The mucoadhesive and gastroretentive properties of hydrophobin-coated porous silicon nanoparticle oral drug delivery systems

Biomaterials 33 (2012) 3353e3362

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The mucoadhesive and gastroretentive properties of hydrophobin-coated porous silicon nanoparticle oral drug delivery systems Mirkka P. Sarparanta a, *,1, Luis M. Bimbo b,1, Ermei M. Mäkilä c, Jarno J. Salonen c, Päivi H. Laaksonen d, A.M. Kerttuli Helariutta a, Markus B. Linder d, Jouni T. Hirvonen b, Timo J. Laaksonen b, Hélder A. Santos b, Anu J. Airaksinen a, ** a

Laboratory of Radiochemistry, Department of Chemistry, University of Helsinki, FI-00014, Finland Division of Pharmaceutical Technology, Faculty of Pharmacy, University of Helsinki, FI-00014, Finland Laboratory of Industrial Physics, Department of Physics and Astronomy, FI-20014 University of Turku, Finland d Nanobiomaterials, VTT Technical Research Centre of Finland, FI-02044 VTT, Finland b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 November 2011 Accepted 11 January 2012 Available online 29 January 2012

Impediments to intestinal absorption, such as poor solubility and instability in the variable conditions of the gastrointestinal (GI) tract plague many of the current drugs restricting their oral bioavailability. Particulate drug delivery systems hold great promise in solving these problems, but their effectiveness might be limited by their often rapid transit through the GI tract. Here we describe a bioadhesive oral drug delivery system based on thermally-hydrocarbonized porous silicon (THCPSi) functionalized with a self-assembled amphiphilic protein coating consisting of a class II hydrophobin (HFBII) from Trichoderma reesei. The HFBII-THCPSi nanoparticles were found to be non-cytotoxic and mucoadhesive in AGS cells, prompting their use in a biodistribution study in rats after oral administration. The passage of HFBII-THCPSi nanoparticles in the rat GI tract was significantly slower than that of uncoated THCPSi, and the nanoparticles were retained in stomach by gastric mucoadhesion up to 3 h after administration. Upon entry to the small intestine, the mucoadhesive properties were lost, resulting in the rapid transit of the nanoparticles through the remainder of the GI tract. The gastroretentive drug delivery system with a dual function presented here is a viable alternative for improving drug bioavailability in the oral route. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Silicon Hydrophobin Nanoparticle Drug delivery Adhesion Biocompatibility

1. Introduction Oral delivery is by far the preferred route for chronic drug therapy due to its higher patient compliance. However, the bioavailability of orally administered drugs is always restricted by their solubility in the variable conditions of the gastrointestinal (GI) tract, obstacles to permeation of the intestinal wall, and in some cases, extensive pre-systemic metabolism in the gut and liver [1]. Several strategies including solid drug dispersions [2,3], prodrugs [4], use of soluble excipients [5], self-emulsifying systems [6] and encapsulation into nanocarriers [7,8] or inorganic mesoporous matrices [9] have been employed to overcome these challenges.

* Corresponding author. Tel.: þ358 9 191 50134; fax: þ358 9 191 50121. ** Corresponding author. Tel.: þ358 9 191 50133. E-mail addresses: mirkka.sarparanta@helsinki.fi (M.P. Sarparanta), anu.airaksinen@ helsinki.fi (A.J. Airaksinen). 1 These authors contributed equally to this work. 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2012.01.029

Bioadhesive drug delivery systems are one way to prolong the gastric residence time in drug formulations, especially for compounds that are intended to be locally active in the stomach, that have a narrow absorption window in the upper GI tract, or are unstable or poorly soluble in the intestinal environment [10]. The rationale behind the design of bioadhesive particle formulations for oral delivery is two-fold. Firstly, mucosal retention by a bioadhesive can be used to increase the transit time of the particulate drug carrier in the GI tract, resulting in prolonged time window for the release of the payload. Secondly, enhanced absorption of the drug could occur as the effective surface area in contact with the intestinal mucosa increases as the bioadhesive swells and fills the crevices of the mucous membrane, yielding a high local concentration of the drug [11]. Traditionally based on polymers, such as crosslinked polyacrylic acids, carboxypolymethylene, carboxymethyl cellulose, alginate and chitosan, oral bioadhesive drug delivery systems are characterized by the presence of strong hydrogen-bonding functionalities and cationic charges, high molecular weight to prevent intestinal absorption, ability to

