Nanodiamonds Mediate Oral Delivery of Proteins for Stem Cell

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Nanodiamonds Mediate Oral Delivery of Proteins for Stem Cell Activation and Intestinal Remodeling in Drosophila Xingjie Hu, Xiaojiao Li, Min Yin, Ping Li, Ping Huang, Lihua Wang, Yiguo Jiang, Hui Wang, Nan Chen, Chunhai Fan, and Haiyun Song ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 16, 2017

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ACS Applied Materials & Interfaces

Nanodiamonds Mediate Oral Delivery of Proteins for Stem Cell Activation and Intestinal Remodeling in Drosophila

Xingjie Hu1,5, Xiaojiao Li2,3,5, Min Yin1, Ping Li2,3, Ping Huang2,3, Lihua Wang1, Yiguo Jiang4, Hui Wang2,3, Nan Chen1,*, Chunhai Fan1,*, Haiyun Song2,3,* 1

Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 201800, China

2

Key Laboratory of Food Safety Research, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of sciences, University of Chinese Academy of Sciences, Shanghai 200031, China

3

Key Laboratory of Food Safety Risk Assessment, Ministry of Health, Beijing 100021, China

4

School of Public Health, Guangzhou Medical University, Guangdong 511436, China

5

These authors contributed equally to this work.

*Emails: [email protected]; [email protected]; [email protected] KEYWORDS: nanodiamonds, protein delivery, oral delivery, stem cells microenvironment, intestinal remodeling

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ACS Paragon Plus Environment


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ABSTRACT Introduction of exogenous bio-macromolecules into living systems is of great interest in genome editing, cancer immunotherapy and stem cell reprogramming. Whereas current strategies generally depend on nucleic acids transfection, direct delivery of functional proteins that provides enhanced specificity, increased safety, fast and temporal regulation is highly desirable. Nevertheless, intracellular delivery of intact and bioactive proteins, especially in vivo, remains poorly explored. In this study, we developed a nanodiamonds (NDs)-based protein delivery system in cultured cells and in Drosophila, which showed high adsorption, high efficiency and effective cytosolic release of fully functional proteins. Through live cell imaging, we observed a novel phenomenon that a substantial amount of internalized NDs-protein complex rejected fusion with the early endosome, thereby evading protein degradation in the lysosome. More significantly, we demonstrated that dietary NDs-RNase induced apoptosis in enterocytes, stimulating regenerative divisions in intestinal stem cells and increasing the number of stem cells and precursor cells in Drosophila intestine. As stem cells are poorly accessible by exogenous agents in vivo, NDs-mediated oral delivery of proteins provides a new approach to modulate the stem cell microenvironment for intestinal remodeling, which has important implications for colorectal cancer therapy and regenerative medicine.

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INTRODUCTION Proteins define fundamental cellular functions within living organisms, such as gene expression,1-2 chromatin remodeling,3-4 cell communication,5 immune recognition and activation,6-7 somatic and stem cell reprogramming.8 Aberrant protein functions are responsible for a variety of diseases including cancers.9-10 Whereas chemical drugs and nucleic acids mediated gene delivery have been widely used to rescue intracellular protein dysfunctions for therapeutic purposes, direct delivery of proteins are of higher specificity and safety that minimizes risks of altering genetic information.11 More significantly, direct introduction of functional proteins into cells can quickly alter cellular activities and rescue cellular functions without delay, which is in contrast to the introduction of DNA or RNA constructs that rely on endogenous transcription and translation machinery to produce appropriate protein products or delete dysfunctional proteins.12-14 Furthermore, the nonreplicable nature of protein is especially useful for transient regulation.15-17 Hence, delivery of functional proteins into cells and organisms can provide enhanced specificity, increased safety, fast and temporal regulation. As one elegant example, Liu and coworkers developed cationic lipid nanoparticle-based agents for delivering the CRISPR-Cas9 system to achieve efficient genome editing.18 Most proteins are unable to spontaneously cross the cell membrane. To circumvent this barrier, many types of nanocarriers for protein delivery have been studied, including liposomes, polymeric carriers, and inorganic nanoparticles (NPs).19-21 In particular, inorganic NPs have proven of high delivering efficiency, which nevertheless are encountered with several challenges.22-24 First, internalized NPs tend to be trapped in the endosome/lysosome pathway that results in degradation of protein cargoes.25 Second, when proteins are chemically 3



