0 mL) was then added dropwise to the solution. The reaction mixture was stirred vigorously at 80 °C for 2 h. The formed nanoparticles were collected by centrifuging the reaction solution at 7,830 rpm for 10 min and were then thoroughly washed with ethanol and toluene for three times. The amino functionalization and the removal of the CTAB template were the same as the abovementioned method. Characterization of nanoparticles. The transmission electron microscopy (TEM) images of nanoparticles were characterized by A TecnaiG2-20- XTWIN microscope. Zeta potentials of MSN and MSN-FITC were measured by a Malvern Zetasizer Nano ZS. Micrometric ASAP 2020 apparatus were used to collect nitrogen adsorption isotherms of MSN before and after expanding the pore at a liquid nitrogen temperature (77 K), Brunauer Emmett Teller (BET) method was applied to calculate the specific surface area, the total pore volume of MSN was measured by-plot method, and the adsorption branch of the isotherms was chosen to calculate pore size distribution. Olympus FV1000 laser scanning confocal microscope was applied to


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get microscopy images. The hydrodynamic diameter of nanoparticles was tested by dynamic light scattering(DLS) (Malvern, Herrenberg, Germany) at room temperature. Cell culture. MDA-MB-231 cells were cultured in high glucose, no phenol red, Dulbecco's Modified Eagle Medium (DMEM, HyClone) containing 10% Fetal Bovine Serum, 1% nonessential amino acids, and 1% Penicillin-Streptomycin. Cells were cultured in a waterjacked humidified incubator at 37 ℃ with 5 % CO2. Measurement of MSN-FITC accumulated in early endosomes. For confocal fluorescence microscopy measurements, MDA-MB-231 cells were seeded at the density of 5 × 105 cells into each 35 mm glass dishes with coverslips at the bottom of the dishes. After 24 h incubation at 37 °C, the medium in the five glass dishes was replaced by 1 mL 300 µg/mL of MSN in the DMEM medium for 2h in 37 °C as MSN pretreatment, for the other five glass dishes, the medium was replaced by 10 µL PBS in the DMEM medium for 2h in 37 °C as non-MSN pretreatment group. Then draw out the DMEM medium and washed once with PBS. After that, all groups were incubated with 1mL DMEM containing 50 µg /mL MSNFITC and 0.5 µg/mL FM 4-64, at each time point (2h, 4h, 8h, 12h, 24h), draw out the medium and washed three times with PBS, fixed cells by 10% formalin at 4 °C for 15min, then washed three times with PBS for confocal fluorescence microscopy. The Pearson’s correlation coefficient was calculated by WCIF ImageJ. We analyzed the colocalization of MSN-FITC and endosomes from the experiment with 3 repeats. In each experiment, we used 4 pictures as to calculate the Pearson's correlation coefficients. The detailed operation followed

the

protocol

according

to:

http://wwwfacilities.uhnresearch.ca/wcif/imagej/colour_analysis.htm.


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Measurement of MSN-FITC accumulated in lysosomes. For confocal fluorescence microscopy measurements, MDA-MB-231 cells were seeded at the density of 5 × 105 cells into each 35 mm glass dishes with coverslips at the bottom of the dishes. After 24h incubation in 37 °C, the media in five glass dishes were replaced by 1 mL 300 µg/mL MSN in the DMEM medium for 2h in 37 °C as MSN pretreatment group, the media in control glass dishes were replaced by 1 µL PBS in the DMEM medium for 2h in 37 °C as non-MSN pretreatment group. Then the DMEM was aspirated and washed once with PBS. After that, all groups were incubated with 1mL 50 µg/mL MSN-FITC in DMEM. At each time point (2, 4, 8, 12and 24h), the spent media were replaced with fresh medium containing 50 nM LysoTracker Red for 30 min. After that, the medium was aspirated and washed three times with PBS. Cells were fixed in 10% formalin at 4 °C for 15 min, then washed three times with PBS for confocal fluorescence microscopy. The Pearson’s correlation coefficient was calculated by WCIF ImageJ. We analyzed the colocalization of MSN-FITC and lysosomes from the experiment with 3 repeats. In each experiment, we used 4 pictures as to calculate the Pearson's correlation coefficients. Flow cytometry analysis. For flow cytometry analysis,1×106 cells/mL were seeded into 6wellplates for 24h. The MSN-FITC group without MSN pretreatment and MSN+MSN-FITC group with 300 µg/mL MSN pretreatment for 2h, then all groups were treated with 50 µg/mL MSN-FITC at different time points of 2, 4, 8, 12 and 24 hours. Cells were washed three times with PBS, detached by 0.05% trypsin/EDTA, harvested by centrifugation, washed again with PBS and resuspended in PBS for flow cytometry analysis. The fluorescence of 15000 cells was detected at FITC channel after excitation at 488 nm laser using a BD FACS (BD


