Protease-functionalized mucus penetrating microparticles: In-vivo evidence for their potential

Accepted Manuscript Title: Protease-functionalized mucus penetrating microparticles: In-vivo evidence for their potential Authors: Arshad Mahmood, Flavia Laffleur, Gintare Leonaviciute, Andreas Bernkop-Schnurch ¨ PII: DOI: Reference:

S0378-5173(17)30833-5 http://dx.doi.org/10.1016/j.ijpharm.2017.08.114 IJP 16967

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

26-5-2017 21-8-2017 24-8-2017

Please cite this article as: Mahmood, Arshad, Laffleur, Flavia, Leonaviciute, Gintare, Bernkop-Schnurch, ¨ Andreas, Protease-functionalized mucus penetrating microparticles: In-vivo evidence for their potential.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2017.08.114 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Protease-functionalized mucus penetrating microparticles: In-vivo evidence for their potential

Arshad Mahmoodab, Flavia Laffleura, Gintare Leonaviciutea and Andreas Bernkop-Schnürcha*

a

Department of Pharmaceutical Technology,

Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria

b

Department of Pharmacy,

COMSATS Institute of Information Technology Abbottabad, Abbottabad, 22060, Pakistan

*Corresponding Author: Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria Tel.: +43-512-507 58601 Fax: +43-512-507 58699 e-mail: [email protected]

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Graphical abstract

ABSTRACT The focus of the current study was to explore whether immobilization of proteases to microparticles could result in their enhanced penetration into mucus. The proteases papain (PAP) and bromelain (BROM) were covalently attached to a polyacrylate (PAA; Carbopol 971P) via amide bond formation based on carbodiimide reaction. Microparticles containing these conjugates were generated via ionic gelation with calcium chloride and were characterized regarding size, surface charge, enzymatic activity and fluorescein diacetate (FDA) loading efficiency. Furthermore, mucus penetration potential of these microparticles was evaluated in-vitro on freshly collected porcine intestinal mucus, on intact intestinal mucosa and in-vivo in Sprague–Dawley rats. Results showed mean diameter of microparticles 2

ranging between 2 to 3 µm and surface charge between -8 to -18 mV. The addition of PAAmicroparticles to porcine intestinal mucus led to a 1.39-fold increase in dynamic viscosity whereas a 3.10- and 2.12-fold decrease was observed in case of PAA-PAP and PAA-BROM microparticles, respectively. Mucus penetration studies showed a 4.27- and 2.21- fold higher permeation of FDA loaded PAA-PAP and PAA-BROM microparticles as compared to PAA microparticles, respectively. Extent of mucus diffusion determined via silicon tube assay illustrated 3.96- fold higher penetration for PAA-PAP microparticles and 1.99- fold for PAABROM microparticles. An in-vitro analysis on porcine intestinal mucosa described up to 16and 7.35-fold higher degree of retention and furthermore, during in-vivo evaluation in Sprague–Dawley rats a 3.35- and 2.07-fold higher penetration behavior was observed in small intestine for PAA-PAP and PAA-BROM microparticles as compared to PAA microparticles, respectively. According to these results, evidence for microparticles decorated with proteases in order to overcome the mucus barrier and to reach the absorption lining has been provided that offers wide ranging applications in mucosal drug delivery. Key words: Microparticles, proteases, mucosal drug delivery, rheology, diffusion, permeation.

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INTRODUCTION Mucosal delivery presents itself as an effective strategy for delivery of a variety of active pharmaceutical ingredients (APIs) from small drugs to peptides and genes. The prominent mucosal surfaces that are easily accessible for drug delivery include gastro-intestinal, respiratory, vaginal and ocular mucosa (Lee, 2000; Zhang et al., 2002). However, the limitation associated with this route is the ability of drugs/drug-carriers to permeate the mucus gel layer and to reach the absorption cell lining. Mucus gel layer covering the mucosal surfaces is constantly being secreted, tattering and finally undergoing digestion. This natural mechanism for mucus clearance partially eliminates the carrier and the encapsulated therapeutic agent even before reaching the absorption lining (Ensign et al., 2012). Moreover, the resolute and sticky network of mucins produces hindrance to diffusion of macromolecules. The reported pore sizes within the mucus gel ranges from 10 to 200 nm (Lai et al., 2010; Pearson et al., 2016) and therefore, particles larger than this size adsorb on the surface as monolayer without penetrating (Ponchel et al., 1997). One promising strategy to penetrate the mucus and reach the absorption lining is via development of mucus penetrating delivery systems utilizing proteases. They cleave mucoglycoprotein substructures resulting in leakier mucus and enable the transport of drugs or even entire delivery systems to the absorption site. The literature describes various nanoparticles up to a size of 285 nm decorated with mucolytic agents such as proteases that are able to enhance the rate and extent of mucus penetration (de Sousa et al., 2015; Köllner et al., 2015; Müller et al., 2013; Müller et al., 2014). However from therapeutic point of view, larger sized particles such as microparticles possess the advantages of a higher drug payload, higher encapsulation efficiency and possibility of sustained drug release. Furthermore, their large scale production is comparatively less challenging than that of nanoparticles. Currently, to best of our knowledge, however, no information about the impact of proteases on the mucus penetration behavior of particles far above the mesh size of the mucus is available. 4

