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Bifunctional Succinylated #-Polylysine Coated Mesoporous Silica Nanoparticles for pH-Responsive and Intracellular Drug Delivery Targeting the Colon Chau Nguyen, Richard Webb, Lynette Lambert, Ekaterina Strounina, Edward Lee, MarieOdile Parat, Michael McGuckin, Amirali Popat, Peter Cabot, and Benjamin P. Ross ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00411 • Publication Date (Web): 02 Mar 2017 Downloaded from http://pubs.acs.org on March 2, 2017

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ACS Applied Materials & Interfaces

Bifunctional Succinylated ε-Polylysine Coated Mesoporous Silica Nanoparticles for pH-Responsive and Intracellular Drug Delivery Targeting the Colon Chau T.H. Nguyen,† Richard I. Webb,‡ Lynette K. Lambert,# Ekaterina Strounina,# Edward C. Lee,† Marie-Odile Parat,† Michael A. McGuckin,φ Amirali Popat,†,φ Peter J. Cabot,† Benjamin P. Ross.*,† †

The University of Queensland, School of Pharmacy, Brisbane, Queensland 4072, Australia.

‡

The University of Queensland, Centre for Microscopy and Microanalysis, Brisbane, Queensland 4072, Australia.

# The University of Queensland, Centre for Advanced Imaging, Brisbane, Queensland 4072, Australia. φ Mater Research Institute – The University of Queensland, Translational Research Institute, Inflammatory Disease Biology and Therapeutics Group, 37 Kent St, Woolloongabba, Queensland 4102, Australia. KEYWORDS: succinylated ε-polylysine, mesoporous silica nanoparticles, pH-responsive, colon targeted drug delivery, intracellular drug delivery, prednisolone.

ABSTRACT: Conventional oral drug formulations for colonic diseases require the administration of high doses of drug to achieve effective drug concentrations at the target site. However, this exposes patients to serious systemic toxicity in order to achieve efficacy. To overcome this problem, an oral drug delivery system was developed by loading a large amount (ca. 34% w/w) of prednisolone into 3-aminopropyl-functionalized mesoporous silica nanoparticles (MCM-NH2), and targeting prednisolone release to the colon by coating the nanoparticle with succinylated ε-polylysine (SPL). We demonstrate for the first time the pH-responsive ability of SPL as a “nanogate” to selectively release prednisolone in the pH conditions of the colon (pH 5.5 to 7.4) but not in the more acidic conditions of the stomach (pH 1.9) or small intestine (pH 5.0). In addition to targeting drug delivery to the colon, we explored whether the nanoparticles could deliver cargo intracellularly to immune cells (RAW 264.7 macrophages) and intestinal epithelial cells (LS 174T and Caco-2 adenocarcinoma cell lines). To trace uptake, MCM-NH2 were loaded with a cell membrane-impermeable dye, sulforhodamine B. The SPL-coated nanoparticles were able to deliver the dye intracellularly to RAW264.7 macrophages and the intestinal epithelial cancer cells, which offers a highly promising and novel drug delivery system for diseases of the colon such as inflammatory bowel disease and colorectal cancer.

INTRODUCTION Targeted drug delivery is of great importance because it can increase efficacy and reduce toxicity.1 Of the many strategies that are available to achieve targeted drug delivery, the development of stimuli-responsive

nanoparticles is one of the most promising. To accomplish this feat, drug-loaded nanoparticles have been coated with polymers that respond to either external stimuli (e.g. ultrasound, light, temperature, or magnetic fields) or internal stimuli (e.g. pH or enzymes), thereby controlling the release of the drug.2-5 In particular, mesoporous silica nanoparticles (MSNs) coated with polymers that undergo physical changes (solubility, ionization, or swelling) in specific pH environments are of great interest.6 This is because these properties can enable targeted drug release from the MSNs based on the distinctive pH conditions of the gastrointestinal (GI) tract, tumors, inflammatory tissues and intracellular compartments (endosomes, lysosomes).6-9

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MSNs are inorganic materials with highly ordered porous interiors (e.g. hexagonal arrangement: MCM-41; cubic arrangement: MCM-48).10 Their large pore volume and adjustable pore size can accommodate substantial amounts of a variety of drugs and protect the drugs from enzymatic degradation.1,11 Moreover, MSNs have a large surface area, relative to their volume, composed of silanol groups1 which can be conjugated with materials such as polymers to form stable and homogenous coatings. In the past decade, MSNs coated with pHresponsive polymers such as chitosan,12 poly(glutamic acid)13 and β-lactoglobulin7 were developed to create targeted delivery systems for drugs including antiinflammatories,7,14 antibiotics12 and cytotoxics.13,15,16 Studies have demonstrated the potential of intracellular drug delivery using nanoparticles.4,17 Despite the success of nanoparticle drug delivery systems, further investigations are needed to achieve higher drug loading and reduce premature drug release. Targeting drug release to treat debilitating diseases of the colon, such as inflammatory bowel disease (IBD), requires that orally administered nanoparticles resist drug release in the acidic conditions of the stomach (pH ca. 1.9) and small intestine (pH ca. 5.0),18 and release drug in the higher pH environment of the colon (pH ca. 5.5-7.4).19 In addition to targeting the drug delivery to tissues, attention should also be directed at delivering drugs to the specific cells involved in disease pathophysiology. The immune response is centrally involved in the pathophysiology of IBD, and intestinal macrophages, in particular, have been recognized to play an instrumental role mediating the pathological inflammatory processes in IBD.20-25 Therefore, focusing on the source of chronic inflammation by delivering anti-inflammatory drugs inside immune cells such as macrophages, is a potential strategy to optimize therapeutic outcomes.20,24,26 In this study, 3-aminopropyl-functionalized MCM-48 nanoparticles (MCM-NH2) were loaded with a

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hydrophobic anti-inflammatory drug, prednisolone, and coated with a pH-responsive polymer (Figures 1A and 1B) to develop a novel oral drug delivery system targeted to the colon for IBD treatment. The polymer SPL was selected as the coating material because it was predicted to prevent drug leaching at stomach pH and release drug at intestinal pH via pH-dependent conformational changes. SPL was obtained by modification of εpolylysine (EPL), a natural, low-cost, monodisperse polymer, that is used as a food preservative and which is classified by the FDA as a generally recognized safe material.27,28 Furthermore, SPL may increase the residence time at the target site due to the electrostatic interaction with GI cells.21,29 Importantly, the uptake of the SPL-coated MSN drug delivery system by macrophages and intestinal epithelial cancer cells was investigated by loading MSNs with a cell membraneimpermeable hydrophilic dye, sulforhodamine B (Figure 1C). Our system demonstrated high loading of hydrophobic drug and hydrophilic dye, and pHresponsive sustained release of cargo with minimal premature release at gastric or small intestinal pH, and extensive drug release in the colonic pH range. To the best of our knowledge, this is the first report of prednisolone-loaded MSNs and the pH-responsiveness of SPL as a “nanogate” controlling drug release from the MSNs in response to different pH conditions of the GI tract. The coated MSNs were readily taken up by both macrophages (RAW 264.7) and intestinal epithelial cancer cells (LS 174T and Caco-2) which resulted in intracellular delivery of sulforhodamine B. In addition to developing a highly promising pH-responsive oral drug delivery system for IBD, we believe the preliminary investigations on the uptake by macrophages and intestinal epithelial cancer cells provides opportunities for intracellular drug delivery, including intracellular delivery of polar hydrophilic drugs with inherently poor cell membrane permeability.

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Figure 1. A novel pH-responsive “nanogate” system proposed for targeted drug delivery to the colon: (A) Construction of the drug delivery system from 3-aminopropyl-functionalized MCM-48 nanoparticles (MCM-NH2), prednisolone, and SPL; (B) pH-Responsive release of drug/cargo under conditions simulating gastrointestinal transit; (C) Cellular uptake by macrophages; it is proposed that the over-activation of pro-inflammatory intestinal macrophages and 21 impaired mucus barrier function in the intestine of patients with IBD enhance intracellular drug delivery.

