Facile Preparation of Delivery Platform of Water-Soluble Low

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Facile Preparation of Delivery Platform of Water-Soluble LowMolecular-Weight Drugs Based on Polyion Complex Vesicle (PICsome) Encapsulating Mesoporous Silica Nanoparticle Akinori Goto, Hung-Chi Yen, Yasutaka Anraku, Shigeto Fukushima, Ping-Shan Lai, Masaru Kato, Akihiro Kishimura, and Kazunori Kataoka ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00562 • Publication Date (Web): 21 Mar 2017 Downloaded from http://pubs.acs.org on March 28, 2017

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ACS Biomaterials Science & Engineering

Facile Preparation of Delivery Platform of WaterSoluble Low-Molecular-Weight Drugs Based on Polyion Complex Vesicle (PICsome) Encapsulating Mesoporous Silica Nanoparticle Akinori Goto†‡, Hung-Chi Yen‡, Yasutaka Anraku‡, Shigeto Fukushima‡, Ping-Shan Lai§, Masaru Katoǁ, Akihiro Kishimura*‡#$ and Kazunori Kataoka*‡⊥¶

†

Kyorin Pharmaceutical CO., LTD., Watarase Research Center, 1848, Nogi, Nogi-machi, Shimotsuga-gun, Tochigi, 329-0114, Japan.

‡

Department of Materials Engineering, Graduate School of Engineering, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.

§

Department of Chemistry, National Chung Shing University, 250 Kuo-Kuang Road, Taichung 402, Taiwan.

ǁ

Graduate School of Pharmaceutical Sciences, the University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo 113-0033 Japan.

ACS Paragon Plus Environment

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Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan $

Center for Molecular Systems, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan

⊥

Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, the University of Tokyo. 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

¶

Innovation Center of NanoMedicne, Kawasaki Institute of Industry Promotion, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki 210-0821, Japan.

Keywords: drug delivery systems, polyion complex, mesoporous silica nanoparticle, polymersome, cancer chemotherapy.


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ABSTRACT

Polyion complex vesicles (PICsomes) are polymeric hollow capsules composed of a unique semipermeable membrane, which may represent a versatile platform for constructing drugloaded nano-formulation. However, it is difficult to retain water-soluble low molecular-weight compounds (LMWCs) in inner space of PICsome because of high permeability of PIC membrane for LMWCs. Herein, we selected mesoporous silica nanoparticle (MSN) as a drugretaining nano-matrix, and we demonstrated successful encapsulation of MSN into the PICsome to obtain MSN@PICsome. The efficacy of MSN loading, a ratio of the amount of MSN encapsulated in the PICsome to the amount of feed MSN, was at most 83%, and the diameter of resulting product was approximately 100 nm. The obtained MSN@PICsome was stably dispersed under the physiological condition, and showed considerable longevity in blood circulation of mice. Furthermore, surface of MSN in MSN@PICsome can be modified without any deterioration of the vesicle structure, obtaining amino-functionalized and sulfonatefunctionalized MSN@PICsomes (A-MSN@PICsome and S-MSN@PICsome, respectively). Both surface-modified MSN@PICsomes were successfully loaded with charged water-soluble low-molecular-weight compounds (LMWCs). Particularly, S-MSN@PICsome kept 8wt% gemcitabine (GEM) per S-MSN, and released it in a sustained manner. GEM-loaded SMSN@PICsome demonstrated marked cytotoxicity against cultured tumor cells, and achieved significant in vivo efficacy to suppress the growth of subcutaneously implanted lung tumor via intravenous administration.


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INTRODUCTION Various nano-formulations have recently been developed in the field of drug delivery.1–8 In particular, those based on vesicular structure have attracted much attention owing to their application versatility, and their submicrometer-scaled formulations have been intensively developed.9–13 Vesicles are generally prepared from self-assembled layer of rationally-designed molecular building blocks.9,10,13 However, tuning method of vesicular membrane permeability is limited, which hamper their biomedical applications.14,15 In fact, most of vesicles are composed of hydrophobic membrane, which is not advantageous for sustained release of water-soluble drugs, and not suitable for chemical modification of loaded materials in aqueous media. Very recently, we have developed novel polymeric vesicles, PICsomes, utilizing polyion complex formation of oppositely charged polyelectrolytes.16,17 PICsomes have many unique characteristics as drug delivery vehicles, such as simple preparation procedure just by mixing oppositely charged pairs of polyelectrolytes in aqueous medium, high and controllable vesicle membrane permeability, tunable size in sub-micrometer scaled range (70–400 nm), with easy loading of water-soluble/dispersible materials, e.g., proteins and nano-particles, into the inner aqueous phase.16,18–20 Furthermore, the PICsome membrane is able to be crosslinked, enhancing the stability in harsh in vivo environments. Indeed, the crosslinked PICsomes with a size of ca. 100 nm showed extended circulation in the blood compartment and appreciable tumor accumulation in a mouse model via systemic injection,18,21,22 and were capable of delivery of water-soluble macromolecules, e.g., enzymes, to the target tissue without deactivation.21 Nonetheless, PICsomes have difficulty in retention of water-soluble low molecular-weight compounds (LMWCs) inside due to high permeability of the PIC membrane for LMWCs.


