Codelivery of Plasmid and Curcumin with Mesoporous Silica

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Biological and Medical Applications of Materials and Interfaces

Co-delivery of Plasmid and Curcumin with Mesoporous Silica Nanoparticles for Promoting Neurite Outgrowth Cheng-Shun Cheng, Tsang-Pai Liu, Fan-Ching Chien, Chung-Yuan Mou, Si-Han Wu, and Yi-Ping Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02797 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019

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ACS Applied Materials & Interfaces

Co-delivery of Plasmid and Curcumin with Mesoporous Silica Nanoparticles for Promoting Neurite Outgrowth Cheng-Shun Cheng,†, ∇ Tsang-Pai Liu,‡,§,∇ Fan-Ching Chien,‖ Chung-Yuan Mou,†,⊥ Si-Han Wu,*,⊥,＃ and Yi-Ping Chen*,⊥,＃ †Department

of Chemistry, National Taiwan University, Taipei 106, Taiwan ‡Mackay Junior College of Medicine, Nursing and Management, Taipei 112, Taiwan §Department of Surgery, Mackay Memorial Hospital, Taipei 104, Taiwan ‖Department of Optics and Photonics, National Central University, Chung-Li 320, Taiwan. ⊥Graduate Institute of Nanomedicine and Medical Engineering, Taipei Medical University, Taipei 110, Taiwan ＃International Ph.D. Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei 110, Taiwan E-mail: [email protected]; [email protected]

KEYWORDS: neurodegenerative diseases, mesoporous silica nanoparticles, neurite growth, codelivery, combining therapy

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Abstract Reactive oxygen species (ROS)-induced oxidative stress leads to neuron damage is involved in the pathogenesis of chronic inflammation in neurodegenerative diseases (NDs), such as Alzheimer’s (AD), Parkinson’s (PD) and amyotrophic lateral sclerosis (ALS). Researchers, therefore, are looking for anti-inflammatory drugs and gene therapy approaches to slow down or even prevent neurological disorders. Combining therapeutics have shown the synergistic effect in the treatment of human diseases. Many nanocarriers could be designed for simultaneous co-delivery of drugs with genes to fight diseases. However, only a few researches have been performed in NDs. In this study, we developed a mesoporous silica nanoparticle (MSN)-based approach for neurodegenerative therapy. This MSNbased platform involved multiple designs in targeted co-delivery of (1) curcumin, a natural antioxidant product, to protect ROS-induced cell damage, and (2) plasmid RhoG-DsRed, which is associated with the formation of lamellipodia and filopodia for promoting neurite outgrowth. At the same time, TAT peptide was introduced onto the plasmid RhoG-DsRed via electrostatic interaction to elevate the efficiency of non-endocytic pathways and nuclear plasmid delivery of RhoG-DsRed in cells for enhanced gene expression. Besides, such plasmid RhoG-DsRed/TAT complex could work as a noncovalent gatekeeper. The release of curcumin inside the channel of MSN could be triggered when the complex was dissociated from MSN surface. Taken together, this MSN-based platform combining genetic and pharmacological manipulations of actin cytoskeleton as well as oxidative stress provides an attractive way for NDs therapy.