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biosciences, Germany). Average fluorescence intensities per cell were calculated for samples at each time point. Saporin loading into MSN with the large pore. The loading of saporin into MSN was performed according to previously reported methods.37 Generally, 1 mg Saporin was dissolved in 1 mL distilled water, then, 5 mg MSN was added to the solution. The solution was stirred for 6 h at room temperature. The suspensions were then centrifuged and the loading efficiency was determined by measuring the difference in absorption at 595 nm in the supernatant before and after the loading using the Bio-Rad Protein Assay. The protein standard curve of BSA was measured by the Bio-Rad Protein Assay. The loading efficiency is about 1.74 %. Cell viability of saporin treatment. MDA-MB-231 cells were cultured in cell culture incubator. Cells were seeded in a 96-well plate with 1×104 per well. After incubation for 24 h, the cells were treated with PBS (non-MSN pretreatment group) or 300 µg/mL MSN (MSN pretreatment group) for 2 h. Then both groups were treated with various concentrations of MSN-saporin, while control group was treated with PBS. MSN group was treated with 300 µg/mL MSN. Saporin group was treated with free saporin. After incubation for 24 h, drew out medium and washed once with PBS, fresh medium containing CCK-8 were added. After 4 h incubation, the absorbance at λ = 450 nm was measured using microplate reader. The percentage of cell viability was calculated by comparing absorbance values. The experiments were repeated 3 times for statistical analysis.


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Statistical analysis. Student’s t-test was applied to examine the differences among variables. Data were shown as mean ± SD. *p values ≤ 0.05, **p values ≤ 0.01were considered to be statistically significant. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx. Additional information on materials and methods are provided. 10 supplementary figures include N2 adsorption–desorption isotherm and pore size distribution of MSN; viability of MDA-MB-231 cells after treatment with MSN-FITC; high magnification fluorescence microscopy images of time-dependent MSN-FITC colocalization with early endosomes; high magnification fluorescence microscopy images of timedependent MSN-FITC colocalization with lysosomes; measurement of MSN-FITC cellular uptake by flow cytometry; imaging processing procedure to determine the intracellular spatial distribution of MSN-FITC; TEM image of MSN after pore expansion; N2 adsorption–desorption isotherm and pore size distribution curve of MSN after pore expansion; viability of MDA-MB-231 cells incubated with MSNCyto C; viability of MDA-MB-231 cells incubated with MSN-DOX at different concentrations for 24 h with PBS and empty MSN pretreatment. Also supplied is a supplementary table of BET and BJH parameters of MSN before and after pore expanding. AUTHOR INFORMATION


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Corresponding Author Si-Yang Zheng Tel: 1-(814) 865-8090 E-mail: [email protected] ORCID Si-Yang Zheng: 0000-0002-0616-030X Wenqing Li: 0000-0002-2460-7945 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. (W-Q, Li and L-P, Sun). Funding Sources We appreciate the National Cancer Institute of the National Institutes of Health for funding this work (Award Number DP2CA174508). Notes The authors declare no competing financial interest. REFERENCES 1. Tyagi, A.; Tuknait, A.; Anand, P.; Gupta, S.; Sharma, M.; Mathur, D.; Joshi, A.; Singh, S.; Gautam, A.; Raghava, G. P., CancerPPD: a database of anticancer peptides and proteins. Nucleic Acids Res 2015, 43 (Database issue), D837-43. 2. Torchilin, V., Intracellular delivery of protein and peptide therapeutics. Drug Discov Today Technol 2008, 5 (2-3), e95-e103.