It was therefore the aim of this study to develop protease-functionalized polymeric microparticles and to evaluate their mucus penetrating potential. Owing to mucolytic properties of proteases, PAP and BROM were immobilized on the polymeric backbone of PAA being formulated to microparticles. PAA was chosen, as particle generation in µm range via ionic gelation method is easily feasible and a negative surface charge of particles could minimize ionic interactions with mucus. These microparticles were characterized with regard to their size, surface charge, enzymatic activity and evaluated in-vitro regarding their impact on rheological changes of mucus, mucus diffusion/penetration, permeation, capability to remain on mucosal surface and in-vivo determination of mucus penetration behavior. 2. MATERIAL AND METHODS 2.1. Materials Poly acrylic acid (Carbopol 971P) was purchased from Lubrizol, Belgium. Papain (from Carica

papaya), bromelain,

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide

hydrochloride (EDAC), N-hydroxysuccinimide (NHS), Bradford reagent, trehalose, Lcysteine, calcium chloride (CaCl2), casein and fluorescein diacetate (FDA) were obtained from Sigma-Aldrich, Austria. Eudragit® L 100-55 was supplied by Röhm GmbH. Germany. PCcaps™ capsules for in-vivo study were acquired from Capsugel, Belgium. All other chemicals were of analytical grade.

2.2. Synthesis of PAA-PAP and PAA-BROM conjugates The polymer-enzyme conjugates were obtained by covalent attachment of either PAP or BROM to PAA. Briefly, 1.0 g of PAA was added in aliquots of 0.1 g to 600 ml of demineralized water and the polymer was allowed to swell for 45 min under continuous stirring. Subsequently components of the bi-catalytic system, 1.0 g of EDAC and 0.3 g of NHS separately dissolved in 50 and 20 ml of demineralized water, respectively, were added to the PAA solution. The mixture was incubated for 1 h under continuous stirring after adjusting 5

pH with 1 M NaOH to 6.0. Afterwards, 500 mg of PAP dissolved in 200 ml of demineralized water was added in in aliquots of 50 ml to the mixture. The reaction mixture was stirred overnight at 10 °C and the resulting PAA-PAP conjugate was dialyzed (Spectra/Por® 6, MWCO 50 kDA) against demineralized water for 2 days at 10 °C in the dark. Thereafter, the dialyzed product was freeze-dried and stored at 4 °C until further use. The same procedure was repeated using 500 mg of BROM, instead of PAP. Moreover, control samples were synthesized and dialyzed in accordance with procedure employed for PAA-PAP and PAABROM conjugates but without addition of EDAC and NHS during conjugation reaction. 2.3. Characterization of enzyme functionalized polymer conjugates The enzyme contents i.e. PAP and BROM covalently attached to PAA were determined photometrically employing Bradford assay (Bradford, 1976). Briefly, 100 μl of each conjugate solution (1 mg/ml) was mixed with 3 ml of Bradford reagent. The mixtures were vortexed for 30 s and incubated for 10 min at room temperature. Later, 100 μl of sample from each mixture was shifted to microplate well and the absorbance of the enzyme-dye complex was measured at 595 nm. The amount of bound PAP and BROM was calculated using a calibration curve with serial dilutions of the enzyme. 2.4. Cytotoxicity studies The cell viability of newly developed PAA-PAP and PAA-BROM was evaluated by Resazurin assay as designated by our research group previously (Mahmood et al., 2015). In brief, Caco-2 cells (density: 1 x 105 cells/mL) were cultured in a 24-well plates in 0.5 ml minimum essential medium (MEM) spiked with 10% fetal bovine serum (FBS) and plate was placed in an incubator at 37°C and 5% CO2. The old MEM was substituted with fresh one at alternate days till an approximately ~100% confluent monolayer was established. For cytotoxicity test, MEM was replaced by 0.5 ml of 0.5% (m/v) unmodified PAA, PAA-PAP and PAA-BROM prepared in MEM (without FBS) and incubated in the same chamber at 6

37°C and 5% CO2 for 3 and 24 h. Untreated cells and 2% solution of Triton X-100 in MEM (without FBS) served as positive and negative control, respectively. At the end of incubation period, polymer solutions were removed and cells were washed twice with 10 mM PBS pH 7.4. Subsequently, 500 µL of resazurin solution was added to each well and the cells were incubated for 2-3 h at 37°C and 5% CO2. Thereafter, samples of 100 µL were taken from each well and transferred to a 96 well microtitration plate. Fluorescence was measured by a microplate reader (Tecan infinite M200 spectrophotometer, Grodig, Austria) at 540 nm excitation and 590 nm emission wavelength. The percentage cell viability was calculated according to equation 1 against the positive control as follows:

(1)

2.5. Preparation of microparticles and fluorescent labeling Microparticles were prepared by ionotropic gelation utilizing calcium chloride (CaCl2) as cross-linker. In order to perform diffusion and permeation studies, all the microparticles were labelled with the fluorescent marker FDA. Same treatment without inclusion of FDA was adapted to obtain non-labeled microparticles. Briefly, 5 mg of the FDA was dissolved in 6 ml of DMSO, followed by addition of 2 mL of demineralized water in aliquots of 100 µl and finally 12 ml of isopropanol was added and mixture was stirred for 30 min at 10 °C. Meanwhile, 20 ml solution of PAA was prepared in demineralized water at a concentration of 2 mg/ml. Afterwards, FDA solution was added to polymer solution under vigorous stirring and incubated at room temperature with continuous stirring under light protection. Microparticles were generated via dropwise addition of CaCl2 until solution turned slightly turbid and the particle suspension was purified by centrifugation at 4000 rpm for 30 min with 1.0% trehalose in order to prevent particle aggregation. Moreover, purification of the labelled microparticles was continued three times with DMSO at 4000 rpm for 10 min. After 7

