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Intracellular Fate of Nanoparticles with Polydopamine Surface Engineering and a Novel Strategy for ExocytosisInhibiting, Lysosome Impairment-Based Cancer Therapy Li Ding, Xianbing Zhu, Yilin Wang, Bingyang Shi, Xiang Ling, Houjie Chen, Wenhao Nan, Austin Barrett, Zilei Guo, Wei Tao, Jun Wu, and Xiaojun Shi Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b03021 • Publication Date (Web): 23 Oct 2017 Downloaded from http://pubs.acs.org on October 23, 2017

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Intracellular Fate of Nanoparticles with Polydopamine Surface Engineering and a Novel Strategy for Exocytosis-Inhibiting, Lysosome Impairment-Based Cancer Therapy Li Ding,[a] Xianbing Zhu,[a] Yiling Wang,[a] Bingyang Shi,[d] Xiang Ling,[b] Houjie Chen,[a] Wenhao Nan,[a] Austin Barrett,[c] Zilei Guo,[c] Wei Tao,* [a,c] Jun Wu,* [b] Xiaojun Shi,* [a]

[a]

School of Life Sciences, Tsinghua University, Beijing 100084, and Graduate School at

Shenzhen, Tsinghua University, Shenzhen 518055, China [b]

Department of Biomedical Engineering, School of Engineering, Sun Yat-sen University,

Guangzhou 510006, China [c]

[d]

Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, United States International Joint Center for Biomedical Innovation, School of Life Sciences, Henan

University, Kaifeng, Henan 475004, China

ACS Paragon Plus Environment

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ABSTRACT Polydopamine (PDA) coating as a bioinspired strategy for nanoparticles (NPs) has been extensively applied in cancer theranostics. However, a cellular-level understanding of nano-bio interaction of these PDA-coated NPs (PDNPs), which drives the fate of them and acts as a critical step to determine their efficacy, still remains unknown. Herein, we utilized the representative mesoporous silica NPs (MSNs) to be coated with PDA and study their nano-bio activities in cancer cells. HeLa cell line was utilized as a model in this study. The PDNPs were discovered to be internalized through three specific pathways, i.e., Caveolae-, Arf6-dependent endocytosis and Rab34-mediated macropinocytosis (55%, 20% and 37% of uptake inhibition by nystatin, Arf6 knockdown and rottlerin, respectively). Autophagy-mediated accumulation of PDNPs in lysosomes was observed, and the formed PDA shells sheded in the lysosomes. Almost 40% of the NPs were transported out of cells via Rab8/10- and Rab3/26-mediated exocytosis pathways at our tested level. Based on these results, a novel combined cancer treatment strategy was further proposed using drug-loaded MSNs-PDA by (i) utilizing naturally intracellular mechanism-controlled PDA shedding for organelle-targeted release of drugs in lysosomes to generate lysosome impairment, and (ii) blocking the demonstrated exocytosis pathways for enhanced therapeutic efficacy.

KEYWORDS: Nanoparticle, Polydopamine coating and shedding, Intracellular fate, Lysosome targeting, Exocytosis-inhibiting


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INTRODUCTION Because of the improvement on hydrophilicity, biocompatibility, colloidal stability and conjugation of multifunctional ligands, surface modification of nanoparticles (NPs) have been extensive investigated in recent biomedical studies.1-11 Among the surface engineering strategies of NPs, polydopamine (PDA) shells could be controllably coated on the NPs through simply dispersing them in the dopamine solution under alkaline condition and mild stirring, regardless of hydrophilic or hydrophobic surfaces.12,

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Due to its easy accessibility, combined with

fascinating properties,14 PDA coating strategy has shown particularly promising potential for various applications.15 Some examples include active reactivity toward functional groups (e.g., amine and thiol groups),12, 16, 17 protein capture and separation,18, 19 free radical scavenging,20 and tissue engineering.21, 22 Notably, the PDA coating strategy of nanomaterials has emerged as a significant field especially for cancer theranostics.23-30 Besides the great benefits brought by the PDA coating strategy, it also greatly impacts the nano-bio interactions of coated NPs with bio-systems, due to the change of surface properties (i.e., the coated PDA shells interact with the bio-systems in fact).4, 31 Understanding the nano-bio interactions of nanoscale materials is of critical importance for their safe and efficient applications, which is well-demonstrated by many works.4, 32-36 Recently, Ji and co-workers have demonstrated that the PDA-coated NPs (PDNPs) have a long circulation time, and the PDA shells are stable enough to reach the target cells after intravenous injection.37 Despite of the large amount of studies performed for their application in cancer theranostics, little is known about the subsequent intracellular fates of the PDNPs when they reach the external milieu of target cells after long-term circulation.23-29 Therefore, a cellular-level understanding of intracellular mechanisms of these PDNPs, which drives the fates of them and acts as a critical step to