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19. Bostad, M.; Olsen, C. E.; Peng, Q.; Berg, K.; Hogset, A.; Selbo, P. K., Light-controlled endosomal escape of the novel CD133-targeting immunotoxin AC133-saporin by photochemical internalization - A minimally invasive cancer stem cell-targeting strategy. J Control Release 2015, 206, 37-48. 20. Tang, F.; Li, L.; Chen, D., Mesoporous silica nanoparticles: synthesis, biocompatibility and drug delivery. Adv Mater 2012, 24 (12), 1504-34. 21. Chen, A. M.; Zhang, M.; Wei, D.; Stueber, D.; Taratula, O.; Minko, T.; He, H., Codelivery of doxorubicin and Bcl-2 siRNA by mesoporous silica nanoparticles enhances the efficacy of chemotherapy in multidrug-resistant cancer cells. Small 2009, 5 (23), 2673-7. 22. Jiao, Y.; Sun, Y.; Tang, X.; Ren, Q.; Yang, W., Tumor-Targeting Multifunctional RattleType Theranostic Nanoparticles for MRI/NIRF Bimodal Imaging and Delivery of Hydrophobic Drugs. Small 2015, 11 (16), 1962-74. 23. Xu, C.; Yu, M.; Noonan, O.; Zhang, J.; Song, H.; Zhang, H.; Lei, C.; Niu, Y.; Huang, X.; Yang, Y.; Yu, C., Core-Cone Structured Monodispersed Mesoporous Silica Nanoparticles with Ultra-large Cavity for Protein Delivery. Small 2015, 11 (44), 5949-55. 24. Slowing, II; Trewyn, B. G.; Lin, V. S., Mesoporous silica nanoparticles for intracellular delivery of membrane-impermeable proteins. J Am Chem Soc 2007, 129 (28), 8845-9. 25. Hudson, S.; Cooney, J.; Magner, E., Proteins in mesoporous silicates. Angew Chem Int Ed Engl 2008, 47 (45), 8582-94. 26. Torney, F.; Trewyn, B. G.; Lin, V. S.; Wang, K., Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nat Nanotechnol 2007, 2 (5), 295-300. 27. Slowing, I.; Trewyn, B. G.; Lin, V. S., Effect of surface functionalization of MCM-41type mesoporous silica nanoparticles on the endocytosis by human cancer cells. J Am Chem Soc 2006, 128 (46), 14792-3. 28. Qu, Q.; Ma, X.; Zhao, Y., Targeted delivery of doxorubicin to mitochondria using mesoporous silica nanoparticle nanocarriers. Nanoscale 2015, 7 (40), 16677-86. 29. Oh, N.; Park, J. H., Endocytosis and exocytosis of nanoparticles in mammalian cells. Int J Nanomedicine 2014, 9 Suppl 1, 51-63. 30.Bae, Y. M.; Park, Y. I.; Nam, S. H.; Kim, J. H.; Lee, K.; Kim, H. M.; Yoo, B.; Choi, J. S.; Lee, K. T.; Hyeon, T.; Suh, Y. D., Endocytosis, intracellular transport, and exocytosis of lanthanide-doped upconverting nanoparticles in single living cells. Biomaterials 2012, 33 (35), 9080-6. 31. Polito, L.; Bortolotti, M.; Mercatelli, D.; Battelli, M. G.; Bolognesi, A., Saporin-S6: a useful tool in cancer therapy. Toxins (Basel) 2013, 5 (10), 1698-722. 32. Gu, Z.; Biswas, A.; Zhao, M.; Tang, Y., Tailoring nanocarriers for intracellular protein delivery. Chem Soc Rev 2011, 40 (7), 3638-55. 33. Liu, X.; Kim, C. N.; Yang, J.; Jemmerson, R.; Wang, X., Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 1996, 86 (1), 147-57. 34. Narita, M.; Shimizu, S.; Ito, T.; Chittenden, T.; Lutz, R. J.; Matsuda, H.; Tsujimoto, Y., Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc Natl Acad Sci U S A 1998, 95 (25), 14681-6. 35. Shi, J.; Kantoff, P. W.; Wooster, R.; Farokhzad, O. C., Cancer nanomedicine: progress, challenges and opportunities. Nat Rev Cancer 2017, 17 (1), 20-37.