centrifugation, the aggregate of the particles was re-suspended in demineralized water, dialyzed against distilled water for 24 h, freeze dried and stored at 4 °C until further use. PAA-PAP and PAA-BROM microparticles were also obtained under the same conditions utilizing calcium chloride solution as cross-linker. 2.6. Particle characterization Mean diameter and size distribution of all microparticles with and without FDA loading was determined directly after their preparation by dynamic light scattering utilizing a PSS Nicomp 380 ZLS particle sizer with a scattering angle of 90°. Moreover, zeta potential was also determined with same equipment at room temperature using electric field strength of 5 V/cm over a period of 180 s. The enzymatic activity of immobilized enzymes on the microparticles was determined via casein assay (Itoyama et al., 1994). Briefly, 500 μl of particle suspensions (1 mg/ml) of PAAPAP and PAA-BROM was mixed with same volume of 1.0% (w/v) casein solution in 50 mM TRIS–HCl buffer pH 7.6 containing 12 mM L-cysteine. The vortexing of mixtures was followed by incubation in a thermomixer (temperature 40 °C, rotation speed 400 rpm) for 30 min. At the end of incubation time, the reaction was stopped by addition of 1.0 ml of a 5.0% (w/v) tri-chloro acetic acid solution. The reaction mixture was centrifuged at 12,500 rpm for 15 min and the absorbance of the supernatant was measured at 280 nm. The enzymatic activity of the microparticles was expressed as percentage of the enzymatic activity of the PAA-PAP and PAA-BROM polymers that were considered as 100% value. The loading efficiency of FDA was quantified by alkaline treatment of the samples of DMSO collected as supernatant during centrifugation. Briefly, 100 µl from each of the three DMSO collected supernatants was treated with 400 µl of 5 M NaOH and incubated for 1 h at room temperature. Afterwards, samples were shifted to 96-well plates and measured photometrically at an excitation wavelength of 485 nm and an emission wavelength of 8

515 nm. All the three values of supernatant were added together and loading efficiency was calculated as per equation 2.

*100% = A 100 µl sample of FDA in DMSO/H2O/isopropanol used during labelling, treated with 400 µl of 5M NaOH served as 100% control.

2.7. Rheological investigations The mucolytic properties of enzyme functionalized microparticle-mucus mixture were determined with a plate-plate rheometer (Haake Mars Rhemeter, 379-0200, Thermo Electron GmbH, Karlsruhe, Germany) (Mahmood et al., 2016a). Briefly, freshly collected porcine intestine was first rinsed with physiological saline (NaCl 0.9%) and then mucus was gently scrapped off from underlying tissue. For the rheological analysis, 500 µl of 1.0 % particle suspension in 100 mM phosphate buffer pH 6.8 was added to 4.5 ml of fresh intestinal mucus. The mixture was vortexed for 60 s followed by incubation at room temperature for 4 h. At time points 2 and 4 h the mixture was vortexed for 10 s and the rheological properties were determined by transferring 500 μl of each mucus-particle mixture to the rheometer. The oscillating measurements were carried out with a shear stress in the range of 0.2–500 Pa and temperature was maintained at 37 ± 0.1°C. Mucus samples mixed with PAA microparticle suspension and buffer alone served as references. Dynamic oscillatory test within the linear visco-elasticity region was performed at 1 Hz frequency.

2.8. Permeation across mucus gel layer The transport of microparticles across the mucus layer was investigated via a 24-transwell plate (Friedl et al., 2013). Briefly, 50 mg of mucus was spread on the transwells with the aim to establish a uniform barrier layer. The donor chamber was filled with 250 μl of FDA labeled PAA, PAA-PAP and PAA-BROM microparticle suspension (1.0 % in 100 mM phosphate 9

buffer pH 6.8) and the acceptor compartment contained 500 μl of same phosphate buffer. The plate wrapped in aluminium foil was placed on a rotating board incubated at 37 °C and continuous shaking at 300 rpm. 100 μl of samples from each acceptor chamber was drawn at specific time points 60, 120, 180 and 240 min and same volume of buffer was refilled in order to maintain the volume. Each collected sample was mixed with 400 μl of 5 M NaOH in order to hydrolyze FDA. The amount of permeated microparticles was calculated with reference to a 100% control value of each particle suspension. The same procedure performed upon the transwell-system without the mucus barrier served as 100% and only buffer without the microparticles across the mucus layer served as 0%.

2.9. Mucus penetration studies using silicon tubes Rotating silicon tube method was employed in order to probe the penetration ability of the microparticles within the mucus layer as described by our research group previously (Mahmood et al., 2016b). Briefly, silicon tubes were filled with 300 μl of mucus and one end was closed with small silicon cap. Afterwards, 50 μl of FDA labeled PAA, PAA-PAP and PAA-BROM microparticle suspension (1.0 % in 100 mM phosphate buffer pH 6.8) was added through the open end of the silicon tubes and sealed with another silicon cap. Buffer without any microparticle suspension served as blank. All the tubes were kept under horizontal rotation in an incubator at 37 °C. After 4 h of incubation, the tubes were frozen by incubating overnight at −20 °C and then cut into slices of 2 mm length. Each slice was treated with 300 μl of 5 M NaOH in order to hydrolyze FDA to sodium fluorescein. After incubation at 37 °C for 30 min, the resulting sodium fluorescein was analyzed and calculated using the 100% value of each particle suspension. 2.10. Determination of mucosal penetration potential Porcine intestinal mucosa obtained from a local slaughterhouse was used for evaluating the mucosal penetration potential of enzyme functionalized microparticles. The experimental set 10

