Polyamino Acid Layer-by-Layer (LbL) Constructed Silica-Supported

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Biological and Medical Applications of Materials and Interfaces

Polyamino acid layer-by-layer (LbL) constructed silica-supported mesoporous titania nanocarriers for stimuli-responsive delivery of microRNA 708 and paclitaxel for combined chemotherapy Biki Gupta, Hima Bindu Ruttala, Bijay Kumar Poudel, Shiva Pathak, Shobha Regmi, Milan Gautam, Kishwor Poudel, Min Hyun Sung, Wenquan Ou, Sung Giu Jin, JeeHeon Jeong, Sae Kwang Ku, Han-Gon Choi, Chul Soon Yong, and Jong Oh Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06642 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 8, 2018

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ACS Applied Materials & Interfaces

Polyamino acid layer-by-layer (LbL) constructed silica-supported mesoporous titania nanocarriers for stimuli-responsive delivery of microRNA 708 and paclitaxel for combined chemotherapy

Biki Gupta1, Hima Bindu Ruttala1, Bijay Kumar Poudel1, Shiva Pathak1, Shobha Regmi1, Milan Gautam1, Kishwor Poudel1, Min Hyun Sung1, Wenquan Ou1, Sung Giu Jin2, Jee-Heon Jeong1, Sae Kwang Ku3, Han-Gon Choi4, Chul Soon Yong1, Jong Oh Kim1*

1

College of Pharmacy, Yeungnam University, 214-1, Dae-Dong, Gyeongsan 712-749, Republic

of Korea 2

Department of Pharmaceutical Engineering, Dankook University, 119 Dandae-ro, Dongnam-gu,

Cheonan, 31116, Republic of Korea 3

College of Korean Medicine, Daegu Haany University, Gyeongsan, 712-702, Republic of

Korea 4

College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang

University, 55, Hanyangdaehak-ro, Sangnok-gu, Ansan 426-791, Republic of Korea

*

Corresponding author: Prof. Jong Oh Kim, Ph.D.

Tel: +82-53-810-2813, Fax: +82-53-810-4654, E-mail: [email protected]

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Abstract Cellular FADD-like interleukin-1β-converting enzyme-inhibitory protein (c-FLIP), often strongly expressed in numerous cancers, plays a pivotal role in thwarting apoptosis and inducing chemotherapy resistance in cancer. An integrated approach combining chemotherapy with suppression of c-FLIP levels could prove paramount in the treatment of cancers with c-FLIP overexpression. In this study, we utilized a polymeric layer-by-layer (LbL) assembly of silicasupported mesoporous titania nanoparticles (MTNst) to co-deliver paclitaxel (PTX) and microRNA 708 (miR708) for simultaneous chemotherapy and c-FLIP suppression in colorectal carcinoma. The resulting LbL miR708/PTX-MTNst showed dose-dependent cytotoxicity in HCT-116 and DLD-1 colorectal carcinoma cell lines, which was remarkably superior to that of free PTX or LbL PTX-MTNst. LbL miR708/PTX-MTNst strongly inhibited c-FLIP expression and resulted in increased expression of proapoptotic proteins. In DLD-1 xenograft tumor-bearing mice, the nanoparticles accumulated in the tumor, resulting in remarkable tumor regression, with the PTX and miR708-loaded nanoparticles showing significantly greater inhibitory effects than the free PTX or PTX-loaded nanoparticles did. Immunohistochemical analyses of the tumors further confirmed the remarkable apoptotic and anti-proliferative effects of the nanoparticles, while organ histology reinforced the biocompatibility of the system. Therefore, the LbL miR708/PTX-MTNst system, owing to its ability to deliver both chemotherapeutic drug and inhibitory miRNA to the tumor site, shows great potential to treat colorectal carcinoma in clinical settings.

Keywords: mesoporous titania nanoparticles, chemotherapy, microRNA-708, paclitaxel, c-FLIP inhibition


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INTRODUCTION Cellular FLICE (FADD-like interleukin-1β-converting enzyme)-inhibitory protein (c-FLIP) is considered to be a master anti-apoptotic regulator protein and resistance factor that inhibits tumor necrosis factor-α (TNF-α)-, Fas-L-, and TNF-related apoptosis-inducing ligand (TRAIL)induced apoptosis, along with chemotherapy-induced apoptosis of cancer cells.1 c-FLIP, by interaction with FADD (Fas-associated protein with death domain) and/or caspase-8 or caspase10 and death receptor 5 (DR5), prevents the formation of DISC (death-inducing signaling complex), and thereby staves off the ensuing caspase cascade activation.2 c-FLIP has been reported to be highly expressed in a large variety of cancers, including colorectal carcinoma,3 cervical cancer,4 hepatocellular carcinoma,5 squamous cell carcinoma,6 gastric cancer,7 pancreatic cancer,8 prostate cancer,9 and ovarian cancer10. In colorectal carcinoma, c-FLIP seemingly plays a significant role in inhibiting chemotherapy-induced apoptotic cell death. siRNA-mediated downregulation of c-FLIP expression has resulted in the synergistic induction of apoptosis induced by chemotherapeutic drugs.11,12 Therefore, a ploy, wherein an inhibitor of cFLIP expression is co-administered with a chemotherapeutic agent, might result in potent enhancement of apoptosis of colorectal carcinoma cells as an effective treatment approach. A myriad of agents have been observed to cause c-FLIP inhibition at the transcriptional or post-transcriptional level. Among these, paclitaxel (PTX) has been reported to inhibit both isoforms of c-FLIP protein (c-FLIPL and c-FLIPS) by a post-transcriptional mechanism.13 Two reports have emphasized that inhibition of c-FLIP expression using microRNA (miRNA) can enhance the sensitivity of cancer cells toward the cytotoxic effects of chemotherapeutic agents.14,15 However, the delivery of miRNA, particularly unmodified miRNA, to the targeted tumor site is faced with several challenges, including mechanical and biological barriers,



degradation in circulation by nucleases, induction of immunotoxicity, off-target effects, and neurotoxicity.16 Carefully designed administration vehicles are, therefore, deemed to be of utmost importance for miRNA delivery. Recently, nanoparticles have gained much popularity in miRNA delivery. Various lipid-based,17-20 polymeric,21-24 and inorganic systems25,26 have been reported to successfully deliver miRNAs to a variety of cancers. Inorganic-organic hybrid nanoplatforms have recently amassed enormous attention as potential clinically-relevant therapeutic carriers for effective cancer therapy. They have been viewed to effectively integrate the major advantages of inorganic (stability, photonics, high payload) and organic (biocompatibility, stimuli responsiveness, cellular targeting, release control) materials as therapeutic carriers.27-30 Titania (titanium dioxide, TiO2) has found extensive utility in solar cells, biosensors, adsorbents, photocatalysts, and electrochromic displays based on its peculiar characteristics, such as its semiconducting trait, UV-photoactivity, thermal stability, chemical inertness, and electrochromism.31-39 Furthermore, mesoporous titaniabased materials are characterized by expansive surface areas with highly tunable pores.40,41 Despite the potentially innumerable advantages of mesoporous titania-based systems, their application in the biomedical field is very limited, with the complexities associated with their synthesis as stable nanoparticulate systems perceived as the major hurdle.42 The major quandary associated with the preparation of mesoporous titania nanoparticles (MTN) is well-highlighted in surfactant-based soft template methods, which produce highly amorphous titania, wherein the uniform mesoporous order is exceedingly prone to collapse upon subsequent crystallization by thermal treatment.43 In an attempt to overcome such shortcomings, inert solid supports can be employed to establish core-shell architecture by utilizing metal, metal oxide, silica, carbon, or graphene oxide nanoparticles.44-49