RESULTS AND DISCUSSION pH-Responsive drug delivery to the colon Synthesis and characterization of 3-aminopropylfunctionalized MSNs. Spherical MSNs (MCM-48) 100-150 nm in diameter were synthesized using a method that was optimized from the 30,31 procedure of Kim et al. To form a surface suitable for conjugation with SPL, MCM-48 were functionalized with 3aminopropyl groups to form MCM-NH2. The reaction generated positively charged nanoparticles, which

corresponds to the presence of protonated 3-aminopropyl groups, and the size and morphology of the nanoparticles were retained with thermogravimetric analysis (TGA) indicating 12% (w/w) incorporation of 3-aminopropyl groups (Table S1, Figures 2A and S3A). The N2 adsorptiondesorption isotherm of MCM-NH2 revealed a characteristic IUPAC-type IV isotherm with a hysteresis loop, which confirmed the porous structure of MSNs synthesized using 31,32 the surfactant template synthesis method (Figure S2). The average pore diameter, based on the Barret-JoynerHalenda (BJH) method, was calculated to be ca. 1.9 nm (Figure S2). X-ray diffraction (XRD) peaks confirmed the

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crystalline mesoporous structure of MCM-NH2 based on a hkl value of 211 (Figure S11A). The surfactant within MCMNH2 was successfully removed by calcination (heating nanoparticles to ca. 550 °C) or solvent extraction. The latter technique produced MSNs with greater surface area and pore volume (Table S2), which may improve drug loading capacity, therefore the nanoparticles produced using this method were used for subsequent experiments. The lower

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surface area and pore volume following calcination may be due to the dehydroxylation and condensation of silanol (Si33 OH) groups at high temperature (ca. 550 ºC). Transmission electron microscopy (TEM) images of MCMNH2 revealed well-defined spherical morphology and a porous structure (Figure 2A).

Figure 2. TEM images of: (A) MCM-NH2; and SPL-MCM of different coating thicknesses formed by conducting the reaction with silica/SPL in a ratio of (B) 1 : 1 and (C) 1 : 2 (w/w) respectively; (D) zeta potential values of EPL, SPL, MCM13 NH2 and SPL-MCM in different pH solutions illustrating the pH-dependent ionization behavior; (E) C solid state 13 NMR ( C SSNMR) spectrum of SPL-MCM showing resonances consistent with the carbon atoms of SPL and MCM-NH2. The chemical structure of SPL-MCM is an approximation because the exact number of amide linkages between SPL and MCM-NH2, and the number of free carboxylic acid groups, is unidentified.

Prednisolone loading. The hydrophobic anti-inflammatory drug, prednisolone, was loaded into the pores of MCM-NH2 prior to coating with SPL. To achieve high loading of prednisolone into MCM-NH2, a series of optimization experiments were

conducted culminating in a method that combined the 7 34 conventional stirring and solvent evaporation methods. The detailed procedure is described in the experimental section; briefly, after stirring MCM-NH2 in a concentrated solution of prednisolone in EtOH, the drug-loaded

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nanoparticles were centrifuged and re-dispersed in a higher concentration of prednisolone solution to minimize drug leaching from the nanoparticles. The solvent was then evaporated in vacuo using a rotary evaporator to allow the deposition of prednisolone inside the mesopores. Based on TGA (Figure S3A), high loading of prednisolone into MCMNH2 was achieved at the level of ca. 34% (w/w). To exclude the possibility that the weight loss observed by TGA was due to the adsorption of prednisolone on the outer surface rather than inside the pores, the crystallinity of prednisolone before and after drug loading were determined using differential scanning calorimetry (DSC) analysis performed in parallel with TGA (Figure S3B). Free prednisolone and prednisolone physically mixed with MSNs exist in the crystalline form which is evident from the sharp peaks in the DSC curve at ca. 238 °C corresponding to the melting point of prednisolone (Figure S3B). After loading prednisolone into MCM-NH2 (PredMCM-NH2), the peak at 238 °C disappeared. The transition of prednisolone from a crystalline to an amorphous state following drug loading indicates the successful loading of drug into the pore channels of MCM-NH2.

property because it can increase residence time in the GI tract, allowing greater time for the delivery system to 29,35 release drug. Following the grafting procedure, the morphology of SPL-coated MSNs (SPL-MCM) was retained 13 (Figures 2B and 2C cf. 2A). The C SSNMR spectrum of 13 SPL-MCM (Figure 2E) was similar to the C NMR spectrum of SPL (Figure S6) which indicated the successful combination of SPL with MCM-NH2. However, the broad 13 peaks of the C SSNMR spectrum made it difficult to confirm the formation of amide linkages between SPL and MCM-NH2 using this technique. Therefore, SPL-MCM were examined by X-ray photoelectron spectroscopy (XPS) which showed the complete absence of NH2 groups thus confirming successful covalent attachment of the NH2 groups with SPL (Figure S10). Based on TGA, the amount of SPL coated on the surface of MCM-NH2 was ca. 19% (w/w) (Figure S3A). The X-ray small angle diffraction peak of SPL-MCM shown in Figure S11A also confirmed that the crystalline mesoporous structure of MSNs was retained after the coating process.

Synthesis and characterization of SPL and SPLcoated MCM-NH2.

SPL was coated onto the surface of Pred-MCM-NH2 to form SPL-Pred-MCM and partial loss of prednisolone was observed based on UV spectrophotometric measurements. This was probably because prednisolone is soluble in both the reaction buffer (MES buffer – pH 6) and the wash solvent (EtOH). Therefore, it was difficult to determine the amount of SPL coated onto SPL-Pred-MCM. To confirm the amount of prednisolone loaded in the drug delivery system, SPL-Pred-MCM and Pred-MCM-NH2 were stirred in 36 aqueous 1 M NaOH which completely dissolved the nanoparticles and resulted in total release of loaded drug. The quantity of released drug was estimated by UV spectrophotometry at 250 nm with reference to a standard curve of prednisolone in aqueous 1 M NaOH. The results indicated ca. 1 mg of Pred-MCM-NH2 or ca. 5 mg of SPLPred-MCM contained 0.34 mg of prednisolone and this was consistent with the TGA result shown in Figure S3A. X-ray small angle and wide angle (Figure S11A) diffraction peaks revealed that MCM-NH2, SPL-MCM, and SPL-Pred-MCM retained their mesoporous structure (hkl at 211) and no impurities were detected. Small diffraction peaks of prednisolone between 12-20 theta from SPL-Pred-MCM and Pred-MCM-NH2 (Figure S11C), are likely due to the small amount of prednisolone that leached out the nanoparticles and adsorbed onto the nanoparticle surface during the coating and loading processes. The colloidal stability of nanoparticles in suspension is an important factor to achieve reproducibility in drug release. Highly aggregated particles may no longer retain the unique properties of nanoparticles. SPL-MCM demonstrated superior colloidal stability compared to MCM-NH2 based on their highly negative zeta potential (-49.9 mV) and a low PDI value of 0.01 (Table S1).

SPL was synthesized by succinylating EPL with succinic anhydride in the presence of 4-dimethylaminopyridine (DMAP) in DMSO. The reaction was conducted at 60 °C to aid the complete dissolution of EPL and completion of the reaction (Scheme S1). The obtained product was purified by dialysis (Snakeskin™ dialysis tubing with 3,500 Da molecular weight cut-off (MWCO)) and then lyophilized to afford SPL. The successful synthesis of SPL was confirmed 1 13 by H and C NMR spectroscopy (Figures S4, S5 and S6). 1 1 Assignments were confirmed for EPL and SPL using H, HCOSY, multiplicity-edited-HSQC and HMBC experiments (Figures S7, S8, and S9). NMR results indicated that EPL and SPL contained 30 monomeric units and EPL was fully succinylated (Figures S4, S5 and S6). Low intensity peaks occurred in the NMR spectra and some of these were assigned to end groups of the polymer. Further extra peaks were present in the SPL spectra and analysis of 2D spectra indicated that these were likely due to cyclisation of some (≤ 6%) of the succinyl side chains to succinimide side chains. SPL was coated onto MCM-NH2 to form SPL-MCM by activating the carboxyl groups of SPL with N-(3dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6) followed by reaction with MCM-NH2 (Scheme S2B). Increasing the proportion of SPL in the reaction from a 1:1 to a 1:2 w/w ratio (silica/SPL) remarkably improved the coating thickness and homogeneity based on TEM images (6.3 nm to 25 nm) (Figures 2B and C, respectively), consequently SPL-MCM tested in subsequent experiments was made using 1:2 w/w ratio (silica/SPL). SPLMCM was negatively charged at pH ˃ 5, which corresponds to the presence of ionized carboxylate groups of SPL and correlates with the zeta potential profile of free SPL (Figure 2D). Negatively charged particles may aid bio-adhesion to the inflamed colonic luminal surface which is rich in 29 positively charged proteins. Bio-adhesion is a useful

Synthesis and characterization of SPL-coated PredMCM-NH2.