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To overcome the abovementioned issues of water-soluble LMWCs delivery, in the present study, a facile method was developed for the preparation of novel hybrid formulations of submicrometer-scaled PICsomes and drug-retaining nano-matrices. We focused on mesoporous silica nanoparticles (MSNs) as nano-matrices for loading of water-soluble LMWCs into the inner aqueous phase of PICsome, because they exhibit features relevant to drug delivery, including a large surface area for drug adsorption, ease of surface functionalization, and tunable particle size (Scheme 1).23–29 Furthermore, the low cytotoxicity of non-calcined MSN, owing to their degradability under physiological conditions, is an advantage for in vivo use.30,31 Moreover, this study was devoted to demonstrate the MSN-encapsulated PICsome as a versatile nanoformulation for LMWCs. Notably, the high permeability of the PICsome membrane allowed in situ surface functionalization of MSN in the PICsome to increase the drug-retaining capacity. 2’,2’-Difluoro-2’-deoxycytidine (gemcitabine, GEM) was selected as a drug for inclusion into MSN-encapsulated PICsome. GEM is a widely used anti-cancer drug, but it is rapidly metabolized into an inactive form in blood, liver, and kidneys after administration,32 and its hematological toxicity and side effects are also serious concerns.33–35 Therefore, there have been strong demands to develop carrier systems of GEM to increase the efficacy with reducing side effects.36–43 Here, feasibility of our MSN-encapsulated PICsome as novel GEM carrier was demonstrated through the effective treatment of xenografted lung tumor model via systemic injection.


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Scheme 1. PICsome-based delivery system of water-soluble LMWCs.

EXPERIMENTAL Materials Cetyltrimethyl ammonium bromide (CTAB), sodium hydroxide, ethyl acetate, hydrochloric acid, sulfuric acid, 30% hydrogen peroxide, sodium hydroxide, acetic acid, and Rose Bengal (RB) were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Tetraethyl orthosilicate (TEOS) and fluorescent isothiocyanate (FITC) were purchased from Sigma−Aldrich (St. Louis, MO, USA). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was purchased from Dojindo Molecular Technologies, Inc (Kumamoto, Japan). 3-Aminoproplytriethoxysilane (APTS) and 3-mercaptoproplytriethoxysilane (MPTS) were purchased from Shin-Etsu Chemical Co., Ltd (Tokyo, Japan). Sulfo-Cy5 NHS ester was purchased from Lumiprobe Corporation (Hallandale Beach, FL, USA). EPON 812 was purchased from TAAB Laboratories Equipment Ltd (Berkshire, UK). GEM and 2,4,6Trinitrobenzenesulfonic acid (TNBS) were purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). All reagents were used without further purification.


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Poly(5-aminopentyl-α,β-aspartamide) (P(Asp-AP), with a degree of polymerization (DP) of 82) and polyethylene glycol (PEG)-block-poly(α,β-aspartic acid) (PEG-PAsp, DP of PAsp = 75; Mn of PEG = 2,000, Mw/Mn of PEG = 1.05), were prepared as reported previously (Scheme 2a).20

Cell lines and animals A human lung adenocarcinoma epithelial cells (A549) (National Cancer Center, Tokyo, Japan) and a murine colon adenocarcinoma cells (C26) (Alexandria Technical & Community College, Alexandria, MN, USA) were cultured in RPMI-1640 (Sigma−Aldrich), supplemented with 100 U/mL penicillin (Sigma−Aldrich), 100 µg/mL streptomycin (Sigma−Aldrich) and 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA). For in vivo therapeutic efficiency and biodistribution experiments, BALB/c nude mice (5weeks-old; female) were purchased from Oriental Yeast (Tokyo, Japan). All animal experiments were carried out in accordance with the guidelines for animal experiments of the University of Tokyo.

Material characterization A Zetasizer-nano (Malvern Instruments Ltd., Worcestershire, UK) was used to measure particle size by Dynamic light scattering (DLS). A Mobius (Wyatt Technology Corporation, Santa Barbara, CA, USA) was used for measuring zeta-potential. Transmission electron micrographs (TEM) were obtained using a JEM-1400 transmission electron microscope (JEOL Ltd., Tokyo, Japan) operating at 120 kV. The samples for TEM observation were dispersed in purified water, and transferred onto copper grids (400 mesh size; coated with a thin film of collodion and carbon), followed by staining with uranyl acetate. For TEM observation of cross-


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sectioned specimens, the sample was purified and concentrated by centrifugal ultrafiltration, followed by fixing with 2% osmic acid solution, embedded in an epoxy resin (EPON 812), and cut with a Sorvall Porter-Blum MT-2 ultra-microtome to a thickness of ca. 50 nm. The ultrathin section was transferred onto a Cu mesh grid, and stained with uranyl acetate for 10 min and lead citrate for 2 min, and dried at RT. Energy dispersive X-ray spectroscopy (EDS) analysis was performed using a JEM-2100 transmission electron microscope (JEOL Ltd.) operating at 200 kV with a JED-2100T detector (JEOL Ltd.). UV−visible spectra were obtained on a Nanodrop ND1000 (Thermo Fisher Scientific Inc.). Absorbance of microplate samples was measured using a Microplate Instrumentation AD200 (Beckman Coulter Inc., Brea, CA, USA). Fluorescence intensity was measured using an FP-6600 fluorescence spectrophotometer (JASCO Corporation, Tokyo, Japan). Energy dispersive X-ray spectrometry-fluorescence spectrometry (EDS-FS) analysis (XGT-5200WR, Horiba Ltd., Kyoto, Japan) was used for the evaluation of sulfonatemodification of MSN.

Preparation of MSN MSN was prepared as reported previously.28 Typically, 0.15 g of CTAB was dissolved in 50 mL of warm distilled water, followed by adjusting of the solution pH to ca. 12 with sodium hydroxide solution. Then, 0.4 mL of TEOS was added to the CTAB solution at 70 °C and vigorously stirred at ca. 300 rpm for 5 min. Subsequently, 3 mL of ethyl acetate was added, and the mixture was stirred for 3 h, resulting in a whitish solution. The resulting mixture was cooled down to room temperature while stirring. Subsequently, the solution was centrifuged at 12,000 ×g for 60 min, and the supernatant was removed. Twenty milliliters of ethanol was added to the residue, followed by dispersion by ultrasonication. The pH of the resulting dispersion was


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adjusted to ca. 1 using hydrochloric acid, and then heated at 60 °C for 3 h while stirring to remove CTAB. The obtained reaction mixture was centrifuged at 12,000 ×g for 60 min, and the supernatant was then removed. The residue was washed 3 times with ethanol, and completely dried under reduced pressure at 40 °C, to give MSN (0.086 g).