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Introduction Neurodegenerative diseases (NDs) such as Alzheimer's disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS), are prevalent human diseases in the 21st century.1 NDs are a heterogeneous group of either hereditary or sporadic conditions all characterized by progressive loss of neuronal function, resulting from the neurons degeneration in the central nervous system (CNS).2 The CNS, especially brain, is highly sensitive and vulnerable to oxidative stress because of its high oxygen consumption. Oxidative stress is generated by reactive oxygen species (ROS) which are emerging as an important factor in the pathogenesis of chronic inflammation in NDs. Hence, radical scavenging by supplemental enzymatic or non-enzymatic antioxidants is an approach against ROS-induced neuron damage and thus prevent or delay the onset of neurodegenerative diseases. Several researches and clinical studies report that reduced neurogenesis occurs in neurodegenerative disorders. For this, gene therapy is particularly attractive for complex NDs caused by genetic mutations and has been conducted due to the absence of pharmacological treatment. Combination of genetic and pharmacological therapy to balance the low levels of antioxidant enzymes or high content of oxidant substrates and to induce neurogenesis have been reported as a practical therapeutic strategy in various NDs presently.3 Curcumin, a natural polyphenol product extracted from the root of the Indian spice turmeric (Curcuma longa), has been demonstrated with the therapeutic effect on various diseases, such as cancer, neuron disease and inflammation.4-5 Curcumin provides neuroprotection and promotes neurite outgrowth and proliferation in vitro and in vivo due to its antioxidant properties.6-10 Curcumin as a neuroprotective or neurotherapeutic agent is very valuable. However, an obstacle in using curcumin in neuron therapy is its poor bioavailability, especially in the brain. The major causes are manifolds: its easy metabolism, low absorption, systemic elimination, and blood brain barrier (BBB) impermeability.11 Over the past decades, nanotechnology has made a great contribution to the development of drug delivery systems. Recently, delivery of curcumin using various nanoparticles (NPs) has been reported as an excellent approach to enhance its bioavailability because NPs are able to decrease the dose requirement and protect curcumin against decomposition.12 Rho family GTPases recently have revealed their roles in the regulation of neuronal network formation, including migration, neurite outgrowth, polarity, axon guidance, dendrite maturation and synapse formation. RhoG, one type of Rho family GTPase, transduces the Ras signal to activate the Racl and Cdc42 involved in the formation of lamellipodia and filopodia growth.13 Previous studies on neuronal cell lines have shown that RhoG is a crucial regulator in nerve growth factor (NGF)-induced neurite outgrowth.14 Recently, gene delivery by viral vectors, such as adeno-associated viruses (AAVs) and lentiviruses (LVs) become a promising tool for treating various genetic diseases. However, in contrast to non-viral vectors, safety concerns regarding viral vectors include those that can induce immune responses, activation of proto-oncogenes, and insertional mutagenesis. Besides, non-viral vectors are capable of delivering larger, multiple payloads and are typically easier to synthesize, reproduce as well as scale up than viral vectors.15 Although non-viral gene-based therapies have yet to be approved by the FDA, developing 3 ACS Paragon Plus Environment


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the types of vectors, especially for synthetic nanoparticle-based ones, are still extremely attractive.16 Strategy for combining multiple therapeutic molecules (chemotherapeutics, DNA, siRNAs and photosensitizers) with synergistic effects is one of the hottest areas in biomedicine, especially in cancer treatment.17-18 For the past few years, nanoparticles offer an excellent approach for co-delivery of biological/chemical agents with synergistic effects into various cancer cells for cancer therapy.19-20 But, only a few studies have been performed in NDs therapy. In addition, multifunctional nanoparticles can respond to stimuli-sensitive drug delivery, also known as “control” or “smart” drug release system through numerous chemical (pH), biological (redox conditions or enzymes) and physical (temperature) stimuli.17 However, for NDs therapy, it is extremely challenging to create such a platform that have all these advanced properties. The main reason is most stimuli of drug release exists only in the tumor microenvironment, and not in the brain. Therefore, the problems need to be overcome before NPs can be used to NDs therapy clinically. Mesoporous silica nanoparticles (MSN) are one of the most widely used nanoparticles and generally considered to be non-cytotoxicity with high biocompatible property in vitro and in vivo.21 MSN as one promising nanomaterial has attracted a lot of attention in biomedical applications that can be designed to deliver multiple molecules, including genetic materials, proteins, and small molecules.22-25 Therefore, functionalized MSN provide opportunities for use widely in the fields of targeting, drug delivery, diagnosis, imaging, and bio-sensing.26-29 In this study, we aim to develop MSN as an advanced nano platform which can co-deliver curcumin drug and RhoG simultaneously to examine their therapeutic efficacy on NDs in N2a cells. Hence, MSN encapsulated curcumin and adsorbed plasmid RhoG-DsRed/TAT peptide complex, forming drug-gene-loaded MSN (Cur@MSNRhoG/TAT). The morphology, size distribution and in vitro curcumin release profile were characterized. Also, plasmid RhoG-DsRed was designed to be a therapeutic agent as well as a drug release switch, which can be dissociated from MSN in the cytoplasmic environment. Then, the release of curcumin from MSN could be triggered successfully. In addition, a driving force from TAT peptide adsorbed on the surface of plasmid RhoG-DsRed could promote the cellular uptake of Cur@MSNRhoG/TAT through non-endocytosis routes, as well as enhance the efficiency of plasmid RhoG-DsRed expression by its nuclear delivery ability. The RhoG expression for enhancing the neurite outgrowth and the therapeutic effect against ROS via curcumin in N2a cells were evaluated. Instead of single drug treatment, simultaneous administration is an attractive way for maximizing the synergetic therapeutic effect and minimizing undesirable side effects for NDs.