up for this study was followed in accordance to method previously described by our group (Ijaz et al., 2015; Mahmood et al., 2017). Briefly, after being cleaned and rinsed with normal saline porcine small intestine was cut into small pieces of 3 × 2 cm and attached to vertically half cut 50 mL falcon tubes. As illustrated in Fig. 1, half tubes along with mucosa were incubated in an environment of 100% relative humidity and 37 oC at an angle of 45o. The intestinal mucosa was rinsed with phosphate buffer (100 mM, pH 6.8) for 5 min. Later, 100 μl of FDA loaded PAA, PAA-PAP and PAA-BROM microparticles (1.0 % in 100 mM phosphate buffer pH 6.8) were applied to the mucosal surface. After an incubation of 2 minutes at room temperature, the mucosa was continuously rinsed with phosphate buffer (100 mM, pH 6.8) serving as artificial intestinal fluid. Utilizing peristaltic pump a constant flow rate (1 ml/min) was provided over a period of 180 min. The buffer flowing down the intestinal mucosa was collected at times points 30, 60, 90, 120 and 180 min. Phosphate buffer flowing over the mucosa without any microparticles served as blank (0%) and FDA loaded microparticles suspension of PAA, PAA-PAP and PAA-BROM (1.0 % in 100 mM phosphate buffer pH 6.8) were employed as a standard (100%). All collected samples were vortexed for 30 s and then 100 μl from each time point elute was incubated with 200 μl of 5 M NaOH for 30 min at room temperature in order to quantitatively hydrolyze FDA to sodium fluorescein. Thereafter, 100 μl of each sample was transferred to microplate reader (Infinite M200, Tecan, Gröding, Austria) and fluorescence was recorded at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The experiment was performed in triplicate.

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Fig. 1 In-vitro experimental setup for evaluation of mucosal penetration potential of microparticles under relatively high humidity (100 %) and 37 oC 2.11. In-vivo study For in-vivo studies, minicapsules were prepared by filling 5 mg of microparticles into PCcaps™ capsules (average diameter ~2.5 mm and length ~7.2 mm). All minicapsules were dipped in 5% Eudragit L 100-55 acetonic solution and air dried for enteric coating. Afterwards, the minicapsules were stored in tightly closed containers at 4oC until use. The protocol employed for in-vivo study was in line with the Principles of Laboratory Animal Care and was approved by Animal Ethical Committee of Vienna, Austria. The in-vivo study for protease-functionalized microparticles was carried out with 16 Sprague–Dawley rats weighing in between 200–300 g. The rats were divided into four cohorts, each cohort comprising 4 animals. The first group was treated orally with minicapsules containing PAA microparticles labeled with FDA. The second group was fed with PAA-PAP microparticles loaded with FDA. The third group received FDA labeled PAA-BROM microparticles and the fourth group was treated with a mixture of mannitol and FDA.

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Rats were kept in fasted state for 2 h before the oral administration of minicapsules and throughout the whole experiment, but free access to water was ensured to them. Minicapsules were placed deep into the throat followed by feeding with 1.0 mL of 0.1% ascorbic acid solution. Ascorbic acid solution not only eases the swallowing but also reduces the pH with the aim to avoid prior disintegration of capsules. After 3 h, rats were euthanized in high concentration CO2 chamber and sacrificed as previously described by our research group (Sakloetsakun et al., 2010). Instantly after removal the gastrointestinal tract was divided into the anatomical segments stomach, duodenum, jejunum I, jejunum II, jejunum III, ileum, cecum and colon. These segments were longitudinally cut to wide them open, gently rinsed with phosphate buffer and then treated with 5 M NaOH overnight in order to assure complete cleavage and extraction of FDA. Subsequently, samples were centrifuged at 13,500 rpm at 25oC for 15 min and absorbance was measured at Ex 485 nm and Em 535 nm via microplate reader (Infinite M200, Tecan, Gröding, Austria).

2.12. Statistical data analysis For in-vitro analysis, all the results are expressed as means (±SD) of at least three experiments. Data were analyzed utilizing the Student’s t-test, two tails with 95% confident interval (p value < 0.05) as the minimal level of significance. For in-vivo analysis, each experiment was conducted four times. The average percentage of FDA remaining in individual segments of gastrointestinal tract after treatment with protease-functionalized microparticles and PAA microparticles were compared via ANOVA. With a confidence interval of 95%, a probability of less than 0.05 (p < 0.05) was considered statistically significant.

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3. RESULTS 3.1. Synthesis and UV-Vis analysis of PAA-PAP and PAA-BROM conjugates By means of carbodiimide based reaction the proteases PAP and BROM were covalently immobilized to PAA. Conjugation takes place via amide bond formation between amino groups of PAP/BROM and carboxylic groups of the polymer. EDAC and NHS, in an appropriate concentration, assured sufficient immobilization of enzymes to the polymer. The synthetic pathway and proposed chemical structure of the developed polymer-enzyme conjugate are depicted in Fig. 2. The amount of PAP and BROM bound to polymer analyzed via Bradford reagent was determined to be 319.01 ± 55.66 µg/mg and 208.33 ± 13.28 µg/mg, respectively. In contrast, the control samples having been prepared without addition of EDAC and NHS exhibited 37.95 µg/mg and 29.54 µg/mg remaining content of PAP and BROM, respectively. The lyophilized final product was an off-white to very light yellow colored amorphous fluffy mass with mild characteristic odor. 3.2. Cytotoxicity studies Resazurin assay revealed that newly synthesized conjugates PAA-PAP and PAA-BROM do not exhibit cytotoxicity at 0.5% (m/v) concentration over an exposure of 24 h. The outcomes of this study are shown in Fig. 3. After 24 h treatment, PAA-PAP exhibited mild to moderate level separation of cells from the bottom of wells. However, at least 70% of the Caco-2 cells were found viable at the end of study demonstrating both PAA-PAP and PAA-BROM to be non-toxic. Statistical analysis illustrated significant difference (p < 0.05) in percentage cell viability of the PAA-PAP after an exposure of 3 and 24 h. 3.3. Physicochemical properties of microparticles An overview on the physicochemical properties of the particles is provided in Table 1. The mean diameter of microparticles remained between 2-3 µm, however, particles beyond this range could also be observed as shown in the size distribution of PAA, PAA-PAP and PAA14