Encapsulation of MSN into PICsome First, P(Asp-AP) and PEG-PAsp were separately dissolved in 10 mM phosphate buffer (PB; pH7.4) at a final concentration of 1 mg/mL. The MSN was dispersed in 10 mM PB (pH7.4) at a final concentration of 10 mg/mL. Five-hundred and forty microliters of the P(Asp-AP) solution was mixed with 18.8 µL of the MSN colloidal solution, and the mixture was subjected to vortex mixing for 2 min and then allowed to stand for 8 min. Then, 400 µL of the PEG-PAsp solution was added to this mixture, followed by vortex mixing for 2 min; this mixture contained the same residual molar concentration of –NH3+ from P(Asp-AP) and –COO– from PEG-PAsp. The weight ratio of MSN to total polymer was 1:5. Next, 550 µL of 10 mg/mL EDC solution was added to the reaction mixture, and stood overnight at room temperature to crosslink the PIC layer for further purification and all the other experiments. The resulting solution was purified using a polyethersulfone (PES) ultrafiltration membrane (molecular weight cut-off (MWCO): 300,000) at 25 °C. The medium exchange was repeated 10 times to completely remove byproducts from the solution to obtain the sample termed MSN*@PICsome, composed of MSN-encapsulated PICsome, empty PICsome, and free MSN.

Evaluation of efficacy of MSN loading into PICsome


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The efficacy of MSN loading, which is defined by a ratio of the amount of MSN encapsulated in the PICsome to the amount of feed MSN, was determined by capillary electrophoresis (CE), performed on a model P/ACE MDQ CE apparatus (Beckman Coulter, Inc.) equipped with a 75-µm capillary (TSP075375, Molex). Electrolyte was 200 mM glycine buffer (pH 8.0). Applied voltage for separation was set to 5 kV. Two samples, i.e., a solution of MSN*@PICsome and a physical mixture of empty PICsome and free MSN, were prepared for analysis. The MSN*@PICsome was dispersed at a total polymer concentration of 0.7 mg/mL. As a control, the mixture solution of free MSN and empty PICsome in a weight ratio of 1:5 was prepared with adjusting the total polymer concentration to 0.7 mg/mL. Both MSN*@PICsome and free MSN were labeled with FITC through adsorption onto the silica surface of the MSN to allow fluorescence detection. One milliliter of the MSN*@PICsome, and the free MSN solutions were treated with 10 µL of 5 mg/mL ethanol solution of FITC for the labeling. Then, this solution was purified by ultrafiltration using PES ultrafiltration membrane (MWCO: 300,000) until excess FITC was removed.

Surface modification of MSN Free MSN fraction was removed from the MSN*@PICsome sample using an Oasis MAX column (Waters corporation, Milford, MA, USA) before modification. Hereafter, the obtained purified sample in this way was termed MSN@PICsome. Surface modifications of MSN were carried out using silane coupling agents. Typical methods are detailed below. Introduction of amino groups. Crosslinked MSN@PICsome containing 2 mg of MSN was dispersed in 5 mL of purified water. Then, APTS was added in a dropwise manner to the solution


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as 1% per water volume, and stirred overnight at room temperature. The product was collected by ultrafiltration using a PES ultrafiltration membrane (MWCO: 300,000) at 25 °C, and washed 10 times with purified water to completely remove unreacted APTS from the solution. The resulting solution contained amino-functionalized MSN@PICsome (A-MSN@PICsome). Bare amino-functionalized MSN (A-MSN) was prepared similarly, but was purified by washing with water 5 times to completely remove unreacted APTS. Introduced amount of amino groups was determined by spectrophotometric analysis based on TNBS assay using empty PICsome as a reference. Introduction of sulfonate groups. Crosslinked MSN@PICsome containing 2 mg of MSN was dispersed in 5 mL of 1% acetic acid. Then, MPTS was added in a dropwise manner to the solution up to a 1% per volume, and stirred overnight at room temperature. The product was collected by ultrafiltration using a PES ultrafiltration membrane (MWCO: 300,000) at 25 °C, and washed 5 times with 1% acetic acid, and another 5 times with purified water, to completely remove unreacted MPTS from the solution. Subsequently, the collected mercaptoMSN@PICsome was dispersed in 5 mL of purified water, followed by mixing with 5 µL of concentrated sulfuric acid and 100 µL of 30% hydrogen peroxide for oxidation of mercaptogroups to sulfonate-groups. The reaction mixture was stirred overnight at room temperature.29 The resulting solution was neutralized using sodium hydroxide. The product was collected by ultrafiltration using a PES ultrafiltration membrane (MWCO: 300,000) at 25 °C, and washed 10 times with purified water, to completely remove salt. The resulting solution contained sulfonatefunctionalized MSN@PICsome (S-MSN@PICsome). Bare sulfonate-functionalized MSN (SMSN) was prepared similarly, except for the purification process as follows: Bare mercaptoMSN was centrifuged and washed with 1% acetic acid 5 times to completely remove unreacted


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MPTS. After the sulfonation, S-MSN was centrifuged and washed with water 5 times to completely remove salt.