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penetrate and interlock the mucus network, and wetting and swelling in contact with mucous membranes [12,13]. Porous silicon (PSi) is a promising material for the development of drug delivery carriers because of its demonstrated biocompatibility, biodegradability, and versatile physicochemical properties including variable surface chemistry, particle and pore size, and degree of porosity [14e16]. PSi particles have been reported to host diverse payloads from poorly soluble drugs to biomolecules and secondary nanoparticles for controlled release and imaging applications [17e20]. We have previously established 18F-radiolabeled thermally hydrocarbonized porous silicon (18F-THCPSi) nanoparticles as a potent tool to study the biodistribution of the particles and their behavior in the GI tract after oral administration [21]. In the previous study, THCPSi particles were shown to associate with Caco-2 cells, a model of intestinal absorption, in culture but without extensive internalization, and the 18F-THCPSi nanoparticles passed intact through the GI tract in 4e6 h after oral administration in rats, leading to the suggestion to use THCPSi as a robust, biologically inert drug delivery carrier for the oral route. Furthermore, because its lack of specific interactions with mucosa of the GI tract, THCPSi is an ideal platform to evaluate biofunctionalizing modifications of nanomaterials intended for oral drug delivery applications. Hydrophobins are small, amphiphilic surface-active proteins from filamentous fungi, known for their capability to self-assemble in monolayers on hydrophobic/hydrophilic einterfaces modifying their wettability [22,23]. In nature, they are present in processes where the fungus escapes from the aqueous environment to air, and attaches to hydrophobic interfaces, i.e. during dispersal via spores and hyphae. Hydrophobins have been shown to disperse nanomaterials such as single-walled carbon nanotubes and graphene sheets efficiently in aqueous media preventing aggregation [24,25], as well as increase the solubility of poorly water-soluble drug nanoparticles [26,27]. In addition, the use of engineered variants of hydrophobins has been reported in the immobilization of drug nanoparticles on nanofibrillar cellulose for enhanced oral bioavailability [28,29], and in fibroblast culture on hydrophobic surfaces [30], illustrating the vast potential of hydrophobinfunctionalized materials. Curiously, hydrophobins have been shown to play a role in the prevention of host immune response to fungal spores [31], a finding that yet awaits further investigation of their potential biocompatibility-enhancing properties in vivo. HFBII used in this work is an 86-amino acid class II hydrophobin from Trichoderma reesei [32]. Class II hydrophobins, in contrast to their class I counterparts, have the advantage of having a high solubility in aqueous media [33]. We have previously shown that functionalization of hydrophobic THCPSi microparticles with HFBII effectively alters the particles’ hydrophilicity, resulting in their efficient dispersal in aqueous solutions minimizing aggregation, and thus easier formulation for biological applications. In addition, the biocompatibility of THCPSi microparticles was shown to be improved by the HFBII functionalization in Caco-2 and HT-29 cell lines. Furthermore, the functionalization was shown not to interfere with the release of a model drug, indomethacin, from THCPSi microparticles in vitro, making the use of HFBII-THCPSi particles as drug delivery carriers feasible [34]. Herein we describe the mucoadhesive and gastroretentive properties of a oral drug delivery system based on HFBIIfunctionalized THCPSi nanoparticles. The stability of the HFBII coating was investigated in vitro in simulated gastric and intestinal fluids and the mucoadhesive properties of HFBII-coated THCPSi nanoparticles were assayed in AGS cells. In vivo biodistribution and mucoadhesion after oral administration was studied in rats using HFBII-THCPSi nanoparticles radiolabeled with a short-lived positron emitter 18F (t½ ¼ 109.8 min).

2. Materials and methods 2.1. Synthesis of HFBII-18F-THCPSi nanoparticles HFBII-18F-THCPSi nanoparticles were prepared from freshly-radiolabeled 18FTHCPSi nanoparticles prepared from boron-doped pþ etype Si wafers according to previously described methods [21,35]. Before the radiolabeling, the nanoparticles were characterized with N2 sorption measurement at 77 K (Tristar 3000, Micromeritics Inc.) and results for the specific surface area, pore volume, diameter and porosity were calculated from the Brunauer-Emmett-Teller (BET) and BarrettJoyner-Halenda (BJH) theories, respectively. HFBII was produced and purified using established methods [36]. All reagents were from Sigma-Aldrich unless otherwise specified and used without further purification. Radiolabeling syntheses were carried out on a semi-automated synthesis unit (DM Automation). 18F was produced in a 18O(p,n)18F reaction on an IBA Cyclone 10/5 cyclotron and eluted as 18  F /Kryptofix2.2.2/Kþ complex. The complex was dried azeotropically, dissolved in 500 mL of anhydrous dimethylformamide with 4% (v/v) glacial acetic acid and added to 1 mg of THCPSi nanoparticles. The nanoparticles were heated to þ120  C for 10 min, cooled and purified with sequential washes in absolute ethanol (99.7%, Altia Corporation), 1  PBS (pH ¼ 7.4), and ultrapure water. In each step, the particles were pelleted with centrifugation at 15000 g for 10 min and resuspended by sonication. 0.24e0.67 GBq of 18F-THCPSi nanoparticles in absolute ethanol were added drop-wise to a solution of 3.9  0.2 mg/mL HFBII in pH 4.0 McIlvaine buffer (0.2 M Na2HPO4e0.1 M citrate) and incubated at þ80  C for 30 min. Particles were separated from the coating solution with centrifugation and purified with 3  5-min sonications in 1 mL of ultrapure water. HFBII-18F-THCPSi nanoparticles were formulated in sterile 0.9% NaCl solution to a final concentration of 19.8  0.5 MBq/ mL (0.16  0.07 mg/mL). Specific radioactivity of the nanoparticles in formulated solution was determined from freeze-dried and weighed 1-mL samples of the formulated batches washed with 1 mL of ultrapure water. 2.2. Nanoparticle characterization Nanoparticle size and zeta potential distributions from dynamic light scattering and electrophoretic mobility were measured on a ZetaSizer Nano instrument (Malvern Ltd.) either in ultrapure water (z-potential) or 0.09% (w/v) NaCl solution (size). HFBII from the nanoparticle coating was dissolved with 48 h incubation in 2.5% (w/v) SDSe0.5% (v/v) ethanol at ambient temperature and quantified with a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. 2.3. Coating stability tests in simulated gastric and intestinal fluids with 125I-labeled HFBII HFBII was radiolabeled with 125I-NaI (in 105 M NaOH, pH 8-10, reductant-free, PerkinElmer) in a standard procedure for Bolton-Hunter radioiodination with SHPP (N-succinimidyl-3-(hydroxyphenyl)propionate) using methods described in the literature [37,38]. 125I-HFBII-coated THCPSi nanoparticles were prepared analogously to HFBII-18F-THCPSi nanoparticles, except that the coating solution was spiked with 0.05 MBq of 125I-HFBII tracer in approximately 300 mL of pH 4.0 McIlvaine buffer. The reaction volume was 1 mL and a 1:4 (w/w) ratio of nanoparticles to HFBII was maintained by supplemental non-radioactive HFBII. Stability of the 125IHFBII coating was investigated in simulated gastric fluid (sGF, 2.0 g/L NaCl and 3.2 g/ L porcine pepsin, pH ¼ 1.2) and fasted-state simulated intestinal fluid (FaSSIF, prepared according to Galia et al. [39]) at þ37  C. Freshly prepared 125I-HFBII coated particles were resuspended in 100 mL of 1  PBS (pH ¼ 7.4) and added to 5 mL of either sGF or FaSSIF and dispersed with gentle shaking. Samples of 200-mL were drawn from the incubation at designated time points and particles were deposited by centrifugation. Radioactivity of the particle pellets and supernatants was counted on an automated gamma counter (Wizard 3, PerkinElmer) for 10 min. In order to identify the radioactive species (i.e. free 125I vs. 125I-HFBII), a 1-mL sample from all supernatants was spotted on Whatman 1 chromatography paper (Millipore Corporation) and developed with 50:50 (v/v) wateremethanol. Chromatographs were analyzed with digital autoradiography. All stability tests were performed in duplicate on two different batches of 125I-HFBII-THCPSi. 2.4. Cell viability and in vitro mucoadhesion The in vitro studies with the 125I-HFBII-THCPSi nanoparticles were done on a human gastric adenocarcinoma (AGS) cell line (CRL-1739, American Type Culture Collection). The cells were cultured in 75 cm2 culture flasks (Corning Life Sciences) in RPMI 1640 medium (EuroClone S.p.A.) supplemented with 10% fetal bovine serum (FBS, Gibco, Invitrogen), 1% nonessential amino acids, 1% L-glutamine, penicillin (100 IU/mL), and streptomycin (100 mg/mL) (all from EuroClone S.p.A.). The cultures were maintained in a BB 16 gas incubator at 37  C (Heraeus Instruments GmbH) in an atmosphere of 5% CO2 and 95% relative humidity. The growth medium was changed every other day until the time of use. AGS cells from passages 7e15 were used in the experiments.

