Accepted Manuscript Title: Interactions of Mussel-inspired Polymeric Nanoparticles with Gastric Mucin: Implications for Gastro-retentive Drug Delivery Authors: Suhair Sunoqrot, Lina Hasan, Aya Alsadi, Rania Hamed, Ola Tarawneh PII: DOI: Reference:

S0927-7765(17)30264-3 http://dx.doi.org/doi:10.1016/j.colsurfb.2017.05.005 COLSUB 8534

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

19-1-2017 6-4-2017 2-5-2017

Please cite this article as: Suhair Sunoqrot, Lina Hasan, Aya Alsadi, Rania Hamed, Ola Tarawneh, Interactions of Mussel-inspired Polymeric Nanoparticles with Gastric Mucin: Implications for Gastro-retentive Drug Delivery, Colloids and Surfaces B: Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.05.005 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.

Interactions of Mussel-inspired Polymeric Nanoparticles with Gastric Mucin: Implications for Gastro-retentive Drug Delivery

Suhair Sunoqrot*, Lina Hasan, Aya Alsadi, Rania Hamed, and Ola Tarawneh

Department of Pharmacy, Faculty of Pharmacy, Al-Zaytoonah University of Jordan, Amman, Jordan *

Corresponding author: Suhair Sunoqrot, PhD, Assistant Professor of Pharmaceutics, Department

of Pharmacy, Faculty of Pharmacy, Al-Zaytoonah University of Jordan, P.O. Box 130, Amman 11733,

Jordan,

Tel:

+962-6-4291511

Ext.

312,

Fax:

+962-6-4291432,

Email:

[email protected]

Present address: Department of Materials Science and Engineering, University of California, Berkeley, Berkeley, CA 94720, USA

Graphical abstract

After 8 h incubation Polydopamine-coated nanoparticles

Uncoated nanoparticles

Stomach mucosa Mucoadhesive polydopamine-coated polymeric nanoparticles as a promising new platform for gastro-retentive drug delivery.

1

Highlights 

Polydopamine-coated polymeric nanoparticles were designed as mucoadhesive platforms.



Nanoparticle interactions with gastric mucin were investigated in vitro and ex vivo.



Favorable mucoadhesion of the nanoparticles validates their use for gastric retention.

ABSTRACT Mussel-inspired polydopamine (pD) coatings have several unique characteristics such as durability, versatility, and robustness. In this study, we have designed pD-coated nanoparticles (NPs) of methoxy polyethylene glycol-b-poly(ε-caprolactone) (mPEG-PCL@pD) as prospective nanoscale mucoadhesive platforms for gastro-retentive drug delivery. Successful pD coating on the NPs was confirmed by Transmission Electron Microscopy and X-ray Photoelectron Spectroscopy. Mucoadhesion of pD-coated NPs was investigated in vitro using commercially available mucin under stomach lumen-mimetic conditions. Mucin-NP interactions were monitored by dynamic light scattering, which showed a significant change in particle size distribution of pD-coated NPs at mucin/NP ratios of 1:1, 1:2, and 1:4 w/w. Turbidity measurements indicated the formation of large mucin-NP aggregates causing a significant increase in turbidity at mucin/NP ratios of 2:1 and 4:1 w/w. pD-coated NPs exhibited a significantly higher mucin adsorption ability compared to uncoated NPs at mucin/NP ratios of 1:4, 1:2, and 1:1 w/w. Zeta potential measurements demonstrated that mucin-pD-coated NP interactions were not electrostatic in nature. An ex vivo wash-off test conducted using excised sheep stomach revealed that 78% of pD-coated NPs remained attached to the mucosa after 8 h of incubation, compared to only 33% of uncoated NPs. In vitro release of rifampicin, used as a model drug, showed a similar controlled release profile from both pD-coated and uncoated NPs. Our results serve to expand the versatility of mussel-inspired coatings to the design of mucoadhesive nanoscale vehicles for oral drug delivery.

KEYWORDS: polydopamine, polymeric nanoparticles, mucin, mucoadhesion, gastric retention.