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A major problem commonly associated with uncapped mesoporous systems is the initial burst release prior to arrival at the required site of action. This is especially pronounced in the case of highly water-soluble payload and may result in sub-therapeutic drug levels at the site of action while increasing secondary toxicity. In order to attain precise control over the release kinetics and to overcome the initial burst release, a capping system at the mesopore entrance is viewed as an optimal approach.50 In this regard, a polyelectrolyte layer-by-layer (LbL) engineered capping system is deemed to be an effective tool to not only control the release of the payload from porous nanocarriers but also to bestow pivotal delivery characteristics, such as pHdependent release.51-53 We have previously reported poly-(L-lysine) (PLL)/ poly(ethylene glycol)-block-poly-(L-aspartic acid) (PEG-b-PLD), chitosan (CT)/ hyaluronic acid (HA) and polyarginine (pARG)/ poly(ethylene glycol)-block-poly-(L-aspartic acid) (PEG-b-PLD)-based LbL-assembled nanoarchitectures with sustained and pH-dependent release characteristics.54-56 Herein, we present a hybrid LbL system comprising a silica-supported mesoporous titania nanoparticle (MTNst) core and an LbL corona composed of PLL and PEG-b-PLD, for the codelivery of paclitaxel (PTX) and microRNA 708 (miR708, to target c-FLIP) to colorectal cancers. While the mesopores in MTNst housed the PTX, the cationic PLL in the corona formed polyplexes with miR708, to yield PTX and miR708-loaded LbL-assembled MTNst (LbL miR708/PTX-MTNst). The practical use of this system is tested in both in vitro cell culture and in vivo animal tumor models.

EXPERIMENTAL METHODS Materials Paclitaxel (PTX) was acquired from Shaanxi Top Pharm Chemical Co. Ltd. (Xi’an, China), and miR708 (hsa-mir-708 mimic) was procured from Bioneer Corp. (Daejeon, South Korea).



Tetraethyl orthosilicate (TEOS, 98%), titanium(IV) butoxide (TBOT, 97%), and (3glycidyloxypropyl)trimethoxysilane (3-GPS) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Ammonia solution (28%) was purchased from Junsei Chemical Co. (Tokyo, Japan). Klucel™ hydroxypropylcellulose (HPC) LF Pharm was obtained from Ashland Inc. (Covington, KY, USA), and poly-(L-lysine) hydrochloride (PLL, MW 1600 Da) and methoxy-poly(ethylene glycol)-block-poly(L-aspartic acid sodium salt) (PEG-b-PLD, MW 2400 Da) were acquired from Alamanda Polymers Co. (Huntsville, AL, USA). Other chemical reagents utilized for miscellaneous purposes were of analytical grade and were used without further purification.

Cancer cell lines The cell lines utilized as model human colorectal carcinoma cell lines were HCT-116 and DLD-1, which were acquired from the Korean Cell Line Bank (KCLB, Seoul, South Korea). Both cell lines were cultured in HyClone RPMI 1640 media (GE Healthcare Life Sciences, Chicago, IL, USA) containing L-glutamine (300 mg/L), 25 mM HEPES (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid) buffer, and 25 mM sodium bicarbonate, and supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin G sodium and 100 µg/mL streptomycin sulfate. The cells were incubated in a 5% CO2 atmosphere at 37 °C.

Preparation of the nanoparticles Preparation of silica templates. The solid silica templates were formulated by employing a modified Stӧber method.57 TEOS (0.428 mL) was added to a solution comprised of 21.7 mL ethanol, 5.6 mL deionized water, and 0.460 mL of 28% aqueous ammonia solution, and the mixture was stirred vigorously at 900 rpm for 3 h. The white dense silica nanoparticles were


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recovered by centrifugation at 10000 ×g for 15 min, and then washed sequentially with ethanol and deionized water.

Preparation of PTX-MTNst. MTNst cores were created by modification of the method for synthesis of mesoporous TiO2 hollow shells reported by Joo et al.58 Briefly, 50 mg of HPC was dissolved in a mixture of 25 mL ethanol and 1 mL deionized water, and the previously synthesized silica templates were added and thoroughly dispersed by stirring at 700 rpm for 30 min. Then, 5 mL ethanolic solution of TBOT (20% v/v) was added dropwise with continuous stirring, followed by refluxing at 85 °C for 100 min with stirring at 900 rpm. The white precipitate was collected by centrifugation at 8000 ×g for 20 min, washed subsequently with ethanol, and dispersed in 10 mL of ethanol. Five milliliters of deionized water was added, and solvothermal treatment was conducted in a polytetrafluoroethylene-lined stainless steel pressure vessel at 150 °C for 6 h. The final MTNst carriers were collected by centrifugation at 8000 ×g for 15 min, washed thoroughly with ethanol and deionized water, and resuspended in 15 mL ethanol. PTX was loaded into the MTNst carriers by mixing them with the drug in ethanolic solution. Specifically, to 150 mg of MTNst dispersed in 15 mL ethanol, 5 mL ethanolic solution of PTX (30 mg/mL) was added, and the mixture was stirred at room temperature for 12 h. The unloaded drug was removed by centrifugation and washing with ethanol.

Preparation of PLL/MTNst. The surface functionalization of MTNst with PLL was performed to introduce strong positive charges to the MTNst surface, and thereby enable effective loading of the negatively charged miR708, as well as provide a platform for the LbL construct. PLL was conjugated to the MTNst surface by a two-step process involving 3-GPS



grafting to the titanol (Ti–OH) groups on the MTNst surface followed by nucleophilic addition of PLL to the epoxide of 3-GPS; this technique was inspired from the method reported by Hartono et al. for functionalization of mesoporous silica nanoparticles with PLL.59 Briefly, 0.35 mL of 3-GPS was added to the MTNst dispersion (15 mL), and the mixture was stirred for 24 h at 70 °C. The resulting epoxy-functionalized MTNst (Ep/MTNst) was collected by centrifugation and then washed with ethanol. Ep/MTNst was then dispersed in 15 mL of 50 mM carbonate buffer. Fifty milligrams of PLL was separately dissolved in 10 mL of 50 mM carbonate buffer. The PLL solution (7.5 mL) was added dropwise to the Ep/MTNst dispersion (15 mL) while continuously stirring. The mixture was allowed to stir at room temperature for 24 h, after which the resulting PLL/MTNst was collected by centrifugation, washed with deionized water, and then redispersed in deionized water. Solid-state cross-polarization magic angle spinning (CP/MAS) 13

C-NMR characterization, by utilizing a Bruker Avance II+ 400 MHz NMR spectrometer, was

employed to verify the successful synthesis of Ep/MTNst and PLL/MTNst.