M.P. Sarparanta et al. / Biomaterials 33 (2012) 3353e3362 For the viability assay, the cells were harvested using 0.25% (v/v) trypsineEDTAePBS and seeded in 96-well plates (PerkinElmer) as 100 mL of the cell solution with a density of 2  105 cells/mL in RPMI 1640 and allowed to attach overnight. The medium was aspirated and 100 mL of uncoated and HFBII-coated THCPSi nanoparticle suspensions with concentrations of 500, 250, 50 and 15 mg/ mL were added to the wells. Incubations with 1  HBSS (Hank’s Balanced Salt Solution) and Triton X-100 detergent were used as positive and negative controls, respectively. After 6 and 24 h of incubation, the wells were washed twice with 1  HBSS and 50 mL of fresh 1  HBSS was then added to the wells along with 50 mL of the CellTiter-GloÔ assay reagent (Promega Corporation). In this assay, the number of viable cells in culture is quantified based on the amount of ATP produced by metabolically active cells. Thus the amount of ATP produced is directly proportional to the number of living cells present in the culture. The plate was then measured for luminescence using a Varioskan Flash fluorometer (Thermo Fisher Scientific). All the assays were carried out at least in triplicate. For the in vitro mucoadhesion studies, the cells were harvested using 0.25% (v/v) trypsineEDTAePBS, seeded to a density of 1.5  105 cells/well on a 12-well plate (Corning Life Sciences) and allowed to attach overnight. The medium was removed and the cells were washed twice with 1  HBSS. The cells were subsequently incubated with 19.3 kBq/mL of 125I-HFBII-THCPSi nanoparticles for 15, 30, 60, 120, 180 and 240 min. At each time point, the particle solution was removed and the cells washed with 1 ml of 1  HBSS and then detached with 1 mL of a 0.25% (v/v) trypsinEDTAePBS. Radioactivity in the fractions was measured on an automated gamma counter for 10 min. For the microscopy and autoradiography experiments, the cells were seeded 1.5  105 cells/well on 13-mm glass coverslips in the 12-well plate. Afterwards, the cells were incubated with 125I-HFBII-THCPSi analogously to the procedure described above, but instead of detaching the cells at the designated time points, they were stained for 5 min with 1 mL of a 15 mg/mL solution of CellMaskÔ (Molecular Probes, Invitrogen). The cells were then fixed with a 2% glutaraldehyde (Sigma-Aldrich) in 0.1 M phosphate buffer (pH ¼ 7.4) for 30 min at room temperature, washed with 1 mL of fresh 1  HBSS and allowed to dry. The coverslips were mounted in VectashieldÔ (Vector Laboratories) and sealed to the glass slide using clear nail polish. For autoradiography, the slides were exposed on a digital imaging plate (SR2040, Fujifilm Corporation) for 72 h and processed on a Fujifilm FLA-5100 scanner. Images were analyzed with AIDA 2.0 imaging software (Raytest Isotopenmessgeräte GmbH). The slides were subsequently imaged under an Olympus IX71 inverted fluorescence microscope (Olympus Corporation). 2.5. Biodistribution of HFBII-18F-THCPSi after oral administration in rats All experimental procedures were approved by the National Board for Animal Experimentation in Finland (State Provincial Office of Southern Finland, Hämeenlinna). Male Wistar:Han rats (250e370 g, 8e14 weeks, either from Harlan, Horst, the Netherlands or the Laboratory Animal Centre of University of Helsinki, Finland) were maintained in groups of two to three animals in standard polycarbonate cages with aspen bedding with food (Harlan Teklad Global Diet 2018) and tap water available ad libitum. Environmental conditions of a 12:12 lighting rhythm, temperature 22  1  C, and relative humidity 60  5% were maintained throughout the study. Animals were single housed and fasted on 10% glucose solution available ad libitum from 18 h before dosing until the end of the experiment. 18.3  0.9 MBq of HFBII-18FTHCPSi nanoparticles in 0.9% NaCl were administered by intragastric gavage (n¼17). Additionally, three rats received uncoated 19.3  1.0 MBq 18F-THCPSi nanoparticles in 5% Solutole1  HBSS (pH ¼ 7.4) to yield a 3 h biodistribution time point for the tracer for comparison [21]. Animals were sacrificed with CO2 asphyxiation and cervical dislocation. Samples from blood, urine and organs outside the GI tract were collected, weighed and their radioactivity counted on an automated gamma counter for 60 s. The lower GI tract was excised from the pyloric sphincter and the rectum and imaged with macroautoradiography. Stomach and distal ileum of selected animals was used for cryosections and processed as described below. 2.6. Tissue autoradiography Radiolabel distribution in the lower GI tract was assessed with digital macroautoradiography using a protocol described elsewhere [21]. For cryosections, the whole stomach and an approximately 2.5-cm segment of the distal ileum was rinsed gently with 1  PBS (pH ¼ 7.4) and 4% neutral buffered formalin (NBF, VWR Life Sciences). The segments were then filled with NBF, immersed in the fixative and fixed for 30 min in order to prevent the tissue from everting. The fixative was squeezed out and the lumen of the specimen dilated with Jung tissue freezing medium (Leica Microsystems) and snap-frozen in isopentane on dry ice. After freezing, the stomach was cut in half at the level of the cardia to separate the forestomach from the glandular. Coronal sections of 25 mm were cut at 12  C on a cryostat microtome (CM1950, Leica Microsystems), thaw-mounted of Superfrost Plus slides (Thermo Fisher Scientific) and air-dried until autoradiography. The slides were exposed on a digital imaging plate (Fujifilm TR2040) for 12 h and the plate scanned on Fujifilm FLA-5100 scanner. Images were analyzed with AIDA 2.0 imaging software. Sections were subsequently stained with hematoxylin and eosin, mounted