INTRODUCTION Oral drug delivery remains the most desirable and convenient route due to the ease of administration, noninvasiveness, and patient compliance [1, 2]. This route is mostly hindered by 2

poor oral bioavailability for poorly soluble drugs and drugs with a narrow absorption window in the upper gastrointestinal tract (GIT). One approach to overcome these limitations is to prolong the gastric residence time of the drug using gastro-retentive dosage forms [3, 4]. These formulations are designed to increase the residence time of drug molecules in the upper GIT via a number of approaches, including the use of floating, expandable, or mucoadhesive formulations [5, 6]. These approaches are particularly advantageous for drugs with low aqueous solubility and narrow absorption windows, as they allow sufficient time for drug dissolution in the stomach to ensure adequate absorption. Additionally, gastric retention has been explored for drugs that act locally in the stomach, such as anti-ulcer medications and antibiotics for H. pylori eradication, weakly basic drugs with low solubility in intestinal fluids, drugs that are unstable in the colon, and drugs that are primarily absorbed in the stomach [3, 7-9]. Polymers such as chitosan and carbomers have been traditionally used as mucoadhesive vehicles for mucosal delivery as they allow intimate contact with the absorbing mucosa, resulting in improved absorption and enhanced bioavailability [8, 10-13]. However, success with these systems and other gastro-retentive approaches has been limited due to rapid mucus turnover and high stomach motility. Moreover, the stomach content is highly hydrated, decreasing the adhesion of most mucoadhesive polymers [5]. This highlights the need to develop a system that is highly resistant to these factors and has strong mucoadhesive properties in aqueous environments in order to allow sufficient time for drug release and subsequent absorption. Mussels and other marine organisms have been investigated as a potential source for water-resistant bioadhesives [14]. Several mussel adhesive proteins have been identified and explored for diverse biomedical applications as bioadhesives due to their biocompatible, biodegradable, and nonimmunogenic nature [15]. Inspired by the composition of mussel adhesive proteins, Messersmith et al. have utilized dopamine self-polymerization in alkaline conditions to form thin surface-adherent polydopamine (pD) films onto a wide range of organic and inorganic substrates [16]. These films have been shown to mediate surface functionalization of the coated substrates with a variety of ligands through interactions with the oxidized catechol moieties of pD. Mussel-inspired chemistry has also been investigated for mucoadhesive applications. For example, 3,4-dihydroxyphenyl-L-alanine (DOPA)-conjugated poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) block copolymers [17] and 4-arm poly(ethylene glycol) (PEG) [18] have been shown to exhibit favorable mucoadhesive 3

interactions in vitro. Recently, Kim et al. have reported enhanced mucoadhesive properties of caffeic acid-modified chitosan due to the formation of irreversible catechol-mucin crosslinks [19]. Thus, mussel-inspired coatings could be considered as a new class of mucoadhesive platforms with promising potential, but they have not yet been properly evaluated for mucosal drug delivery applications. Among the various types of nanoparticulate systems, polymeric nanoparticles (NPs) based on amphiphilic block copolymers such as methoxy poly(ethylene glycol)-b-ε-caprolactone (mPEG-PCL), have gained a great deal of attention due to their biodegradable and biocompatible properties, low toxicity, and ability to encapsulate a wide variety of molecules in their matrix [20-22]. Polymeric NPs have been widely investigated for drug delivery through a variety of routes of administration, including the oral route. Additionally, NP fabrication can be readily achieved using simple methods such as emulsion-solvent evaporation and nanoprecipitation, without the need for sophisticated equipment [23-26]. Building upon the versatility of polymeric NPs and mussel-inspired adhesives, we designed pD-coated mPEG-PCL NPs as novel mucoadhesive platforms targeting the gastric mucosa. Mucoadhesive properties of the NPs were evaluated by investigating their interactions with gastric mucin as well as the stomach mucosa, providing the first evidence on the potential use of mussel-inspired biodegradable NPs for oral drug delivery.