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conjugated to NPs, their biological activities and cellular functions are often hampered. Third, most studies focus on protein delivery in cultured cells, whereas their in vivo applications are less explored. Therefore, convenient protein delivery strategies with easy assembly, high efficiency of intracellular delivery, rapid endosomal escape and improved in vivo efficacy are highly desirable. Nanodiamonds (NDs) are a type of carbon nanoparticles with a truncated octahedral architecture. Previous studies have demonstrated that NDs have good biocompatibility in cell-based assays and animal models.26 Therefore, NDs have been widely used for bioimaging, biodiagnostic and therapeutic purposes.27-29 For example, fluorescent NDs are utilized as long-term in vivo imaging agents in mice and in Caenorhabditis elegans.30-31 NDs have also proven to be efficient delivery agents for anti-cancer drugs, siRNAs, immunostimulatory CpG oligonucleotides and circulating proteins such as insulin.32-36 In this study, we explored the use of NDs as nanocarriers for direct intracellular delivery of proteins. We showed that NDs could efficiently deliver different types of proteins into cultured cell lines without perturbing their biological activities. More importantly, our in vivo study indicated that oral delivery of functional proteins could activate intestinal stem cells. As in vivo stem cells are located in niches and are poorly accessible by exogenous drugs or biomolecules,10 our results reveal an effective method to modulate stem cells by targeting the stem cell microenvironment. RESULTS AND DISCUSSION Characterization of NDs-Protein Complex NDs synthesized by detonation techniques are usually functionalized with hydroxyl and carboxyl groups, which enable them to physically interact with various molecules via electrostatic forces. By exploiting this property, we explored 4


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to load target protein cargoes. The red fluorescent protein (RFP) was employed as the first model protein since it provides readily observable red fluorescence in cells. Analysis with transmission electron microscopy (TEM) and dynamic light scattering (DLS) revealed that bare NDs and NDs-RFP complex formed clusters with similar hydrodynamic diameters. In addition, RFP adsorption reduced the zeta potential of NDs from 31 mV to 12 mV (Figure S1).To examine the loading capacity of NDs, we incubated 0.5 mg/mL NDs with 0.125-2 mg/mL RFP and measured adsorbed protein levels. The adsorption capacity of NDs increased proportionally to the concentration of RFP between 0.125 and 1 mg/mL, and plateaued out afterwards (Figure S2a). To achieve maximum protein loading capacity, we fixed the ratio of NDs to protein at 1:2 (w/w) in the following experiments. Cellular Internalization of NDs-Protein We evaluated cellular uptake of NDs-RFP with a confocal microscope. Red fluorescence signal was not visualized in Hela cells incubated with NDs or RFP alone. In contrast, it was readily observed in those incubated with NDs-RFP (Figure S2b). We further tested intracellular delivery of other types of fluorescent proteins, including cyan fluorescent protein (CFP), green fluorescent protein (GFP) and yellow fluorescent protein (YFP). Similar results were obtained and shown in Figure 1. These fluorescent proteins appeared as punctate structures in cells. This suggests that the large portions of proteins are stably adsorbed on NDs after cellular uptake, which avoids dispersion in the cytoplasm. We tracked the dynamic process of internalization of single NDs-RFP particle with a total internal reflection fluorescence (TIRF) microscope (Figure 2 and Movie S1). After the NDs-RFP particle came into contact with the cell membrane, its moving velocity slowed down significantly. The retention of the NDs-RFP particle on the cell 5