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determine their efficacy, is urgently needed. Herein, we utilized the mesoporous silica NPs (MSNs) as one of the most representative NPs to prepare PDNPs (i.e., MSNs-PDA), and preliminarily access their intracellular mechanisms and nano-bio activities in cancer cells. Different vesicle trafficking markers were applied to identify the intracellular pathways involved in the transportation of MSNs-PDA. Our results revealed MSNs-PDA were internalized through three different endocytosis pathways, i.e. “Arf6 → early endosome → lysosome”, “caveolae → early endosome → lysosome” and “macropinocytosis → late endosome → lysosome”. We also observed autophagy-mediated accumulation of MSNs-PDA in lysosomes, and the shedding of the PDA shells in the lysosomes due to the acid environment (pH≈5.2). The NPs were transported out of cancer cells via Rab8/10- and Rab3/26-mediated exocytosis pathways. Based on the revealed intracellular trafficking networks and the fates of MSNs-PDA, we proposed an intelligent and novel strategy for effective exocytosis-inhibiting, lysosome impairment-based cancer therapy. Paclitaxel (PTX) can induce lysosomal-membrane permeabilization (LMP),38, 39 which would cause the release of lysosomal enzymes, further damage lysosomes either directly or through triggering phospholipases,40 and sequentially induce apoptosis. Therefore, we utilized the PTX-loaded MSNs-PDA (Figure 1a) to targeted and triggered release of PTX in lysosomes to generate lysosome damage to effectively kill cancer cells, through the naturally intracellular mechanism-controlled PDA shedding in lysosomes. Furthermore, by blocking the demonstrated exocytosis pathways to increase the drug concentrations in cancer cells, we also achieved an enhanced cancer therapeutic effect. This work provided not only comprehensive insights into the fate of PDNPs, but also a novel exocytosis-inhibiting, lysosome impairment-based strategy for effective cancer therapy.


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RESULTS AND DISCUSSION MSNs were prepared by dropping tetraethyl orthosilicate (TEOS) to a mixture of cetyltrimethyl ammonium bromide (CTAB) and NH4F. The surfactant CTAB was removed by refluxing with ethanol and HCL.41 We carried out field emission scanning electron microscope (FESEM) and transmission electron microscopy (TEM) to examine the morphology of the MSNs. As presented in Figure 1b-1c, MSNs have nearly spherical shapes and porous surfaces, with a diameter of approximately 130 nm. Dynamic light scattering measurements indicated that the size of MSNs was uniformly distributed (133.2±4.58) (Figure 1d), being consistent with the results of TEM and FESEM. The pore size of the synthesized MSNs is 2.7 ± 0.3 nm. MSNs-PDA were prepared by surface modification with PDA using a previously reported method.12, 23 TEM and FESEM were further performed to study the morphology of MSNs-PDA. As shown in Figure 1e-1f, the surface of MSNs-PDA is rougher than that of MSNs. Additionally, there is a clear layer of PDA shell on the periphery of MSNs, indicating the successful coating of PDA. The efficacy of theranostic PDNPs depends on the successful internalization and sustained retention by cancer cells. Therefore, the FITC-labelled MSNs were used to prepare MSNs-PDA and investigate the cellular uptake of the PDNPs. HeLa cells, the most commonly used human cervical cancer cells, were chosen as representative cells. Figure 2a shows the confocal images of HeLa cells after incubation with FITC-labelled MSNs-PDA for 3 h. The data suggests MSNs-PDA can be ingested by HeLa cells and internalized into the cytoplasm. Moreover, the internalization of MSNs-PDA is in a time dependent manner (Figure 2b-2c). It was reported that nanomaterials usually enter cells by an energy-dependent manner termed endocytosis, rather than passive diffusion.42, 43 In order to determine if the internalization of PDA-coated NPs was in an


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energy-dependent manner, different metabolic inhibitors, including sodium azide (which depletes intracellular ATP)44 and bafilomycin A (which inhibits the v-ATPase function),45 were used to examine the effect of energy on MSNs-PDA uptake. Pre-incubation with different metabolic inhibitors dramatically suppressed the uptake of MSNs-PDA into cells (Figure 2d-2e). It is reported that proteins involved in endocytosis are sensitive to temperature, the endocytosis processes should be reduced at low temperature.46,

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Then, we incubated HeLa cells with