EXPERIMENTAL SECTION Materials Poly(ethylene glycol) monomethyl ether MW 5,000 Da (mPEG5K), ε-caprolactone, tin(II) 2ethylhexanoate (Sn(Oct)2), dopamine hydrochloride, Trizma® base, mucin from porcine stomach type III, nile red (NR), and rifampicin (RF) were obtained from Sigma-Aldrich (St Louis, MO, USA). Dichloromethane (DCM) and acetone were obtained from Fisher Scientific (Loughborough, UK); diethyl ether was obtained from Tedia (Fairfield, OH, USA).

Synthesis of mPEG-PCL mPEG-PCL copolymer was synthesized by ring opening polymerization of ε-caprolactone in the presence of mPEG5K as previously described [24]. Briefly, mPEG5K (1 g) was weighed and transferred to a 50 mL Schlenck flask connected to a vacuum pump and dried under vacuum for 4

1 h. ε-caprolactone (0.5 g, 500 μL) was then added to the flask and dried for an additional 1 h. Sn(Oct)2 (30 mg, approximately 3 drops) was added to the flask and dried under vacuum for 2 min. The flask was placed in an oil bath pre-heated to 120 °C, and polymerization was carried out under gentle stirring at 120 rpm for 24 h. The flask was then cooled to room temperature (RT) and 5 mL DCM was added to dissolve the product. DCM was partially evaporated to adjust the solution viscosity, and the copolymer was precipitated in cold diethyl ether, vacuum filtered, and

stored

in

a

dessicator.

1

H

NMR

(500

MHz,

CDCl3):

1.38

ppm

(m,

O=C−CH2−CH2−CH2−CH2−CH2−O−), 1.65 ppm (m, O=C−CH2−CH2−CH2−CH2−CH2−O−), 2.25 ppm (t, O=C−CH2−CH2−CH2−CH2−CH2−O−), 3.38 ppm (s, CH3−O−), 3.62 ppm (s, −O−CH2−CH2−O−), 4.06 ppm (t, O=C−CH2−CH2−CH2−CH2−CH2−O−). The MW of the PCL block was estimated to be 9,200 Da based on the relative integration ratio of the peak corresponding to the ethylene oxide repeating units at 3.62 ppm and that of the PCL backbone at 2.25 ppm.

Preparation of mPEG-PCL@pD NPs mPEG-PCL@pD NPs were prepared by the nanoprecipitation method as previously described [27]. Briefly, 60 mg of mPEG-PCL was weighed and dissolved in 3 mL acetone. This solution was added dropwise to 6 mL Tris buffer (10 mM, pH 8.5) under stirring at 300 rpm. The NP dispersion was stirred overnight at RT to evaporate acetone. The next day, dopamine HCl (6 mg) was added to the NP dispersion and vigorously stirred for 24 h to produce mPEG-PCL@pD NPs. The NPs were then centrifuged at 13,000 rpm (9,960 × g) for 30 min at RT to remove excess dopamine HCl, and the supernatant was resuspended in 6 mL double distilled water (ddH2O) for subsequent experiments. mPEG-PCL NPs were prepared as above except ddH2O was used instead of Tris buffer without the addition of dopamine HCl. NR- and RF-loaded NPs were similarly prepared by dissolving either NR (600 μg) or rifampicin (6 mg) in acetone together with mPEG-PCL (60 mg) in the first step. Unencapsulated NR and RF were removed by centrifugation (13,000 rpm for 30 min at RT) and repeated washing with ddH2O. For the preparation of dye- and drug-loaded mPEG-PCL@pD NPs, NPs were redispersed in 6 mL Tris buffer (10 mM, pH 8.5) containing 1 mg/mL dopamine HCl and vigorously stirred at RT for 24 h. Excess dopamine HCl was removed as described above.