BROM in Fig. 4. All the microparticles were characterized via transmission electron microscopy (TEM). The available resolution demonstrated no absolute differences in morphology of individual microparticles. All microparticles appeared irregular shaped, however, PAA microparticles were relatively compact and smooth compared to PAA-PAP microparticles and PAA-BROM microparticles (data shown as supplementary Fig. 1(s)). Zeta potential measurements in demineralized water showed overall negative values for all microparticles. FDA payload for all microparticles was found to be in between 7-9 %. FDA loading resulted in little increase in the particle size of all microparticles but this was statistically insignificant. In addition, enzymatic activity was determined after microparticles preparation and was found to be at least 75% compared to enzyme functionalized polymer that was supposedly sufficient for the penetration of the mucus layer. 3.4. Microparticle-mucus interaction Treatment of mucus with both PAA-PAP and PAA-BROM microparticles caused its liquefaction that could be observed by simply tilting/inverting the sample tubes at the end of study. As illustrated in Fig. 5, the enzyme functionalized microparticles significantly (p < 0.05) decreased viscosity of mucus over a study period of 4 h as compared to blank. In case of PAA microparticles the viscosity of the microparticles-mucus mixture slightly increased (1.39-folds). However, this increase was statistically insignificant (p > 0.05) and no difference in flow could be observed when compared to blank via tilting tube method. 3.5. Mucus permeation The influence of proteases on permeation characteristics was evaluated via trans-well diffusion chamber and outcomes of the study are illustrated in Fig. 6. All the microparticles exhibited a fast permeation up to 2 h, followed by a slow permeation stage. By the end of 4 h, PAA-PAP microparticles exhibit a 4.3-fold higher permeation as compared to PAA 15

microparticles and statistically, this enhancement was found significant (p < 0.05). Moreover, PAA-BROM microparticles also exhibited 2.1- folds higher permeation across mucus gel layer as compared to PAA microparticles. However, this permeation was ~50% to that of PAA-PAP microparticles. 3.6. Mucus diffusion As the trans-well method gives idea about the improvement in permeation through a mucus layer, the deepest mucus penetration was revealed via rotating silicon tube method and results are displayed in Fig. 7. Due to protease functionalization the transport capacity of microparticles was enhanced. Particularly the difference is noticeable in the initial five segments, each segment being 3 mm. After being in direct contact with mucus PAA-PAP, PAA-BROM and PAA exhibited 62.23%, 28.66% and 12.20% of microparticles penetrated through these segments collectively. Statistically, the difference in penetration ability of modified microparticles is significantly different to that of unmodified polymer microparticles. 3.7. Mucosal residence The in-vitro mucus penetration potential was further studied over porcine intestinal tissue. During initial incubation, the polymeric microparticles were allowed to diffuse the mucus and then rinsed continuously for 3 h with phosphate buffer. Polymeric microparticles remaining on porcine intestinal mucosa were evaluated by measuring the amount of fluorescence in the rinsed buffer at defined time points. Microparticles that penetrated deeper into the mucus gel exhibited a prolonged residence time. The percentage of FDA loaded microparticles remaining on intestinal mucosa is shown in Fig. 8. During the initial 30 min a major proportion of all microparticles that could not penetrate mucus were washed away. Moreover, the results revealed that PAA-BROM microparticles could not penetrate as deep as PAA-PAP microparticles and were therefore washed away in higher proportion within 30 min. By the 16

end of study, PAA-BROM microparticles demonstrated ~45% penetration potential to that of PAA-PAP microparticles. The results of all the experiments with mucus are very much in line and support each other. 3.8. In-vivo study After oral ingestion of minicapsules, microparticles were allowed to penetrate into mucus for 3 h. Their distribution along the gastrointestinal tract after this incubation time period is illustrated in Fig. 9. Although minicapsules were enteric coated yet a little proportion of microparticles was found in stomach. Control (FDA + mannitol) faced fast excretion and resulted in lowest amount of fluorescent marker recovered from all segments. PAA microparticles were found in minor quantities in the most distal segment of the small intestine and colon. However, a significant amount of FDA was recovered from proximal part of small intestine (duodenum and jejunum I) in case of PAA-PAP microparticles and a considerable amount was residing in distal part of small intestine (jejunum III and ileum) for PAA-BROM microparticles. Statistically, the intestinal retention via protease-functionalization was found significant and therefore, the results provide strong evidence for the potential of protease functionalized microparticles to penetrate the intestinal mucus gel layer. 4. DISCUSSION Within the current study newly developed mucus permeating polymeric microparticles comprising PAP and BROM were evaluated regarding an enhanced diffusion/permeation across the porcine intestinal mucus gel layer and a prolonged residence time at mucosal surface compared to microparticles of unmodified polymer. Apart from the surface charge and concentration, the size of the particles plays a crucial role for penetration into the mucus. Generally nanoparticles with size < 200 nm to some extent hold tendency to permeate through the mucus. However, the larger particles (> 1µm) adsorb in the form of a monolayer on the mucosal surface that further acts like smooth flat surface for the following particles. In such a case, these large particles when orally administered can have 17