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membrane lasted for about 90 seconds before it moved into the cytoplasm. Interestingly, we previously showed that the same type of NDs loaded with CpG oligodeoxynucleotides had a retention time of about 5 minutes on the cell membrane34, suggesting that the internalization speed of NDs was also affected by assembled cargoes. Intracellular Trafficking of NDs-Protein Previous studies showed that many types of NPs were often entrapped in endosomes and lysosomes after their cellular internalization.37-38 As a result, cargoes on these NPs might not be efficiently delivered to their destinations. Moreover, biological activities of protein cargoes might be destroyed by the acidic environment and various proteases in these vesicular compartments. Therefore, it was necessary to examine the subcellular localizations of NDs-RFP particles after they were internalized into cells. Rab5 is a molecular marker for the early endosome.39 We found that intracellular NDs-RFP had steadily low levels of co-localization with GFP-Rab5. Only about 10% NDs-RFP co-localized with the early endosomes after the cells were incubated with NDs-RFP for 2 hours or 6 hours (Figure 3a,c). These observations revealed that intracellular NDs-RFP particles were not entrapped in these vesicles. The early endosome serves as a sorting station for endocytic biomolecules. Except that transmembrane receptors can be recycled back to the plasma membrane, other endocytic molecules are either delivered to the lysosome, or escape from this compartment.40-41 Next, we investigated whether endocytic NDs-protein adopted the endosome/lysosome pathway. Lysotracker is a dye that labels acidic organelles including endosomes and lysosomes.42-43 We measured the levels of co-localization between NDs-RFP and the Lysotracker Green dye. About 45% NDs-RFP were localized in the endosomes/lysosomes after 2-hour 6


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incubation, whereas the co-localization level was reduced to 35% after 6-hour incubation (Figure 3b,c). These results suggested that NDs-protein particles only partially entered the endosome/lysosome pathway and could adopt an alternative trafficking pathway. To explore the mechanism through which NDs-protein particles evaded the lysosomal destination, we traced intracellular transport of NDs-RFP particles via live cell imaging and focused on their dynamic interaction with the early endosomes. Earlier studies speculated that endosomal escape might occur after fusion of internalized NDs with the early endosomes.44 Surprisingly, we found that some of the endocytic NDs-RFP particles did not fuse with the early endosomes after their initial contact (Figure 3d-f, indicated by a yellow circle; Movie S2). Although the NDs-RFP particle continuously interacted with the early endosome during the live imaging, internalization of NDs-RFP into the early endosome was not

observed.

In

addition,

many

NDs-RFP

particles

showed

early

endosome-independent movements during the live imaging (Figure 3e, indicated by white circles; Movie S2). Fluorescence intensity time traces of signals from the NDs-RFP particle and GFP-Rab5 also exhibited an asynchronous manner, indicating that they were not in the same compartment (Figure 3g). These observations revealed that NDs-RFP particles avoided the endosome/lysosome pathway by preventing endosomal fusion, a previously not described phenomenon. The failure to fuse with the early endosome after contact suggested that the inner environment of the endocytic vesicle was altered by internalized NDs-protein complex. Acidification of the endocytic vesicle was required for subsequent fusion.45 Other studies suggested that cationic polymer based nanocarriers could resist acidification of endocytic vesicles via their pH-buffering property.46 7