5

Characterization of mPEG-PCL@pD NPs NP morphology before and after pD coating was examined by Transmission Electron Microscopy (TEM). One drop of uncoated and pD-coated NP suspension was placed onto 400 mesh carbon-coated copper grids (Electron Microscopy Sciences, Hatfield, PA, USA) and left to dry overnight at RT. Each sample was stained with uranyl acetate and then analyzed using a Tecnai 12 TEM (FEI, Hillsboro, OR, USA) at an accelerating voltage of 120 kV. Successful pD coating on the NP surface was verified by X-ray Photoelectron Spectroscopy (XPS). TiO2-coated silicon substrates were cleaned by consecutive sonication in ddH2O, acetone, and isopropanol for 10 min each, followed by drying under a stream of N2 and then plasma discharge (Harrick Plasma Cleaner, Ithaca, NY, USA). A drop of each NP suspension was then placed onto the surface of the substrates and left to dry overnight. Substrates were completely dried under vacuum prior to analysis using a PHI 5600 spectrometer (PerkinElmer, Waltham, MA, USA) equipped with an Al monochromated 2 mm filament and a built-in charge neutralizer. The X-ray source operated at 350 W power and 15.0 V voltage. Survey scans were performed between 0 and 1100 eV electron binding energies. High resolution spectra of the N1s region were obtained between 395 and 410 eV. Charge correction was performed setting the C1s peak at 285.0 eV. Data analysis was conducted using MultiPak software version 9.6.015.

Preparation of mucin solutions A stock solution of mucin was prepared at 20 mg/mL in ddH2O and stored at 4 °C throughout the experiments. Mucin working solutions were freshly prepared prior to each experiment by diluting the stock in HCl (pH 2) to 2 mg/mL followed by sonication in a bath sonicator for 30 min.

Preparation of mucin-NP mixtures Mixtures of mucin with mPEG-PCL@pD and mPEG-PCL NPs were prepared at various mucin: NP ratios (0, 1:4, 1:2, 1:1, 2:1, and 4:1 w/w) by adding the appropriate volume of presonicated mucin working solution (2 mg/mL in HCl pH 2) to 200 μL aliquots of 10 mg/mL NP dispersions and completing the volume up to 6 mL with HCl (pH 2). The mixtures were then incubated in a shaking water bath (37 °C, 100 rpm) for 1 h and then removed for analysis.

6

Particle size and zeta potential measurements Mucin-NP mixtures were prepared and incubated for 1 h as described above and then the particle size and zeta potential of the mixtures were measured by dynamic light scattering (DLS) using a Nicomp Nano Z3000 particle size/zeta potential analyzer (Particle Sizing Systems, Santa Barbara, CA, USA). Samples were vortexed briefly before each measurement. Each measurement was repeated at least three times using freshly prepared batches of the NPs.

Turbidity measurements Turbidity of mucin-NP mixtures prepared as described above was determined by measuring the UV absorbance (Shimadzu UV-1800 spectrophotometer, Kyoto, Japan) of the mixtures at λ = 500 nm. Results were plotted as the difference in absorbance (ΔA) between mucin-NP mixtures and pure mucin and NP dispersions prepared at the same concentration. Each measurement was repeated at least three times using freshly prepared batches of the NPs.

Adsorption of mucin by NPs Mucin-NP mixtures were prepared as above. After the incubation, 1 mL aliquots of each mixture were centrifuged (13,000 rpm for 30 min at RT). The UV absorbance of the supernatants was measured at λ = 263 nm to calculate the amount of unadsorbed mucin based on a standard curve of mucin solutions in HCl (pH 2) centrifuged under the same conditions. Mucin adsorption was determined as follows: Mucin adsorbed (%) = (Total amount of mucin – amount of unadsorbed mucin)/Total amount of mucin × 100%

Each measurement was repeated at least three times using freshly prepared batches of the NPs.