Drug absorption and release test MSN@PICsome (without any modification), A-MSN@PICsome, and S-MSN@PICsome were used for absorption and release of drugs. These solutions were prepared using 10 mM phosphate-buffered saline (PBS; 10 mM PB with 150 mM NaCl, pH7.4) at 2 mg/mL of MSN. For absorption of RB, 5 mL of each solution (containing 10 mg of MSN) was added to 20 mL of RB solution (2.0 mg/mL) separately, and allowed to stand overnight. Then, the PICsome was removed using a PES ultrafiltration membrane (MWCO: 300,000) at 25 °C, and the collected solutions were analyzed by absorption spectrophotometry at a fixed wavelength of 550 nm to determine the residual amount of RB in the solution. The absorption amount was calculated as the difference between the initial and residual RB concentration. Absorption tests of GEM were performed similarly. Solutions of MSN@PICsome, AMSN@PICsome, and S-MSN@PICsome were prepared using 10 mM PBS (pH7.4) at 2 mg/mL of MSN. Ten milliliters of each solution (containing 20 mg of MSN) was added to 20 mL of GEM solution (1.5 mg/mL), separately, and allowed to stand overnight. Each formulation was washed to remove excess GEM by ultrafiltration. Then, the collected filtrates were analyzed by absorption spectrophotometry to determine the residual amount of GEM in the solution. Purification was repeated until GEM was not detected in the filtrate. The absorption amount was calculated as the difference between the initial and residual GEM concentration. For release test of GEM, 2 mL of the resulting solution of GEM-absorbed S-MSN@PICsome (GEM-SMSN@PICsome) was placed into a dialysis bag, and release test was performed at 37 °C using


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20 mL of 10 mM PBS (pH7.4) or simulated body fluid (SBF, pH7.4) as an outer solution. SBF (pH 7.4) contains Na+, K+, Mg2+, Ca2+, Cl–, HCO3–, HPO42–, SO42– at the same level as human body.48 GEM concentration was determined by absorption spectrophotometry using a fixed wavelength of 270 nm (n=3).

Evaluation of cytotoxicity To evaluate cytotoxicity, C26 or A549 cells were seeded in 96-well plates (2.2 × 103 cells/well) and pre-incubated for 24 h at 37 °C under a humidified atmosphere of 5% CO2. Six preparations, viz., GEM-S-MSN@PICsome, GEM, S-MSN@PICsome, GEM-S-MSN, S-MSN and empty PICsome were used for cytotoxicity evaluation as solutions in 10 mM PBS (pH 7.4), as listed in Table S2 (supporting information). The final GEM concentration of GEM-SMSN@PICsome, GEM, and GEM-S-MSN was 0.1 µg/mL for A549 cells, and 0.5 µg/mL for C26 cells. Concentration of MSN contained in S-MSN@PICsome and S-MSN were adjusted to that of GEM-S-MSN@PICsome and GEM-S-MSN, respectively. The cells were exposed to each of the 6 preparations for 48 or 72 h, followed by analysis using cell counting kit-8 (Dojindo Molecular Technologies, Inc.), involving incubation for 1 h at 37 °C. To prepare a blank and a negative control sample, 10 mM PBS (pH 7.4) and the cell culture medium were added to the cells. Finally, the absorbance was measured at 450 nm using a Microplate Instrumentation plate reader.

Blood retention of MSN and MSN*@PICsome For fluorescence labeling of MSN, Cy5 was adsorbed onto the silica surface of the MSN. To obtain Cy5-labeled MSN*@PICsome, 5 mg of Cy5 was added to 1 mL of the


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MSN*@PICsome solution containing 4 mg of the MSN, and stood overnight. The product was washed by 10 mM PBS (pH7.4) 20 times using a PES ultrafiltration membrane (MWCO: 300,000) at 25 °C to remove the residual Cy5. Cy5-labeled MSN was prepared by the same method using the MSN instead of the MSN*@PICsome. Cy5-labeled empty PICsome was prepared using Cy5-conjugated PEG-PAsp (PEG-PAsp-Cy5) instead of non-labeled PEG-PAsp, as previously reported.20 Two hundred microliters of solutions of the Cy5-labeled MSN*@PICsome (1 mg/mL as PEG-PAsp), the Cy5-labeled MSN (the same MSN concentration to the Cy5-labeled MSN*@PICsome), and the Cy5-labeled empty PICsome (1 mg/mL as PEG-PAsp-Cy5) in 10 mM PBS (pH 7.4) were systemically administered, respectively, into BALB/c mice by tail vein injection. Mice were sacrificed 1 h after the administration, and blood was collected from the postcaval vein using heparinized syringe. After centrifugation of the collected blood at 12000 × g for 5 min at 4 °C, fluorescence of the resulting supernatant was measured (Ex/Em = 650/670 nm). As a positive control, each sample solution was diluted 10-fold by mouse plasma. All measurements were repeated 3 times for each sample.

Evaluation of in vivo therapeutic efficacy and biodistribution of GEM-MSN@PICsome Each BALB/c nude mouse was subcutaneously inoculated with 50 µL of a suspension of A549 cells in RPMI-1640 with 10% Fetal Bovine Serum (FBS, 3 × 107 cells/mL). Tumors were allowed to grow for 14 days (Average tumor volume is 8.7 mm3). GEM-S-MSN@PICsome, SMSN@PICsome, GEM-S-MSN, S-MSN, PICsome, GEM (Table S2), and 10 mM PBS (pH7.4) were prepared for administration as described above. Two-hundred microliters of each solution were injected into the tail vein of mice. The dose of GEM was 5 mg/kg, and formulations