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Therefore, it was likely that the positively-charged NDs-protein complex affected vesicle fusion via a similar mechanism. As a result, these NDs-protein particles might bypass the endosome/lysosome pathway and are released into the cytosol. Intracellular Delivery and Desorption of Multiple Proteins Having successfully delivered RFP via NDs, we next aimed to deliver several types of proteins simultaneously, which had important implications for biological studies or therapeutic purposes. Therefore, we incubated four types of fluorescent proteins, CFP, GFP, YFP and RFP, with NDs and tested the efficacy of concurrent delivery in Hela cells. Four types of fluorescence signals were concurrently detected in most cells, suggesting the high effectiveness of NDs in simultaneous delivery of multiple proteins (Figure 4b, left panel). Similar to our observation in the delivery of single type of protein, we also found that these different types of fluorescent proteins appeared as punctate structures and showed heterogeneous distribution in cells. This phenomenon suggested that significant amount of proteins were not desorbed from NDs in the cytosol, which might consequently affect biological activities of delivered proteins. To overcome this potential disadvantage, we tested the notion that whether Poly-D-Lysine (PDL) functionalization of NDs, which served as bridges between NDs and proteins, could facilitate protein desorption in the cytosol. NDs-PDL and bare NDs showed comparable capacity in adsorbing RFP (Figure 4a, upper panel). TEM and DLS analysis showed that PDL functionalization did not significantly alter the sizes of NDs and NDs-protein complex, and slightly increased their zeta potentials (Figure S1). In the cell lysate, RFP proteins displayed much faster desorption rate from NDs-PDL compared to that from NDs (Figure 4a, lower panels). When we utilized NDs-PDL for intracellular delivery, four types of fluorescent proteins were efficiently and concurrently delivered into Hela cells, which displayed more 8


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homogenous cellular distribution and less punctate structures (Figure 4b, right panel). We further measured percentages of overlapped and non-overlapped fluorescence signals from four types of fluorescent proteins. The statistical data indicated that multiple fluorescent protein delivery by NDs-PDL had significantly lower levels of non-overlapped signals than that by bare NDs (Figure 4c and Figure S3). These results suggested that NDs-PDL might be superior to bare NDs in promoting protein desorption in the cytosol. Next, we explored whether NDs could be used to deliver functional proteins to interfere with cellular activities. RNases catalyze the degradation of RNAs. Excessive amount of intracellular RNase can affect cell viability, which has been utilized for cancer research.47-48 Here, we tested the efficacy of delivering functional RNase A into cancer cells by NDs or NDs-PDL. Unexpectedly, RNase A showed low adsorption to bare NDs. In contrast, it was abundantly adsorbed to NDs-PDL (Figure 4d). Therefore, we examined the viability of two types of cancer cells, Hela and MCF7, after incubation with NDs-PDL-RNase, NDs-PDL, or RNase alone. Whereas incubation with NDs-PDL or RNase had minimal effect on the viability of Hela cells, incubation with NDs-PDL-RNase triggered cell death in about 70% Hela cells after two days (Figure 4e and Figure S4). Similar results were observed when MCF7 cells were incubated with NDs-PDL-RNase (Figure S5), suggesting that the RNase A proteins were successfully delivered into these cancer cells and were fully functional in the cytosol. Oral Delivery of Protein into Intestinal Cells Further, we examined whether the PDL functionalized NDs could be used for oral delivery of proteins into intestinal cells, which may have diagnostic or therapeutic potential for intestinal diseases including colorectal cancer.49 We tested this idea in Drosophila (Figure 5a). Flies were fed with food containing RFP or 9