Release of drug from SPL-Pred-MCM in vitro. The study of drug release from SPL-Pred-MCM was performed in vitro using a “universal” buffer which was initially prepared as a solution at pH 1.9 using a mixture of acetic acid and phosphoric acid. Titration with small

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volumes of 8 M NaOH generated buffers at pH 5.0 and 7.4. This meant that the release experiment could be conducted in one buffer suspension, thus maintaining the concentration of the nanoparticles. To simulate GI transit following oral administration, the nanoparticles were tested at pH 1.9 for 2 h (stomach), pH 5.0 for 1 h (small intestine), and pH 7.4 for 41 h (colon), at 37 °C with stirring at 100 rpm 37 under sink condition (Figure 3). The amount of prednisolone released was quantified by measuring absorbance at 250 nm with reference to a standard curve. Suspending Pred-MCM-NH2 in pH 1.9 solution led to the burst release of prednisolone (ca. 80% release) during the first 10 min and the majority of drug (ca. 95%) was released within 2 h (Figure 3). In contrast, SPL-Pred-MCM released less than 14% of prednisolone within 2 h at pH 1.9, indicating that the SPL polymeric coating was able to minimize premature drug release in the pH condition of the stomach. At pH 1.9, SPL is unionized and covers the drug-loaded-nanoparticles, which inhibits drug release. The small amount of prednisolone that was released from the coated nanoparticles may be due to prednisolone that was adsorbed on the surface of the polymer during the coating process. In addition to ensuring high drug concentration at the target site, minimizing drug release in the stomach may 38 also reduce the upper GI side effects of prednisolone.

Figure 3. Drug release profiles of Pred-MCM-NH2 and SPL-Pred-MCM in the simulated variable pH conditions of transit through the GI tract. The nanoparticles were stirred in pH 1.9 solution for 2 h, pH 5.0 for 1 h, and pH 7.4 for 41 h at 37 °C under sink conditions. Only the first 8 hours of the drug release experiment are shown here; refer to Figure S12 for the complete profile. Values are expressed as the mean ± SD of n = 3 independent experiments. In the simulated pH condition of the small intestine (pH 5.0), the release of prednisolone from SPL-Pred-MCM was also minimal at just 6% w/w. In the pH range 4 to 6, the zeta potential of SPL-MCM transitioned from positive to negative (Figure 2D), hence SPL is apparently only partially ionized, leading to low drug diffusion. At the simulated pH condition of the colon (pH 7.4), prednisolone was released steadily from SPL-Pred-MCM as SPL is seemingly fully ionized with anionic carboxylate groups in this pH environment, as indicated by the strongly negative zeta potential of SPL-MCM at pH 7.4 (Figure 2D). The resulting increase in solubility and repulsive forces between the anionic polymeric chains may cause swelling, which promotes drug diffusion. Furthermore, the release profile of

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prednisolone from SPL-Pred-MCM was more reproducible compared to the uncoated nanoparticles (Pred-MCM-NH2) (Figures 3 and S12). This may be due to the higher colloidal stability of SPL-Pred-MCM at pH 7.4 leading to a more uniform size distribution of nanoparticles in suspension. Complete (ca. 94%) drug release of SPL-Pred-MCM was observed after ca. 27 h. Overall, the in vitro drug release study demonstrated that coating the prednisolone-loadedMCM-NH2 with SPL provided pH-responsive controlled drug release at simulated colonic pH.

Inherent toxicity of SPL-MCM and SPL-Pred-MCM. The inherent toxicity of the drug delivery carrier, SPLMCM was evaluated using the MTT assay for RAW 264.7 macrophages and intestinal epithelial cancer cells (LS 174T and Caco-2) at concentrations of up to 1000 µg/mL. Comparing the toxicity of MCM-NH2 and SPL-MCM as a weight/volume measurement cannot evaluate the influence of the SPL coating on biocompatibility due to the different concentrations of silica. Therefore, the concentration of MCM-NH2 and SPL-MCM was adjusted to give an equivalent concentration of silica. After exposing the cells to the nanoparticles for 48 h, the plates were incubated with the MTT reagent for 4 h and the formazan crystals were dissolved in DMSO to measure the absorbance at 595 nm. It was found that SPL-MCM were less toxic than their uncoated counterparts to RAW 264.7 macrophages and Caco-2 cells at the highest tested concentration (Figure S13). The toxicity of the SPL-Pred-MCM nanoparticles towards RAW 264.7 macrophages was also evaluated using the MTT assay. The concentrations of MCM-NH2, SPL-MCM, and SPL-Pred-MCM nanoparticles were adjusted and compared based on the concentration of silica. The results revealed that MCM-NH2, SPL-MCM, and SPL-Pred-MCM resulted in close to 100% cell viability in RAW 264.7 macrophages at concentrations of up to 100 µg/mL silica following 48 h incubation (Figure S14). Moreover, SPL-Pred-MCM led to similar cell viability compared to SPL-MCM at 500 µg/mL silica. This indicates that prednisolone does not significantly reduce cell viability when it is loaded inside nanoparticles at this concentration. Additionally, the similar toxicity profile of MCM-NH2 and SPL-MCM indicates that the SPL polymer does not significantly affect cell viability of RAW 264.7 macrophages at the tested concentrations.

NF-κB inhibition by SPL-Pred-MCM. NF-κB is a pro-inflammatory transcription factor, its activation is a marker of inflammation, and activation is suppressed by anti-inflammatory steroids like 39 prednisolone. Therefore, to confirm if the drug released from SPL-Pred-MCM can retain its anti-inflammatory effects, SPL-Pred-MCM was evaluated for its ability to inhibit NF-κB activation compared to free prednisolone using an NF-κB reporter RAW 264.7 macrophage cell line. SPL-Pred-MCM, or an equivalent concentration of free prednisolone, were incubated with the cells for 1 h. The cells were then activated by the addition of 2.5 ng/mL LPS for 4 h and NF-κB-dependent green fluorescent protein (GFP) expression was measured by fluorimetry at λex 485

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nm and λem 520 nm. The results showed that prednisolone released from SPL-Pred-MCM had an equivalent ability to inhibit GFP expression compared to the free drug (Figures 4A and 4B). This suggests that prednisolone was released from SPL-Pred-MCM during the course of the assay and that the processes of drug loading and nanoparticle coating did not adversely affect the pharmacological activity of prednisolone, namely inhibition of LPS-induced NF-κB 25 activation. Importantly, the concentration of SPL-PredMCM used for the NF-κB assay (ca. ≤ 2.5 µg/mL silica) was much lower than the concentrations that induced toxicity in the inherent toxicity study of the SPL-Pred-MCM nanoparticles (Figure S14). Therefore, it is unlikely that the nanoparticles caused cell death in the NF-κB assay and hence the results represent inhibition of NF-κB activation.

fluorimetry at λex 485 nm and λem 520 nm. (A) GFP fluorescence intensity as a function of prednisolone concentration. Fluorescence was not detected without LPS activation. Values are expressed as the mean + SD of n = 3 independent experiments. Significance was determined by two-way ANOVA followed by Sidak’s multiple comparison test, ** p < 0.01 and *** p < 0.001. (B) Images of NF-кB reporter RAW 264.7 macrophages: (a and b) Cells under normal light source; (c-f) Cells under λex 485 nm and λem 520 nm; (c) LPS activated cells without drug treatment; (d) Cells without drug treatment and without LPS activation; (e) LPS activated cells treated with 100 ng/mL free prednisolone; and (f) LPS activated cells treated with SPL-Pred-MCM at a concentration equivalent to 100 ng/mL free prednisolone. The scale bar represents 100 µm.