Results and Discussion PEGylated FITC fluorescent MSN with an average diameter of 50 nm was synthesized with our previously reported method based on a sol-gel synthetic strategy in the presence of cationic surfactant template.30 Here, for adsorbing the negatively charged plasmid RhoG-DsRed (plasmid RhoG expressing DsRed), MSN was also grafted with a positively charged molecule (Ntrimethoxysilylpropyl-N,N,N-trimethylammonium chloride, TMAC) by co-condensation. 4 ACS Paragon Plus Environment

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Transmission electron microscope (TEM) images displayed a well-ordered hexagonal structure with a statistical diameter of 46.6  6 nm (Figure 1a). Figure 1b showed the nitrogen adsorption-desorption isotherms of the extracted MSN at 77K. These isotherms could be categorized as type IV behaviors without obvious hysteresis loop. The Brunauer-Emmett-Teller (BET) surface area and Barrett-JoynerHalenda (BJH) maximum pore diameter of MSN were around 682 m2/g and 1.90 nm, respectively. The measurement of hydrodynamic diameter by dynamic light scattering (DLS) and surface charge by zeta potential analysis at room temperature were shown in Table S1. The resulting MSN carried a net positive charge of 32.7  2.4 mV due to the quaternary ammonium groups of TMAC on the surface. DLS determined the average hydrodynamic diameter of MSN in H2O was around 63 nm. Neuro-2a (N2a) cells can be provided as a homogeneous population of cells in large numbers and exhibit unique neuronal property, allowing its use as a cellular model for neuron related diseases studies. Hence, the cytotoxicity was evaluated in N2a cells treated with various amounts of MSN for 4 h. WST-1 assay revealed that there was no significant cell death on the cell viability, indicating MSN was suitable as a dug carrier for neuron therapy (Figure 1c). To investigate the cellular uptake efficiency, MSN-treated N2a cells were assessed by flow cytometry. The results showed that cell uptake was in a dose-dependent manner (Figure 1d). Then, MSN was used as a carrier to adsorb plasmid RhoG-DsRed via electrostatic adsorption to perform the RhoG delivery. The interactions between the MSN and plasmid RhoG-DsRed were analyzed by electrophoretic mobility in 1% agarose gel with various weight/weight (w/w) ratios of plasmid DNA to nanoparticles (Figure 1e). The results revealed that plasmid RhoG-DsRed could bind with MSN to form stable DNA-MSN complexes MSNRhoG at weight ratios larger than 1:64 because no free plasmid RhoG-DsRed was detected. We therefore chose the RhoG/MSN ratio of 1:128, to ensure complete adsorption between RhoG and MSN, in further gene delivery experiments. However, from the results of Figure 2a, no RhoG expression was observed in the MSN-RhoG-treated N2a cells. Therefore, we introduced TAT peptide on the MSNRhoG surface to enhance the RhoG gene expression efficiency. Recently, gene delivery by nanoparticles becomes a powerful technology for manipulating gene expression and a multifunctionally therapeutic strategy for treating various human diseases. But, the issues of low gene expression and significant cytotoxicity are still a challenge for neuron cells presently. The TAT peptide (AYGRKKRRQRRR) derived from the primary region of the transactivator of transcription of HIV-1 is a well-known cell penetrating peptides. It has been shown to freely transport across the biological barriers, such as cellular and nuclear membranes and BBB.31-33 Currently, conjugating TAT on nanoparticles is able to provide a non-endocytic cell uptake, enhance drug/gene nuclear delivery and overcome multidrug resistance (MDR).34-35 Here, to prevent the influence of plasmid RhoG-DsRed expression from TAT peptides, instead of covalent binding, positively charged TAT peptides were adsorbed with negatively charged plasmid RhoG-DsRed by electrostatic interaction first and subsequently incubated with MSN to form MSN-RhoG/TAT. As shown in Figure 2a, we found that neither MSN-RhoG nor RhoG/TAT treated N2A cells showed red fluorescent RhoG-DsRed expression, probably due to insufficient delivery and expression. However, RhoG-DsRed expression 5 ACS Paragon Plus Environment