Loading miR708 onto PLL-MTNst. Agarose gel electrophoresis was utilized to determine the appropriate quantity of PLL-MTNst carrier for efficient loading of miR708. Briefly, 5-µL aliquots of 5 µM miR708 solution (25 pmol miR708) were placed in five separate Eppendorf tubes, and calculated amounts of PLL-MTNst carriers were added to each tube to yield N:P (nitrogen-to-phosphate) ratios of 1:1, 2:1, 5:1, 10:1, and 20:1. The volume in each tube was brought to 16 µL by the addition of diethyl pyrocarbonate (DEPC)-treated water. The mixture in each tube was vortexed briefly, incubated at room temperature for 20 min, and then subjected to electrophoresis on a 2% (w/v) agarose gel, using GelRed™ nucleic acid gel stain (Biotium Inc., Fremont, CA, USA) for visualization. The lowest N:P ratio corresponding to full efficiency


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encapsulation was used to determine the optimum quantity of miR708 for a given amount of PLL/MTNst. Hence, the optimum quantity of 5 µM miR708 was added to 5 mL of 10 mg/mL PLL/MTNst, and the mixture was vortexed briefly, followed by incubation at room temperature for 20 min to produce miR708/PTX-MTNst.

Layer-by-layer assemblage of miR708/PTX-MTNst. PLL/PTX-MTNst, following miR708 loading, was used as the platform for LbL assemblage. Initially, PEG-b-PLD (1 mL, 10 mg/mL) was added to PLL/MTNst (5 mL, 10 mg/mL) dropwise under continuous stirring, followed by mild sonication for 20 min and undisturbed standing for 120 min. Thereafter, the mixture was centrifuged to remove excess PEG-b-PLD and the sediments comprising PEG-bPLD/PLL/miR708/PTX-MTNst or LbL miR708/PTX-MTNst(1) were collected. Likewise, PLL (1 mL, 10 mg/mL) was added to the above LbL miR708/PTX-MTNst(1), followed by mild sonication and centrifugation to eventually yield LbL miR708/PTX-MTNst(2). Alternate layers of PLL and PEG-b-PLD were consolidated in similar fashion to eventually yield LbL miR708/PTX-MTNst(7). For LbL miR708/PTX-MTNst, the numbers in parentheses indicate the total number of coating layers. The nanoparticles (NPs) with optimal numbers of layers were selected based on dynamic light scattering (DLS) characterization and were referred to as LbL miR708/PTX-MTNst for further purposes.

Loading capacity and loading efficiency The loading capacity (LC) and loading efficiency (LE) of PTX in PTX-MTNst were determined by the centrifugation method. Briefly, 1 mL of PTX-MTNst, before washing to remove the free PTX, was centrifuged, and the unloaded PTX in the supernatant was quantified



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by HPLC, using an Agilent 1260 Infinity HPLC system based on a previously reported method.60 An Inertsil ODS-3® (5 µm, 4.6 × 150 mm) column was used as the stationary phase, and a mixture of acetonitrile and 20 mM aqueous potassium dihydrogen phosphate (60:40, v/v) was employed as the mobile phase, with a flow rate of 1 mL/min and a column temperature of 25.0 ± 0.5 °C. Twenty-microliter aliquots of the samples were injected and detection was conducted at 230 nm. Calibration curves were established by plotting peak areas corresponding to known concentrations of PTX against their respective concentrations, and the HPLC method was validated by measuring interday and intraday variances. Finally, LC and LE were computed based on following equations: LC = (WT – WU) / (WC + WT – WU) × 100 LE = (WT – WU) / WT × 100

-------------------

eq. [1]

----------------------------

eq. [2]

Where WT, WU, and WC denote the weight of the total drug input, weight of the unloaded drug, and weight of the carrier, respectively.

Size and morphological characterization Particle size and ζ-potential. DLS characterization was employed to estimate the hydrodynamic particle size, size distribution and ζ-potential of the nanoparticles. The DLS measurements were conducted on aqueous dilutions of the nanoparticles by utilizing a ZetaSizer Nano S90 (Malvern Instruments, UK) at a scattering angle of 92° and temperature of 25 °C. Triplicate measurements were performed for each sample and the results were recorded as means ± standard deviations (n = 3). Transmission electron microscopy. The size and morphology of MTNst carriers and LbL miR708/PTX-MTNst were further investigated by transmission electron microscopy (TEM)


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imaging. The nanoparticles were mounted on carbon film-coated copper grids and dried by exposure to mild infrared radiation. The images were recorded with an H7600 transmission electron microscope (Hitachi, Tokyo, Japan). Porosity and surface area determination. Nitrogen adsorption-desorption isotherms were utilized to analyze the textural characteristics of MTNst carriers, including surface area, pore size, and pore volume. The conventional Brunauer-Emmett-Teller (BET) and Barrett-JoynerHalenda (BJH) methods were employed to determine the specific surface area and porosity, respectively. The nitrogen adsorption-desorption isotherms were resolved using a Micromeritics ASAP 2010 BET/Porosimeter (Micromeritics Instrument Corp., Norcross, GA, USA) at 77.35 K. The samples were degassed at 150 °C for 24 h prior to measurement.

Physicochemical characterization Thermogravimetric analysis. Differential thermal degradation profiles of MTNst, PLL/MTNst, LbL MTNst(1), and LbL MTNst(5) were investigated by thermogravimetric analysis (TGA), based on a previously reported method.61 Briefly, the NPs (~2 mg) were placed in a platinum crucible and heated at a constant rate of 10 °C/min from 30 to 1000 °C under a nitrogen atmosphere in an SDT Q600 thermogravimetry-differential thermal analysis (TG-DTA) instrument (TA Instruments, New Castle, DE, USA). Differential scanning calorimetry. The thermal behaviors of the free drug (PTX) powder, free nanocarrier (LbL MTNst), and miR708 and PTX-loaded nanocarrier (LbL miR708/PTXMTNst) were investigated by DSC. The samples were heated over temperature ranges of 40 to 240 °C at 10 °C per min under a dynamic nitrogen environment in a DSC Q200 instrument (TA Instruments, DE, USA).