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in DPX and imaged under an Axioplan 2 microscope fitted with Axiocam HRc camera and AxioVision 3.1 software (all from Carl Zeiss MicroImaging GmbH). 2.7. Statistical analysis When appropriate, statistical analysis of the results was carried out using the Student’s t-test for HFBII-THCPSi compared to their uncoated counterparts with the level of significance set at p  0.05. Analyses were performed on either PASW Statistics (version 18.0.0, IBM Corporation) or GraphPad Prism software (version 5.01, GraphPad Software).

3. Results and discussion 3.1. Synthesis and characterization of HFBII-18F-THCPSi nanoparticles The THCPSi nanoparticles used in the study had a specific surface area of 323  2 m2/g, pore volume of 0.48  0.01 cm3/g, average pore diameter of 7.4  0.2 nm and porosity of 53%. The nanoparticles were successfully labeled with 18F in a direct onestep reaction with 18F/Kryptofix2.2.2/Kþ complex followed by post-radiosynthetic functionalization with T. reesei HFBII. The HFBII coating procedure was adapted to 18F-radiolabeled nanoparticles from a protocol described earlier for HFBII-THCPSi microparticles [34]. Characterization data for the radiolabeled HFBII-18F-THCPSi nanoparticles is given in Table 1. A total of four batches of HFBII-18FTHCPSi nanoparticles were produced in the study, with batch sizes ranging from 0.36 to 1.56 mg. The radiochemical yield from 18F/ Kryptofix2.2.2/Kþ to formulated HFBII-18F-THCPSi was 37.5  4.3% (corrected for the decay of the 18F isotope) and total synthesis time 189  5 min, illustrating that the functionalized particles could be prepared without compromises to radiolabeling yield or product specific radioactivity in a time frame shorter than two times the half-life of 18F. The synthesis was reproducible in terms of the radiochemical yield and the variation in batch size can be attributed to the variation in specific radioactivity (MBq/mg) of the prepared nanoparticles. 3.2. Coating stability Stability of the HFBII coating on the THCPSi nanoparticles was evaluated in vitro in simulated conditions of the GI tract. Because of the minute amount of HFBII present in the coated particles and the tendency of PSi for strong UV absorption prompting limitations in the detection sensitivity of protein quantification methods based on absorbance, we chose to use radiolabeled 125I-HFBII as a tracer in the stability experiments, which allows the use of more sensitive radiometric methods. Results of the stability experiments are depicted in Fig. 1. The HFBII coating showed good stability in simulated gastric fluid (sGF) for 6 h, with only a slight decline from 94% to 88.5% over the duration of the experiment. In contrast, the stability of the HFBII coating was dramatically reduced in fastedstate simulated intestinal fluid (FaSSIF), with almost 45% detachment of the coating immediately upon suspension of the nanoparticles in the medium. 125I-HFBII continued to leach out slowly from the particle surface over time decreasing to 40% of the radioactivity particle-bound at the last time point. In both sGF and

Table 1 Characterization data for the HFBII-18F-THCPSi nanoparticles synthesized in this study. Size [nm] z-potential [mV] Specific radioactivity [MBq/mg] HFBII content [mg protein/mg 18F-THCPSi]

243.1 25.78 203.7 0.095

   

40.9 13.03 127.0 0.078

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sGF

FaSSIF

A

90.0

140

80.0

120

70.0

100

HBSS

THCPSi

Triton X-100

HFBII-THCPSi

60.0 80

50.0 40.0

60

30.0

40

20.0 10.0 0.0 0

100

200 Time [min]

300

Fig. 1. Stability of the coating in 125I-HFBII-coated THCPSi nanoparticles in simulated gastric fluid (sGF) and fasted-state simulated intestinal fluid (FaSSIF) at þ37  C. Values denote mean  s.d. (n ¼ 2).