FITC-labelled MSNs-PDA at either 37 or 4 ℃ for 3 hours. Figure S1 shown the intracellular fluorescence of HeLa cells, which was incubated with FITC-labelled MSNs-PDA at 4 ℃ for 3 h, diminished significantly. These results clarified the cellular uptake mechanism of PDNPs used by HeLa cells is through energy-dependent endocytosis, rather than simple passive diffusion. Then, we examined the endocytosis pathways of PDNPs. Endocytosis is usually divided into macropinocytosis, clathrin-dependent, caveolae-dependent, and clathrin-independent and caveolae-independent endocytosis.42, 48 Rab34 plays a very important role in macropinocytosis and is generally considered a biomarker of this process.49 The clathrin-independent and caveolae-independent endocytosis mainly include Afr6-, Cdc42-, RhoA-, and Flotillin-dependent pathways. In order to detect the pathways through which the PDNPs enter the cells, HeLa cells were incubated with FITC-labelled MSNs-PDA for 3 h. Then immunofluorescence assay was then performed to identify the localization of FITC-labelled MSNs-PDA with Clathrin-, Caveolae-, Arf-6-, Flotillin-, Cdc42- and RhoA-positive vesicles. We found the FITC-labelled MSNs-PDA co-localized with Caveolae- and Arf6-positive vesicles, but not with Clathrin-, Flotillin-, Cdc42- and RhoA-positive vesicles (Figure 3a-3b and Figure S2). In the EGFP-Rab34 transfected HeLa cells, we also found perfect co-localization between Rab34-marked macropinocytosis and FITC-labelled MSNs-PDA (Figure 3c). These data indicate HeLa cells


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Arf6-dependent

endocytosis,

and

Rab34-dependent macropinocytosis. Moreover, we also further checked the effect of the uniformity of PDNPs on the internalization pathways using highly uniform FITC-labelled MSNs-PDA (Figure S3a). As shown in our results (Figure S3b-i), the uniformity does not affect the internalization pathways involved in the uptake of DPNPs (i.e., the same Caveolae-, Arf6-dependent endocytosis, and Rab34-mediated micropinocytosis), but may affect the percentage of uptake in each pathway. In order to further confirm our conclusion, we employed different pharmacological inhibitors of different pathways to understand the contribution of different endocytic pathways in HeLa cells. We used nystatin to prevent caveolae-mediated endocytosis,47, 50 and rottlerin to avoid intake by macropinocytosis.51 HeLa cells were exposed to different inhibitors for 2 h, and then incubated with fluorescent MSNs-PDA for another 3 h. We used fluorescent-activated cell sorting (FACS) to measure the cytoplasmic fluorescence and found internalization of fluorescent MSNs-PDA was markedly blocked by these inhibitors (Figure 3d). The macropinocytosis pathway inhibitor, rottlerin, inhibited about 37% uptake of fluorescent MSNs-PDA in HeLa cells. The caveolae-dependent endocytosis inhibitor, nystatin, also significantly reduced the uptake of fluorescent MSNs-PDA in HeLa cells. More than 55% of the fluorescent MSNs-PDA were blocked by nystatin. However, the cell metabolic activity of HeLa cells was not influenced by these inhibitors (Figure S4). Additionally, we also used siRNA to knockdown Arf6 (Figure 3e), and found the internalized fluorescent MSNs-PDA were obviously reduced in the siRNA treated HeLa cells (Figure 3f and Figure S5). However, Clathrin-dependent endocytosis pathway, which was reported involving in internalization of nanomaterials, does not participate in the internalization of PDNPs (Figure S6). Taken all together, there are three endocytosis pathways


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involved in the internalization of PDNPs in HeLa cells (Figure 3g). These endocytic vesicles are responsible for the internalization of PDNPs and subsequently transport them to endosomes. After internalization, nanomaterials are generally transported to early endosomes and then late endosomes.52 This process results in accumulation of nanomaterials in endosomes. We then examined whether these endocytic vesicles could deliver their cargo to endosomes and result in accumulation of PDNPs in endosomes. As Rab5 is widely used as a marker of early endosomes, and Rab7 is widely used as a marker of late endosomes, we then detected the co-localization between these markers and Caveolae-, Arf6- and Rab34-positive vesicles. The co-localization between these markers and fluorescent MSNs-PDA was also identified. We found Caveolae- and Arf6-positive vesicles co-localized with Rab5-positive early endosomes (Figure 4a-4b). Rab5-positive early endosomes also co-localized with fluorescent MSNs-PDA (Figure 4c). This implies Caveolae- and Arf6-positive vesicles might deliver their cargos to early endosomes. However, we did not find co-localization between Rab34-positive vesicles and Rab5-positive early endosomes (Figure S7). Instead, Rab34-posotive vesicles merged perfectly with Rab7-labelled late endosomes (Figure 4d). This indicates Rab34-positive vesicles prefer to deliver their cargos late endosomes rather than early endosomes, which is consistent with our previous work.53 As expected, we also found Rab7-labelled late endosomes merged well with fluorescent MSNs-PDA (Figure 4e). Taken all together, in the Caveolae- and Arf6-dependent endocytosis pathways, Caveolae- and Arf6-positive vesicles would transport fluorescent MSNs-PDA to early endosomes and then deliver them to late endosomes. However, in macropinocytosis, Rab34-positive vesicles would directly deliver their cargos to late endosomes. These pathways result in accumulation of PDNPs in both early and late endosomes (Figure 4f). In the classical endocytosis pathways, nanomaterials would be transported to early endosomes,