X-ray diffraction analysis. The crystallinities of the free drug (PTX) powder, free nanocarrier (LbL MTNst), and miR708 and PTX-loaded nanocarrier (LbL miR708/PTX-MTNst) were analyzed by using X-ray diffraction (XRD) scans. The scans were recorded using an X-ray diffractometer (X’pert PRO MPD Diffractometer, Almelo, Netherlands) coupled with a vertical goniometer. The scan speed was set at 5°/min over a diffraction angle (2θ) ranging from 10 to 60°, and the voltage and current were fixed at 40 kV and 30 mA, respectively. Energy dispersive X-ray spectroscopy. The elemental composition of MTNst was investigated by energy dispersive X-ray spectroscopy (EDS). The freeze-dried powder of MTNst was mounted on a brass stub and the target region for EDS was selected using a scanning electron microscope (S-4100, Hitachi, Japan). Then, EDS analysis was performed using an EDX analyzer (Horiba, Japan) coupled with a PCI image analyzer (Hitachi, Japan). The regions for analysis within the sample were defined by point analysis mode.

Serum stability of miR708 in nanoparticles Agarose gel electrophoresis was employed to investigate the stability of the NP-bound miR708. Free miR708 (25 pmol) and LbL miR708/PTX-MTNst (equivalent to 25 pmol miR708) were incubated in 20% FBS for 0 and 24 h at 37 °C. The samples were then incubated in 1% sodium dodecyl sulfate (SDS) for 5 min at 60 °C and then heparin was added (2% final concentration) to each of the samples. Electrophoresis on 2% agarose gels was utilized, as previously discussed, to determine the stability of the NP-bound miR708 compared to that of free miR708.

In vitro drug release study


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The in vitro release behavior of PTX from LbL MTNst was analyzed in release media (either pH 7.4 phosphate-buffered saline (PBS, 0.14 M NaCl) or pH 5.0 acetate-buffered saline (ABS, 0.14 M NaCl)), based on a previously reported method.62 Briefly, 1-mL samples of LbL MTNst were placed inside dialysis bags. The bags were clipped at both ends and placed inside 50-mL tubes containing 25 mL of release media. The tubes were subjected to synchronized agitation in a water bath shaker maintained at 37 ± 0.5 °C. Aliquots (0.5 mL) of the release media were withdrawn at predetermined time points and analyzed for PTX by using the HPLC quantification method described earlier.

In vitro cytotoxicity assay The capabilities of free PTX, LbL PTX-MTNst, and LbL miR708/PTX-MTNst to inhibit in vitro proliferation of HCT-116 and DLD-1 human colorectal carcinoma cells were investigated using an MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. Both HCT-116 and DLD-1 cell lines were seeded in 96-well plates at 1×104 cells/well and incubated for 24 h at 37 °C. The cells were then treated with sequential dilutions of PTX, LbL PTXMTNst, or LbL miR708/PTX-MTNst, and were incubated under the specified conditions. After 48 h of incubation, the cells were washed twice with PBS, treated with the MTT reagent (100 µL/ well; 1.25 mg/mL in FBS-free media), and incubated for 3 h. Eventually, dimethyl sulfoxide (100 µL) was added to dissolve the resulting formazan crystals and the viable proportion of cells was calculated relative to that of untreated control cells based on the equation: cell viability = (Asample/Acontrol) × 100%, where A is the absorbance at 570 nm. Additionally, the intrinsic cytotoxicity of drug-free LbL MTNst carrier was also determined in a similar fashion.



In vitro cellular uptake evaluation Fluorescence-activated cell sorting. The cellular uptake of coumarin 6-labeled LbL MTNst (LbL Cou-MTNst) by HCT-116 and DLD-1 cells was demonstrated via fluorescence-activated cell sorting (FACS) analysis. The cells (2 × 105 cells/well) were seeded in 12-well plates and incubated for 24 h at 37 °C, after which they were treated with LbL Cou-MTNst at different concentration (0.5, 1, or 2 µg/mL) and incubation times (0.5, 1, or 2 h). The cells were washed twice with PBS, detached from the surface of the plates, and prepared as a cell suspension in PBS. The extent of cellular uptake of the NPs in each of the cell samples was determined by using a BD FACSCalibur™ (BD Biosciences, San Jose, CA, USA). HCT-116 and DLD-1 cells receiving no treatment were used as respective internal control cells, and the acquisition and analysis of 10 000 events per sample was fixed for each analysis. Confocal laser scanning microscopy. Confocal laser scanning microscopy (CLSM) was utilized to provide additional information regarding the cellular uptake of LbL Cou-MTNst by the HCT-116 and DLD-1 cell lines. The cells (3 × 105 cells/well) were seeded on glass coverslips inside 12-well plates and incubated for 24 h at 37 °C, and then were treated with LbL CouMTNst (1 µg/mL, 2 h). The cells were washed with PBS, incubated with 100 nM of LysoTracker® Red (Thermo Fisher Scientific, MA, USA) for 10 min, rewashed, and subsequently fixed with 4% paraformaldehyde at room temperature. The glass coverslips were mounted on glass slides using Gel/Mount solution (M01, Biomeda, CA, USA) and confocal images of the cells were captured using a Nikon A1 confocal microscope system (Nikon Instruments Inc., Japan). Western blot analysis. Western blotting was employed to evaluate the expression levels of c-FLIP and apoptosis marker proteins (i.e., cleaved caspase-7 and cleaved poly (ADP-ribose)


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polymerase (PARP)) in HCT-116 and DLD-1 cell lines following PTX, LbL PTX-MTNst, LbL miR708-MTNst, or LbL miR708/PTX-MTNst treatments compared to those in untreated control cells, as reported earlier.63 The cells (6 × 105 cells/well) were seeded in 6-well plates, incubated for 24 h at 37 °C, and treated (25 ng/mL equivalent of PTX and 25 nM equivalent of miR708) for 24 h. The cells were then washed twice with PBS, detached from the plate surface, and lysed with M-PER protein extraction reagent (Thermo Fisher Scientific Inc., Rockford, IL, USA) in the presence of protease inhibitors. The proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes, pre-blocked with 5% skim milk in Tris-buffered saline with 1% Tween® 20 (TBST), were incubated with the specific antibodies (at supplier-recommended dilutions) against the intended marker proteins, as well as antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody as a loading control. Following primary antibody incubation, the membranes were washed three times with TBST, incubated with the recommended secondary antibody, washed with TBST, and then soaked in SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Rockford, IL, USA). The specific protein expressions were detected using an ‘LAS-400 mini’ automated image acquisition system (Fujifilm, Tokyo, Japan).

