Adhesion of thiolated silica nanoparticles to urinary bladder mucosa: effects of PEGylation, thiol content and particle size of thiolated silica nanoparticles to urinary bladder

10 Intravesical drug administration is used to deliver cytotoxic agents through a catheter to 11 treat bladder cancer. One major limitation of this approach is poor retention of the drug 12 in the bladder due to periodic urine voiding. Mucoadhesive dosage forms thus offer 13 significant potential to improve drug retention in the bladder. Here, we investigate thiolated silica nanoparticles retention on porcine bladder mucosa in vitro , quantified 15 through Wash Out 50 (WO 50 ) values, defined as the volume of liquid necessary to 16 remove 50% of the adhered particles from a mucosal tissue. Following irrigation with 17 artificial urine solution, the thiolated nanoparticles demonstrate significantly greater 18 retention (WO 50 up to 36 mL) compared to non-mucoadhesive dextran (WO 50 7 mL), but 19 have weaker mucoadhesive properties than chitosan (WO 50 89 mL). PEGylation of 20 thiolated silica reduces their mucoadhesion with WO 50 values of 29 and 8 mL for 21 particles decorated with 750 and 5000 Da PEG, respectively. The retention of thiolated 22 silica nanoparticles is dependent on their thiol group contents and physical dimensions.


INTRODUCTION
bladder via a catheter (Malmstrom, 2003, Gasion and Cruz, 2006, Nirmal et al., 2012, 35 Haupt et al., 2013. This provides localized treatment, minimizes systemic side effects 36 and allows direct exposure of the affected tissue to therapeutic agents. However, 37 intravesical drug delivery also has some limitations. The normal capacity of the bladder 38 is 400-600 mL, but filling to 150-300 mL causes the urge to urinate. Due to periodical 39 voiding of urine from the bladder, instilled drug formulations can be rapidly washed out, 40 requiring frequent repeated administration (Guhasarkar and Banerjee, 2010). 41 Additionally, frequent use of catheters is inconvenient for the patients and may cause 42 inflammatory reactions and infections. 43 The residence time of a dosage form in the bladder can potentially be improved by 44 using mucoadhesive materials, which could adhere to the epithelial mucosa and resist 45 drug washout. Mucoadhesive formulations for intravesical drug delivery must satisfy 46 three main criteria: they should adhere rapidly to the bladder mucosa, should not 3 interfere with the normal functions of the bladder and should be retained in situ even 48 after urination (Tyagi et al., 2006). 49 Hydrophilic polymers are traditionally used as mucoadhesive materials in many 50 formulations for transmucosal drug delivery (Peppas, 1996;Andrews, 2009;51 Khutoryanskiy, 2011) and commonly used are chitosan and carbomers (weakly cross-52 linked poly(acrylic acid). The adhesion of these polymers to mucosal surfaces is through 53 non-covalent interactions such as hydrogen bonding, electrostatic attraction, 54 hydrophobic effects and diffusion and interpenetration (Khutoryanskiy, 2011). Recently, 55 a number of chemical approaches have been reported to enhance mucoadhesive 56 properties of polymers including the introduction of thiol groups 57 2004), acrylate groups (Davidovich-Pinhas, 2011), catechols (Kim, 2015) and boronates 58 (Liu, 2015). 59 The literature contains few reports on chemically modified and enhanced mucoadhesive 60 materials for intravesical drug delivery. Barthelmes et al (2011Barthelmes et al ( , 2013 used thiolated 61 particles based on chitosan and demonstrated that retention in rat bladder in vivo was 62 approximately 170-fold greater than for a small-molecular weight fluorescent marker. 63 Storha et al (2013) developed thiolated nanoparticles using thiol-ene click chemistry and 64 studied their retention on porcine urinary bladder mucosa in vitro. Zhang et al (2014) 65 reported the synthesis of a series of β-cyclodextrin modified mesoporous silica 66 nanoparticles with hydroxyl, amino, and thiol groups. They demonstrated that retention 67 of thiol-functionalized nanoparticles on the urothelium was significantly higher than the 68 hydroxyl-and amino-functionalized materials.

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NaH 2 PO 4 •H 2 O (1.00 g), and Na 2 HPO 4 (0.11 g). The pH of the resulting solution was 6.2, 164 8 which is in agreement with Chutipongtanate and Thongboonkerd (2010 placed onto the mucosal surface, fluorescence microscopy images were again taken, 179 followed by 7 washing cycles, for each of which the bladder tissue was irrigated with 10 180 mL of artificial urine solution at 5 mL/min using a syringe pump. Fluorescence  The fluorescently labelled nanoparticles were characterized using dynamic light 232 scattering, fluorescent spectroscopy and Ellman's assay. Figure 1 shows size 233 distributions of the nanoparticles formed in DMSO, DMF and AcN, determined using 234 dynamic light scattering, illustrating the influence of changing solvent polarity on particle 235 size, but with similar dispersities.

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Previously (Irmukhametova et al, 2011) it was demonstrated that PEGylation prevents 237 the adhesion of thiolated silica to intact bovine cornea, but could facilitate their 238 penetration into more porous stroma in de-epithelialized cornea (Mun et al, 2014).

239
Clearly, deeper penetration of particles into a biological tissue could also improve their 240 retention, which means that PEGylation may have various effects on particle behavior 241 on different mucosal surfaces. In this work the effect of thiolated silica PEGylation was 242 studied in relation of urinary bladder mucosa.  showing that the greater the molecular weight of PEG shell, the larger the nanoparticles.

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Analysis of the fluorescent images using ImageJ software allows the retention of 288 fluorescent species on mucosal surfaces to be quantified (Figure 3). FITC-chitosan is 289 retained on the bladder surface even after 7 washes (total volume 70 mL) with artificial 290 urine solution and illustrates its strong interaction with the mucosal surface. However, 291 for FITC-dextran, a significant decrease in fluorescence was observed after the first 292 wash (10 mL) with urine solution, confirming its poor mucoadhesive properties. Retention of thiolated silica nanoparticles on the bladder mucosa was significantly 296 higher than for FITC-dextran (p<0.05): approximately 15% of the fluorescence, hence 297 particles, remains on the mucosal surface even after 7 washing cycles with 10 mL of 298 artificial urine solution. However, their retention was significantly lower than FITC-299 chitosan (p<0.05). This may be due to the polymeric nature of chitosan, whose 300 positively-charged macromolecules are able to penetrate into the mucosal layer of the 301 bladder epithelium, form non-covalent interactions (e.g. electrostatic attraction and 302 hydrogen bonding) with mucins and generate an interpenetration layer (Sogias, 2008).

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This interpenetration could potentially facilitate better retention of chitosan on the 304 bladder mucosa compared to thiolated nanoparticles.

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PEGylated silica nanoparticles were washed from the mucosal surface more rapidly  (Table 1).

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The poorer mucoadhesive performance of PEGylated nanoparticles compared to the 317 thiolated silica is in good agreement with our previous study of retention of similar 318 nanoparticles on the ocular surfaces (Irmukhametova, 2011). However, both thiolated

327
Retention studies were conducted with differing sizes of thiolated silica nanoparticles, 328 synthesized in different aprotic solvents (DMSO, DMF and AcN, Table 1). Figure 4   329 shows the retention profiles for these nanoparticles.     * WO 50 is the volume of artificial urine required to wash out 50% of the particles