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late endosomes soon afterwards, and finally lysosomes after being internalized into the cells.52 We detected the accumulation of PDNPs in lysosomes and found MSNs-PDA-contained late endosomes could merge with lysosomes, which implies late endosomes delivers MSNs-PDA to lysosomes (Figure 5a). We further found fluorescent MSNs-PDA merged well with the lysosomes while the Lyso-Tracker Red probes were used to mark lysosomes (Figure 5b). The results suggested MSNs-PDA could be transported to lysosomes and accumulate in lysosomes. Because late endosome and lysosome are dynamic acidic organelles (pH 5.0-5.5)54, 55 and PDA coating is sensitive to pH,56, 57 the PDA shells may shed or be dissociated in acidic environment. Therefore, we examined the effect of pH on PDA layer of MSNs-PDA to simulate their behaviors in lysosomes. The PDA layer was relatively intact in neutral PBS (pH 7.4), while the PDA layer was obviously peeled off from the surface after being treated with acidic media (Figure 5c-5d). These data confirmed that the PDA layers on NP surface are sensitive to the pH in late endosomes and lysosomes, indicating their shedding starts from late endosomes and ends in lysosomes. Based on this naturally intracellular trafficking-controlled shedding strategy, we assumed that using PTX-loaded MSNs-PDA (PTX-MSNs-PDA) could trigger the release of PTX in lysosomes and target the damaged lysosomes to effectively kill cancer cells,58, 59 which could further lead to a novel lysosome impairment-based strategy for effective cancer therapy. To validate our ideas, we moved on to detect the drug release profiles of PTX-MSNs-PDA and non-coated PTX-MSNs at different pH values. Without the PDA coat, PTX freely diffused from the pores of MSNs to the solution, exhibiting quick release at all tested pH values (Figure 5e). With PDA shells, PTX-MSNs-PDA exhibited totally different release profiles (Figure 5f). At neutral pH (pH=7.4), which is similar to normal physiological conditions of interstitial fluid or blood, less than 10% of drug was released in 6 days. However, in acidic conditions (pH=6.2 and


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pH=5.2) that simulated those of early endosomes and lysosomes (or late endosomes), about 22% and 36% of drugs were released in 24 h, respectively. The release rate of PTX increased with rising acidity. These results could be explained by the fact that PDA shells blocked the pores of MSNs and acted as gatekeepers in the physiological conditions (pH 7.4), while would shed and break in acidic conditions (pH 5.0-5.5; i.e., start from late endosomes and end in the lysosomes), thus triggering the release of loaded drugs. After confirming the triggered release of PTX in lysosomes, we then went on to verify the potential and efficacy of PDA shedding-mediated lysosome impairment strategy. As shown in Figure 5g, we found PTX-MSNs-PDA significantly damaged the lysosomes after accumulation in lysosomes. In the PTX-MSNs-PDA treated cells, there is markedly loss of lysosome membrane permeability which is a potentially lethal event in cells.38, 39 However, in the PTX-MSNs treated cells, the deleterious effect was not as obvious as that of PTX-MSNs-PDA treated cells. This could be attributed to the non-target release of PTX for PTX-MSNs which could not concentrate the drug concentrations in lysosomes. In the MSNs-PDA treated cells (positive control group), there was no impairment of lysosomes detected. Taken together, the PDA shells of PDNPs shed after accumulating in the lysosomes, and in our case of drug-loaded MSNs-PDA, a novel lysosome impairment-based strategy potential for effective cancer therapy is proposed. In recent years, autophagy is widely investigated in the field of nanomedicine. Since nanomaterials could induce autophagy; autophagy could sequester nanomaterials and transport them to lysosomes.60 Then we investigated the effect of PDNPs on autophagy. After incubation with MSNs-PDA for 24 h, autophagosomes were significantly increased within the cells (Figure 6a). We further used chloroquine, which could disrupt lysosomes and block the autophagy flux, to examine the protein level of LC3-II. The protein level of LC3-II, which is widely used as a


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marker of autophagy, were increased in the cells (Figure 6b). We concluded that MSNs-PDA could induce autophagy. Furthermore, we found P62, which is a marker of sequestosome1 (SQSTM1) and an adapter molecule that selectively recognizes and binds the substrates of autophagy, merged well with fluorescent MSNs-PDA-containing vesicles (Figure 6c). LC3 could interact directly with P62/SQSTM1 and capture P62 on the isolation membrane.61,