Ex vivo mucoadhesion test Adhesion of NR-loaded NPs to stomach mucosa was investigated by an ex vivo wash-off test adapted from a previous report [28]. Briefly, sheep stomach freshly obtained from a local slaughterhouse was rinsed in normal saline to remove excess debris and cut into 2 cm × 2 cm 7

pieces with a surgical blade. Pieces were placed in 2-well chamber slides (Nunc Lab-Tek, Thermo Scientific, Rochester, NY, USA) with the mucosa side facing up. NR-loaded mPEGPCL@pD and mPEG-PCL NPs were each redispersed in HCl (pH 2) at 5 mg/mL, and 1 mL of each dispersion was added to the wells in triplicates. The slides were incubated at 37 °C for 1 h under static conditions to allow NPs to adhere to the mucosa. Excess NPs were then removed (T0) and 1 mL HCl (pH 2) was gently added to each well. The slides were then placed in a shaking water bath at 37 °C and 100 rpm. At predetermined time points (1, 2, 4, 6, and 8 h), the entire medium was removed from each well and replaced with 1 mL fresh HCl (pH 2). Media collected at T0 and at every time point thereafter was centrifuged (13,000 rpm, 30 min, RT), the supernatants were discarded and the pellets were redispersed in 1 mL DMF to dissolve the NPs and extract NR. The amount of NPs washed-off from the mucosa at each time point was determined by measuring the UV absorbance of the extract at λ = 545 nm based on a standard curve of NR in DMF. % Mucoadhesion was calculated as follows: % Mucoadhesion = (Original amount of NPs added – amount washed off)/Original amount of NPs added × 100%

In vitro release of RF RF-loaded mPEG-PCL@pD and mPEG-PCL NPs were each redispersed in HCl (pH 2) at 5 mg/mL, and 1.5 mL aliquots of each dispersion were placed in microcentrifuge tubes in triplicate. Samples were then placed in a shaking water bath (37 °C and 100 rpm). At predetermined time points (30 min, 1, 2, 4, 6, 8, 24, 48, 72, and 96 h), samples were centrifuged at 14,300 rpm (12,000 × g) for 15 min at RT. One milliliter of the supernatants was collected and replaced with 1 mL HCl (pH 2), and then the pellets were redispersed and placed back in the water bath. The amount of RF in the supernatant was measured by UV absorbance based on a standard curve of RF in HCl pH 2 at λ = 474 nm, and cumulative % released was plotted against time.

Statistical analysis Statistical analysis was performed in GraphPad Prism 6 using a 2-way ANOVA followed by Sidak’s multiple comparison test, where p < 0.05 was considered statistically significant. 8

RESULTS Characterization of mPEG-PCL@pD NPs mPEG-PCL copolymer was synthesized by ring-opening polymerization of ε-caprolactone in the presence of mPEG5K as a macroinitiator. The copolymer was then used to prepare mPEGPCL@pD NPs by the nanoprecipitation technique followed by oxidative self-polymerization of dopamine in mild alkaline conditions to produce the pD coating (Fig. 1). There have been several proposed mechanisms for the formation of pD layers on solid substrates. For example, oligomeric dopamine can first form nanoaggregates, which assemble into microaggregates and then deposit on the polymer surface [29]. Alternatively, Park et al. have shown that pD is deposited on polymeric NPs as a thin film [30]. In the case of mPEG-PCL NPs, we hypothesize that pD coating can be initiated in spite of the PEG corona since dopamine is a small molecule (189.6 g/mol) that can penetrate the outer PEG layers resulting in direct contact with the hydrophobic core surface of PCL. As the polymerization of dopamine proceeds, the pD layer starts to grow from the inner surface of the PCL core toward the PEG chains. Given the branched nature of pD, it can intercalate between the PEG chains as the polymerization continues. Note that the polyphenolic structure of pD can be further stabilized within the PEG corona by Hbonds. A similar polyphenolic compound, tannic acid, has been known to form H-bond directly with PEG, resulting in the formation of PEG hydrogels [31].