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without GEM were administered at a concentration equivalent to the polymers or MSN included in 5 mg/kg GEM-S-MSN@PICsome. Each formulation was administered 3 times with a 7-day interval (days 0, 7, and 14 days). Tumor size was measured twice a week using a digital vernier caliper across both perpendicular diameters, and its volume, V, was calculated using the following formula: V = (a2 × b)/2, where a and b are the minor and major diameter of the tumor. For the biodistribution study, A549-tumor bearing mice (BALB/c nude, female; 5 weeks old; n = 5) were prepared as above. GEM-S-MSN@PICsome and PICsome (a negative control) were prepared using Cy5-labeled polymer instead of non-labeled polymer.20 Polymer concentration was adjusted to the same concentration with the in vivo therapeutic experiment. GEM-S-MSN was prepared as follows: Cy5 was added to the S-MSN dispersed solution for fluorescence labeling. The resulting solution was purified using a PES ultrafiltration membrane (MWCO: 300,000) at 25 °C. The PBS buffer (pH7.4) was exchanged repeatedly until excess Cy5 was removed completely from the sample to obtain Cy5-loaded S-MSN. Then, GEM was absorbed in Cy5-loaded S-MSN similarly. MSN concentration was adjusted to contain the same amount of MSN with GEM-S-MSN@PICsome. Two-hundred microliters of 10 mM PBS solutions of each formulation were injected into the tail vein of mice. As a positive control of plasma clearance evaluations, each sample solution was diluted 10-fold by mouse plasma. Blood was collected from the postcaval vein using heparinized syringes, and the main organs (lung, kidney, liver, and spleen) and A549 tumor tissues were excised. After centrifugation of the collected blood at 12000 ×g for 5 min at 4 °C, fluorescence of the resulting supernatant was measured using a plate reader. The main organs and tumor tissues were homogenized using 5 ×


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Passive Lysis Buffer (Promega, Tokyo, Japan), and the resulting colloidal solutions were analyzed by spectrofluorometry using a microplate reader (Ex/Em = 643/667 nm).

RESULTS AND DISCUSSION Encapsulation of MSN into PICsome Synthesized MSN was characterized as previously reported.26 DLS measurements revealed that the MSN had a monodispersed size distribution with an average diameter of ca. 70 nm with a polydispersity index (PdI) of 0.09. TEM images showed that the MSN possessed a spherical shape and mesoporous structure (Figure S1). The zeta-potential of the synthesized MSN in 10 mM PBS (pH 7.4) was –18.1 mV due to the negative charge of the silanol groups on their surface. Thus, the obtained MSN has a suitable size for encapsulation into PICsome and for further drug delivery applications. Assuming the mechanism of PICsome formation, charged materials may interact with oppositely charged constituent polymers of PICsome during their loading process, hampering the vesicle formation.19,21 In fact, MSN has negative charge in nature, which may cause this issue. Actually, direct mixing of the solutions of PEG-PAsp, P(Asp-AP) and MSN dispersion led to visible aggregation without any sign of the formation of monodispersed PICsomes (data not shown). Thus, to circumvent this non-specific aggregation, the sequential method described in the EXPERIMENTAL was adopted here: positively charged P(Asp-AP) was added to MSN, followed by the addition of negatively charged PEG-PAsp (Scheme 2b). Then, the product was cross-linked to enhance their stability, and subjected to further purification to obtain the sample of MSN*@PICsome as described in the section “Encapsulation of MSN into PICsome” of the EXPERIMENTAL. The obtained sample has averaged size of 105 nm with a PdI of 0.15.


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Moreover, the averaged zeta-potential was –2.8 mV in 10 mM PBS (pH 7.4), which is significantly smaller absolute value compared to –18.1 mV observed for bare MSN. TEM images of the sample revealed that there were two types of particles with differing contrast, in which dark contrast particles can be considered as MSN-encapsulated PICsome (Figure 1a). There were some PICsome that did not encapsulate MSN (light contrast particles), although little amount of free MSN was observed (data not shown). The particle number ratio of dark contrast PICsome is ca. 30%, which was estimated based on TEM images (Figure S2 and Table S1). Cross-sectional TEM images verified that MSN particles, which are seen as dark-contrast spherical particles (indicated by arrows) were encircled by a vesicular membrane (Figure 1b). This result clearly shows successful loading of MSN into PICsome. Loading of MSN into PICsome was also confirmed by EDS analysis (Figure 1c). Bare MSN particles were clearly visualized as dots with intense signal from Si atoms on a Si-mapping image as well as on a bright-field image (Figure 1c-i and ii). Signals from Si atoms were also observed for MSNencapsulated PICsome, although there was a fraction of empty PICsomes lacking Si signal, which were only visualized in a bright-field image (Figure 1c-v, vi).


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Scheme 2. Chemical structures of P(Asp-AP) and PEG-PAsp (a), Schematatic illustration of preparation method of MSN-encapsulated PICsome (b), and the proposed mechanism of MSNencapsulated PICsome formation (c).


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Figure 1. A conventional TEM image (a), and cross-sectional TEM images (b) of MSN*@PICsome. Arrows indicate MSN. Bright-field and Si-mapping images of bare MSN (i, ii), PICsome (iii, iv), and MSN*@PICsome (v, vi). Si-mapping was determined by energy dispersive X-ray spectroscopy (EDS) analysis (c-ii,iv,vi).

To estimate the efficacy of MSN loading, the prepared samples were analyzed by CE after labeling of the MSN with FITC as the fluorescent marker. The peak of empty PICsome was not observed in the electropherograms of Figure 2, because empty PICsome has no capability to retain FITC unless it was loaded with MSN. Three major peaks were found in the electropherogram of the MSN*@PICsome solution (Figure 2a, indicated by arrows). In this experimental condition, eluting time should be correlated with the net negative charge of analytes. The averaged zeta potentials of MSN and empty PICsome in 10 mM PBS (pH7.4) were –18.1 mV, and –0.3 mV, respectively. As aforementioned, averaged zeta potential of the MSN@PICsome was –2.8 mV. Accordingly, peak (1), eluted at ca. 3 min is assignable to MSNencapsulated PICsome having only a slightly negative zeta potential. The peaks (2) and (3) were assigned to free-FITC and bare MSN, respectively, by comparing to the result obtained for a physical mixture of FITC-labeled MSN and non-labeled PICsome shown in Figure 2b. Here, no peak was observed at the elution time of ca. 3 min, indicating negligible physical interaction of FITC-labeled MSN with PICsome surface. Bare FITC-labeled MSN was assignable to peak (3’), considering its large negative zeta potential, and appeared at the same elution time with peak (3) in Figure 2a. Sharp peaks found at ca. 4 min of elution time in Figures 2a and 2b, i.e., peaks (2) and (2’), correspond to free-FITC. From the area-ratio of peaks (1) and (3), the efficacy of MSN loading was calculated to be ca. 83%. Such efficient loading of MSN without forming any