Intracellular trafficking of SPL-MCM.

Figure 4. NF-κB activation assay comparing the antiinflammatory activity of prednisolone released from SPL-Pred-MCM to that of free prednisolone. NF-κB reporter RAW 264.7 macrophages were treated with SPL-Pred-MCM or free prednisolone for 1 h then activated with LPS (2.5 ng/mL) for 4 h and NF-κBdependent GFP expression was monitored by

Many drugs exert their pharmacological activity by acting on intracellular components, hence intracellular drug delivery systems are a useful therapeutic strategy especially 40 for drugs with poor membrane permeability. Furthermore, drug targeting via intracellular delivery to specific cells may help to overcome the side effects of some drugs. To achieve intracellular drug delivery, the drug delivery system must cross the cell membrane which is 40 composed of a lipid bilayer. The passage of materials to and from cells is tightly regulated by the cell membrane and occurs by passive diffusion processes or energydependent active transport mechanisms such as 40 Many nanoparticles enter cells by endocytosis. endocytosis, namely clathrin-mediated or caveolaemediated endocytosis whereby the nanoparticles that pass through the cell membrane are enclosed in a vesicle which 41 is released into the cytoplasm. Recently, studies have utilized MSNs as drug delivery systems for intracellular 4,42,43 drug release. The immune response has a critical involvement in the pathophysiology of IBD. Intestinal macrophages have been recognized to play an instrumental role mediating the pathological inflammation in IBD and are highly abundant at inflamed sites in the colon (Figure 20-25 Therefore, delivering anti-inflammatory medicines 1C). directly to immune cells such as intestinal macrophages 20,24,26 may improve therapeutic outcomes. Interestingly, negatively charged materials were found to have preferred 44 uptake by macrophages. Other pathologies of the intestine include colorectal cancer which is the third most 45 common cancer worldwide and a major cause of death. Since the simplified goal of cancer chemotherapy is to selectively induce the death of cancer cells while leaving healthy tissues unharmed, cytotoxic drugs have been loaded inside MSNs to induce apoptosis of cancer cells 4,43,46 following intracellular uptake. In this study, the uptake of SPL-MCM by macrophages and intestinal epithelial cancer cells was investigated. To visualize cellular transport, sulforhodamine B was loaded inside SPL-MCM to create SPL-Sul-MCM. Sulforhodamine B (Figure S15) is a fluorescent hydrophilic dye (λex 565 nm, λem 586 nm) that

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cannot penetrate cell membranes. Therefore the presence of cell membrane-impermeable sulforhodamine B inside cells indicates uptake of the dye-loaded nanoparticles. The uptake of SPL-MCM loaded with cargo was evaluated using confocal microscopy, flow cytometry, and TEM techniques as discussed below.

Loading of sulforhodamine B into MCM-NH2 and coating with SPL. Sulforhodamine B was loaded into the pores of MCM-NH2 to form Sul-MCM-NH2 and then coated with SPL to create SPL-Sul-MCM. The amount of sulforhodamine B loaded into Sul-MCM-NH2 was ca. 26% (w/w) based on TGA (Figure S16A). The DSC curve of free sulforhodamine B had a sharp peak at the sulforhodamine B melting point of 374 °C, due to the crystalline form of sulforhodamine B (Figure S16B). Sulforhodamine B transitioned to an amorphous state in Sul-MCM-NH2 and SPL-Sul-MCM, confirming that sulforhodamine B was loaded inside the mesopores. SPL was coated onto the surface of Sul-MCM-NH2 using a similar method to that used to coat SPL onto Pred-MCMNH2. However, after completing the coating reaction, SPLSul-MCM was washed more times with EtOH compared to SPL-Pred-NH2. The number of washing steps was increased because hydrophilic sulforhodamine B was expected to leach from the nanoparticles into the aqueous pH 6 MES buffer, during the coating reaction, much more than hydrophobic prednisolone. Analysis of SPL-Sul-MCM by XPS showed the complete absence of NH2 groups thus confirming successful covalent attachment of MCM-NH2 with SPL (Figure S17). Due to the leaching of sulforhodamine B from the nanoparticles, it was difficult to quantify the SPL content of SPL-Sul-MCM but the value seemed to be ca. 8% w/w based on the TGA result of SPL-MCM (21% w/w) and MCM-NH2 (12% w/w) (Figure S16A). Moreover, TEM images of the nanoparticles before and after coating with SPL (Figure S18) clearly show the formation of a homogenous coating layer on SPL-Sul-MCM of similar thickness to SPL-MCM (28 nm cf. 25 nm) (Figure 2C). The release of sulforhodamine B from SPL-Sul-MCM demonstrated that sulforhodamine B was preferentially released at the pH of the colon (Figure S19). However, a greater proportion of sulforhodamine B was released in the early stages of the experiment compared with prednisolone release (Figure S19 cf. Figure 3). This may be attributed, at least in part, to the hydrophilic sulforhodamine B leaching out of the nanoparticle and adsorbing onto the outer surface during the SPL-coating procedure. Due to this, the pH-responsive controlled-release properties of SPL in SPLSul-MCM are less apparent compared to SPL in SPL-PredMCM within the first 8 h. However, sulforhodamine B from uncoated nanoparticles (Sul-MCM-NH2) was fully released within the first 2 h whereas sulforhodamine B was completely released from coated nanoparticles SPL-SulMCM only after 28 h (Figure S19). Therefore, the SPL coating layer of SPL-Sul-MCM still provided controlledrelease of sulforhodamine B albeit less controlled than prednisolone release from SPL-Pred-MCM. The uptake studies discussed below were conducted for ≤ 2 h, so burst release of sulforhodamine B from SPL-Sul-MCM is unlikely to be of concern, since at least ca. 50% of sulforhodamine B

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would remain in the nanoparticles over the duration of the uptake experiments.

Live cell imaging using laser scanning confocal microscopy. Laser scanning confocal microscopy (LSCM) was used to visualize the uptake of sulforhodamine B by intestinal epithelial cancer cells (LS 174T and Caco-2) and RAW 264.7 macrophages after 2 h of incubation with SPL-Sul-MCM (Figure 5). The cell membrane of RAW 246.7 macrophages, Caco-2 cells, and LS 174T cells was stained with DiD which fluoresces at far-red wavelength and the cell nuclei were stained with Hoechst 33334 which exhibits blue fluorescence. Sulforhodamine B fluoresces red but it is represented in Figure 5 in green to avoid overlap with DiD. As discussed above and illustrated in Figure S20, sulforhodamine B cannot pass through the cell membrane unassisted. Furthermore, since the concentration of nanoparticles (90 µg/mL of silica) was shown previously to be well tolerated (Figures S13 and S14), cell membrane damage is unlikely to occur under these conditions. Hence detection of intracellular sulforhodamine B signifies that it was delivered intracellularly via uptake of SPL-Sul-MCM. To exclude the possibility that sulforhodamine B or SPLSul-MCM was simply adsorbed onto the surface of the cells rather than being located intracellularly, images of the samples were taken at multiple focal depths (z-sections) and sections that passed through the cells demonstrated that treatment with SPL-Sul-MCM resulted in sulforhodamine B internalized within RAW 264.7 macrophages, Caco-2 cells and LS 174T cells with no evidence of cellular death or damage. Confocal microscopy of RAW 264.7 macrophages incubated with uncoated nanoparticles (Sul-MCM-NH2) (Figure S21) showed sulforhodamine B signal on the cell membrane surface but not inside the cells. This suggests that Sul-MCM-NH2 was adsorbed onto the cell membrane with no evidence of cellular uptake. Therefore, the SPL coating may play a key role in enabling uptake of sulforhodamine B into RAW 264.7 macrophages.