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observed co-localization between P62-positive SQSTM1 and LC3-positive autophagosome (Figure 6d). As expected, we also observed that fluorescent MSNs-PDA-containing vesicles merged perfectly with LC3-positive autophagosomes (Figure 6e). P62-positive SQSTM1 selects target and LC3-positive autophagosomes select P62. Finally, fluorescent MSNs-PDA-containing autophagosomes would translocate to fuse with lysosomes and deliver the contents to lysosomes. Autophagosomes well co-localized with lysosomes (Figure 6f). These results advocate P62-positive sequestosome1 targeted fluorescent DPNPs and transported them to LC3-positive autophagosomes. The autophagosomes then delivered them to lysosomes. Autophagy participate in delivery of PDNPs to lysosomes. It is reported that trafficking vesicles could also deliver contents from early and late endosomes to Golgi body, then some specific secretory vesicles would transport these contents out of cell from Golgi body.49, 63 Rab22 positive vesicles are responsible for delivery of contents from early endosomes to Golgi, and Rab24 positive vesicle involves in transportation from late endosomes to Golgi.49,

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The classic secretory vesicles (marked by Rab3 and Rab26) and the GLUT4

translocated vesicles (marked by Rab8 and Rab10) could help to secret the contents out of cells from Golgi.49, 63 Therefore, we continued to examine whether these pathways involved in the exocytosis of PDNPs. We found Rab22-positive vesicles merged with both fluorescent MSNs-PDA and Rab5-labelled early endosomes, while Rab24-positive vesicles co-localized


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with both fluorescent MSNs-PDA and Rab7-labelled late endosomes (Figure S8). These results suggested Rab22- and Rab24-positive vesicles may transport MSNs-PDA from endosomes to Golgi. We further detected the co-localization between excretory vesicles and fluorescent MSNs-PDA. We found the fluorescent MSNs-PDA merged with EGFP-Rab8 and Rab10 positive GLUT4 translocated vesicles (Figure 7a-7b), as well as EGFP-Rab3 and Rab26 marked classic secretory vesicles (Figure 7c-7d) in HeLa cells. These results indicate that MSNs-PDA could be secreted to extracellular space via classic secretory vesicles and GLUT4 translocated vesicles. Our FACS result further confirmed our conclusions (Figure 7e). After incubation with fluorescent MSNs-PDA for 12 h, the media was renewed and the cells were cultured for another 14 h. Then the intracellular fluorescence was measured by FACS. After 14 h, we found the intracellular fluorescence was reduced to 62% due to exocytosis. To further confirm our result, we utilized a lysosome inhibitor chloroquine, which was used to block the degradation of NPs by lysosomes, to examine the role of degradation in fluorescence decrease. However, we found the intracellular fluorescence did not increase in chloroquine-treated cells (Figure S9). The TEM result also revealed the exocytosis of MSNs-PDA in HeLa cells (Figure 7f). In conclusion, Rab22-positive vesicles could deliver MSNs-PDA from early endosomes to Golgi, Rab24-positive vesicles could deliver MSNs-PDA from late endosomes to Golgi. Then classic secretory vesicles and the GLUT4 translocated vesicles would help to vomit the ingested nanomaterials out of cell via exocytosis (Figure 7g). Endosome-based exocytosis results in reduced accumulation of MSNs-PDA in cells. Exocytosis causes reduced accumulation of MSNs-PDA in cancer cells and puts a brake on therapeutic effects when acting as drug delivery platforms. Therefore, it is possible to use some inhibitors to block exocytosis and increase the accumulation of drug-loaded MSNs-PDA in


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cancer cells. 2-(4-Fluorobenzoylamino)-benzoic acid methyl ester (Exo1) is a widely used chemical inhibitor of the exocytosis pathways, which could induce collapse of the Golgi apparatus.65 We examined the intracellular fluorescent MSNs-PDA when Exo1 is used to inhibit the exocytosis pathways. As expected, after 2 h of pre-incubation of cells with Exo1, the cytoplasmic fluorescence increased about 20% after another 3 h-incubation of fluorescent MSNs-PDA in HeLa cells (Figure 8a). The inhibition of exocytosis pathway could significantly increase the accumulation of MSNs-PDA in cells, without influencing cell metabolic activity (Figure S10). To further verify this, we renewed the culture medium after treated with fluorescent MSNs-PDA and incubated cells in the presence or absence of Exo1 for 2 h. We renewed the culture medium and incubated the cells for 14 h, and afterwards detected the cytoplasmic fluorescence. We found the cytoplasmic fluorescence reduced about 31% compared with the control group. However, that of Exo1-treated group reduced only about 17% (Figure 8b). The data refers to that MSNs-PDA could be greatly transported out of cells via exocytosis in HeLa cells, and the block of exocytosis pathways could significantly increase the accumulation of these PDNPs in cells. The in vitro therapeutic effect of PTX-MSNs-PDA was evaluated in HeLa cells. Figure 9a-9b show the in vitro cell viability of the drug formulated in PTX-MSNs-PDA and PTX at equivalent concentrations of 0.05, 0.5, 5 µg/ml, respectively. PTX-MSNs-PDA showed higher therapeutic effect than free PTX at the same equivalent concentration at 24 and 48 h, respectively. The MSNs and MSNs-PDA did not show any obvious toxicity against cancer cells. Additionally, PTX-MSNs-PDA could markedly induce apoptosis in cells. Incubation with PTX-MSNs-PDA activated caspase-3 and PARP in cells (Figure 9c), which are widely used as maker of apoptosis. The effect of PTX-MSNs-PDA on apoptosis is much more obvious than that of free PTX. As