In vivo animal studies Development of DLD-1 xenograft tumor model. BALB/c nude mice (female, 6-week old) were used for the DLD-1 xenograft tumor model. Throughout the study period, the mice were kept in a segregated animal facility at Yeungnam University, South Korea under ambient temperature and relative humidity (20 ± 2 °C and 50-60%, respectively), and were handled



strictly according to the approved protocols of the Institutional Animal Ethical Committee of Yeungnam University. For the DLD-1 xenograft tumor model, 1 × 107 cells, dispersed in 100 µL of serum-free media, were injected into the right flanks of the mice. In vivo and ex vivo fluorescence imaging. A Fluorescence-labeled Organism Bioimaging Instrument (FOBI; NeoScience, South Korea) was used to investigate the in vivo biodistribution and ex vivo tissue distribution of LbL MTNst in the DLD-1 xenograft tumor-bearing mice. Cy5.5-labeled LbL MTNst (1 mg/mL, 100 µL) was administered by intravenous injection into the tail vein of the DLD-1 tumor-bearing mice and in vivo images of the mice were captured using the FOBI 6, 12, 24, and 48 h post-injection. Afterward, the mice were euthanized and their principal organs (liver, lungs, heart, kidneys, and spleen), along with the tumor, were excised, and their ex vivo fluorescence images were acquired. In vivo anticancer investigation. The DLD-1 xenograft tumor-bearing mice were randomly assigned to 5 groups (n = 6 per group), each of which were identified as CONTROL, MTNst, PTX, LbL PTX-MTNst, and LbL miR708/PTX-MTNst, based on the treatment received. The mice were administered the respective treatments by intravenous tail vein injection on days 0, 3, 7 and 10, with 5 mg/kg and 10 nmol/kg equivalents of PTX and miR708, respectively. The tumor sizes and mouse body weights were investigated until 28 days post-injection. The tumor volumes were estimated using the formula: volume = ½ × {(longest dimension) × (shortest dimension)2}. Histological and immunohistochemical analysis. After completion of the in vivo anticancer study, the mice were euthanized and representative tumor masses and principal organs (liver, lungs, heart, kidneys and spleen) of the mice (n = 3 per treatment) were excised. The tumors and organs were fixed in 10% buffered formalin and histology and immunohistochemistry (IHC)


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were performed. Histopathological and histomorphometric changes in the tumor masses and principal organs were evaluated by general hematoxylin and eosin (H&E) staining, followed by observation under an Eclipse 80i light microscope (Nikon, Japan). IHC of the tumor samples to identify tumor cell apoptosis and angiogenesis/ proliferation was performed by primary antisera and avidin-biotin-peroxidase complex (ABC) methods. Cleaved caspase-3 and cleaved PARP were evaluated to identify apoptosis, CD31 was evaluated to identify angiogenesis, and Ki-67 was evaluated to identify tumor cell proliferation. Statistical analysis. Results are presented as arithmetic means ± standard deviations. A Student’s t-test was utilized to compare mean values, where applicable. Statistical significance was considered at P < 0.05.

RESULTS AND DISCUSSION Fabrication and characterization of LbL miR708/PTX-MTNst A schematic illustration of LbL miR708/PTX-MTNst synthesis is presented in Figure 1. LbL miR708/PTX-MTNst comprised MTNst at its core, which was constructed by overlaying mesoporous titania on solid silica nanospheres (~60 nm diameter). Over the MTNst core, an initial layer of PLL was conjugated via nucleophilic addition to yield PLL/MTNst, which was preceded by 3-GPS grafting to the surface titanol groups of MTNst. The MTNst surface underwent

epoxy-functionalization

upon

3-GPS

grafting

(Ep/MTNst).

The

surface-

functionalized epoxy groups facilitated nucleophilic addition of PLL by epoxy ring opening.59 The proposed reaction mechanisms as well as the results of the solid-state CP/MAS

13

C-NMR

characterization verifying the successful synthesis of Ep/MTNst and PLL/MTNst are included in the supporting information, Figures S1–S4. Characteristic chemical shift peaks in


13

C-NMR


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confirmed the successful preparation of Ep/MTNst and PLL/MTNst. Modification of the MTNst surface with PLL had a dual purpose. First, the positive charges introduced by the polycationic polymer, PLL, on the MTNst surface allowed for effective loading of the negatively charged miR708 by electrostatic interactions. Second, PLL provided a platform for LbL modification of the nanoparticles with alternate layers of the polyanionic polymer, PEG-b-PLD, and the polycationic polymer, PLL.

Figure 1. Schematic illustration of the step-by-step preparation of polyamino acid layer-by-layer constructed solid silica-supported mesoporous titania nanoparticles carrying paclitaxel and microRNA

708

(LbL

miR708/PTX-MTNst).

PTX,

paclitaxel;

3-GPS,

(3-

glycidyloxypropyl)trimethoxysilane; PLL, poly(L-lysine); PEG-b-PLD, methoxy-poly(ethylene glycol)-block-poly(L-aspartic acid); MTNst, silica-supported mesoporous titania nanoparticles; LbL, layer-by-layer.


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The changes in particle size, polydispersity index (PDI), and ζ-potential with sequential addition of each polymeric layer are shown in Figure 2A. The progressive increase in particle size coupled with the reversal of ζ-potential with alternate layers of polycationic PLL and polyanionic PEG-b-PLD provide strong evidence for successful LbL assembly. After initial nucleophilic addition of PLL to the MTNst core, the average hydrodynamic particle size increased from 119.3 ± 3.1 nm for the MTNst core to 141.5 ± 4.9 nm for the PLL/MTNst, while the ζ-potential switched from negative (–39.9 ± 1.9 mV) to positive (+26.7 ± 1.8 mV), respectively. The hydrodynamic particle size further increased to 155.9 ± 3.5 nm after modification of PLL/MTNst with PEG-b-PLD, while the ζ-potential reverted back to a negative value (–29.6 ± 1.8 mV). Flipping back and forth between negative and positive values of the ζpotential provides strong evidence for LbL modification. Further assembly with successive layers of PLL and PEG-b-PLD revealed that each PLL/PEG-b-PLD pair increased the particle size by ~20 nm. The LbL assembly with 3 and 5 polymeric layers over PLL/PTX-MTNst led to hydrodynamic particle sizes less than 200 nm, while that with 7 polymeric layers formed nanoparticles over 200 nm in diameter. From our previous experiences with LbL assemblies, we have learned that the greater the number of layers, the lesser is the leakage of the nanoparticle contents at normal physiologic pH.54 Hence, our target was to maximize the polymeric layers in the LbL assembly while ensuring that the eventual particle size remains less than or close to 200 nm. Consequently, we regarded 5 polymeric layers as the optimum number of polymeric layers for the LbL assembly. The average hydrodynamic particle size of the eventual LbL PEG-bPLD/miR708/PTX-MTNst(5), or simply LbL miR708/PTX-MTNst, was 194.3 ± 6.6 nm, with a PDI of 0.072 ± 0.023 and a ζ-potential of –27.6 ± 1.8 mV. TEM and SEM images of the initial



MTNst and the eventual LbL miR708/PTX-MTNst were recorded (Figure 2B, S5), which revealed spheroid particles with sizes consistent with the DLS measurements. The MTNst appeared as dark-contrasted particles as a result of uniform MTN coating over the less-contrasted dense silica particles, while the LbL miR708/PTX-MTNst appeared as larger and darker particles as a consequence of the LbL coatings of PLL and PEG-b-PLD as well as the presence of the therapeutic agent.