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NDs-PDL-RFP for 2 hours and were transferred to normal food for 24 hours. We then evaluated intestinal uptake of RFP or NDs-PDL-RFP with a confocal microscope. As a control, dietary RFP proteins did not produce red fluorescence signal in intestinal cells. In contrast, RFP signal was readily observed in intestinal cells from flies fed with NDs-PDL-RFP, suggesting that the RFP proteins were effectively delivered into intestinal cells via oral administration (Figure 5b). We also tested whether functional RNase A could be delivered into intestinal cells in the same manner. Cleaved Caspase-3, a marker for apoptosis,50 was observed in fly intestinal cells one day after the flies were fed with food containing NDs-PDL-RNase. As a control, dietary NDs-PDL-BSA did not trigger apoptosis in intestinal cells (Figure S6). We further investigated the consequences of RNase delivery on intestinal homeostasis. The fly midgut is an equivalent of the mammalian small intestine. The midgut epithelium consists of four types of cells. Enterocytes (ECs) are absorptive cells and enteroendocrine cells (EEs) are hormone-secreting cells, both of which are differentiated from enteroblasts (EBs). EBs come from intestinal stem cells (ISCs), which go through asymmetric divisions to produce EBs and for self-maintenance.51 ECs can be distinguished from the other three types of cells by their significantly larger cell and nuclear sizes. In addition, ECs are located at the apical surface of the midgut epithelium and are responsible for nutrient uptake, whereas ISCs, EBs and EEs are located at the basal side of the midgut epithelium and do not expose to the intestinal lumen. Delta (Dl) is a marker for ISCs, while Escargot (Esg) is a marker for both ISCs and EBs (Figure 6a).52 Here, flies were separated into four groups and fed with food containing NDs-PDL-BSA, NDs-PDL-RNase, RNase protein or normal food, respectively. Cell numbers of ISC, EC, EB and EE were counted according to their corresponding markers (Figure 6b,c). In flies fed with NDs-PDL-RNase, the number of ECs was significantly reduced, whereas the numbers of both ISCs 10


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and EBs were significantly increased. Homeostatic regulation of intestinal cell types was further analyzed with the linear discriminant analysis (LDA), a powerful statistical technique.53 Four replicates were tested for each group, and the raw data were subjected to LDA to generate three canonical factors (97.7, 2.28, and 0.02% of the variation), which represented linear combinations of the intestinal homeostatic response matrix (four cell types × four treatments × four replicates). The first two most significant discrimination factors were employed to generate a 2D plot, in which each point represented the response pattern of an individual cell type against dietary treatment (Figure 6d). Importantly, we were able to cluster the 16 response patterns (4 treatments × 4 replicates) into four distinct groups. Intestines from flies fed with NDs-PDL-RNase showed clearly distinct homeostatic response in pattern recognition, whereas three other groups (control, NDs-PDL-BSA, and RNase) exhibited significant overlaps among their 95% confidence ellipses. ISCs are located in a microenvironment that is surrounded by ECs and EBs. It is difficult for exogenous agents to directly regulate ISCs.10 However, alterations in the stem cell microenvironment can produce signals to affect the state of stem cells.51 Therefore, this observation indicated that oral delivery of RNase A caused apoptosis in ECs and consequently triggered regenerative divisions of ISCs for tissue repair. These effects were not observed in flies fed with either RNase A or NDs-PDL-BSA, confirming that NDs served as effective carriers to deliver RNase A into intestinal cells (Figure 6c,d). CONCLUSION In this study, NDs and PDL functionalized NDs have proven to be highly effective in delivering proteins into cultured cells and in vivo. Imaging analyses show that less than half of internalized NDs enter the endosome/lysosome pathway, 11



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whereas the rest do not fuse with the early endosome. As a result, significant amounts of NDs-protein stay in the cytosol and thereby avoid protein destruction in the lysosome. We further find that NDs-PDL can not only efficiently load proteins but show superior desorption of proteins in the cytosol, which ensures normal biological functions of delivered proteins. NDs-PDL mediated delivery of RNase A can efficiently induce apoptosis in cancer cell lines. Significantly, this strategy is effective for oral delivery of proteins and can be used to regulate intestinal stem cell microenvironment. We show that dietary NDs-PDL can target a fluorescent protein and a functional RNase into intestinal cells. Oral delivery of RNase A causes apoptosis in enterocytes, stimulates regenerative divisions in intestinal stem cells and triggers intestinal remodeling, which significantly increases the number of stem cells and precursor cells in the intestine. In summary, our study provides a convenient tool for cellular and in vivo delivery of functional proteins, which will greatly benefit biological research as well as disease diagnosis and therapy. METHODS Chemicals and Materials NDs with individual sizes ranging from 2-10 nm were purchased from Gansu Gold Stone Nano. Material. Co. Ltd., China. Poly-D-Lysine (PDL) and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. RNase A was from TaKaRa Co. Ltd. Dulbecco’s modified Eagle’s medium (DMEM) and Lysotracker Green DND-26 (L7526) were purchased from Invitrogen. HeLa cells and MCF-7 cells were purchased from Shanghai Institute of Biological Sciences. Cells were grown in DMEM medium supplemented with 10% heat inactivated fetal bovine serum (Gibco), 100 units/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine at 37ºC in humidified air containing 5% CO2. 12