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was observed in cells after the treatment of MSN-RhoG/TAT, indicating MSN could carry plasmid RhoG-DsRed efficiently into cells probably because TAT peptides could trigger the non-endocytosis routes as well as enhance nuclear delivery of plasmid RhoG-DsRed for performing gene expression. The transfection efficiency of plasmid RhoG-DsRed analyzed by using flow cytometry was 15.8 %. In addition, confocal microscopy imaged the enhanced neurite outgrowth 4 days after treatment, suggesting that MSN-RhoG/TAT can deliver plasmid RhoG-DsRed into N2a cells for gene expression, followed by enhancing neurite outgrowth (Figure 2b). To understand the effect and mechanism of TAT peptide on the delivery of MSN-RhoG/TAT, the internalization of NPs into cells was carried out by flow cytometry. The uptake of nanoparticles into the cells may be facilitated by endocytosis (energy-dependent) and nonendocytosis (energyindependent) routes.36-37 As shown in Figure 2c, all the NPs (256 g/ mL) had 100 % of cell uptake at 37°C for 4 h. Because endocytosis is an energy-dependent process, we delivered the NPs at 4°C for 4 h.38 The results showed that MSN and MSN-RhoG had only around 5 % cell uptake. However, MSNRhoG/TAT obviously enhanced the uptake of N2a cells (around 15%) through a TAT-mediated nonendocytosis delivery. The increased non-endocytosis delivery could explain MSN@RhoG/TAT displayed plasmid RhoG-DsRed gene expression and neurite outgrowth successfully. Furthermore, we used several inhibitors of endocytosis to elucidate the specific routes of cellular uptake of MSNRhoG/TAT into N2a cells. To decrease the endocytosis energy, ATP depletion by treating with sodium azide (10 mM) and 2-deoxyglucose (6 mM) was employed, resulting in the inhibition of endocytosis uptake.38 Additionally, Amiloride (10 M), Filipin III (5 g/mL), and Chlorpromazine (30 M) are common endocytosis pathway inhibitors, which are associated with the macropinocytosis, caveolaemediated uptake, and clathrin-mediated endocytosis, respectively.39-41 Hence, N2a cells were pretreated with ATP-depletion solution and various types of inhibitors for 1 h at 37 °C and subsequently treated with 256 g/ mL of MSN-RhoG/TAT for another 4 h under different kinds of endocytosis inhibitors. The results demonstrated that MSN-RhoG/TAT was involved in the cell internalization via both energy-independent and dependent process (Figure 2d). Regarding the curcumin-loaded MSN-RhoG/TAT (Cur@MSN-RhoG/TAT), the amount of curcumin was quantitatively assayed using multi-well plate ELISA reader (Bio-Red) by measuring the fluorescence intensity of curcumin at 420 nm (excitation) and 520 nm (emission). Table S2 summarized the results of loading efficiency and loading amount of curcumin on Cur@MSNRhoG/TAT. Furthermore, the release behavior of curcumin was conducted in PBS buffer at a pH of 7.4 within 48 h, which exhibits that ∼60% loaded curcumin has been released from the Cur@MSNRhoG/TAT after 4 h (Figure 3a). One expects curcumin could achieve similar release behavior inside the cells. We then check the influence of Cur@MSN-RhoG/TAT to cell uptake and RhoG enhanced neurite outgrowth after the loading of curcumin in N2a cells (Figure 3b and c). The results showed Cur@MSN-RhoG/TAT with the concentration of 256 g/mL still exhibited highly cell uptake for 4 h and exhibited RhoG expression at day 4 after the delivery, indicating curcumin loading did not affect the function of plasmid RhoG-DsRed as well as enhanced neurite outgrowth. The transfection 6 ACS Paragon Plus Environment