Figure 2. (A) Dynamic light scattering (DLS) characterization: (a) particle size and polydispersity index (PDI), and (b) ζ-potential of the initial and final nanoparticles. (B)


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Transmission electron microscopy (TEM) images of (a) MTNst, and (b) LbL miR708/PTXMTNst. The prefix “PLL” or “PEG-PLD” in (A) indicates the outermost layer and the number in parentheses indicates the total number of coating layers, including both PLL and PEG-b-PLD coating. Values are expressed as means ± SD (n = 3). The scale bars in (B) represent 100 nm.

PTX was loaded into the MTNst carriers by incubating equal amounts of the drug and the carrier in ethanolic solution. The unloaded fraction of PTX was estimated by HPLC, followed by calculation of the loaded amount of PTX in the PTX-MTNst to determine the LC and LE of the PTX in the PTX-MTNst. Consequently, an LC of 13.16 ± 2.10% (w/w) and an LE of 15.19 ± 2.80% were obtained, which were excellent values compared to those in previous reports of mesoporous titania-based systems.42,64 Notably, the loaded amount of PTX was not significantly affected by LbL assembly. Following preliminary studies to determine the appropriate inhibition dose of miR708 (Supporting information, Figure S6), agarose gel electrophoresis was used to determine the appropriate ratio of miR708:PLL/MTNst to ensure efficient loading of miR708 onto the polycationic PLL/MTNst and to investigate the stability of the NP-bound miR708. As shown in Figure 3A, when PLL/MTNst and miR708 were incubated at an N:P ratio of 2:1, the electrophoretic migration of miR708 was completely prevented. This implies that, at an N:P ratio of 2:1, the miR708 was efficiently loaded in PLL/MTNst. Considering that we intended to further perform LbL assembly post-miR708 loading, an N:P ratio of 5:1 was deemed suitable for our purpose in order to ensure that no miR708 leakage transpired during or after LbL assembly. Following a 24-h incubation in FBS, the free miR708 showed no band on the agarose gel, while the miR708 associated with LbL miR708/PTX-MTNst showed a clear band (Figure 3B). This



alluded to the stability of the microRNA in serum when bound to the LbL nanocarriers, because the microRNA is readily degraded in an unbound form.

Figure 3. Agarose gel electrophoresis: (A) determination of optimum N:P ratio for miR708 and PLL/MTNst complexation, (B) stability of miR708 inside LbL miR708/PTX-MTNst carriers. PTX, paclitaxel; PLL, poly(L-lysine); MTNst, silica-supported mesoporous titania nanoparticles; LbL, layer-by-layer.

Further characterization of the NPs was performed by using BET/BJH, EDS, TGA, DSC, and XRD analyses. Nitrogen adsorption and desorption isotherms were utilized to investigate the surface area and pore configuration of MTNst (Figure S7). The MTNst carriers revealed a type IV isotherm with P/Po inflection at ~0.6, which is representative of mesoporous materials.65 The BET surface area of MTNst was observed to be ~203 m2/g. BJH adsorption cumulative pore volume and BJH desorption cumulative pore volume were 0.736 cm3/g and 0.739 cm3/g, respectively. Likewise, BJH adsorption average pore diameter and BJH desorption average pore diameter were 152 Å and 147 Å, respectively. Elemental analysis by EDS (Figure S8) revealed 16.04 ± 1.60%, 47.25 ± 6.43%, 22.04 ± 2.90%, and 14.67 ± 2.04% by weight of C, O, Si, and Ti, respectively. Furthermore, TGA analyses (Figure 4A) of dried samples of MTNst, PLL/MTNst, LbL PEG-b-PLD/MTNst(1), and LbL PEG-b-PLD/MTNst(5) at temperature ranges of 25–1000


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°C were performed to estimate the amount of PLL conjugated to the MTNst surface to yield PLL/MTNst, and the total amount of the polyanionic and polycationic polymers that comprised the LbL assembly in the final NPs. MTNst exhibited a weight loss of ~18% owing to incomplete condensation of TBOT and TEOS and the presence of residual solvents. Assuming that the residual solvent and moisture contents were constant, a comparison of the weight loss of MTNst with that of PLL/MTNst and LbL MTNst showed that PLL/MTNst was comprised of ~8.5% (w/w) PLL and LbL MTNst was comprised of ~35% (w/w) aggregate coating polymers. DSC (Figure 4B) and XRD (Figure 4C) analyses of PTX, LbL MTNst, and LbL miR708/PTXMTNst unveiled the crystalline/amorphous state of the NP bound and unbound drug.66,67 The DSC thermogram of PTX was characterized by an endothermic peak at 216 °C, which corresponds to the intrinsic melting endotherm of PTX (Tm = 216–217 °C). An absence of the melting endotherm of PTX was observed in the DSC thermogram of LbL miR708/PTX-MTNst, which implied that PTX remained inside the mesoporous structures of the NPs in the amorphous state. These findings concurred with those of the XRD analysis. The diffractogram of free, powdered PTX displayed numerous sharp peaks at 2θ values ranging from 10–30°, which is characteristic of highly crystalline PTX.68 These distinctive PTX peaks were discernably absent in the diffractogram of LbL miR708/PTX-MTNst. The in vitro release behavior of PTX from LbL miR708/PTX-MTNst was examined in PBS (pH 7.4) and ABS (pH 5.0) (Figure 4D). The in vitro release profile of PTX from the NPs at physiologic pH (7.4) exhibited sustained-release characteristics throughout the 48-h release period, without an initial burst release, which suggested the efficient encapsulation of the PTX inside the MTNst by the LbL assembly, as well as its controlled-release characteristics. The in vitro release appeared highly pH-dependent, with the 48-h cumulative release of PTX showing a



substantial rise from ~33% at pH 7.4 to ~72% at pH 5.0. This pH-dependent release behavior of the PEG-b-PLD/PLL-based LbL engineered system is consistent with that shown in our previous report.54 PEG-b-PLD, being a weakly acidic polyanion (pKa = 3.9), protonates at the lower pH. This reduces the ionization potential and charge density for its interaction with PLL, which ultimately leads to disassembly of the LbL construct and subsequent higher release of PTX from the MTNst mesopores.69

Figure 4. (A) Thermogravimetric analysis (TGA) scans of MTNst, PLL/MTNst, LbL PEG-bPLD/MTNst(1), and LbL PEG-b-PLD/MTNst(5); (B) Differential scanning calorimetric (DSC) thermograms of PTX, LbL MTNst, and LbL miR708/PTX-MTNst; (C) X-ray diffraction (XRD)


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scans of PTX, LbL MTNst, and LbL miR708/PTX-MTNst; and (D) In vitro release profiles of LbL PTX-MTNst at pH 7.4 and pH 5.0. PTX, paclitaxel; PLL, poly(L-lysine); MTNst, silicasupported mesoporous titania nanoparticles; LbL, layer-by-layer. Values are expressed as means ± SD (n = 3).