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exocytosis results in reduced accumulation of PTX-MSNs-PDA in cells, inhibition of exocytosis pathways will increase the accumulation of PTX-MSNs-PDA in cancer cells, which will further lead a higher drug concentration for more effective therapy. Based on all these results, we could further develop an exocytosis-inhibiting strategy plus PDA shedding-mediated lysosome impairment for enhanced cancer therapy. To verify the proposed strategy, we treated cells with PTX-MSNs-PDA for 12 h, then the medium was renewed and Exo1 was used to incubate cells for 2 h. 12 h later, the cytotoxicity was tested. Block of exocytosis significantly increased the cytotoxicity of PTX-MSNs-PDA in cells (Figure 9d). The FACS results further confirmed the enhanced cytotoxicity of PTX-MSNs-PDA by Exo1 (Figure 9e). In the PTX-MSNs-PDA and Exo1 treated cells, the level of apoptosis increased about 11% than that of PTX-MSNs-PDA treated cells. To further verify the potential of this novel strategy, we also carried out in vivo antitumor studies in a HeLa xenograft tumor model. The tumor-bearing nude mice were differently treated with Saline (control group), MSNs-PDA (drug-free PDNPs group), PTX (free drug group), PTX-MSNs-PDA (drug-loaded PDNPs group, i.e., lysosome impairment-based strategy), or PTX-MSNs-PDA + Exo1 (Exo1 pre-treated and drug-loaded PDNPs group, i.e., exocytosis-inhibiting, lysosome impairment-based combined strategy), respectively. As shown in Figure 10a-c, the tumor growth of PTX-MSNs-PDA group was significantly inhibited compared with Saline, MSNs-PDA, and free PTX groups. Moreover, with the effect of Exo1 on inhibiting the exocytosis of therapeutic PDNPs, the combined treatment group (i.e., PTX-MSNs-PDA + Exo1) showed the most significant antitumor efficacy (i.e., more than 2-fold of efficacy compared with the PTX-MSNs-PDA group without Exo1 pre-treatment). No significant difference in body weight was observed in all these groups (Figure 10d). Additionally, no apparent inflammation or tissue damage was observed through histology analysis (Figure S11),


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strategy.

Taken

all

together,

this

exocytosis-inhibiting, lysosome impairment-based strategy will be a promising and novel method for effective cancer therapy.

CONCLUSION In summary, we utilized the MSN as one of the most representative NPs, and systematically studied their intracellular mechanisms and nano-bio activities after PDA coating (as summarized in Figure 11) in HeLa cells. The PDNPs were discovered to be internalized through three specific endocytosis pathways (i.e., Caveolae-, Arf6-dependent endocytosis, and Rab34-mediated macropinocytosis). Autophagy-mediated accumulation of PDNPs in lysosomes was also observed, and the formed PDA shells shedded in the lysosomes. The bare NPs were then transported out of HeLa cells via Rab8/10-and Rab3/26-mediated exocytosis pathways. Lysosomes are dynamic acidic organelles which involve in the cell death pathways in cancer cells,66,

67

thus lysosomes are excellent pharmacological target for killing cancer cells.59

Additionally, since the internalized PDNPs could be secreted out of HeLa cells via endosome-based exocytosis pathways, the inhibition of exocytosis significantly reduced the exocytosis-induced loss of drug-loaded PDNPs and enhanced the therapeutic effects in cancer cells. Therefore, based on these factors and results, we further proposed a novel combined cancer treatment strategy using drug-loaded MSNs-PDA by (i) utilizing naturally intracellular mechanism-controlled PDA shedding for organelle-targeted release of drugs in lysosomes to generate lysosome impairment (as summarized in Figure S12),

and (ii) blocking the

demonstrated exocytosis pathways for enhanced therapeutic efficacy. This novel combined strategy was confirmed both in vitro and in vivo. With these preliminary and promising results,


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this work provided not only comprehensive insights into the fate of PDNPs, but also a potential exocytosis-inhibiting, lysosome impairment-based strategy for enhanced cancer therapy.