three pathways to follow; being captured by gut-associated lymphoid tissue, mucoadhesion and direct fecal elimination but no mucosal penetration to the absorption layer (Ponchel et al., 1997). In order to overcome the mucosal barrier and reach the absorption lining, the prospective approaches described in literature include dense particle coating with muco-inert polymer such as ultra-low molecular weight PEG (2 kDa) (Lai et al., 2007) and deployment of proteases on the surface of drug carriers (Müller et al., 2013). Proteases are reported to be stable in in-vitro environment of simulated intestinal fluid. They are able to cleave the complex mucosal structure thereby decreasing the mucus viscosity and making way for drug carriers. The way the proteases reduce the cross-linking capacity of the mucus is yet to be exhaustively understood. However, the broad specificity of PAP and BROM could give us a clue. On one hand, at position P2 PAP likely prefers the presence of lipophilic amino acids such as Phenylalanine (Phe) and Tyrosine (Tyr) and at position P1, the presence of Glycine (Gly), Methionine (Met), Cysteine (Cys) and Arginine (Arg) and on the other hand the acceptance of Valine (Val) is to minimum at position P10. Similarly, BROM possesses the ability to cleave the carbonyl terminal sites of Lysine (Lys), Alanine (Ala), Glycine (Gly) and Tyrosine (Tyr) (Davy et al., 2000; Kennedy and White, 1984). Müller et al. illustrated through an in vivo study that PAP functionalized nanocarriers show a prolonged residence time in the intestinal tract of rats, as compared to non-functionalized nanocarriers (Müller et al., 2014). In another study Pereira de Sousa et al. demonstrated that nanocarriers decorated with BROM exhibited more significant effect in altering the mucus structure and the higher performance in permeating a fresh mucus layer compared to PAP conjugated nanocarriers (de Sousa et al., 2015). As far as all these studies are concerned they described the impact of mucolytic agents immobilized on the carriers that are <300 nm size. Typically, nanoparticles possess large surface to volume ratio that often causes a fast release of the drugs. As the size of drug-loaded particles increases, drug-release kinetics are usually greatly improved, and sustained release 18

of therapeutics over days can be achieved with enhanced therapeutic efficacies. Therefore, a successful development of mucus penetrating microparticles is therapeutically important in a number of ways; particularly the release of drugs could efficiently be controlled as compared to nanoparticles. With the aim to achieve a higher concentration of drug at the absorption lining via microparticles, mucolytic properties were harvested to microparticles by immobilizing PAP and BROM on polymeric backbone of PAA. Conjugation was based on carbodiimide chemistry; however, the reaction was performed under mild condition that preserved the functionality of both enzymes. PAP immobilization on PAA was 319.0 µg/mg being in the same range as in previous studies with 230.1 µg/mg [7], 361.1 µg/mg [10] and 396.6 µg/mg [9]. In contrast, BROM conjugation having been determined to be 208.3 µg/mg was to some extent lower than in a previous study where 338.4 µg/mg were immobilized to the polymer [7]. As in these previous studies the efficacy of PAP and BROM to modify mucus in these concentration range on polymers were already demonstrated, it can be assumed that they were also effective in this study. The cytotoxicity analysis of polymer conjugates by resazurin test demonstrated mild detachment of cells from the plate in 24 h study, possibly due to the action of the proteolytic agents but it did not affect the overall results of the study. Afterwards, microparticles were prepared utilizing ionic gelation method, with relatively mild conditions it would allow the maintenance of the enzymatic activity of incorporated enzyme. Physicochemical characterization revealed that mean diameter of particles comprising conjugates of both enzymes was slightly higher than that of PAA microparticles. Moreover, all microparticles bear negative zeta potential owing to the carboxylic acid moieties of PAA and embedding of PAP and BROM slightly decreased the negative surface charge in comparison to PAA microparticles. The negative zeta potential of microparticles ensures no ionic interactions with 19

the negatively charged mucosa. The ability of protease-functionalized microparticles to cleave mucoglycoprotein substructures was evaluated initially by rheological measurements. The viscoelastic properties of mucus are strongly dependent on the content of mucin and these immobilized enzymes are able to attack the non-glycosylated regions of the shell protecting the protein (Sellers et al., 1988). On one hand, decrease in viscosity via reduction in crosslinking efficiency of mucus by incubating it with PAP and BROM functionalized microparticles reveals enzymatic efficiency and on the other hand, it leads to an undesired mild liquefaction of mucus, however, this liquefaction was not visually observable and all mucus-microparticle mixture samples appeared very similar. Afterwards, three different methods were carried out in order investigate the penetration behavior of microparticles in intestinal mucus. Outcomes of all methods illustrated higher permeation and deeper penetration of protease-functionalized microparticles as compared to PAA microparticles. All methods were performed over at 37 oC in order to ensure comparability. Moreover, in-vivo study was performed that comprised of oral treatment of rats with FDA labelled PAA, PAA-PAP and PAA-BROM microparticles in order to evaluate the impact of proteases on mucus penetration potential and ultimately gastrointestinal residence time. The experiment was based on the fact that microparticles that are able to diffuse deeper into mucus exhibit a prolonged residence time and therefore can be analyzed via fluorescent labelling. From the higher concentration of PAA-PAP and PAA-BROM microparticles in the intestine, it may be concluded that immobilization of proteases leads to an enhanced penetration of the microparticles into the mucus and therefore a prolonged residence time. Moreover, PAP decorated microparticles not only demonstrated higher decrease in viscosity but also higher permeation through porcine intestinal mucus and prolonged retention as mucosal surface compared to PAA-BROM microparticles. This overall better performance of PAA-PAP microparticles could be ascribed to the higher degree of 20