TEM was used to confirm the spherical morphology of the NPs, which did not change upon pD coating (Fig. 2A). In addition, pD-coated NPs appeared with a dark rim surrounding the NP core under TEM, indicative of the presence of the electron-rich pD layer as previously reported [32, 33]. XPS survey and high resolution scans also confirmed successful pD coating, as indicated by the appearance of N1s peak at ~400 eV in the XPS spectrum of mPEG-PCL@pD NPs, which was absent in uncoated NPs (Fig. 2B). DLS analysis of the NPs under stomach-mimetic pH is summarized in Table 1, which showed that particle size of coated and uncoated NPs ranged between 55.4 – 58.1 nm with no significant increase in size upon coating. The surface charge was close to neutral for both types of NPs. PEGylated NPs typically result in neutral to slightly negative zeta potential values [24, 34]. No significant change in surface charge was observed after pD coating, due to the full protonation of the catechol protons at pH 2 [35]. 9

Monitoring mucin-NP interactions by DLS Fixed concentrations of the NPs were incubated with increasing concentrations of mucin at pH 2, which is within the physiological pH range of the stomach, and particle size of the mixtures was monitored by DLS. Representative particle size distributions of mucin-NP mixtures are depicted in Fig. 3. Initially, incubation of pD-coated NPs with mucin did not lead to a significant change in their mean diameter and particle size distribution, as evidenced by the minor shifts and changes in peak position and frequency at mucin/NP ratios of 1:4 and 1:2 w/w (Fig. 3A). As the ratio of mucin increased to 1:1, 2:1, and 4:1 w/w, particle size distribution of pD-coated NPs showed a significant reduction in NP peak frequency and the appearance of peaks most probably attributed to mucin and mucin/NP aggregates. These observations strongly suggest the occurrence of concentration-dependent mucin-NP interactions attributed to the pD coating. On the other hand, mPEG-PCL NPs exhibited minor changes in peak frequency and insignificant changes in their mean particle size throughout the study, indicating little to no interactions with mucin (Fig. 3B).

Monitoring mucin-NP interactions by zeta potential measurements As shown in Table 1, pD-coated and uncoated NPs exhibited almost neutral surface charges. Zeta potential for mucin itself was recorded as -2.2 mV at pH 2 since the sialic acid residues are present in their unionized form, which imparts a slightly negative surface charge [36]. Due to the small differences in their zeta potential values, the surface charge of pD-coated NPs was not expected to change significantly upon interacting with mucin. Interestingly, Fig. 4A shows a significant drop in the zeta potential of mPEG-PCL NPs at mucin/NP ratio of 4:1 w/w, where the reading (-2.3 mV) was closer to that of pure mucin, while pD-coated NPs retained their neutral surface charge throughout the mucin concentrations tested. This strongly suggests that pD-coated NPs, by interacting with mucin, can mask some of its surface charge, which resulted in a neutral zeta potential reading even at high mucin/NP ratios. mPEG-PCL NPs, which were less able to form mucin-NP aggregates, remained separated in solution so that as the concentration of mucin increased, the zeta potential measurement resembled that of pure mucin dispersions.

10

Turbidity as a measure of mucin-NP interactions The increase in particle size and the formation of large mucin-NP aggregates can be detected by turbidity measurements [36-38]. In this study, turbidity of mucin-NP mixtures was determined by measuring the UV absorbance at 500 nm after adjusting for turbidity of pure mucin dispersions. As depicted in Fig. 4B, pD-coated NPs caused a significant increase in turbidity (ΔA) of mucin-NP mixtures at mucin/NP ratios of 2:1 and 4:1 w/w compared to uncoated mPEG-PCL NPs. These findings further support our argument that pD-coated NPs capable of achieving more favorable mucoadhesive interactions than uncoated NPs.

Adsorption of mucin by NPs Mucoadhesive properties of pD-coated mPEG-PCL NPs were further investigated by measuring their ability to adsorb mucin. As shown in Fig. 5, mPEG-PCL@pD NPs were able to adsorb a significantly higher % of mucin compared to mPEG-PCL NPs even at low mucin/NP ratios, with 95.8, 72.7, and 56.4% of the amount of mucin initially added being adsorbed onto the NPs at ratios of 1:4, 1:2, and 1:1 w/w, respectively. The decline in the mucin adsorption ability of pDcoated NPs was attributed to the saturation of the NP surface as the mucin concentration increased, reaching a minimum of 41.4% at mucin/NP ratio of 4:1 w/w. mPEG-PCL NPs demonstrated a limited ability to adsorb mucin and only at relatively high mucin/NP ratios (1:1, 2:1, and 4:1 w/w), likely attributed to nonspecific interactions. In addition, no significant difference in % adsorbed mucin was observed between pD-coated and uncoated NPs at mucin/NP ratios higher than 1:1 w/w.