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FIGURES

Figure 1. Characterization of MSN and MSN-FITC. (A) TEM images of MSN and MSNFITC. Scale bar = 100 nm. (B) Zeta potential of MSN and MSN-FITC in H2O. (C) Particle size distribution of MSN and MSN-FITC nanoparticles measured by DLS.


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Figure 2. Time-dependent intracellular transport of MSN-FITC with and without MSN treatment and colocalization of MSN-FITC with early endosomes. Bright field and confocal fluorescence images of MDA-MB-231 cells exposed to MSN-FITC (green) and endosome dye FM 4-64 (red) in the control group without (Non-MSN Pretreatment group) and with (MSN Pretreatment group) MSN pretreatment at different time points of 2, 4, 8, 12 and 24 hours.


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Figure 3. Time-dependent intracellular transport of MSN-FITC with and without MSN treatment and colocalization of MSN-FITC with lysosomes. Bright field and confocal fluorescence images of MDA-MB-231 cells exposed to MSN-FITC (green) and lysosome dye Lyso-Tracker Red in the control group without (Non-MSN Pretreatment group) and with (MSN Pretreatment group) MSN pretreatment at different time points of 2, 4, 8, 12 and 24 hours.


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Figure 4. Colocalization of MSN-FITC with early endosomes and lysosomes. (A) Pearson’s correlation coefficients of MSN-FITC with early endosomes in the experimental groups without (blue) and with empty MSN pretreatment (red). (B) Pearson’s correlation coefficients of MSN-FITC with lysosomes in the experimental groups without (blue) and with empty MSN pretreatment (red). *: p < 0.05; **: p < 0.01. n = 3.


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Figure 5. The distributions of normalized sum of fluorescence (FITC) intensity as a function of distance to cell membrane in (A) MSN-FITC (non-MSN pretreatment) and (B) MSN +MSN-FITC (MSN pretreatment). The fluorescence images were quantified for MNS-FITC treatment of 2, 8, and 24 hours, respectively. For this analysis, 36, 44, 46, 40, 47 and 42 cells were used for the cases of 2 h MSN-FITC, 8 h MSN-FITC, 24 h MSN-FITC, 2 h MSN+MSN-FITC, 8 h MSN+MSN-FITC, and 24 h MSN+MSN-FITC, respectively.


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Figure 6. Proposed diagram of endo/lysosomal occupation due to empty MSN pretreatment and the consequent reduced endo/lysosome escape. (A) normal process. (B) endo/lysosome occupation process. After pretreating a high concentration of MSN, the occupation of the endosomes and lysosomes inside the cells prevents the subsequent endocytosis of the MSNFITC process, which keeps it inside the endocytic vesicles or early endosomes.


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Figure 7. Viability of MDA-MB-231 cells incubated with free saporin, MSN-saporin with PBS and empty MSN pretreatment at saporin concentration of 1 µg/mL and 10 µg/mL for 24 hours. Cells in culture medium (Control) and cells treated with empty MSN are served as negative controls. *: p < 0.05; **: p < 0.01, n = 3.


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TOC


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Lysosome Escape

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