conjugation of PAP upon polymer conjugation and slightly lower enzymatic activity loss during the microparticle generation. The scope of these protease functionalized microparticles ranges from variety of lipophilic drugs irrespective of molecule size to hydrophilic macromolecular drugs such as low molecular weight heparins or DNA/RNA-based drugs. Merely peptide drugs that are substrate for the conjugated protease will not be appropriate. Bernkop-Schnürch et al. compared mucolytic activity of various compounds and their interactions with the model peptide (Bernkop-Schnürch et al., 1999). In the light of the outcomes of their study the use of proteases is significantly limited for (poly)peptide drug delivery due to degradation. Moreover, there will rarely be a (poly)peptide drug that is not a substrate for papain, therefore, possibly sulfhydryl compounds could be useful option in such cases depending on the chemical structure of the (poly)-peptide drug. Nevertheless, proteasefunctionalized microparticles exhibiting potential for mucus penetration in both in-vitro and in-vivo experiments are therefore a significant development in mucus penetrating systems and mucosal drug delivery. 5. CONCLUSION The concept of immobilizing proteases to drug delivery systems in order to gain higher mucus penetration has successfully been extended from nanoparticles in the past to polymeric microparticles of size 2-3 µm in this study. PAP and BROM functionalized PAA microparticles are not only able to permeate the mucus gel layer but also their residence time at mucosal surface is prolonged due to deeper penetration into the mucus. Moreover, it was observed that extent and type of enzyme conjugation significantly affected the mucus penetration potential of polymeric microparticles. Accordingly, mucosal drug delivery inparticular oral bioavailability of a number of drugs may be improved via this novel mucus permeating microparticles approach.

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Conflict of interest The authors report no conflicts of interest.

Acknowledgement The authors are thankful and would like to extend acknowledgement to Higher Education Commission of Pakistan (HEC) and the Austrian Agency for International Cooperation in Education and Research (ÖAD) for their support and funding.

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REFERENCES Bernkop-Schnürch, A., Valenta, C., Daee, S.M., 1999. Peroral Polypeptide Delivery. Arzneimittelforschung 49, 799-803. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248254. Davy, A., Sørensen, M.B., Svendsen, I., Cameron-Mills, V., Simpson, D.J., 2000. Prediction of protein cleavage sites by the barley cysteine endoproteases EP-A and EP-B based on the kinetics of synthetic peptide hydrolysis. Plant Physiol. 122, 137-146. de Sousa, I.P., Cattoz, B., Wilcox, M.D., Griffiths, P.C., Dalgliesh, R., Rogers, S., BernkopSchnürch, A., 2015. Nanoparticles decorated with proteolytic enzymes, a promising strategy to overcome the mucus barrier. Eur. J. Pharm. Biopharm. 97, 257-264. Ensign, L.M., Cone, R., Hanes, J., 2012. Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers. Adv. drug deliv. Rev. 64, 557-570. Friedl, H., Dünnhaupt, S., Hintzen, F., Waldner, C., Parikh, S., Pearson, J.P., Wilcox, M.D., Bernkop‐Schnürch, A., 2013. Development and evaluation of a novel mucus diffusion test system approved by self‐nanoemulsifying drug delivery systems. J. Pharm Sci. 102, 44064413. Ijaz, M., Matuszczak, B., Rahmat, D., Mahmood, A., Bonengel, S., Hussain, S., Huck, C.W., Bernkop-Schnürch, A., 2015. Synthesis and characterization of thiolated β-cyclodextrin as a novel mucoadhesive excipient for intra-oral drug delivery. Carbohydr. Polym. 132, 187-195. Itoyama, K., Tanibe, H., Hayashi, T., Ikada, Y., 1994. Spacer effects on enzymatic activity of papain immobilized onto porous chitosan beads. Biomaterials 15, 107-112. Kennedy, J.F., White, C.A., 1984. Industrial enzymology: The application of enzymes in industry. Edited by Tony Godfrey and Jon Reichelt, Macmillan, The Nature Press, London, 1983. Pp x+ 582, Price£ 40.00. ISBN 0943818001. Wiley Online Library. 23

Köllner, S., Dünnhaupt, S., Waldner, C., Hauptstein, S., de Sousa, I.P., Bernkop-Schnürch, A., 2015. Mucus permeating thiomer nanoparticles. Eur. J. Pharm. Biopharm. 97, 265-272. Lai, S.K., O'Hanlon, D.E., Harrold, S., Man, S.T., Wang, Y.-Y., Cone, R., Hanes, J., 2007. Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus. Proceedings of the National Academy of Sciences 104, 1482-1487. Lai, S.K., Wang, Y.-Y., Hida, K., Cone, R., Hanes, J., 2010. Nanoparticles reveal that human cervicovaginal mucus is riddled with pores larger than viruses. Proceedings of the National Academy of Sciences 107, 598-603. Lee, V., 2000. Mucosal drug delivery. Journal of the National Cancer Institute. Monographs, 41-44. Mahmood, A., Bonengel, S., Laffleur, F., Ijaz, M., Idrees, M.A., Hussain, S., Huck, C.W., Matuszczak, B., Bernkop-Schnürch, A., 2016a. Can thiolation render a low molecular weight polymer of just 20-kDa mucoadhesive? Drug Dev.Ind.Pharm. 42, 686-693. Mahmood, A., Bonengel, S., Laffleur, F., Ijaz, M., Leonaviciute, G., Bernkop-Schnürch, A., 2015. An in-vitro exploration of permeation enhancement by novel polysulfonate thiomers. Int. J. Pharm. 496, 304-313. Mahmood, A., Lanthaler, M., Laffleur, F., Huck, C.W., Bernkop-Schnürch, A., 2017. Thiolated chitosan micelles: Highly mucoadhesive drug carriers. Carbohydr. Polym. 167, 250-258. Mahmood, A., Prüfert, F., Efiana, N.A., Ashraf, M.I., Hermann, M., Hussain, S., BernkopSchnürch, A., 2016b. Cell-penetrating self-nanoemulsifying drug delivery systems (SNEDDS) for oral gene delivery. Exp. Opinion Drug Deliv. 13, 1503-1512. Müller, C., Leithner, K., Hauptstein, S., Hintzen, F., Salvenmoser, W., Bernkop-Schnürch, A., 2013. Preparation and characterization of mucus-penetrating papain/poly (acrylic acid) nanoparticles for oral drug delivery applications. J. Nanoparticle Research 15, 1353.