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Protein Expression and Purification The full-length sequences of fluorescence proteins (CFP, GFP, YFP and RFP) were cloned into pGEX-6p-1 vector (GE Healthcare), respectively. The protein was expressed as a glutathione-S-transferase (GST)-fusion protein in E. coli. Cells harboring the GST–fusion proteins were lysed by incubation with lysozyme in a lysis buffer (20 mM Tris–HCl (pH 7.6), 500 mM NaCl, 2 mM DTT, 1 mM EDTA, 0.1 mM PMSF, Proteinase inhibitor cocktail) followed by sonication. The lysate was separated by centrifugation at 13000rpm and the supernatant was loaded onto a glutathione-Sepharose 4B column (Amersham). After extensive washing with the lysis buffer, pure GST-fusion protein was eluted with 20 mM glutathione in phosphate buffer saline (PBS) and was dialyzed in PBS. Functionalization and Characterization of NDs and NDs-protein complex To prepare poly-D-lysine (PDL)-coated NDs, NDs were mixed with PDL (average MW 30000-70000) at a ratio of 7:3 (w/w) in a boric acid buffer (pH8.5). After 24-hour vortexing, NDs were isolated by centrifugation at 13000 rpm for 20 minutes, and washed with Millipore water six times to remove excessive PDL. NDs or NDS-PDL were incubated with protein solution at a ratio of 1:2 (w/w) in PBS for 2 hours. For transmission electron microscopy, solution containing NDs or NDs-protein mixture was dropped onto carbon coated copper grids to evaporate excess solvent, and examined with TEM (Jeol 2010, 200KV).54 The apparent hydrodynamic size was measured with a Delsa Nano C analyzer (Beckman Coulter). Adsorption and Desorption of Proteins NDs or NDs-PDL were mixed with RFP or RNase A at indicated ratio in PBS for 2 hours and separated by centrifugation. The amount of adsorbed RFP was 13



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determined by the reduction of fluorescence in the supernatant. Alternatively, the pellet was boiled in SDS loading buffer after washing, and the amount of NDs-bound RFP or RNase was determined by SDS-PAGE and Coomassie Blue staining. To evaluate protein desorption, NDs-RFP and NDs-PDL-RFP were incubated in cell lysate for indicated time. After centrifugation, the amount of bound RFP was determined. Imaging of Intracellular Fluorescent Proteins Observations of individual fluorescence proteins were carried out with a Leica TCS sp5 confocal microscope. (RFP: λex 594 nm, λem 600-650 nm; GFP: λex 488 nm, λem 500-520 nm; YFP: λex 514 nm, λem 520-570 nm; CFP: λex 458 nm, λem 465-500 nm). For concurrent delivery of multiple fluorescent proteins, cells were fixed and imaged 24 hours after NDs-protein or NDs-PDL-protein complex (50 µg/mL) was added. Quantification of colocalization of fluorescence signals was performed with the ImageJ software. Colocalization of NDs with Early Endosomes and Lysosomes For early endosome labeling, plasmid expressing the early endosome marker protein (GFP-Rab5) was transfected into HeLa cells one day before addition of the NDs-RFP complex. For endosome/lysosome staining, Hela cells were incubated with Lyso Tracker Green DND-26 (0.5 µM) for 5 minutes. Then, NDs-RFP (50 µg/mL) were added. After 2-hour or 6-hour incubation, cells were fixed and imaged at channels for GFP and RFP, respectively. Single-Particle Tracking of NDs-RFP Total internal reflection fluorescence (TIRF) microscopy was carried out using an inverted Leica AM TIRF MC Imaging system equipped with a live cell incubator. The internalization process of NDs-RFP was continuously monitored and each 14