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ACS Applied Materials & Interfaces Quantification cytometry.

of

cellular

uptake

using

flow

To provide quantitative confirmation of the results of the confocal microscopy studies, flow cytometry was used to calculate uptake of sulforhodamine B by all three cell lines. The sulforhodamine B dye alone and physically mixed with MCM-NH2 was minimally internalized by all three cell lines (Figure 6), in agreement with the confocal microscopy results (Figure S20) and confirming that it is a cell 47 membrane-impermeable dye. When sulforhodamine B was loaded into MCM-NH2 nanoparticles without SPL coating (Sul-MCM-NH2), uptake was low for all three cell lines (Figure 6). Importantly, when Sul-MCM-NH2 nanoparticles were coated with SPL (SPL-Sul-MCM), uptake of sulforhodamine B by RAW 264.7 macrophages and LS 174T cells was significantly increased (Figure 6). These results correlate with the LSCM studies (Figure 5) and support the notion that the SPL coating may promote nanoparticle uptake by the two cell lines. However, it needs to be considered that some dye will leach from the uncoated nanoparticles during the uptake experiment, and this may confound the quantitative direct comparison of Sul-MCM-NH2 with SPL-Sul-MCM. Overall, two-way ANOVA analysis determined that the nature of the nanoparticles and the cell type both affected the efficiency of the dye internalization (p < 0.0001). To determine the involvement of active transport in cellular uptake of the nanoparticles, the experiment described above was conducted at 4 °C instead of 37 °C (Figure S22). The low temperature inhibits active transport mechanisms while maintaining the ability of cells to transport nanoparticles via passive diffusion. The uptake of sulforhodamine B administered via Sul-MCM-NH2 and SPL-Sul-MCM was substantially impaired at 4 °C for all three cell lines especially for RAW 264.7 macrophages and LS 174T cells. For example, the fluorescence intensity of RAW 264.7 macrophages incubated for 2 h with SPL-Sul-MCM at 37 °C was over 15,000 arbitrary units (Figure 6), however it reduced to ca. 200 when incubation was performed at 4 °C (Figure S22). This indicates that SPL-Sul-MCM is taken up predominantly by active transport by the cell lines tested in this study.

Figure 5. LSCM images of the uptake of SPL-Sul-MCM (90 µg/mL of silica) following 2 h incubation with (A) RAW 264.7 macrophages, (B) Caco-2 cells, and (C) LS 174T cells. The cell membranes were stained with DiD and are shown in red. The cell nuclei were stained with Hoechst 33334 and are shown in blue. Sulforhodamine B fluoresces red but it is represented here in green to avoid overlap with DiD. Sections through the cells showed that the sulforhodamine B signal was located intracellularly rather than being adsorbed onto the cell surface.

Figure 6. Quantification of the uptake of sulforhodamine B by RAW 264.7 macrophages, Caco-2

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cells, and LS 174T cells. The samples (MCM-NH2, sulforhodamine B, sulforhodamine B physically mixed with MCM-NH2, Sul-MCM-NH2, and SPL-Sul-MCM; equivalent to 90 µg/mL of silica and/or 6 µg/mL of sulforhodamine B) were incubated with the cells for 2 h at 37 °C then analyzed by flow cytometry. Values are the mean + SD of n = 3 independent experiments. Significance was determined using two-way ANOVA followed by Tukey's post hoc test, **p < 0.01 and **** p < 0.0001.

Time-dependent cytometry.

cellular

uptake

using

flow

The time-course of sulforhodamine B uptake by the cells was investigated by incubating SPL-Sul-MCM with the three cell lines for 30 min, 1 h, or 2 h, and the extent of uptake was measured using flow cytometry (Figure 7). All three cell lines displayed an increase in uptake of

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sulforhodamine B over time. This was most pronounced for RAW 264.7 macrophages which showed a substantial increase in fluorescence intensity at each time point, whereas for Caco-2 cells, there was only a subtle increase in fluorescence intensity between 30 min and 2 h. The fluorescence intensity of LS 174T cells did not change much between 30 min and 1 h, however a substantial increase occurred by 2 h. The FSC-A signals of the three different cell lines alone (Figure 7, green signals) were similar to cells incubated with SPL-Sul-MCM (Figure 7, yellow, purple and blue signals). This provides additional evidence that the detected sulforhodamine B was associated with the cells. If sulforhodamine B was not associated with the cells, a low FSC-A signal similar to that observed for the SPL-Sul-MCM delivery system alone (Figure 7, red signals) would be observed due to the smaller size of the nanoparticles compared to the cell bodies.

Figure 7. Flow cytometry analysis providing quantitative data on the uptake of SPL-Sul-MCM by RAW 264.7 macrophages, LS 174T cells, and Caco-2 cells. (A) Uptake of SPL-Sul-MCM with increasing incubation time (30 min, 1 h, and 2 h) was measured based on the fluorescence intensity of sulforhodamine B. (B) FSC analysis was used to confirm

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that sulforhodamine B from SPL-Sul-MCM was associated with the cells by comparing the signal of the cell lines and SPL-Sul-MCM alone. Three independent experiments were conducted to obtain all flow cytometry data. Black spherical dots, identified as nanoparticles, were observed within vesicular intracellular components. As shown in Figure 8, these nanoparticles were engulfed by Determining intracellular location using TEM. the plasma membrane which suggests uptake by We next studied the intracellular location of drug-loaded endocytosis. The cell membrane then pinched off to form a nanoparticles after internalization. RAW 264.7 macrophage vesicle enveloping the nanoparticles. The composition of cells were selected for further investigations to evaluate the the black spherical dots was further analyzed, as described uptake of the SPL-Pred-MCM drug delivery system. Thin below, using scanning transmission electron microscopy sections of RAW 264.7 macrophages exposed to SPL-Pred(STEM) with high angle annular dark field (HAADF) to MCM were examined using TEM to determine the confirm that it was SPL-Pred-MCM inside the cells and not ultrastructural location of SPL-Pred-MCM in the cell an artefact. Energy-dispersive X-ray spectroscopy (EDS) (Figure 8). The cell nuclei, Golgi apparatus, and the was used to detect all elemental peaks. endoplasmic reticulum of the cell were clearly identified.

Figure 8. TEM images of RAW 264.7 macrophages showing uptake of SPL-Pred-MCM nanoparticles into vesicles; red arrows indicate the location of silica nanoparticles.

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Figure 9. HAADF STEM mapping of SPL-Pred-MCM nanoparticles inside a RAW 264.7 macrophage. (A) The location of nanoparticles, detected by HAADF STEM, appeared as bright white dots as shown in the yellow box. (B) Magnified position of crystalline silica nanoparticles detected in (A). Elemental mapping of: (C) O, shown in blue; (D) C, shown in green; and (E) Si, shown in red. (F) Overlapping images of O, C, and Si confirmed the identity of the silica drug delivery system in the cell. (G) EDS spectrum showing each elemental peak of intracellular SPL-Pred-MCM.