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Müller, C., Perera, G., König, V., Bernkop-Schnürch, A., 2014. Development and in vivo evaluation of papain-functionalized nanoparticles. Eur. J. Pharm. Biopharm. 87, 125-131. Pearson, J.P., Chater, P.I., Wilcox, M.D., 2016. The properties of the mucus barrier, a unique gel–how can nanoparticles cross it? Therapeutic Deliv. 7, 229-244. Ponchel, G., Montisci, M.-J., Dembri, A., Durrer, C., Duchêne, D., 1997. Mucoadhesion of colloidal particulate systems in the gastro-intestinal tract. Eur. J. Pharm. Biopharm. 44, 25-31. Sakloetsakun, D., Perera, G., Hombach, J., Millotti, G., Bernkop-Schnürch, A., 2010. The impact of vehicles on the mucoadhesive properties of orally administrated nanoparticles: a case study with chitosan-4-thiobutylamidine conjugate. AAPS Pharm. Sci. Tech. 11, 11851192. Sellers, L.A., Allen, A., Morris, E.R., Ross-Murphy, S.B., 1988. Mucus glycoprotein gels. Role of glycoprotein polymeric structure and carbohydrate side-chains in gel-formation. Carbohydr. Research 178, 93-110. Zhang, H., Zhang, J., Streisand, J.B., 2002. Oral mucosal drug delivery. Clinical Pharmacokinetics 41, 661-680.

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Table 1 Characterization of microparticles

Polymer

Enzyme content polymer (µg/mg)

PAA

Microparticle size (µm)

Zeta potential

-

2.279 ± 2.249

-18.02

PAA-PAP

319.01 ± 55.66

2.416 ± 1.998

-8.24

84.19

8.89

PAA-BROM

208.33 ± 13.28

2.685 ± 1.769

-12.71

76.86

8.97

of

Microparticle‘s enzyme activity (%)

FDA loading efficiency (%)

7.16

26

Fig. 2 Schematic diagram describing covalent attachment of proteases to polyacrylates in presence of bi-catalytic system and presumptive chemical substructure of protease functionalised PAA conjugates.

27

Cell viability (%)

100 75 50 25

-B

R

O

M

P PA A

-P A PA A

PA A

X10 0

Tr ito n

M EM

0

Fig. 3 Resazurin assay performed on Caco-2 cells monolayer after incubation with PAA, PAA-PAP and PAA-BROM for 3 h (white bars) and for 24 h (Black bars) applied in a concentration of 0.5% (m/v). Triton® X-100 (2%; m/v) serves as negative control and white MEM as positive control. Results are expressed as means ±S.D. of three experiments.

28

Relative intensity (%)

100 75 50 25 0 0

2 4 6 M icroparticles size (m)

8

Fig. 4 Particle size distribution of PAA microparticles (○), PAA-PAP microparticles (□), and PAA-BROM microparticles () generated via ionic gelation with CaCl2.

29

Viscosity [Pas]

5 4 3 2 1

R O M PA A -B

P A PA A -P

PA A

B la

nk

0

Fig. 5 Overview of changes in porcine intestinal mucus viscosity after incubation with an equal volume of 0.5% (m/v) PAA, PAA-PAP and PAA-BROM microparticles within 4 h at room temperature. Indicated values represent the mean (±SD) of at least three experiments.

30

20

FDA [%]

15 10 5 0 0

60

120

180

240

Time (min)

Fig. 6 Percentage of FDA labeled PAA microparticles (○), PAA-PAP microparticles (□), and PAA-BROM microparticles () having permeated a layer of porcine intestinal mucus within 4 h. The donor compartment contained 1% (m/v) of microparticles in 100 mM phosphate buffer pH 6.8 at 37oC. Indicated values are means of at least three experiments (±SD).

31

40

FDA [%]

30 20 10

8

7

6

5

4

3

2

1

0 Segments

Fig. 7 Penetration capacity of FDA labeled PAA-PAP microparticles (white bars), PAABROM microparticles (grey bars) and PAA microparticles (black bars) analyzed via rotating silicon tube method at 37oC for 4 h. Indicated values are means ± SD (n = 3).

32

100

FDA [%]

80 60 40 20 0 0

30

60

90

120

150

180

Time (min)

Fig. 8 Percentage of FDA loaded to microparticles remaining on excised porcine intestinal mucosa being continuously rinsed with 100 mM phosphate buffer pH 6.8 for 3 h; ( ○) PAA microparticles, (□) PAA-PAP microparticles and () PAA-BROM microparticles. Presented data is mean ± standard deviation of three experiments.

33

12

FDA [%]

9 6 3

D

St om

ac h uo de nu Je m ju nu m Je 1 ju nu m Je 2 ju nu m 3 Ile um C ol on

0

Fig. 9 Percentage of FDA incorporated mannitol (dark grey bars), PAA microparticles (light grey bars), PAA-PAP microparticles (black bars) and PAA-BROM microparticles (white bars) remaining on rat gastrointestinal mucosa within 3 h. Microparticles were administered orally as enteric coated minicapsules to rats. Indicated values are means (±SD) of four animals.

34