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efficiency of plasmid RhoG-DsRed analyzed by using flow cytometry was 12.3 %. In addition, TAT peptides also enhanced the cell uptake efficiency of Cur@MSN-RhoG/TAT, as compared to the Figure 1d results of MSN at the same concentration. According to previous studies, curcumin, which was found from the rhizome of turmeric with anti-cancer and anti-inflammatory properties, could display its therapeutic potential in the inflammatory-related neurodegenerative disease, especially in AD.6 With low-dose treatment of curcumin could reduce the ROS levels to protect the neural cells damage, which plays an important role in the regulation of neurodegenerative disease. Therefore, we evaluated the intracellular ROS levels and its potential of Cur@MSN-RhoG/TAT as ROS scavenger for neurodegenerative disease therapy. N2a cells were added 256 g/mL of MSN-RhoG/TAT or Cur@MSN-RhoG/TAT for 4 h, and subsequently treated with 150 μM of paraquat (N, N’-dimethyl-4, 4’-bipyridinium dichloride, PQ), a generator of superoxide anion radicals, to simulate the inflammation, for another 48 h. Next, ROS generation detected by dihydroethidium (DHE, 5 M), a fluorescent indicator for intracellular ROS, was imaged by using fluorescence microscopy and quantified by using flow cytometry.42 As shown in Figure 4a and 4b, no significant ROS were produced in N2a cells treated with MSN-RhoG/TAT. Not surprisingly, we found an obvious increase after the treatment of PQ as comparted to control of cells. MSN-RhoG/TAT cannot prevent ROS production under the PQ stimulation. However, the treatment with Cur@MSN-RhoG/TAT dramatically suppressed the PQ-induced DHE fluorescent signal. The early release of curcumin after detaching of plasmid RhoG took an important role in scavenging ROS to protect N2a cells as well as RhoG from ROS-mediated damage. After that, the effective protein expression could be generally achieved 24 h post-transfection. In this situation, the effect of two different agents, curcumin and RhoG, could be happened at different time points. ROS are not only toxic to the cells, but also may behave as second messengers to active mitogenactivated protein kinase (MAPK) signaling pathway which has been implicated in the oxidative stress and apoptosis. To determine whether the Cur@MSN-RhoG/TAT could regulate p-p38 protein, an important biomarker of MAPK pathway responded to intracellular oxidative stress, through decreasing the production of ROS, western blot analysis was carried out at the same condition.43 As shown in Figure 4c, following treatment with MSN-RhoG/TAT (256 g/mL) could not induce the expression of p-p38. The expression of p-p38 was significantly activated by 150 M of PQ and was not suppressed by MSN-RhoG/TAT co-treatment. However, decreased expression of p-p38 can be detected when we treated with curcumin loaded nanoparticles, Cur@MSN-RhoG/TAT (256 g/mL). Several studies have revealed that ROS cause inflammation by modulating the nuclear factor kappa-B (NF-B) expression that has been associated with the pathogenesis of neurodegenerative disorders.44-45 During normal condition, NF‐κB exist in the cytoplasm as an inactive form consisting of the subunits p50/p65 bound to inhibitory proteins known as inhibitory kappa-B (IκB). When activated, the phosphorylated IκB is ubiquitinated and proteolytically degraded, leading to NF‐κB translocation into the nucleus and then binding to its downstream genes involved in inflammation, cell survival, proliferation, invasion, and angiogenesis.46 Therefore, we isolated the cytosolic and nuclear 7 ACS Paragon Plus Environment


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proteins respectively and further study the expression levels of NF-B. As shown in Figure 4d, stimulation with 150 M of PQ markedly induced the activation of NF-B with nuclear translocation of NF-B p65 protein. The nuclear translocation of NF-B p65 by PQ could be attenuated significantly in N2a cells treated with Cur@MSN-RhoG/TAT (256 g/mL). The results further support that Cur@MSN-RhoG/TAT could decrease neuron cell damage and inflammation via the decrease of ROS generation, followed by the inhibition of p-p38 and NF-B. To date, the non-viral vector systems by various nanoparticles not only avoid the risk of using viral vector systems, but have demonstrated the enhanced efficacy of combining therapy in cancer treatment.47 But, only a few studies have been performed in NDs therapy. Here, we have demonstrated that the benefits of Cur@MSN-RhoG/TAT with multiple therapeutic functions including (1) curcumin release to scavenge ROS, and (2) RhoG delivery to enhance neuron outgrowth. For NDs therapy, a combined therapy strategy by Cur@MSN-RhoG/TAT may be an option. One important indicator for assessing neuron protection is neurite outgrowth, such as neurite number and length. Neurite outgrowth is a critical process in the differentiation of neurons, which shows the morphological phenotype of neuronal cells that correlates with their health and function. To investigate the neuroprotective effect of combining therapy with synergistic effect by Cur@MSN-RhoG/TAT on neuronal differentiation, we further studied the neurite length under the PQ-induced ROS condition. According to neurite length, the level of neurite outgrowth of differentiated cells was estimated and divided into four groups (L0, L1, L2, and L3). L0 indicates the cells with no neurites; L1 indicates the neurite length of differentiated cells is shorter than the size of the cell body; L2 indicates the neurite length of differentiated cells is between the L1 and L3; L3 indicates the neurite length of differentiated cells is longer than twice the size of the cell body. Details were in the Experimental Section.48 Retinoic acid (RA) is a well-known agent to promote neuronal differentiation and neurite outgrowth.49 After the treatment for 4 days, neurite length was affected by 150 M of PQ (Figure 5a). Not surprisingly, RA (20 M) only can enhance the neurite outgrowth because of the increase of L2 and L3 portions, as compared to PQ only. Also, we found that all the groups treated with RA showed a significant increase in the percentage of L2 and L3 even if under the condition of PQ treatment. If we compared the difference of L3, N2a cells treated with Cur@MSN-RhoG/TAT (256 g/mL), RA and PQ (Cur@MSN-RhoG/TAT +RA +PQ) still have the highest percentage of L3 (56%), which was approximately twofold higher than that of treatment with RA and PQ (+RA +PQ). The morphological images were observed shown in Figure 5b. The neurite outgrowth of RA-differentiated N2a cells was obviously inhibited by PQ. At the same condition, the percentage of the cells with longer neurite increased with an increase in the treatment of Cur@MSN-RhoG/TAT, which was consistent with the results of Figure 5a. Therefore, the results indicated that Cur@MSN-RhoG/TAT protected neurite outgrowth against PQ-induced ROS damage to N2a cells through its successful delivery of the anti-inflammatory drug, curcumin. In addition, RhoG also can enhance the neurite outgrowth with a synergistic effect. We proposed that the combining therapy based on the Cur@MSN-RhoG/TAT is a promising approach for NDs.