B

Cell viability [%]

125I-HFBII

retained in particles [%]

100.0

20 0

500

250

50

15

140 120 100 80

FaSSIF, the detached radioactivity was shown to correspond to intact 125I-HFBII with paper chromatography (data not shown). The rapid desorption and solubilization of HFBII from the nanoparticle surface in FaSSIF is likely due to the presence of sodium taurocholate, a surfactant, in the simulated fluid. As HFBII is bound to the THCPSi surface most likely through hydrophobic interactions, it is no surprise that the adsorption of HFBII would be hampered by the competing adsorption of the smaller surfactant molecules to the nanoparticle surface resulting in the detachment of the HFBII layer. In physiological conditions the intestinal fluid is a much more complex solution, but nevertheless contains a pool of compounds with surfactant properties (e.g. bile salts, bicarbonate and lipolysis products), making the coating prone to disruption in the intestine also in vivo [40]. In addition, previous investigations on the pH stability of the hydrophobin coating in hydrophobin-functionalized PSi microparticles have showed that the coating is stable at pH 1.2 for up to 8 days, whereas the stability is reduced at neutral pH [34]. Together, the results indicate that the HFBII coating likely remains intact in the stomach and is possibly detached from the nanoparticle surface upon entry to the duodenum, which is of particular importance for the targeting of the therapeutic payload to the gastric mucosa yet allowing fast clearance of the particles from the gut after the release of the payload. Furthermore, the isoelectric point (pI) of HFBII is reported to be 4.8 [41], indicating that the HFBII-coating of the nanoparticles would have a cationic charge in the low pH of the stomach and would be turned anionic as the pH rises when the nanoparticles enter the small intestine, thus possibly weakening their affinity to the mucosa of the small intestine. These local changes in the surface charge could play a role in the onset and offset of mucoadhesion at the cellular level in vivo. 3.3. Mucoadhesive properties of HFBII-THCPSi Mucoadhesion of HFBII-THCPSi was investigated in vitro in AGS cells which excrete a mucous layer on their surface in culture. As it has been previously shown that particle size is a critical parameter for cytotoxicity of both PSi and non-PSi particles [42,43], the effect of the HFBII-THCPSi nanoparticles on AGS cell viability was first investigated. Results of the cell viability assay on AGS cells incubated with both uncoated and HFBII-coated THCPSi showed that

60 40 20 0

500 250 50 15 Nanoparticle concentration [µg/mL]

Fig. 2. Cell viability of AGS cells incubated with HFBII-THCPSi nanoparticles for 6 h (A), and 24 h (B). The results are expressed as percentage of live cells compared to the positive control. Values represent mean  s.d. (n  3), *p < 0.05.

the viability was not reduced by the HFBII coating (Fig. 2). In fact, the HFBII-coating was shown to even increase the cell viability after 6 and 24 h compared to uncoated THCPSi at the higher concentrations tested (250 and 500 mg/mL), possibly reflecting the greater biocompatibility of HFBII-functionalized nanoparticles compared to uncoated THCPSi. The plausible explanation for the reduced cytotoxicity of HFBII-THCPSi nanoparticles is that the nanoparticles are hydrophilic, which favors hydration and might contribute to indirect cellenanoparticle interactions with cell surface proteins mediated by the water layer surrounding the nanoparticle instead of strong and direct interactions with the particles that can hamper cell viability [42]. Interestingly, no significant biocompatibility-promoting effect was shown in a previous study with HFBII-THCPSi microparticles in AGS cells [34], illustrating that the nanosized THCPSi are possibly inherently more cytotoxic than microparticles in this cell model. This is likely due to the larger surface area and the higher number of particles that are in contact with the cell in the case of the nanoparticles and thus contributing to the suggested strong nanoparticleecell interactions. The observed improved biocompatibility of hydrophobin-functionalized THCPSi particles together with the previous report on the lack of immunogenicity of hydrophobincontaining fungal spores alludes to the potential safety of the drug delivery system in vivo [31,34]. When incubated with AGS cells, THCPSi nanoparticles coated with 125I-radiolabeled HFBII (125I-HFBII-THCPSi) showed a gradual adhesion to the cells over time in autoradiography (Fig. 3A). Quantified from radioactivity measurements of the cells and the extracted medium, 35% of the 125I-HFBII-THCPSi was found to be

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Fig. 3. (A) Autoradiographs of individual coverslips with AGS cells incubated with 125I-HFBII-THCPSi nanoparticles for 15e240 min. The results are expressed as units of photostimulable luminescence (PSL) per mm2. (B) Quantification of in vitro mucoadhesion to AGS cells as a function of time. Values denote mean  s.d. (n ¼ 2 per time point). (C) AGS cells incubated with 125I-HFBII-THCPSi nanoparticles imaged in brightfield and fluorescence. Arrows indicate clusters of 125I-HFBII-THCPSi nanoparticles seen in the brightfield images.

retained in the cells at the last time point at 4 h (Fig. 3B). A pronounced adhesion effect can be seen from 120 min onwards, indicating that it takes 30e60 min for the nanoparticles to become in contact with the cells and attach in this system. The cells appear normal in morphology after a stain for the plasma membrane (Fig. 3C) and their examination in brightfield confirmed that their ability to secrete mucus was not impaired upon contact with the nanoparticles. Furthermore, clusters of THCPSi nanoparticles (indicated by arrows in Fig. 3C) appear in the brightfield images at 30 min and increase in numbers for the subsequent time points, further corroborating the adhesion of HFBII-THCPSi to mucussecreting AGS cells in vitro. Possible mechanisms of mucoadhesion in vitro could include hydrophobic and electrostatic interactions mediated by specific amino acid residues on the HFBII layer. Furthermore, the cysteine residues in hydrophobin could form disulfide bonds with thiol groups in the glycoproteins of mucus, prompting strong adhesion [36,44]. Assessment of the contributions of these interactions to the observed mucoadhesion, however, warrants further mechanistic studies.