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aggregates can be explained by assuming the adsorption of cationic P(Asp-AP) on the surface of negatively-charged MSN in the first step of MSN-encapsulated PICsome preparation shown in Scheme 2b. Subsequent addition of PEG-PAsp induces PIC formation preferentially at the proximity of the surface of P(Asp-AP)-adsorbed MSN, leading to the formation of PICsome loaded with MSN (Scheme 2c). PEG-PAsp also interacts with excess P(Asp-AP), remaining in the aqueous phase without adsorbing onto MSN, to form “unit PIC (uPIC)”, a unit ioncomplexed pair of P(Asp-AP) and PEG-PAsp, according to electrostatic interaction as reported previously.19,44 These uPICs in aqueous phase then be integrated into the preformed PIC membrane surrounding MSN particles to facilitate the formation of the vesicular structure. 43 Thus, it is reasonable to assume that the key step to increase the efficacy of MSN loading may be the smooth initial PIC formation at the proximity of the surface of P(Asp-AP)-adsorbed MSN upon the addition of PEG-PAsp. This may be the feasible mechanism involved in the efficient encapsulation of MSN into PICsomes (Scheme 2c).

Figure 2. Electropherograms of as-prepared solution of MSN*@PICsome (a) and the mixture of MSN and PICsome solutions (b). Surface modification of MSN included in PICsome


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To enhance the drug-retaining ability of MSN, we examined surface modification of MSN encapsulated in PICsome. Here, as described in the section of “Surface modification of MSN” of the EXPERIMENTAL, free MSN fraction was removed from the MSN*@PICsome by Oasis MAX column. The obtained column-purified sample (MSN@PICsome), composed of MSN-encapsulated PICsome and empty PICsome (particle number ratio, 3:7) without free-MSN, was subjected to the surface modification. The surface of MSN was modified with amino groups or sulfonate groups as described in the EXPERIMENTAL, to obtain A-MSN@PICsome and SMSN@PICsome, respectively. Bare A-MSN and S-MSN were prepared as control. The averaged zeta-potentials of non-modified MSN, A-MSN, and S-MSN in 10 mM PBS (pH 7.4) were –18.1 mV, 7.9 mV, and –40.2 mV, respectively. These changes in zeta-potential values are consistent with

respective

surface

modification.

In

contrast,

the

averaged

zeta-potentials

of

MSN@PICsome, A-MSN@PICsome, and S-MSN@PICsome in 10 mM PBS (pH 7.4) were –2.8 mV, –3.3 mV, and –3.7 mV, respectively. Thus, the zeta-potentials of modified MSN@PICsomes were not significantly different even after the chemical treatment. Such zetapotential values are consistent with the MSN encapsulation into PICsome surrounded by chargeneutral PEG palisades. Nevertheless, direct evidence of MSN modification even in the interior of PICsome is needed. Thus, the presence of amino-groups and sulfur atoms of sulfonate-groups of chemically treated MSN@PICsomes was confirmed by TNBS assay and EDS-FS, respectively. The TNBS assay showed that A-MSN@PICsome contained 8.9 × 1019 amino groups for 1 mg of MSN, whereas a negligible amount of amino groups was detected for the original MSN@PICsome. Moreover, after the treatment of empty PICsome with APTS using the same procedure of MSN@PICsome modification, negligible increase in the amount of amino groups was observed by TNBS assay (less than detection limit). These results exclude the possibility of


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PICsome membrane modification by APTS. EDS-FS showed a clear difference in the intensity ratio of the S peak and the Si peak between the S-MSN-@PICsome and original MSN@PICsome (Figure S3). A markedly higher S signal of the S-MSN@PICsome clearly supported the successful incorporation of sulfur-containing moieties. Note that the empty PICsome subjected to the MPTS treatment using the same method of MSN@PICsome modification gave almost the same EDS spectrum with those before the treatment (Figure S3), excluding the chemical modification of PICsome membrane during MPTS treatment. Furthermore, particle size was unchanged even after the modification (MSN@PICsome: 101 nm with a PdI of 0.08; A-MSN@PICsome: 105 nm with a PdI of 0.09; S-MSN@PICsome: 104 nm with a PdI of 0.08). These results demonstrate that surface modification of MSN can be performed without substantial change in the original PICsome structure.

Absorption and release of LMWCs Next, the potential utility of A- and S-MSN@PICsomes for LMWCs carriers was investigated. RB was used as an anionic model compound with a carboxyl group. MSN@PICsome and S-MSN@PICsome absorbed negligible amount of RB (0.5 and 0.2 w/w%, respectively; the values were defined by [RB weight]/[MSN weight]). In contrast, the absorbed amount of RB was increased markedly, to 3.2w/w% for A-MSN@PICsome, which is in consistency to the positively-charged nature of A-MSN. Then, GEM, a deoxycytidine nucleoside analogue widely used as cytotoxic reagent for the treatment of non-small cell lung cancer, was selected as a model compound possessing a cationic moiety. The amount of GEM absorbed into both MSN@PICsome and A-MSN@PICsome was less than the detection limit. In contrast, the GEM absorption was significantly improved using S-MSN@PICsome, and the loading amount


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reached 7.9w/w% (GEM weight per MSN weight). In addition, neither RB nor GEM could be retained in the empty PICsome after purification. This result is also consistent with our previous findings.21,45 Then, we examined the release profiles of GEM-S-MSN@PICsome in 10 mM PBS (pH 7.4; Figure 3). The amount of GEM released in 72 h was only 10%. Notably, GEM release underwent a remarkable increase in SBF (pH 7.4), and the release amount reached ca. 40% of the loaded GEM in 72 h. This may be a feasible property from a practical viewpoint, because GEM release can be suppressed in low ionic condition under the purification and storage, while it may be accelerated under physiological condition.