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Conclusions In conclusion, we have developed MSN as an advanced nano platform which can co-deliver curcumin drug combined with RhoG simultaneously to examine their therapeutic efficacy on NDs in N2a cells. Here, we successfully encapsulated curcumin and adsorbed plasmid RhoG-DsRed/TAT peptide complex, forming drug-gene-loaded MSN (Cur@MSN-RhoG/TAT). The morphology, size distribution and in vitro curcumin release profile were characterized. We demonstrated that the plasmid RhoG-DsRed expression can enhance the neurite outgrowth, as well as the therapeutic effect against ROS via curcumin in N2a cells by using MSN mediated combining therapy strategy. For preclinical studies and clinical translation, approaches are needed for overcoming the expected brain barrier beyond delivering the hybrid MSN in the vicinity of neuronal cells.

Experimental Section Synthesis of Green Fluorescent Mesoporous Silica Nanoparticles (MSN) First, the FITC-APTMS solution was prepared by mixing of 2 mg of fluorescein isothiocyanate (Sigma) and 10 L of APTMS in 5 mL of ethanol under continuous agitation at room temperature for 24 hours. Then, 0.29 g cetyl-trimethylammonium bromide (CTAB) was dissolved in 150 mL of 0.51 M ammonia solution at 50 °C with vigorous stirring in a sealed beaker. After 15 min, 2.5 mL of FITC-APTMS solution and 2.5 mL of 0.88 M ethanolic TEOS solution were sequentially added dropwise to the reaction solution. After 1 h, 250 L of N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (TMAC) and 450 L of silane-PEG were added to the solution under stirring for an additional 30 min. Then, the solution was kept static at 50 °C for 24 h. Next, hydrothermal treatment of the solution was applied at 70 °C and 90 °C, respectively, for 24 h. The resulting as-synthesized samples were collected by centrifugation and washed by 99.5% ethanol twice. To remove surfactant, the as-synthesized samples were dispersed in 100 mL of ethanol containing 50 L of 37 wt% HCl and stirred at 60 °C for 1 h. Finally, the MSN nanoparticles were collected by centrifugation, washed by ethanol several times and stored in ethanol. Plasmid RhoG-DsRed/MSN Binding and Agarose Gel Electrophoresis For plasmid RhoG-DsRed binding affinity analysis, 2 g of plasmid RhoG-DsRed was mixed with various amounts of MSN at different weight/weight (w/w) ratios of plasmid DNA to nanoparticles (1/8, 1/16, 1/32, 1/64, 1/128, and 1/256) in DI water. After incubation for 30 min, the complexes were analyzed by electrophoretic mobility in 1% agarose gel at a voltage of 110 V for 30 min. Finally, the DNA bands were visualized by staining with ethidium bromide. Images were taken using a UV Transilluminator (Major Science). DNA Ladder, plasmid RhoG, and MSN were used as references. Preparation of Cur@MSN-RhoG/TAT Two micrograms of plasmid RhoG-DsRed was first mixed with 2 g of TAT peptide in DI water. 9 ACS Paragon Plus Environment