In vitro cytotoxicity and cellular uptake behavior In vitro cytotoxicity analyses of LbL miR708/PTX-MTNst, free PTX, and LbL PTX-MTNst were conducted in HCT-116 and DLD-1 cells as the two model colorectal carcinoma cell lines (Figure 5). All three agents elicited dose-dependent cytotoxicity in both cell lines. There was no clear demarcation between the cytotoxic effects of PTX and LbL PTX-MTNst in both cell lines. However, the cytotoxicity of LbL miR708/PTX-MTNst was distinctly superior to that of PTX or LbL PTX-MTNst, which was reaffirmed by the respective IC50 values for each treatment (Table S1). The cytotoxicity elicited by the drug-free carriers (LbL MTNst) was practically non-existent in both cell lines, with cell viabilities > 80%, even at a carrier concentration of 1000 µg/mL (insets of Figure 5). This reflected the high biocompatibility of the LbL NP system.



Figure 5. In vitro cytotoxicity profiles of PTX, LbL PTX-MTNst, and LbL miR708/PTX-MTNst in (A) HCT-116 and (B) DLD-1 cell lines. The equivalent concentration of miR708 in LbL miR708/PTX-MTNst at each treatment point was 25 nM. Figure insets: cytotoxicity profiles of drug-free LbL MTNst carriers in the respective cell lines. PTX, paclitaxel; MTNst, silicasupported mesoporous titania nanoparticles; LbL, layer-by-layer. Values are expressed as means ± SD (n = 8).


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The cellular uptake of LbL MTNst by HCT-116 and DLD-1 cells was investigated by FACS analysis (Figure 6A) and CLSM-based fluorescence analysis (Figure 6B). Both HCT-116 and DLD-1 cells were incubated with 0.5, 1, or 2 µg/mL LbL MTNst for 0.5, 1, or 2 h. Quantification of the fluorescence associated with the cells that had internalized the fluorescent coumarin 6-tagged NPs by FACS analysis showed that both cell lines displayed concentrationand time-dependent uptake of the NPs. An attempt to further elaborate the internalization mechanism of LbL MTNst by HCT-116 and DLD-1 cells was made using CLSM imaging following treatment with coumarin 6-loaded LbL MTNst (1 µg/mL, 2 h). The appearance of the high intensity green fluorescence of coumarin-6 in the cytosolic regions of both the cells could be ascribed to excellent cellular uptake of a high quantity of LbL MTNst. Co-localization of LbL MTNst with the acidic lysosomal compartments of the cancer cells could be attributed to clathrin-mediated endocytosis of the NPs.70 The LbL NPs prepared in the current study exhibited pH-dependent release behavior attributed largely to protonation of PEG-b-PLD, which ultimately results in disassembly of the LbL construct. Hence, in the acidic tumor microenvironment, the negatively-charged PEG-b-PLD might wear off, which might result in NPs chiefly having the positively-charged PLL on their surface that is capable of undergoing internalization by the clathrin-mediated pathway.



Figure 6. In vitro cellular uptake of LbL MTNst by HCT-116 and DLD-1 cells: (A) Representative FACS analysis showing (a,c) concentration- and (b,d) time-dependent cellular uptake; (B) Representative CLSM images showing co-localization of the nanoparticles with the lysosomal vesicles. The scale bars in (B) indicate 5 µm. Coumarin 6 was used as a fluorescence dye. FACS, fluorescence-activated cell sorting; CLSM, confocal laser scanning microscopy; Cou, coumarin 6; MTNst, silica-supported mesoporous titania nanoparticles; LbL, layer-bylayer.

Apoptosis and inhibition of c-FLIP expression


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Western blot analysis was employed to determine the expression level of c-FLIP, along with the proapoptotic proteins, cleaved caspase-7 and cleaved PARP, in the colorectal carcinoma cell lines following 24-h treatment with PTX, LbL PTX-MTNst, LbL miR708-MTNst, and LbL miR708/PTX-MTNst (Figure 7). All of the treatment agents appeared to inhibit c-FLIP expression in both cell lines in the order of PTX < LbL PTX-MTNst < LbL miR708-MTNst < LbL miR708/PTX-MTNst. Accordingly, the levels of cleaved caspase-7 and cleaved PARP in both cell lines increased following PTX, LbL PTX-MTNst, LbL miR708-MTNst, and LbL miR708/PTX-MTNst treatment, with the LbL miR708/PTX-MTNst treatment showing the greatest effect. This suggests a highly superior proapoptotic activity of the co-loaded NPs, compared to not only the free drug but also PTX or miR708 delivered as sole therapeutic agents via the LbL MTNst. This demonstrates the advantages of integrating a combined chemotherapeutic and microRNA approach in a single nanoparticle delivery system.

Figure 7. Western blot analysis showing inhibited expression of c-FLIP and enhanced levels of proapoptotic proteins (cleaved caspase-7 and cleaved PARP) in (A) HCT-116 and (B) DLD-1 cells following treatment with PTX, LbL PTX-MTNst, LbL miR708-MTNst, or LbL miR708/PTX-MTNst.

c-FLIP,

(FADD-like

interleukin-1β-converting

enzyme)-inhibitory

protein; PARP, poly (ADP-ribose) polymerase; GAPDH, glyceraldehyde 3-phosphate



dehydrogenase; PTX, paclitaxel; MTNst, silica-supported mesoporous titania nanoparticles; LbL, layer-by-layer.

In vivo biodistribution of nanoparticles The in vivo biodistribution of the NPs in DLD-1 xenograft tumor-bearing mice were evaluated using an FOBI analyzer following intravenous administration of Cy5.5-labeled LbL MTNst (Figure 8A). Evaluation of in vivo fluorescence images at 6, 12, 24, and 48 h demonstrated that a large proportion of LbL MTNst reached the tumor, and was well-retained within the tumoral region, even 48 h post-injection when the NPs in the non-tumoral sites appeared to diminish. This alluded to the enhanced permeation and retention (EPR) of the LbL MTNst in the tumors. Subsequently, the tumor bearing mice were euthanized, and the tumors and principal organs (liver, lungs, kidneys, heart, and spleen) were excised. The ex vivo fluorescence imaging of the tumors and organs (Figure 8B) and its quantification (Figure 8C) demonstrated that a significant proportion of the LbL NPs was retained in the tumors, which appeared similar to the amount that infiltrated the lungs and spleen; the majority of the NPs targeted the liver.