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frame of the fluorescence image series was analyzed using the ImageJ software with a manual tracking plugin. To observe intracellular trafficking of NDs-RFP and the interaction with GFP-Rab5, HeLa cells were transfected with plasmid encoding GFP-Rab5. After 24 hours, cells were incubated with NDs-RFP (50 µg/mL) for 2 hours and washed three times with cell culture medium before confocal imaging. Image Analysis Fluorescence images were analyzed using ImageJ software (US National Institutes of Health). To quantify the co-localization ratio of two fluorescent signals, tMr values (the thresholded Mander's coefficients) indicating the percentage of green signals colocalized with red signals in merged images was calculated. Values represent mean ± SE based on analysis of randomly selected cells. For single particle tracking, the trajectories of red signals and green signals were built by pairing spots in each frame using single-particle tracking plug-in of ImageJ. Cytotoxicity Assay Cytotoxicity was measured by MTT assay. Briefly, cells were seeded in 96-well plates and incubated overnight to reach ~80% confluency. NDs-PDL, RNase A and NDs-PDL-RNase (50 µg/mL) were incubated with cells for 24, 48 or 72 hours at 37°C. 20 µL MTT solution (5 mg/mL) was then added to each well, followed by 4-hour incubation at 37°C. Cells were lysed with 10% acid SDS solution (pH 2~3). After centrifugation, the absorbance of supernatant was determined at 570 nm using a microplate reader (Bio-Rad 680, USA). Fly Culture and Midgut Fluorescence Staining

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Wild-type yw strain and transgenic Escargot-Gal4, uas-GFP (Esg>GFP, which labels ISCs and EBs) strain were raised at 25°C with 60% humidity and a 12/12 hour, light/dark cycle. 50 µL NDs-PDL-RFP, NDs-PDL-RNase, NDs-PDL-BSA, RFP or RNase A Solution (1 mg/mL) was dispersed and absorbed on the surface of fly food. For delivery of RFP, yw flies were fed with food containing RFP or NDs-PDL-RFP for 2 hours and were transferred to normal food for 24 hours. For delivery of RNase A, three-day-old Esg>GFP flies were fed with food containing NDs-PDL-RNase or NDs-PDL-BSA for 24 hours and were transferred to normal food for 24 hours. Flies were anesthetized with CO2, dissected in PBS and were fixed in PBS containing 4% formaldehyde and 0.3% Triton X-100 for 20 minutes at room temperature. They were then washed three times in PBS containing 0.1% Triton X-100 (PBS-T) and blocked with PBS-T containing 5% BSA. Primary antibodies were incubated at 4°C overnight and secondary antibodies were incubated at room temperature for 2 hours, followed by DAPI staining and confocal fluorescence imaging. Rabbit anti-Caspase3 (32183, 1:400) was from Cell Signalling Technology. DAPI (D9542, 1:10000) was from Sigma. Mouse anti-Delta (C594.9B, 1:50) was from Developmental Studies Hybridoma Bank (DSHB). Phalloidin Alexa Fluor568 (A12380, 1:50), goat anti-mouse Alexa Fluor594 (A11012, 1:500) and goat anti-rabbit Alexa Fluor594 (A11005, 1:500) were from Invitrogen. Statistic results on the ratio of each cell type under different dietary treatments were subjected to the linear discriminant analysis (LDA) using the Origin software. Briefly, the ratio of each cell type in each food treatment was used to generate three canonical factors, and the first two most significant discrimination factors