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After 30 min incubation, 256 g of Cur@MSN was added to the mixture under stirring for an additional 30 min to form Cur@MSN-RhoG/TAT. In Vitro Drug Loading Study The loading of curcumin onto the MSN could be performed through hydrogen bonding between the phenolic/enolic hydroxyl groups of curcumin and the hydroxyl groups present on the surface of MSN. First, 36.8 mg of curcumin was dissolved in 10 mL of 99.5% ethanol (10 mM curcumin) for use. Then, 2 mg of MSN suspended in 0.95 mL ethanol was mixed with 50 L of 10 mM curcumin solution and stirred at room temperature for 24 h. Afterward, curcumin loaded MSN (Cur@MSN) was collected by centrifuging at 11,000 rpm for 90 min and washed twice using ddH2O to remove the loosely attached curcumin. The supernatant was collected, and the amount of unloaded curcumin was quantitatively assayed using multi-well plate ELISA reader (Bio-Red) by measuring the fluorescence intensity of curcumin at 420 nm (excitation) and 520 nm (emission). The curcumin loading efficiency (%) and loading amount (g curcumin/mg NPs) were calculated according to the following formulation: 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑐𝑢𝑟𝑐𝑢𝑚𝑖𝑛 𝑖𝑛 𝐶𝑢𝑟@𝑀𝑆𝑁 The loading efficiency(%) = × 100% 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡𝑜𝑡𝑎𝑙 𝑐𝑢𝑟𝑐𝑢𝑚𝑖𝑛

The loading amount =

𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑐𝑢𝑟𝑐𝑢𝑚𝑖𝑛 𝑖𝑛 C𝑢𝑟@𝑀𝑆𝑁 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝐶𝑢𝑟@𝑀𝑆𝑁

In vitro Drug Release Profile One mL of Cur@MSN-RhoG/TAT dispersed in PBS (pH 7.4) was added into a regenerated cellulose dialysis tubing (MWCO 12-14 kDa). The dialysis bag was dialyzed against 50 mL of PBS under gentle stirring at 37°C. At predetermined time intervals, 1 mL of the dialysate was removed, and the same volume of fresh PBS was added to keep a constant volume. The removed samples were then assayed for curcumin by an ELISA reader. Cell Line and Cell Culture Neuro-2a (N2a) cells are derived from spontaneous neuroblastoma of mouse and are extensively used to study neuronal differentiation. N2a cells were cultured in DMEM medium (GIBCO) containing 10% fetal bovine serum (GIBCO), 100 μg/mL streptomycin and 100 U/mL penicillin (GIBCO) under an atmosphere of 5% CO2 at 37 °C. In vitro Cell Uptake N2a cells were seeded in 6-well plates at a density of 3 × 105 cells per well. After 24 h incubation at 37℃, different concentrations of NPs were treated for 4 h. Then, the treated cells were washed twice with PBS and harvested by trypsinization. After centrifugation, the cells were dispersed in 0.25 mL of PBS and the extracellular FITC signals from the MSN were quenched by using trypan blue. FACS Calibur flow cytometer (BD Biosciences) was used to determine the cellular uptake of NPs by 10 ACS Paragon Plus Environment

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detecting the FITC signals inside the N2a cells. For cell uptake mechanism study, N2a cells were pretreated at 4 °C or various inhibitors (filipin III: 5 μg/mL; chlorpromazine: 30 μM; amiloride: 10 μM) or ATP-depletion solution (sodium azide: 10 mM; 2-deoxyglucose: 6 mM) at 37 °C for 1 h and subsequently treated with 256 μg/ mL of MSN-RhoG/TAT for another 4 h under 4 °C or different kinds of inhibitors condition. After that, the cells were washed with PBS and trypsinized. Finally, the extracellular FITC signals were quenched by using trypan blue for FACS Calibur flow cytometer analysis. Measurement of Neurite Length of N2a cells To identify the neuroprotective effect of Cur@MSN-RhoG/TAT on PQ induced ROS stress, neurite length of N2a cells was measured. N2a cells were seeded onto the glass slide and cultured at 37℃ for 24 h. After the attachment, N2a cells were treated with 256 g/mL of Cur@MSN-RhoG/TAT for 4 h at 37℃. Then, the cells were washed with PBS and incubated with or without 150M paraquat (PQ) and 20 M retinoic acid for 4 days. After that, N2a cells were washed with PBS and fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 5 min at room temperature, followed by 1 h of blocking in blocking buffer (Tris-buffered saline containing 0.1% Tween 20 and 5% w/v non-fat dry milk). After washing with TBST buffer (Tris-buffered saline containing 0.1% Tween 20), the cells were incubated at 4℃ overnight with an actin primary antibody diluted into blocking buffer. The cells were extensively washed with TBST buffer and incubated with Alexa Fluor 568 secondary antibody diluted into blocking buffer (1:200, Thermo Fisher Scientific) for 2 h at room temperature, followed by nuclear staining with DAPI for 5 min. Finally, the neurite outgrowth of N2a cells were imaged by fluorescence microscopy and then the neurite length was measured and calculated by Image J analysis. According to neurite length, the level of neurite outgrowth of differentiated cells was defined as L0, L1, L2 and L3. L0 indicates the cells with no neurites; L1 indicates the neurite length of differentiated cells is shorter than the size of the cell body; L2 indicates the neurite length of differentiated cells is between the L1 and L3; L3 indicates the neurite length of differentiated cells is longer than twice the size of the cell body.48