ASSOCIATED CONTENT Supporting Information. Synthesis and characterization of the PDNPs, characterization of the endocytosis pathways, Drug loading content, In vitro drug release study, Immunofluorescence, Intracellular fluorescence assay, Lysosome membrane permeability assay, cell viability, apoptosis assay are presented in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author [email protected] (W.T.); [email protected] (J.W.); [email protected] (X.S.). Author Contributions L.D., X.Z. and Y.W. contributed equally to this work. W.T., L.D., X.Z. and Y.W. conceived the idea and designed the study. W.T. directed the project. L.D. X.Z. and Y.W. performed all the in vitro experiments and analyzed data. X.Z. performed all the in vivo experiments. B.S., X.L., H.C. and W.N. helped in nanoparticle preparation, in vitro and in vivo experimental assays. W.T., J.W., B.S., A.B. and Z.G. provided technical support and corrections of manuscript. W.T. and X.S. provided reagents and conceptual advice. L.D. X.Z. and Y.W. wrote the manuscript and revised according to the comments of W.T. Correspondence and requests for materials should be addressed to W.T.


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ACKNOWLEDGMENT This work was supported by the grants Natural Science Foundation of Guangdong Province 2016A030310023; China Postdoctoral Science Foundation 2016M600676; Tsinghua Scholarship for Overseas Graduate Studies 2013159.

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Figure 1. (a) Schematic of PTX-loaded MSNs-PDA. (b) FESEM and (c) TEM images of MSNs. (d) DLS size distribution of MSNs. (e) FESEM and (f) TEM images of MSNs-PDA.


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Figure 2. The effect of temperature on the uptake of PDNPs. (a) The uptake of fluorescent MSNs-PDA (50 μg/ml) was tested by confocal laser scanning microscopy after 3 h-incubation with HeLa cells. The images above are the enlarged ones in the white box on the below images. Cell membrane was labled with molecular probe (Red). (b) The uptake of fluorescent MSNs-PDA (50 μg/ml) was measured by flow cytometer at indicated incubation time in HeLa cells. (c) Statistical analysis of intracellular fluorescence in HeLa cells. (d) Cells were pretreated with or without metabolic inhibitor sodium azide (0.1%) and bafilomycin A (0.05 μM) for 30 min, then cell were incubated with fluorescent MSNs-PDA (50 μg/ml) for 14 h later, Cytoplasmic fluorescence was measured by flow cytometer. (e) Histogram of intracellular fluorescence. (Scale bar: 10 µm; ** P < 0.01)


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Figure 3. PDNPs enter cells through Caveolae-, Arf6-dependent endocytosis, and macropinocytosis. Confocal images of HeLa cells, which were incubated with fluorescent MSNs-PDA (50 μg/ml) for 3 h, and then the immunofluorescence experiment was performed with the primary antibody against (a) Caveolae, (b) Arf6. (c) Confocal images of DsRed-Rab34 transfected HeLa cells incubated with fluorescent MSNs-PDA for 3 h. (d) Cells were pretreated with indicated inhibitors (nystatin, 100 µg/ml; rottlerin, 2.6 µg/ml) for 2 h and then incubated


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with fluorescent MSNs-PDA for another 3 h. The cytoplasmic fluorescence was measured by flow cytometer. (e) Cells were pretreated with siRNA against Arf6 for 48 h, and then the protein and mRNA level were examined. (f) Cells were pretreated with siRNA against Arf6 for 48 h and then incubated with fluorescent MSNs-PDA (50 μg/ml) for another 3 h. The cytoplasmic fluorescence was measured by flow cytometer. (g) Schematic representation of how PDNPs enter cells. (Scale bar: 10 µm; * P < 0.05)


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Figure 4. Accumulation of PDNPs in endosomes. Confocal images of DsRed-Rab5 transfected HeLa cells incubated with MSNs-PDA (50 µg/ml) for 3 h, and then the immunofluorescence experiment was performed with the primary antibody against (a) Arf6, (b) Caveolae. (c) Confocal images of DsRed-Rab5 transfected HeLa cells incubated with fluorescent MSNs-PDA for 3 h. (d) Confocal images of DsRed-Rab7 and EGFP-Rab34 transfected HeLa cells incubated


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with MSNs-PDA for 3 h. (e) Confocal images of DsRed-Rab7 transfected HeLa cells incubated with fluorescent MSNs-PDA for 3 h. (f) Schematic representation of how PDNPs are transported to late endosomes. (Scale bar: 10 µm)


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Figure 5. Accumulation of PDNPs in lysosomes and novel strategy for targeting lysosome damages. (a) Confocal images of EGFP-Rab7 transfected HeLa cells incubated with MSNs-PDA (50 µg/ml) for 3 h, then lysosome was detected by Lyso-Tracker. (b) Confocal images HeLa cells incubated with fluorescent MSNs-PDA (50 µg/ml) for 3 h, then lysosome was detected by Lyso-Tracker. (c) TEM image of MSNs-PDA treated with pH 7.4 for 12 h. (d) TEM image of MSNs-PDA treated with pH 5.2 for 12 h. (e) In vitro release profiles of PTX from MSNs. (f) In vitro release profiles of PTX from MSNs-PDA. (g) HeLa cells were incubated with different NPs