3.4. Biodistribution and gastric retention of HFBII-18F-THCPSi in rats Performance of HFBII-THCPSi nanoparticles in vivo was studied with 18F-labeled nanoparticles, in which the radiolabel is covalently bound to the THCPSi surface under the HFBII coating [21,45]. This radiolabeling strategy was selected to ensure that we could follow the particle biodistribution regardless of the possible loss of the HFBII functionalization through solubilization or erosion of the coating in the GI tract. For the in vivo study, HFBII-18F-THCPSi nanoparticles were orally administered to male Wistar rats. The passage of the nanoparticles in the GI tract was monitored with intestinal macroautoradiography. Macroautoradiography is a powerful technique for the visualization of intestinal distribution of radiolabeled nanoparticles, as it allows for the precise quantification of the nanoparticle concentration in different parts of the GI tract at high resolution within the same image. Macroautoradiography revealed that during the first 2 h after administration the entry of HFBII-18F-THCPSi from the stomach to the intestines was markedly slower than that of uncoated 18F-THCPSi

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and that the effect persists for up to 3 h after administration (Fig. 4A). Representative macroautoradiographs for animals dosed with HFBII-18F-THCPSi are given in Fig. 4B. At 3 h, HFBII-18F-THCPSi nanoparticles can be seen in all the parts of the small intestine and

appearing the cecum, but the total amount of radioactivity in the intestines is lower than for 18F-THCPSi at the same time point because the bulk of the HFBII-18F-THCPSi nanoparticles are still retained in the stomach. Based on the in vitro findings on the

Fig. 4. (A) Distribution of HFBII-18F-THCPSi and 18F-THCPSi nanoparticles in the rat lower GI tract as a function time quantified from macroautoradiography. The results are expressed as units of photostimulable luminescence (PSL) per mm2 normalized to the administered dose of 18F-radiolabeled particles in MBq. Values represent mean  s.d. (n ¼ 3 per time point), *p < 0.05. (B) Representative macroautoradiographs of the rat GI tract at 1e6 h after oral administration of HFBII-18F-THCPSi nanoparticles. A schematic diagram on top shows the arrangement of the intestines in the autoradiography cassette.

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coating stability in FaSSIF, we would expect the HFBII-THCPSi nanoparticles to start to lose the HFBII-coating upon the entry into the duodenum and therefore start behaving analogously to the uncoated THCPSi nanoparticles, which is corroborated by the similar patterns of the respective nanoparticle distributions depicted at 4 and 6 h in Fig. 4A. Although the large inter-individual variation arising from pica and coprophagy exhibited by some of the animals during the course of the experiment hampers the observation of significant differences in the passage of coated versus uncoated particles in this experimental setup, the sloweddown passage of HFBII-18F-THCPSi can nevertheless be seen. Furthermore, no reappearance of HFBII-18F-THCPSi in the small intestine as a result of coprophagy is seen at 6 h, in contrast to 18FTHCPSi, suggesting that the particles had not been excreted in feces in high amounts yet. Similarly to 18F-THCPSi nanoparticles, the HFBII-18F-THCPSi were not taken up from the intestinal lumen and solely a small fraction of free 18F detached from the nanoparticles and absorbed from the gut is detected in organs outside the GI tract [21,46]. Biodistribution of 18F released from HFBII-18F-THCPSi in organs and fluids outside the GI tract is given in Fig. 5A. As

A

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expected, the HFBII coating seems to stabilize the 18F-radiolabel towards hydrolytic cleavage even further, as decreased accumulation of 18F to bone is seen in animals dosed with the HFBII-coated nanoparticles compared to animals receiving uncoated ones Fig. 5B. The 18F-radiolabel in THCPSi itself has been shown to have excellent stability in both sGF and FaSSIF under dynamic conditions in vitro [45]. As the results of the macroautoradiography strongly suggested that the entry of HFBII-18F-THCPSi nanoparticles from the stomach into the intestines was delayed, we wanted to investigate in detail whether the mechanism of this was truly a gastric retention effect due to mucoadhesion. Quantified from the emptied stomach of the animals, HFBII-coated nanoparticles were found to be retained in the stomach up to 3 h after administration, whereas uncoated THCPSi were released in 2 h in the same conditions (Fig. 6). Curiously, the amount of HFBII-18F-THCPSi nanoparticles retained in the stomach seems to peak at 2e3 h after administration, suggesting that the nanoparticles are not immediately attached to the gastric mucosa, and are leached out as loose particles when the dissected stomach is emptied prior to the radioactivity counting with a gentle rinse. This could also be reflective of the dynamics of ingestion and mixing of gastric contents in the rat in which the stomach is divided into two parts performing distinct functions: the forestomach, where ingested, salivated food is stored, and the glandular stomach where digestion continues [47]. In this case the delay observed in the onset of gastric retention could be explained with the preferential adhesion of the HFBIITHCPSi nanoparticles to the gastric mucosa of the glandular stomach. It is plausible that the HFBII coating is stable during the storage period in the forestomach. Autoradiography of cryosections from the stomach and ileum (Fig. 7A) showed that bulk of the radioactivity is found in the lumen of both parts of the stomach at 2 h after administration, and only little radioactivity can be seen in the ileum of the animal. Unfortunately, the resolution of digital autoradiography with 18F does not permit the delineation of the gastric mucosa in the autoradiography images alone, and therefore the sections were subsequently stained with hematoxylineeosin (H&E) and examined with microscopy along the mucosa (Fig. 7BeE). In the forestomach, only scattered sub-micron nanoparticle aggregates were seen adhering to the mucosa (Fig. 7BeC). The majority of the particles were found associated to debris of the material the animal had ingested during the duration of the experiment (e.g. hair, bedding chips) in the