ASSOCIATED CONTENT Supporting Information. DLS, zeta potential, TEM, N2 Adsorption-desorption Isotherms, XRD, cell viability assay, confocal microscopy, superoxide detection, western blot analysis, loading efficiency and loading amount of curcumin.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected] 11 ACS Paragon Plus Environment


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Author Contributions ∇ C.-S.

Cheng and T.-P. Liu contributed equally to this work.

ACKNOWLEDGMENTS This work was supported by Ministry of Science and Technology (MOST 106-2113-M-038-001-MY2 and MOST 106-2113-M-038-006-MY2). The authors thank Ms. C.-Y. Chien of Ministry of Science and Technology (National Taiwan University) for the assistance in TEM experiments. Some elements in TOC designed by Freepik.

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Figure 1. Characterization of MSN. (a) TEM image and size distribution calculated from TEM. (b) Nitrogen adsorption/desorption isotherms and pore size distribution (inset). (c) Cytotoxicity and (d) cell uptake of MSN at different concentrations for 4 h incubation in N2a cells. (e) Gel electrophoresis examined the binding ability of plasmid RhoG-DsRed and MSN in 1% agarose gel. DNA ladder, plasmid RhoG, and MSN were references. 16 ACS Paragon Plus Environment

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Figure 2. N2a cells were treated with 256 g/mL of NPs for 4 h. (a) RhoG-DsRed gene expression after 24 h treatment and (b) enhanced neurite outgrowth after 4 days treatment of MSN-RhoG/TAT were imaged by using confocal laser scanning microscopy (CLSM). BF indicates bright field; Red (DsRed) and blue (DAPI) fluorescence represent RhoG and nucleus, respectively. Flow cytometry analyzed (c) TAT enhanced cell uptake and (d) the mechanism of cell uptake of MSN-RhoG/TAT in the presence of chemical inhibitors in N2a cells. ATP depletion: sodium azide (10 mM) and 2deoxyglucose (6 mM); Filipin III (5 g/mL); CPZ: chlorpromazine (30 M); Amiloride (10 M). 17 ACS Paragon Plus Environment


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Figure 3. (a) In vitro release behavior of curcumin from Cur@MSN-RhoG/TAT in PBS buffer (pH 7.4). After the treatment with 256 g/mL of Cur@MSN-RhoG/TAT, (b) cell uptake for 4 h was assayed by using flow cytometry and (c) enhanced neurite outgrowth after 4 days treatment was imaged by using confocal laser scanning microscopy (CLSM). BF indicates bright field; Red (DsRed) and blue (DAPI) fluorescence represent RhoG and nucleus, respectively.


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Figure 4. Protective effect of Cur@MSN-RhoG/TAT on paraquat-induced ROS production and its signaling pathway. N2a cells were treated with various NPs (256 g/mL) for 4 h and then incubated with and without PQ (150 M) for another 48 h. (a) Fluorescence microscopy imaged and (b) flow cytometry quantified the intracellular ROS levels stained by DHE (5 M). Western blotting was used to determine the protein levels of (c) p-p38 and (d) NF-B activation. NE and CE indicate the nuclear and cytoplasmic extraction, respectively. Lamin B and -actin were used as a loading control.



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Figure 5. Therapeutic effect of Cur@MSN-RhoG/TAT on neurite growth. (a) Distribution of the neurite length and (b) morphological images after 4 days of treatment with and without PQ (150 μM), RA (20μM), and Cur@MSN-RhoG/TAT (256μg/mL). (*p < 0.05 , **p < 0.01, and ***p

Codelivery of Plasmid and Curcumin with Mesoporous Silica

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