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(2.5 µg/ml) for 24 h, then the lysosome membrane permeability was measured by flow cytometer. (Scale bar: 10 µm; * P < 0.05; ** P < 0.01)


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Figure 6. Autophagy mediated accumulation PDNPs in lysosome. (a) Representative images and quantification of HeLa cells with EGFP-LC3 vesicles (autophagosomes). EGFP-LC3 transfected cells were treated with MSNs-PDA (50 µg/ml) for 24 h. (b) LC3I/II protein levels were analyzed by western blotting in the HeLa cells treated with MSNs-PDA (50 µg/ml) for 24 h. (c) Confocal images of HeLa cells, which were incubated with fluorescent MSNs-PDA (50 µg/ml) for 24 h, and then the immunofluorescence experiment was performed with the primary antibody against p62. (d) Confocal images of DsRed-LC3 transfected HeLa cells incubated with MSNs-PDA (50 µg/ml) for 24 h, then the immunofluorescence experiment was performed with the primary


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antibody against p62. (e) Confocal images of DsRed-LC3 transfected HeLa cells incubated with fluorescent MSNs-PDA (50 µg/ml) for 24 h. (f) Confocal images of EGFP-LC3 transfected HeLa cells incubated with MSNs-PDA for 24 h, then lysosome was detected by Lyso-Tracker. (Scale bar: 10 µm; ** P < 0.01)


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Figure 7. Exocytosis of PDNPs. Confocal images of (a) DsRed-Rab8, (b) DsRed-Rab10, (c) DsRed-Rab3, (d) DsRed-Rab26 transfected HeLa cells incubated with fluorescent MSNs-PDA (50 µg/ml) for 3 h. (e) Cells were incubated with fluorescent MSNs-PDA (50 µg/ml) for 12 h, then the culture medium was renewed. 14 h later, Cytoplasmic fluorescence was measured by flow cytometer. (f) Cells were incubated with fluorescent MSNs-PDA (50 µg/ml) for 12 h, then


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the culture medium was renewed. 14 h later, cells were fixed and TEM experiment was performed. (g) Schematic representation of Exocytosis pathways. (Scale bar: 10 µm; * P < 0.05)


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Figure 8. Block of exocytosis increases accumulation of PDNPs in cells. (a) HeLa cells were pretreated with Exo1 for 2 h, then cells were incubated with fluorescent MSNs-PDA (50 µg/ml) for 3 h. The cytoplasmic fluorescence was measured by flow cytometer. (b) HeLa cells were incubated in the presence or absence of Exo1 (50 µg/ml) for 2 h after treating with fluorescent MSNs-PDA (50 µg/ml) for 3 h. After that, we renewed the culture medium with fresh DMEM, and incubated the cells for 14 h. Cytoplasmic fluorescence was then measured by flow cytometer. (* P < 0.05)


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Figure 9. Block of exocytosis enhances the in vitro therapeutic effect of drug-loaded PDNPs. Viability of HeLa cells cultured with PTX-MSNs-PDA compared with that of PTX at the same drug dose and that of MSNs and MSNs-PDA with the same nanoparticles concentration: (a) 24 h, (b) 48 h. (c) Cells treated with MSNs, MSNs-PDA, PTX-MSNs-PDA and PTX for 24h, then caspase3 and PARP protein levels were analyzed by western blotting. PTX-MSNs-PDA compared with that of PTX at the same drug dose (0.5 µg/ml). (d) Cells treated with MSNs and PTX-MSNs-PDA for 12 h, then media was renewed and Exo1 (50 µg/ml) was added. 2 h later,


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media was changed and cells were cultured for another 12 h. Cell viability was measured by MTT assay. (e) Cells treated with MSNs and PTX-MSNs-PDA for 12 h, then media was renewed and Exo1 (50 µg/ml) was added. 2 h later, media was changed and cells were cultured for another 12 h. cell apoptosis was measured by flow cytometer. (* P < 0.05; ** P < 0.01; *** P < 0.001)


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Figure 10. (a) Photographs and (b) weights of the tumors after excision at day 24 (n =5). (c) Tumor sizes and (d) body weights during the treatment period. HeLa-tumor bearing mice were treated with Saline, MSNs-PDA, PTX, PTX-MSNs-PDA, or PTX-MSNs-PDA+Exo1, respectively. (** P < 0.01)


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Figure 11. Schematic representation of the intracellular trafficking network of PDNPs. MSNs-PDA were internalized through three different endocytosis pathways: Caveolae-, Arf6-dependent endocytosis and Rab34-mediated macropinocytosis. MSNs-PDA first accumulated in endosome, then lysosome. Exocytosis vesicles helped to transport MSNs-PDA out of cells, and inhibition of exocytosis reduced loss of MSNs-PDA in cells. Autophagy also involved in delivering MSNs-PDA to lysosome.


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