B F-THCPSi

HFBII- F-THCPSi

35.00

ID% in stomach

30.00 25.00 20.00 15.00 10.00 5.00 0.00 Fig. 5. (A) Biodistribution of 18F released from the HFBII-18F-THCPSi nanoparticles in organs outside the GI tract after oral administration to rats. Values represent mean  s.d. (n ¼ 3 per time point). (B) Accumulation of 18F to bone in animals dosed with HFBII-18F-THCPSi and 18F-THCPSi nanoparticles. Values represent mean  s.d. (n ¼ 3 per time point).

0

1

2

3 Time [h]

4

5

6

Fig. 6. Gastric clearance of HFBII-18F-THCPSi nanoparticles versus uncoated 18F-THCPSi nanoparticles. Values denote mean  s.d. (n ¼ 3 per time point), *p < 0.05.

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Fig. 7. Autoradiography and H&E stain of rat stomach and ileum (A) and photomicrographs of the H&E stained sections showing forestomach (BeC) and glandular stomach mucosa (DeE) at 2 h after administration of HFBII-18F-THCPSi nanoparticles by intragastric gavage. Scale bar 20 mm. Arrows indicate HFBII-18F-THCPSi nanoparticle clusters and sheets.

lumen of the forestomach, very likely interfering with the adhesion of the nanoparticles to the forestomach mucosa. In the glandular stomach, however, sheets of nanoparticles and nanoparticle aggregates were found in the mucosa (Fig. 7DeE), indicating that they are strongly adhered, thus supporting the finding of the

difference in gastric clearance time seen in the in vivo study. Conversely, no nanoparticles were found in the H&E-stained sections of the ileum (data not shown), indicating that as the nanoparticles lose their HFBII-coating upon entry to the small intestine, they also lose their mucoadhesive properties and are

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consequently washed away during the sample preparation for H&E-staining despite being visualized in the autoradiography. Therefore, we can conclude that the gastric retention observed in vivo in this study is a result of the mucoadhesion of the HFBIITHCPSi nanoparticles, possibly taking place in the mucosa of the glandular stomach, resulting in an increase in the transit time of the drug carrier in the GI tract of the rat. Consequently the question for the driving force of THCPSi-HFBII nanoparticle adhesion and subsequent detachment from the gastric mucosa arises. The interactions of HFBII-THCPSi nanoparticles with the gastric mucosa are most probably non-specific, i.e. hydrogen bonds and van der Waals forces, as the hydrophilic domain of the HFBII amphiphile monolayer is presented to the mucosa. In vivo, the GI tract is a much more dynamic system than what can ever be simulated in vitro. The initial contact time with the mucous membrane, shear forces, pH, and local mucin turnover rate are all factors influencing the strength and duration of mucosal bioadhesion [48]. The rate of gastric mucin synthesis or turnover in rats has not been explicitly reported in the literature, but it is most likely higher than values measured for the small intestine in situ, ranging from 47 to 270 min [49] as the mucous layer turnover is most intense in the stomach due to high proteolytic activity and mechanical erosion by gastric movements [50]. The mucous gel layer of the GI tract is also the thickest in the stomach [51], likely resulting in somewhat restricted penetration of the HFBII-THCPSi nanoparticles into the layer and consequent release when the topmost loose mucus is shed as a result of the churning of gastric contents and entry of liquid ingested by the animal during the course of the experiment. Our findings on the in vivo behavior of HFBII-THCPSi nanoparticles after oral administration in rats highlight the potential utility of using them as a gastroretentive drug delivery system for compounds that are either targeted to the stomach for localized action (such as antibiotics against Helicobacter pylori) or that have a narrow absorption window in the upper GI tract, such as L-DOPA that suffers from poor bioavailability due to rapid transit through its primary absorption site in the proximal small intestine [52]. In addition, the rapid detachment of HFBII upon entry to the small intestine is envisioned to be advantageous in the design of multiparticulate, biodegradable drug delivery systems. 4. Conclusions One known caveat in the oral administration of drug-loaded nanoparticles is their rapid passage from the stomach to the intestine and subsequent elimination in excreta that prevents drug release or nanoparticle uptake from occurring to a sufficient degree. In this work, we have described a bioadhesive gastroretentive drug delivery system based on THCPSi functionalized with T. reesei HFBII, capable of increasing the biocompatibility of the material in vitro and the transit time of the nanoparticles in the rat GI tract. The HFBII-THCPSi nanoparticles were found to be mucoadhesive and non-cytotoxic in AGS cells in vitro, and the HFBII coating was stable in simulated gastric fluid for up to 4 h, rendering mucoadhesion plausible also in vivo. Biodistribution of HFBII-18FTHCPSi nanoparticles in rats after oral administration showed that the nanoparticles are retained in the stomach by mucoadhesion to the glandular stomach mucosa up to 3 h, after which they are released to the small intestine where the HFBII monolayer is shed in the presence of surface-active compounds. The results presented here comprise a framework for future delivery studies with drugs that would benefit from the dual function of the carrier: the solubility-enhancing and protective effects of PSi towards the payload drug and the bioadhesive and gastroretentive properties of the HFBII coating.