Elemental mapping using STEM/EDS. The thin sections showing cellular uptake of SPL-PredMCM by RAW 264.7 macrophages were examined using STEM/EDS to identify the nature of the nanoparticle-like shapes observed in the dark field TEM studies (Figure 9). To do this, HAADF STEM was used to image thin sections with atomic number thickness contrast. Following the detection of the crystalline nanoparticles located intracellularly which appeared as white dots (Figures 9A and 9B), EDS was used to determine the elemental composition of individual nanoparticles. The white nanoparticles were determined to be composed of oxygen, carbon, nitrogen and silicon using elemental mapping (Figure 9G). Therefore, the white silica nanoparticles detected using HAADF STEM mapping (Figure 9) and the black silica nanoparticles seen inside the vesicles in TEM (Figure 8) are indicative of SPL-Pred-MCM. Overall, the results demonstrate that MSNs coated with SPL such as

SPL-Pred-MCM can potentially intracellular drug delivery.

be

used

to

provide

CONCLUSIONS This study demonstrated that coating cargo-loaded-MSNs with the polymer SPL achieved both pHresponsive drug release at simulated colonic pH, and superior intracellular drug delivery to RAW 264.7 macrophages and LS 174T cells compared to uncoated MSNs. The mechanism of cellular uptake appeared to be predominantly active transport since poor cellular uptake was observed at 4°C. These results provide opportunities to utilize SPL-coated nanoparticles to deliver drugs, including those with inherently poor cell membrane permeability, intracellularly to macrophages and colorectal cancer cells for the improved treatment of IBD and colorectal cancer. Further in vivo studies should be conducted to demonstrate the value of SPL and this novel drug delivery system for

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cellular uptake and intracellular delivery of cargo because it has the potential to increase efficacy and reduce toxicity. EPL is cheap and easy to modify which facilitates the development of a cost-efficient drug delivery system. Moreover, SPL may become a robust excipient for drug delivery and drug targeting more broadly.

MATERIALS AND METHODS Materials and general methods All chemicals and solvents were of analytical grade and were used without further purification. Cetyltrimethylammonium bromide (CTAB), Pluronic® F127, tetraethylorthosilicate (TEOS), (3-aminopropyl)triethoxysilane (APTES), EDC, MES powder, succinic anhydride, DMSO, DMAP, and prednisolone were purchased from Sigma Aldrich. Phosphoric acid (H3PO4), HCl 32% (w/w), and NaOH were purchased from Chem-Supply. Glacial acetic acid (CH3COOH) and NH4OH 25% (w/w) was purchased from Merck. EPL was purchased from BOC Sciences, Creative Dynamics Inc. Sulforhodamine B was purchased from TCI. Hoechst 33334 and 1,1'-dioctadecyl-3,3,3',3'tetramethylindodicarbocyanine perchlorate (DiD) stains were purchased from AAT Bioquest®. Mouse monocyte macrophage cells transfected with a NF-κB GFP-reporter was a gift from Associate Professor Matthew Sweet, Institute for Molecular Bioscience, UQ. Dulbecco’s modified eagle medium (DMEM) and streptomycin sulfate/penicillin were purchased from Invitrogen, fetal bovine serum (FBS) from JRH Biosciences, and L-glutamine from ThermoTrace. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) reagent and lipopolysaccharide (LPS) were purchased from Sigma Aldrich. The 96-well plates were purchased from PerkinElmer Inc. All experiments were conducted at room temperature (ca. 22 °C) unless stated otherwise.

Preparation of SPL ΕPL was fully succinylated to SPL by reaction with succinic anhydride, using DMAP as the base and DMSO as the solvent (Scheme S1). In a typical reaction, 0.500 g EPL, 0.803 g succinic anhydride and 0.990 g DMAP was dissolved in 10 mL of DMSO. This solution was stirred at 500 rpm at 60 °C for 19 h. The dark brown solution was purified using Snakeskin™ dialysis tubing with 3,500 Da MWCO in an aqueous solution of HCl (pH 3) and type 2 pure water. The dialysate was analyzed with UV spectrophotometry and when no absorbance was detected the purification process was deemed to be completed. The obtained dialysis solution was freeze-dried for 48 h to afford SPL, a white lyophilized powder.

Preparation of MSNs (MCM-48) MSNs (MCM-48) were prepared by using the procedure 30,31 adapted from Kim et al. In a typical reaction, 0.500 g of CTAB was added to 107 mL NH4OH (NH3 2.8% w/w) and 43 mL EtOH. This mixture was stirred at 1000 rpm until CTAB was solubilized. Then 2.05 g of Pluronic® F127 was added and stirred until solubilized. TEOS (1930 µL) was added into the

solution and stirred at 850 - 875 rpm for 60 s. The reaction mixture was kept for 24 h under quiescent conditions. The obtained white suspension was centrifuged at 22,000 rcf for 10 min to collect the nanoparticles, which were washed twice with type 2 pure water and twice with EtOH. The white solid nanoparticles were then dried in an oven at 80 °C overnight. These MCM-48 nanoparticles containing surfactant (template) were used in the preparation of MCM-NH2 (see below), however for the purpose of characterization, surfactant was removed by solvent extraction. Unextracted MCM-48 (150 mg) were dispersed in a solution of 20 mL MeOH and 1 mL aqueous HCl 32% (w/w), stirred at 1000 rpm and 60 °C for 24 h, and collected by centrifugation (22,000 rcf for 10 min). This extraction process was repeated with fresh MeOH/HCl solution, followed by washing four times with type 2 pure water and four times with EtOH. The white solid nanoparticles were finally dried in an oven at 80 °C overnight.

Preparation of MCM-NH2 Unextracted MCM-48 (2.0 g) was dispersed in 125 mL MeOH by stirring the suspension at 750 rpm. APTES (7.5 mL) was added to the stirred suspension and was left to react for 24 h. The white solid nanoparticles were collected by centrifuging at 22,000 rcf for 10 min, washed twice with MeOH and dried in an oven at 60 °C overnight. The reaction is described in Scheme S2A. Surfactant was removed using the solvent extraction procedure described above for MCM-48 nanoparticles. 3-Aminopropyl-functionalised mesoporous silica nanoparticles were denoted MCM-NH2.

Prednisolone loading into MCM-NH2 MCM-NH2 (100 mg) was mixed with prednisolone solution (25 mL solution at a concentration of 20 mg/mL prednisolone in EtOH) and the suspension was dispersed using a bath sonicator for 10 min then stirred at 100 rpm for 24 h. The suspension was centrifuged at 22,000 rcf for 5 min to collect the nanoparticles then dried in an oven at 60 °C overnight. The dried nanoparticles (100 mg) were then redispersed in 5 mL prednisolone solution at a concentration of 30 mg/mL in EtOH and stirred for 2 h at 100 rpm. The solvent was removed in vacuo using a rotary evaporator and the dried powder was washed twice with 50 mL EtOH to remove excess prednisolone adsorbed on the surface of drugloaded nanoparticles. These prednisolone-loadednanoparticles were denoted Pred-MCM-NH2.

Coating SPL onto Pred-MCM-NH2 SPL (40 mg) was dissolved in 4 mL of 0.1 M MES buffer (pH 6), and stirred at 500 rpm. EDC (4.8 mg) was added and the solution was stirred for 5 min to activate the carboxylic acid groups of SPL. Pred-MCM-NH2 (equivalent to 20 mg of MCM-NH2) was dispersed in 2 mL of MES buffer then added to the reaction mixture and stirred at 200 rpm for 40 min. The mixture was centrifuged at 22,000 rcf for 10 min to collect the nanoparticles which were then washed once with EtOH to remove any impurities and prednisolone that may have leached out of the nanoparticles during the coating

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process. The nanoparticles were finally freeze-dried for 48 h. This sample was denoted SPL-Pred-MCM. In a separate experiment, MCM-NH2 without loaded drug was grafted with SPL to obtain SPL-MCM using the procedures described previously. SPL-MCM was used to determine the amount of polymer coated onto nanoparticles.

Prednisolone release experiment The pH-dependent release of prednisolone from Pred-MCMNH2 with and without SPL coating was investigated using a “universal” buffer. Aqueous solutions of 0.05 M H3PO4 and 0.05 M CH3COOH were mixed in a 1:1 (v/v) ratio to form a solution with a pH of 1.9. This solution was titrated at defined time points during the release experiment using 8 M NaOH to produce buffers with a pH of 5.0 and 7.4. Pred-MCM-NH2 and SPL-Pred-MCM, both containing the same amount of prednisolone (0.34 mg), were added separately to a large volume (50 mL) of pH 1.9 solution (50 mL of pH 1.9 solution contained 25 mL of 0.05 M H3PO4 and 25 mL of 0.05 M CH3COOH) and stirred at 100 rpm at 37 °C under sink conditions. After 2 h, the pH was titrated to pH 5.0, and after one more hour (3 h since the start of experiment), to pH 7.4 by adding 250 µL and 150 µL of aqueous 8 M NaOH, respectively. Samples (1.5 mL) were withdrawn at predetermined time intervals and immediately replaced with an equal volume of fresh buffer solution of equivalent pH to maintain constant volume. The withdrawn samples were centrifuged (12,000 rcf, 4 min) and the concentration of prednisolone was determined by UV-visible spectrophotometry using the prednisolone λmax value of 250 nm. Linear calibration curves of absorbance at 250 nm versus prednisolone concentrations were obtained for each pH 2 solution with R values > 0.99. The release experiment was conducted in total 44 h.