Figure 3. Release profiles of GEM-S-MSN@PICsome in 10 mM PBS (pH7.4) () and in the SBF (pH7.4) ().

Cytotoxicity of GEM-formulations Cytotoxicity of GEM-S-MSN@PICsome was evaluated in A549 and C26 cells using the samples listed in Table S2. GEM-S-MSN@PICsome showed cytotoxicity to both A549 and C26 cells in time dependent manner, to a level as high as or slightly lower than that of GEM alone and GEM-S-MSN (Figure 4). Formulations without GEM (S-MSN, S-MSN@PICsome, and


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empty PICsome) showed negligible cytotoxicity to both cell lines, indicating that the cytotoxicity of GEM-S-MSN@PICsome is indeed due to GEM payload.

Figure 4. Cytotoxicity against A549 cells (a), and C26 cells (b) over 48 h (open bar) and 72 h (slashed bar) periods. GEM concentration was adjusted at 0.01 µg/mL for C26 cells, 0.05 µg/mL for A549 cells, respectively. Sample 1: GEM-S-MSN@PICsome, sample 2: S-MSN@PICsome, sample 3: GEM-S-MSN, sample 4: S-MSN, sample 5: Empty PICsome, and sample 6: GEM. *: p < 0.05 Blood retention of MSN and MSN*@PICsome To confirm the potential utility of MSN-encapsulated PICsome for in vivo applications, plasma clearance of the MSN*@PICsome was evaluated as a feasibility study (Figure 5). Here, blood retention was compared among Cy5-labeled free MSN (the sample 1), Cy5-labeled MSN*@PICsome (the sample 2), and Cy5-labeled empty PICsome (the sample 3). Of note, all the PICsome formulations were crosslinked for enhancement of stability in in vivo environment. It should be noted that the Cy5-labeled MSN*@PICsome (the sample 2) contained both empty PICsome (MSN-encapsulated PICsome : empty PICsome = 3:7 in the number ratio based on the TEM result shown in Figure 1) and free MSN (MSN-encapsulated PICsome : free MSN = 83:17 in the weight ratio based on the CE result shown in Figure 2). All the PICsome-based formulations showed markedly higher blood retention than MSN alone. In the sample 2, Cy5 was adsorbed only onto the silica surface of the MSN, but not accumulated in the PICsome


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membrane. Thus, the data of the sample 2 in Figure 5 reflects the blood retention profile of free MSN and MSN-encapsulated PICsome, excluding an influence from empty PICsome fraction. Apparently, the sample 2 (free MSN + MSN-encapsulated PICsome) revealed the longevity in blood retention comparable to the empty PICsome (sample 3). Further to notice is that free MSN (sample 1) underwent the fast clearance from the blood compartment (76% exclusion within 1h), indicating that the longevity of the sample 2 is mainly due to the contribution from the MSNencapsulated PICsome fraction. Slight decrease in blood retention observed for the sample 2 compared to the sample 3 (87% vs. 100%) can be explained by the fast elimination of free MSN fraction from the sample 2 in the initial stage of blood circulation. Consequently, it is reasonable to conclude that the MSN-encapsulated PICsome indeed has long-circulating property comparative to the empty PICsome.

Figure 5. Plasma clearance of Cy5-labeled free MSN (sample 1), Cy5-labeled MSN*@PICsome (mixture of Cy5-labeled MSN-encapsulated PICsome, Cy5-labeled free MSN, and empty PICsome without labeling) (sample 2), and Cy5-labeled empty PICsome (sample 3) measured at 1 h after administration into BALB/c mice. Evaluation of biodistribution and in vivo therapeutic efficacy of GEM-MSN@PICsome For estimating the feasibility of GEM-S-MSN@PICsome for systemic treatment of solid


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tumors, its biodistribution was examined using mice subcutaneously inoculated with A549 tumor by quantification of fluorescence of Cy5 attached to the terminus of PEG–PAsp or adsorbed onto the silica surface of the MSN. It is worthy of note that release of Cy5 was suppressed under the SBF condition (Figure S4, supporting information). Note that all the PICsome formulations were crosslinked. The amount of GEM-S-MSN@PICsome remaining in the blood at 24 h after injection was 10.8 ± 0.4% (Figure 6a). On the other hand, the amount of GEM-S-MSN remaining in the blood at 24 h after injection was negligible (0.9 ± 0.1%). These results indicate that the retention of GEM-S-MSN in blood compartment was improved markedly by the encapsulation into PICsome. The organ distribution and tumor accumulation are summarized in Figure 6b. Tumor accumulation was remarkably improved for GEM-S-MSN@PICsome compared to GEM-S-MSN. This improved tumor accumulation can be explained by the enhanced permeability and retention (EPR) effect45–47, which was observed for previous longcirculating PICsomes so far reported.16,18,20–22 Of note, tumor to liver selectivity was also significantly improved by loading GEM-S-MSN into PICsome, which is apparently an advantage to circumvent potential toxicity against liver.

Figure 6. The residual amount in blood (a) and biodistribution (b) of GEM-S-MSN@PICsome (open square) and GEM-S-MSN (slashed square) 24 h after i.v. injection.