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Acknowledgements M.Sc. Saara Tegelberg, M.Sc. Jaakko Sarparanta and Dr. Anna Naukkarinen of the Folkhälsan Institute of Genetics and Haartman Institute, University of Helsinki, are gratefully acknowledged for their help with the H&E staining and microscopy. The AGS cells were a generous gift from Prof. Outi Monni (Genome-Scale Biology Research Program, Institute of Biomedicine, University of Helsinki). Mrs. Helena Jauho is thanked for assistance with the size and zeta potential measurements. Financial support from the Academy of Finland (decision numbers 127099, 123037, 122314, 136805, 140965, 252215 and 256394), the University of Helsinki Research Funds (grant number 490039), the Jenny and Antti Wihuri Foundation, and the Drug Discovery Graduate School is acknowledged. References [1] Han C, Wang B. Factors that impact the developability of drug candidates. In: Wang B, Siahaan T, Soltero R, editors. Drug delivery e principles and applications. Hoboken: Wiley Interscience; 2005. p. 1e14. [2] Chiou WL, Riegelman S. Pharmaceutical applications of solid dispersion systems. J Pharm Sci 1971;60:1281e302. [3] Janssens S, Van den Mooter G. Review: physical chemistry of solid dispersions. J Pharm Pharmacol 2009;61:1571e86. [4] Li F, Maag H, Alfredson T. Prodrugs of nucleoside analogues for improved oral absorption and tissue targeting. J Pharm Sci 2008;97:1109e34. [5] Loftsson T, Brewster ME. Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization. J Pharm Sci 1996;85:1017e25. [6] Pouton CW. Lipid formulations for oral administration of drugs: nonemulsifying, self-emulsifying and ‘self-microemulsifying’ drug delivery systems. Eur J Pharm Sci 2000;11:S93e8. [7] Cai Z, Wang Y, Zhu L, Liu Z. Nanocarriers: a general strategy for enhancement of oral bioavailability of poorly absorbed or pre-systemically metabolized drugs. Curr Drug Metab 2010;11:197e207. [8] Makhlof A, Werle M, Tozuka Y, Takeuchi H. A mucoadhesive nanoparticulate system for the simultaneous delivery of macromolecules and permeation enhancers to the intestinal mucosa. J Control Release 2011;149:81e8. [9] Vallet-Regí M, Balas F, Arcos D. Mesoporous materials for drug delivery. Angew Chem Int Ed Engl 2007;46:7548e58. [10] Streubel A, Siepmann J, Bodmeier R. Gastroretentive drug delivery systems. Expert Opin Drug Deliv 2006;3:217e33. [11] Ponchel G, Irache J. Specific and non-specific bioadhesive particulate systems for oral delivery to the gastrointestinal tract. Adv Drug Deliv Rev 1998;34: 191e219. [12] Peppas NA, Buri PA. Surface, interfacial and molecular aspects of polymer bioadhesion on soft tissues. J Control Release 1985;2:257e75. [13] Lee JW, Park JH, Robinson JR. Bioadhesive-based dosage forms: the next generation. J Pharm Sci 2000;89:850e66. [14] Salonen J, Kaukonen AM, Hirvonen J, Lehto V. Mesoporous silicon in drug delivery applications. J Pharm Sci 2008;97:632e53. [15] Prestidge CA, Barnes TJ, Lau C, Barnett C, Loni A, Canham L. Mesoporous silicon: a platform for the delivery of therapeutics. Expert Opin Drug Deliv 2007;4:101e10. [16] Canham LT. Bioactive silicon structure fabrication through nanoetching techniques. Adv Mater 1995;7:1033e7. [17] Salonen J, Laitinen L, Kaukonen AM, Tuura J, Björkqvist M, Heikkilä T, et al. Mesoporous silicon microparticles for oral drug delivery: Loading and release of five model drugs. J Control Release 2005;108:362e74. [18] Bimbo LM, Mäkilä E, Laaksonen T, Lehto V, Salonen J, Hirvonen J, et al. Drug permeation across intestinal epithelial cells using porous silicon nanoparticles. Biomaterials 2011;32:2625e33. [19] Tanaka T, Mangala LS, Vivas-Mejia PE, Nieves-Alicea R, Mann AP, Mora E, et al. Sustained small interfering RNA delivery by mesoporous silicon particles. Cancer Res 2010;70:3687e96. [20] Tasciotti E, Liu X, Bhavane R, Plant K, Leonard AD, Price BK, et al. Mesoporous silicon particles as a multistage delivery system for imaging and therapeutic applications. Nat Nanotechnol 2008;3:151e7. [21] Bimbo LM, Sarparanta M, Santos HA, Airaksinen AJ, Mäkilä E, Laaksonen T, et al. Biocompatibility of thermally hydrocarbonized porous silicon nanoparticles and their biodistribution in rats. ACS Nano 2010;4:3023e32. [22] Wösten HAB. Hydrophobins: Multipurpose proteins. Annu Rev Microbiol 2001;55:625e46. [23] Linder MB. Hydrophobins: proteins that self assemble at interfaces. Curr Opin Colloid Interface Sci 2009;14:356e63. [24] Kurppa K, Jiang H, Szilvay G, Nasibulin A, Kauppinen E, Linder M. Controlled hybrid nanostructures through protein-mediated noncovalent functionalization of carbon nanotubes. Angew Chem Int Ed Engl 2007;46:6446e9. [25] Laaksonen P, Kainlauri M, Laaksonen T, Shchepetov A, Jiang H, Ahopelto J, et al. Interfacial engineering by proteins: Exfoliation and functionalization of graphene by hydrophobins. Angew Chem Int Ed Engl 2010;49:4946e9.

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