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were used for the 2D plot. About 200 cells were counted in each midgut, and four replicates were performed in each food condition. AUTHOR CONTRIBUTIONS H.S., C.F. and N.C. developed the concept, designed experiments and wrote the manuscript. X.H., X.L., M.Y., P.L., P.H. and L.W. performed cell-based experiments and analyzed the results. X.L., P.L., Y.J. and H.W. performed Drosophila-based experiments and analyzed the results. ACKNOWLEDGEMENTS We would like to dedicate this article to Professor Qing Huang. This study was supported by the National Basic Research Program of China (2016YFA0201200), National Natural Science Foundation of China (31371493, 31571498, 31470970, 31322039 and U1332119), the Youth Innovation Promotion Association from Chinese Academy of Sciences (2015211) and the Key Research Program of Frontier Sciences, CAS (QYZDJ-SSW-SLH019). COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. SUPPORTING INFORMATION Figures S1−S6: characterization of functionalized NDs; assembly and cellular uptake of NDs-protein; colocalization of intracellular fluorescent proteins; delivery of active RNase A into Hela cells and MCF7 cells; apoptosis induced by oral delivery of RNase A (PDF) Movie: S1-S2: videos of internalization and intracellular trafficking of NDs-protein complex (AVI) 17



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Figure 1. Intracellular delivery of fluorescent proteins by NDs. (a) 3D molecular structures of CFP (PDB ID: 1OXD), GFP (PDB ID: 1EMA), YFP (PDB ID: 2YFP), RFP (PDB ID: 2H5Q) generated with the PyMOL software. (b) Emission plots of four fluorescent proteins. (c) Confocal images of HeLa cells after incubation with NDs-fluorescent proteins. Scale bars: 10 µm.

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Figure 2. Dynamic internalization of NDs-protein complex. Live cell imaging of NDs-RFP internalization with a TIRF microscope (also see Movie S1). The initial position of the NDs-RFP particle was indicated by a white circle. A representative trajectory of the internalization of single NDs-RFP particle was divided into three stages and delineated by different colors (I: approaching the membrane, red; II: staying on the membrane, green; III: inside the cytoplasm, blue; IV: the whole trajectory; V: bright field). Scale bars: 10 µm. Velocity analysis of the internalization trajectory was shown below each stage.

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Figure 3. Intracellular trafficking of NDs-RFP. (a-c) Colocalization of NDs-RFP with the endosome and the lysosome. Confocal fluorescence imaging showed the subcellular localizations of NDs-RFP and (a) early endosomes (GFP-Rab5) or (b) endosomes/lysosomes (Lysotracker Green) at indicated time after NDs-RFP was added to Hela cells. Scale bars: 10 µm. (c) Quantitative analysis for (a) and (b). n=10. (d-g) Dynamic interaction of NDs-RFP with the early endosome. (d) Live cell imaging of a single NDs-RFP particle (in yellow circle). Scale bar: 10 µm. (e) Enlarged view of the square region in (d) showed the whole trajectory, starting and end point. White circles indicated other NDs-RFP particles. Scale bars: 2 µm. (f) Snapshots acquired at different time points (also see Movie S2). (g) Fluorescence time traces of signals from the NDs-RFP particle (red) and the early endosome (black) in (e).

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Figure 4. Intracellular release of proteins. (a) Adsorption of RFP to NDs or NDs-PDL in PBS, and desorption of RFP from NDs or NDs-PDL in cell lysate. (b) Confocal fluorescence imaging showed concurrent delivery of CFP, GFP, YFP and RFP into Hela cells by NDs or NDs-PDL. Scale bars: 25 µm. (c) Quantitative analysis of fluorescence signals in Hela cells (n=20). The ratio of non-overlapped signals from each type of protein was indicated in corresponding quadrant, and overlapped signals were scattered near the origin of coordinates. (d) Adsorption of RNase A to NDs or NDs-PDL in PBS. (e) Viability assays of Hela cells after incubation with RNase, NDs-PDL or NDs-PDL-RNase (50 µg/mL) for indicated time. n=3. Student’s t-test, *: p

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