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Finally, GEM-S-MSN@PICsome was exploited for cancer treatment in mice model bearing subcutaneous A549 lung tumor. All of the formulations were administered every 7 days, and 3 times in total. All the drug-loaded formulations contained 5 mg/kg of GEM. At this dose of GEM, there was no significant body weight loss observed for all the formulations (Figure S5b). The formulations of GEM alone, GEM-S-MSN, S-MSN, S-MSN@PICsome, and PICsome did not show any significant tumor growth suppression compared to the PBS-treatment group (Figure 7). In contrast, mice treated with GEM-S-MSN@PICsome revealed suppression in tumor growth from 7 days after the initial administration. The difference in tumor volume between GEM-S-MSN@PICsome and other formulations became significant with time. As GEM release from GEM-S-MSN@PICsome occurred in sustained manner (Figure 3) at physiological condition, it may be reasonable to assume that enhanced retention of GEM-S-MSN@PICsome in tumor led to an increased cumulative concentration of GEM in tumor (or increased tumor AUC of GEM), achieving appreciable in vivo anti-tumor efficacy compared to free GEM injection.

Figure 7. Change in tumor volume with time for A549 bearing BALB/c nude mice after i.v. injection of GEM-S-MSN@PICsome (◊), S-MSN@PICsome (♦), GEM-S-MSN (□), S-MSN (■),


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PICsome (●), GEM (▲), and 10 mM PBS (pH7.4) (×). Each group included 5 mice. Administration started on Day 0 and repeated on Days 7 and 14 (indicated as an arrow in the figure). Tumor volume was measured twice a week until 28 days after injection. *: p < 0.05 (between ◊ and ▲). Individual data are given in the Supplementary Information as Figure S5a.

Conclusion A novel nano-formulation of water-soluble LMWCs was developed here based on PICsomes encapsulating surface-modulated MSN in high efficacy. S-MSN@PICsomes thus prepared retained and sustainably released GEM in physiological condition, and revealed prolonged blood circulation, thereby achieving enhanced tumor accumulation compared to bare MSN. Eventually, appreciable in vivo antitumor efficacy was obtained, appealing the feasibility of PICsome formulations in targeted cancer chemotherapy. Moreover, semi-permeable nature of PICsome membrane enables the in situ modulation of entrapped MSN just by simple treatment with silane coupling agents in aqueous medium. Accordingly, MSN@PICsomes with desired functionality can be obtained to trap a wide variety of drugs with different chemical properties. A remaining issue from the standpoint of practical application of PICsome in the present form for drug carrier is the necessity to stabilize PIC membrane by crosslinking reagent such as EDC. Nevertheless, our recent study revealed that the stability of PICsome in physiological medium can be improved by modulating side chain structure of constituent polyelectrolytes without crosslinking process20, suggesting a promising trend to construct MSN@PICsome carrier working in vivo with programmable disintegrating property to ultimately be excreted from the body.


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ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publication website at DOI: XXXXXXXXXX. TEM images, EDS-FS analysis, release profiles, list of samples for biological evaluation, and results of in vivo therapeutic efficacy and body weight loss. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This research was supported in part by the Core Research Program for Evolutional Science and Technology and the Center of Innovation (COI) Program from the Japan Science and Technology Agency (JST), Grants-in-Aid for Scientific Research (No. 25107009 and 26288082 to A. K.) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan and JSPS Core-to-Core Program, A. Advanced Research Networks. We thank Mr. H. Hoshi, Research Hub for Advanced Nano Characterization at the University of Tokyo, for his valuable support in the TEM measurements. We would like to thank Dr. A. Shimojima, Waseda University, School of Advanced Science and Engineering, for his helpful advice regarding MSN preparation.


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ABBREVIATIONS A-MSN, amino-functionalized MSN; A-MSN@PICsome, amino-functionalized MSN@PICsome; APTS, 3-Aminoproplytriethoxysilane; CE, capillary electrophoresis; CTAB, cetyltrimethyl ammonium bromide; GEM, 2’,2’-Difluoro-2’-deoxycytidine, gemcitabine; DLS, dynamic light scattering; EDC, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; EDS, energy dispersive X-ray spectroscopy; EDS-FS, Energy dispersive X-ray spectrometryfluorescence spectrometry; EPR, enhanced permeability and retention; FBS, fetal bovine serum; FITC, fluorescent isothiocyanate; GEM-S-MSN@PICsome, GEM-absorbed S-MSN@PICsome; MSN*@PICsome, a mixture of the MSN-encapsulated PICsome, empty PICsome, and free MSN; LMWCs, low-molecular-weight compounds; MPTS, 3-mercaptoproplytriethoxysilane; MSN, mesoporous silica nanoparticles; MSN@PICsome, a mixture of the MSN-encapsulated PICsome and empty PICsome; PB, phosphate buffer; PBS; phosphate-buffered saline; PdI, polydispersity index; PEG, polyethylene glycol; PEG-PAsp, polyethylene glycol-block-poly(α,βaspartic acid); PES, polyethersulfone; PICsomes, Polyion complex vesicles; RB, Rose Bengal; SBF, simulated body fluid; S-MSN, sulfonate-functionalized MSN; S-MSN@PICsome, sulfonate-functionalized MSN@PICsome; TEM, Transmission electron micrographs; TEOS, tetraethyl orthosilicate; TNBS, 2,4,6-trinitrobenzenesulfonic acid; P(Asp-AP) , poly(5aminopentyl-α,β-aspartamide);

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For Table of Contents Use Only Facile Preparation of Delivery Platform of Water-Soluble Low-Molecular-Weight Drugs Based on Polyion Complex Vesicle (PICsome) Encapsulating Mesoporous Silica Nanoparticle Akinori Goto, Hung-Chi Yen, Yasutaka Anraku, Shigeto Fukushima, Ping-Shan Lai, Masaru Kato, Akihiro Kishimura and Kazunori Kataoka


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Facile Preparation of Delivery Platform of Water-Soluble Low

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