Cell viability assay MCM-NH2 and SPL-MCM were tested for their potential cytotoxic effect on RAW 264.7 macrophages, and intestinal epithelial cancer cells (Caco-2 and LS 174T cells) using the MTT assay. RAW 264.7 macrophages, and Caco-2 cells and LS 174T cells were cultured in high glucose DMEM medium, containing 100 µg/mL streptomycin sulfate and 100 units/mL penicillin, 10% (v/v) FBS, and 4 mM L-glutamine. The cells were grown at 37 °C in a humidified incubator with 5% CO2. 4 RAW 264.7 macrophages (2 × 10 cells/well), Caco-2 cells (1 × 4 4 10 cells/well), and LS 174T cells (2 × 10 cells/well) were seeded into 96-well plates and grown for 24 h. The weighed mass of MCM-NH2 and SPL-MCM were adjusted to give equivalent concentrations of 25, 50, 100, 250, 500, and 1000 µg/mL of silica in media in which cells were incubated for 48 h. Cells with medium only were used as controls for each plate. After incubation, 25 µL/well of MTT reagent was added for a further 4 h at 37 °C. The cell culture medium was then aspirated, and the formazan crystals were dissolved by adding 100 µL/well DMSO. The absorbance signal of formazan was measured at 595 nm using a microplate reader. Equivalent volumes of nanoparticles dispersed in DMEM 10% FBS at different concentrations were also placed in wells

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without cells to correct for any interference of absorbance that may occur due to the nanoparticles such as light scattering. The absorbance due to any interference of nanoparticles was subtracted from the sample absorbance value. Three independent experiments (n = 3) were conducted with triplicate wells per treatment for each cell line and concentration. In a separate experiment, the MTT assay of SPL-Pred-MCM, MCM-NH2, and SPL-MCM nanoparticles at concentrations of 25, 50, 100, and 500 µg/mL of silica in media towards RAW 264.7 macrophages were also evaluated using the same method.

NF-κB inhibition assay Macrophages (RAW 264.7 stably expressing GFP under NF48 4 κB control) were seeded at 2 × 10 cells/well in 96-well plates and grown for 24 h in a 37 °C incubator with 5% CO2. The weighed mass of free prednisolone and SPL-Pred-MCM were adjusted to prepare concentrations of 5, 10, 25, 50, 100, and 250 ng/mL of prednisolone in medium. Cells were incubated with SPL-Pred-MCM for 1 h. Cells with medium only were used as controls. The cells were then activated by adding 2.5 ng/mL LPS per well for 4 h. The cells with and without LPS activation were used as positive and negative control, respectively, for each plate. Cell stimulation by LPS led to NF-κB-dependent GFP expression. A reduced fluorescence intensity of GFP was indicative of a suppressed NF-κB activation due to the action of prednisolone. GFP expression was measured using a fluorescence plate reader (FLUOstar Omega) at excitation and emission wavelengths of 485 nm and 520 nm, respectively. Three independent experiments (n = 3) were conducted with triplicate wells for each treatment. Nanoparticles dispersed in media at different concentrations without cells were prepared in 96-well plates to correct for background fluorescence of the 49 nanoparticles. The fluorescence of experimental wells (treated cells) was subtracted with the background fluorescence of wells containing only nanoparticles and media.

Sulforhodamine B loading into MCM-NH2 MCM-NH2 (400 mg) was mixed with 25 mL of sulforhodamine B solution prepared in EtOH at a concentration of 32 mg/mL. The suspension was using a bath sonicator for 15 min and then stirred at 200 rpm for 24 h. The nanoparticles were collected by centrifuging the suspension at 22,000 rcf for 10 min and were dried at 60 °C in an oven overnight. The dried nanoparticles then were re-dispersed in 15 mL of sulforhodamine B solution (32 mg/mL in EtOH) and stirred at 100 rpm for 2 h. The solvent was evaporated in vacuo using a rotary evaporator to obtain a dry powder. To remove excess sulforhodamine B adsorbed on the surface of dye-loaded nanoparticles, the dried nanoparticles were washed twice by redispersing in 50 mL type 1 ultrapure water and centrifuging at 22,000 rcf for 10 min. MCM-NH2 loaded with sulforhodamine B was denoted Sul-MCM-NH2.

Coating SPL onto Sul-MCM-NH2

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SPL (40 mg) was dissolved in 4 mL of 0.1 M MES buffer (pH 6) and stirred at 500 rpm. Then EDC (4.8 mg) was added and the solution was stirred for 5 min to activate the carboxylic acid groups of SPL. Sul-MCM-NH2 (equivalent to 20 mg of MCM-NH2), was dispersed in 2 mL of MES buffer and added to the reaction and stirred at 200 rpm for 40 min. The suspension was centrifuged at 22,000 rcf for 10 min to collect the nanoparticles which were washed multiple times with EtOH to remove any impurities and sulforhodamine B that may have leached out of the nanoparticles and adsorbed on the surface of the SPL-coated nanoparticles during the coating procedure. The obtained nanoparticles were freezedried for 48 h. This sample was denoted SPL-Sul-MCM. Before grafting SPL onto Sul-MCM-NH2, the grafting experiment was initially conducted using MCM-NH2 and the obtained coated nanoparticles were named SPL-MCM. This sample was used to determine the amount of SPL coated onto the nanoparticles using TGA.

Sulforhodamine B release experiment Sul-MCM-NH2 and SPL-Sul-MCM, both containing the same mass of sulforhodamine B (0.33 mg), were added separately to a large volume (100 mL) of pH 1.9 (100 mL of pH 1.9 solution prepared by mixing 50 mL of 0.05 M H3PO4 and 50 mL of 0.05 M CH3COOH) and stirred at 100 rpm at 37 °C under sink condition. After 2 h, the pH was titrated to pH 5.0 by adding 500 µL of aqueous 8 M NaOH, and after one more hour (3 h since the start of experiment), to pH 7.4 by adding 300 µL of aqueous 8 M NaOH. Samples (1.5 mL) were withdrawn at the predetermined time intervals and immediately replaced with an equivalent volume of fresh buffer solution of equivalent pH to maintain constant volume. The samples were centrifuged (12,000 rcf, 4 min) and the concentration of sulforhodamine B in the supernatant was determined by UV-visible spectrophotometry at a wavelength of 565 nm. Calibration curves of absorbance at 565 nm versus sulforhodamine B concentrations were 2 obtained at each pH value and R values were ≥ 0.99.

Cellular uptake of silica nanoparticles studied using TEM and STEM/EDS 5

RAW 264.7 macrophages were seeded at 2 ×10 cells/well in a small round Corning® dish and grown for 24 h. To determine intracellular location of the SPL-MCM drug delivery system, SPL-Pred-MCM (100 µg/mL) were added into the dish and incubated for 30 min. The sample was washed twice with PBS to remove nanoparticles in the extracellular environment. The sample was fixed with 2.5% (v/v) glutaraldehyde in PBS and then post fixed with 2% (w/v) osmium tetroxide and 1.5% (w/v) potassium ferricyanide for 1 h. The sample was treated with 1% (w/v) thiocarbohydrazide for 20 min, 2% (w/v) osmium tetroxide for 30 min, 1% (w/v) uranyl acetate overnight at 4 °C and lead aspartate for 1 h at 60 °C. Between each of these steps, the samples were washed three times with type 1 ultrapure water. Then, a graded series of EtOH solutions were used to dehydrate the sample for 5 min in TM each step. The sample was then embedded in Durcupan resin and ultrathin sections were cut on a Leica Ultracut 6

ultramicrotome. Sections were viewed using a JEOL 1010 transmission electron microscope. Scanning transmission electron microscopy with energy-dispersive X-ray spectroscopy (STEM/EDS) analysis was conducted on these same sections using a JEOL 2100 TEM instrument.