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Figure 8. (A) In vivo biodistribution of Cy5.5-labeled LbL MTNst in DLD-1 xenograft tumorbearing mouse at (a) 6, (b) 12, (c) 24, and (d) 48 h following intravenous injection. (B) Ex vivo tissue distribution of Cy5.5-labeled LbL MTNst in (a) liver, (b) spleen, (c) kidneys, (d) heart, (e) lungs, and (f) tumor excised from DLD-1 xenograft tumor-bearing mice. (C) Quantification of fluorescent signals from part B. All values indicate mean ± SD (n = 3). MTNst, silica-supported mesoporous titania nanoparticles; LbL, layer-by-layer.

Regression of tumors in DLD-1 tumor-bearing mice The in vivo tumor inhibition potential of LbL miR708/PTX-MTNst was evaluated over a 28day period in DLD-1 xenograft tumor-bearing mice after intravenous administration at 0, 3, 7, and 10 days. The representative images of the excised tumors after completion of the study have been shown in the Supporting Information file, Figure S9. LbL miR708/PTX-MTNst caused conspicuous inhibition of tumor growth, which was significantly greater (p < 0.001) than that of PTX or LbL PTX-MTNst (Figure 9A). The drug-free LbL MTNst showed no apparent in vivo



antitumor activity, which could be well-correlated with its low in vitro cytotoxicity against the cancer cell lines. Additionally, the body weights of the tumor-bearing mice in all treatment groups were evaluated to identify any signs of toxicity. The mice belonging to the PTX group showed a marked loss in body weight in response to drug treatment, which could be correlated with the toxic effects of this chemotherapeutic drug. The mice appeared to recover their weight loss upon cessation of drug treatment. Most importantly, the LbL miR708/PTX-MTNst and LbL PTX-MTNst triggered no apparent weight loss, which was also true for the drug-free LbL MTNst (Figure 9B). This led us to conclude that our LbL MTNst carriers were biocompatible and capable of shielding normal tissues from the inherent toxic effects of chemotherapeutic agents.


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Figure 9. (A) Tumor growth inhibition, and (B) body weight alterations in DLD-1 xenograft tumor-bearing mice following treatment with LbL miR708/PTX-MTNst, free PTX, LbL PTXMTNst, or drug-free LbL MTNst carriers. Untreated control mice are also shown for comparison. The arrows indicate the four treatment points at 0, 3, 7, and 10 days. (*) Tumor growth inhibition of LbL miR708/PTX-MTNst was significantly greater than that of control, LbL MTNst, PTX, or LbL PTX-MTNst mice (p < 0.001). PTX, paclitaxel; MTNst, silicasupported mesoporous titania nanoparticles; LbL, layer-by-layer. All values indicate mean ± SD (n = 6).

Histological and immunohistochemical characteristics The histology of the tumors and chief organs of the mice and IHC staining of the tumors were analyzed to verify the overall anticancer activity and signs of acute toxicity (Figure 10, S10; Table S2, S3). The tumor histology demonstrated obvious decreases in tumor cell volumes in all drug-containing treatment groups compared to the tumor volumes in the untreated control group, in the order of LbL miR708/PTX-MTNst > LbL PTX-MTNst > PTX. However, no significant decrease in tumor cell volume was observed with LbL MTNst treatment. Tumor IHC analysis showed increased cleaved caspase-3 and cleaved PARP immunoreactivity and decreased CD31- and Ki-67-positive cells in all drug-containing treatment groups compared to that of the untreated control group, in the order of LbL miR708/PTX-MTNst > LbL PTX-MTNst > PTX; no significant changes were observed with LbL MTNst. The organ histopathological analysis revealed no signs of organ damage in any of the principal mouse organs, which suggested a lack of acute toxicity following treatment of the DLD-1 tumor-bearing mice. These observations further strengthen our belief that LbL miR708/PTX-MTNst is capable of providing effective tumor treatment benefits.



Figure 10. (A) Histological and immunohistochemical analyses of tumors, and (B) histological analysis of five principal organs excised from DLD-1 tumor-bearing mice belonging to (a) untreated control, (b) LbL MTNst, (c) PTX, (d) LbL PTX-MTNst, and (e) LbL miR708/PTXMTNst groups. All scale bars indicate 120 µm. H&E, hematoxylin and eosin; PARP, poly (ADPribose) polymerase; PTX, paclitaxel; MTNst, silica-supported mesoporous titania nanoparticles; LbL, layer-by-layer.


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CONCLUSIONS The LbL miR708/PTX-MTNst was successfully utilized to efficiently encapsulate miR708 and PTX as combined chemotherapeutic agents that exhibit pH-dependent in vitro release with exceedingly higher release at low pH conditions mimicking tumor pH, as compared to normal physiologic pH conditions. These nanotherapeutic agents showed excellent in vitro cytotoxicity against HCT-116 and DLD-1 colorectal carcinoma cells, with superb cellular uptake profiles, which were superior to that of free PTX or PTX-loaded NPs. The exceptionally pronounced cytotoxic effects were consistent with the inhibition of c-FLIP expression in the cell lines, as well as with clear signs of apoptosis that were conveyed by increases in proapoptotic marker proteins. When administered intravenously into DLD-1 xenograft tumor-bearing mice, the NPs showed substantial accumulation in tumors. LbL miR708/PTX-MTNst significantly inhibited tumor growth in the DLD-1 tumor-bearing mice compared to that of free PTX or PTX-loaded NPs. In addition to these exemplary in vitro and in vivo efficacy results, the NPs showed no signs of acute toxicity in the DLD-1 tumor-bearing mice. This further emphasizes the enormous potential of the LbL miR708/PTX-MTNst system in its prospective application for combined chemotherapy of colorectal carcinoma in clinical settings.

ASSOCIATED CONTENT Supporting Information Mechanism of 3-GPS grafting over MTNst surface, mechanism of nucleophilic addition of poly(L-lysine) (PLL) to epoxy-grafted MTNst, solid-state C13-NMR spectrum of epoxy-grafted MTNst, solid-state C13-NMR spectrum of PLL-modified MTNst, scanning electron microscope (SEM) images of MTNst and LbL miR708/PTX-MTNst, preliminary study for appropriate



inhibition dose of miR708, BET and BJH plots for surface area and porosity analysis of MTNst, energy dispersive spectrograph for elemental analysis of MTNst, representative images of excised tumors, H&E staining images of tumors, IC50 values of various treatment groups of HCT-116 and DLD-1 colorectal carcinoma cells, histomorphometric analysis of tumor masses taken from DLD-1 xenograft athymic nude mice, histopathologic-histomorphometric analysis of principal organs taken from athymic nude mice with DLD-1 xenograft.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]

Notes The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2018R1A2A2A05021143), and by the Medical Research Center Program (2015R1A5A2009124) through the NRF funded by MSIP.


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Graphical Abstract


Polyamino Acid Layer-by-Layer (LbL) Constructed Silica-Supported

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