Quantitative assay of cellular uptake using flow cytometry 5

5

RAW 264.7 macrophage (2 × 10 cells/well), Caco-2 (1 × 10 5 cells/well), and LS 174T (2 × 10 cells/well) cells were seeded into 6-well plates and grown for 24 h. The weighed mass of samples (MCM-NH2, sulforhodamine B, sulforhodamine B physically mixed with MCM-NH2, Sul-MCM-NH2, and SPLSul-MCM) were adjusted to give the same concentration of silica for Sul-MCM-NH2 and SPL-Sul-MCM (90 µg/mL silica), and the same concentration of sulforhodamine B for SPL-SulMCM and free sulforhodamine B (6 µg/mL sulforhodamine B). For the physical mixture of sulforhodamine B and MCMNH2, 90 µg/mL silica and 6 µg/mL sulforhodamine B was used. Cells were incubated in these suspensions for 2 h. Cells with medium only were used as control. After 2 h, all samples were washed once with medium to remove excess nanoparticles, and then trypsinized, harvested and suspended in 1 mL PBS to measure the fluorescence intensity using a LSRFortessa™ flow cytometer. Data was analyzed using the FlowJo FJXv10.0.8 software. All data were gated using a scatter gate (forward scattering - FSC-A and side scattering - SSC-A) to exclude debris, followed by a singlet gate using both SSC-W and FSC-W; SSC-W and FSC-H to exclude doublets. In a separate experiment, SPL-Sul-MCM (100 µg/mL) were added to cells with different duration of incubations (30 min, 1 h and 2 h) to evaluate the extent of cellular uptake with increasing incubation time for each cell line at 37 °C. For cellular uptake studies conducted at 4 °C, cell plates, media and PBS were kept in a cold room for at least 30 min before adding the samples. The experiments were maintained in cold conditions except during the trypsinization procedure. Cells treated with samples were harvested and analyzed using the flow cytometer as described above.

Visualization microscopy

cellular

uptake 5

using

confocal 5

RAW 264.7 macrophage (2 × 10 cells/well), Caco-2 (1 × 10 5 cells/well), and LS 174T (2 × 10 cells/well) cells were seeded into glass bottom dishes (Thermo Scientific™ Nunc Glass Bottom Dishes) and grown for 24 h. SPL-Sul-MCM (90 µg/mL equivalent to 6 µg/mL of sulforhodamine B) prepared in medium was added to the dish and incubated for 2 h. Cells with medium only were used as control. After 2 h, cells were washed three times with serum-free DMEM. A solution (1 mL/well) of DiD at a concentration of 25 µg/mL in serumfree DMEM was added and incubated with the cells for 30 min. Cells were then washed three times with serum-free DMEM to remove excess DiD. Hoechst 33334 in phosphate buffered saline (PBS) (1 mL/well) at a concentration of 5 µg/mL was added and incubated for a further 10 min. Cells were then washed with PBS twice and DMEM with 10% FBS

15



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was added for cell imaging using a ZEISS LSM 510 META confocal scanning microscope with a 63× objective lens immersed in oil. DiD was used to label the cell membrane (λex-em 644-665 nm – far red) and Hoechst 33334 was used to label the cell nuclei (λex-em 361-497 nm – blue). The sulforhodamine B signal was recorded at λex-em 565-586 nm – red. Since the color of DiD and sulforhodamine B overlap in images, the color of sulforhodamine B in the image was pseudocolored green. In a separate experiment, free sulforhodamine B and SulMCM-NH2 were prepared (equivalent to 6 µg/mL sulforhodamine B) as controls using the same procedures as above to investigate the cellular uptake by RAW 264.7 macrophages.

Characterization of nanoparticles The morphology of uncoated and coated nanoparticles was visualized by TEM using a JEOL 1010 instrument operated at 100 kV with samples prepared on GSCu200 mesh copper grids. The hydrodynamic diameter, polydispersity index, and zeta potential of nanoparticles were determined by dynamic light scattering (DLS) using Malvern Nano ZS Zetasizer. To quantify the loading of cargo into nanoparticles, coating of nanoparticles with polymer, and to identify the physical state of cargo, TGA and DSC measurements were performed using a Mettler Toledo TGA star system with a heating rate of 5 °C/min. Compressed air was used as a protective gas with at flow rate 50 mL/min, and samples were placed in alumina 13 crucibles. C SSNMR was conducted using the Bruker Avance III spectrometer operating at 300 MHz, to identify the structure of the polymer on the surface of the nanoparticles. To determine the structure of MSNs, XRD at small and wide angle was used and operated at 40 kV with Cu and Kα radiation sources at 4 °C/min time per step. Nitrogen adsorption-desorption isotherm analysis was used to determine pore size and structure of MSNs and MCMNH2 using a Tristar II 3020 instrument operated at 77 K. XPS data was acquired using a Kratos Axis ULTRA X-ray photoelectron spectrometer incorporating a 165 mm hemispherical electron energy analyzer. The incident radiation was monochromatic Al Kα X-rays (1486.6 eV) at 225W (15 kV, 15 ma). Survey (wide) scans were taken at analyzer pass energy of 160 eV and multiplex (narrow) high resolution scans at 20 eV. Survey scans were carried out over 1200-0 eV binding energy range with 1.0 eV steps and a dwell time of 100 ms. Narrow high-resolution scans were run with 0.05 eV steps and 250 ms dwell time. Base pressure in the -9 analysis chamber was 1.0 × 10 torr and during sample -8 analysis 1.0 × 10 torr. Atomic concentrations were calculated using the CasaXPS version 2.3.14 software and a Shirley baseline with Kratos library Relative Sensitivity Factors (RSFs). Peak fitting of the high-resolution data was also carried out using the CasaXPS software. Live cell imaging was performed using a LSCM ZEISS confocal microscope at 63× oil. HAADF/EDS was performed using a ZEOL 2100. Flow cytometry was performed using a LSRFortessa flow cytometer, all data was gated using a scatter gate (FSC-A &

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SSC-A) to exclude debris, followed by a singlet gate using both SSC-W and FSC-W; SSC-W and FSC-H to exclude cell doublets.

ASSOCIATED CONTENT Supporting Information Available: Chemical structures of prednisolone and sulforhodamine B; hydrodynamic size, polydispersity and zeta potential values of MCM-48, MCMNH2 and SPL-MCM; surface area, pore volume, and pore size of MCM-48 and MCM-NH2 after surfactant removal by calcination or extraction; N2 adsorption-desorption isotherm of MCM-NH2; schemes showing syntheses of: SPL, MCMNH2, and SPL-MCM; TGA and DSC data; NMR spectra of EPL and SPL; XPS and XRD spectra; cargo release profiles of: Pred-MCM-NH2, SPL-Pred-MCM, Sul-MCM-NH2, and SPLSul-MCM; cell viability data; TEM images of: MCM-NH2, SPL-Sul-MCM, and SPL-Pred-MCM; confocal images of RAW 264.7 macrophages incubated with sulforhodamine B or SulMCM-NH2; and quantification of the uptake of sulforhodamine B at 4 °C by RAW 264.7 macrophages, Caco2 cells, and LS 174T cells. This supporting information is available free of charge on the ACS Publication website at “http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Email: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Chau T. H. Nguyen thanks The University of Queensland (UQ), the UQ School of Pharmacy, and the Vietnamese Government for scholarship funding. The authors acknowledge the facilities, and the scientific and technical assistance, of: the Australian Microscopy & Microanalysis Research Facility at the UQ Centre for Microscopy and Microanalysis; the UQ Centre for Advanced Imaging; and the Translational Research Institute. The authors thank Robyn Chapman, Dr. Graeme Auchterlonie, Dr. Sandrine Roy, and Dr. Dalia Khalil for their kind assistance while conducting the research.

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