11

Ex vivo mucoadhesion to stomach mucosa To confirm the mucoadhesive properties imparted by the pD coating, NR-loaded NPs were incubated with excised stomach mucosa for 1 h in HCl (pH 2) under static conditions. This step was important to determine the ability of the NPs to establish initial binding and attachment to the gastric mucosa. Fig. 6 demonstrates the superior ability of pD-coated NPs to adhere to the mucosa, as 79.1% of the NPs remained attached after 1 h of static incubation (T0), compared to only 33.4% of uncoated mPEG-PCL NPs. Interestingly, both types of NPs displayed similar gastric retention, with 77.9% and 33.0% of pD-coated and uncoated NPs, respectively, remaining attached to the mucosa after periodically replacing the incubation medium with fresh HCl up to 8 h under dynamic conditions. However, due to their enhanced initial binding ability, pD-coated NPs are expected to exhibit improved gastro-retention compared to uncoated NPs in vivo.

12

In vitro release of RF from NPs RF was encapsulated into the NPs as a model drug candidate for gastro-retentive delivery, since it is mostly absorbed in the stomach and is one of the common treatments for H. pylori infections [39]. Drug-loaded pD-coated and uncoated NPs were incubated under stomach-mimetic conditions (low pH, shaking) up to 4 days and the amount of drug released was measured over time. The constructed release profiles shown in Fig. 7 were biphasic, as typically observed in biodegradable polymeric NPs, with an initial burst release during the first 8 h and a sustained release profile throughout the period of the study. Notably, the presence of the pD layer did not significantly impede drug release from mPEG-PCL NPs, with both types of NPs displaying similar release profiles. During the burst release phase, % RF released was 27.8% and 24.7% for mPEG-PCL and mPEG-PCL@pD NPs, respectively. The highest % RF released after 4 days was 36.6% and 31.5% for mPEG-PCL and mPEG-PCL@pD NPs, respectively.

DISCUSSION Several approaches to increase gastric retention have been reported to date, including the use of mucoadhesive systems. Adhikary et al. investigated ranitidine HCl mucoadhesive tablets using a combination of hydrophilic polymers such as carbopol and hydroxypropyl methyl cellulose to prolong gastric retention [7]. Mucoadhesive acyclovir formulations using chitosan [8] and alginate microspheres [9] have shown significant improvement in its oral bioavailability. Mucoadhesive microspheres of poly(acrylic acid) and poly(vinyl pyrrolidone) were also investigated as carriers for antimicrobial agents for the eradication of H. pylori to improve their residence time in the stomach [11]. Biodegradable NPs have also been explored for oral drug delivery [40-44], but so far there has been no report on the use of biodegradable mucoadhesive NPs to improve gastric retention. Catechol-functionalized polymers have been investigated as bioadhesive platforms, where their adhesive properties were evaluated with or without the presence of mucin [17-19, 45]. However, mussel-inspired adhesive coatings have not yet been explored for oral drug delivery, which creates the need to probe their bioadhesive interactions with mucin, the major component of mucosal membranes in the GIT. The objectives of this study were to conduct a series of experiments investigating the interactions of pD-coated polymeric NPs with gastric mucin and the stomach mucosa, in order to validate their use as mucoadhesive platforms for gastro-retentive drug delivery. Our design takes 13

advantage of the larger surface area-to-volume ratio of NPs compared to microparticles. Combined with the mucoadhesive properties of pD, our platform provides a greater surface area for mucoadhesion and drug dissolution, and thus has the potential to enhance the bioavailability and absorption of poorly soluble drugs with narrow absorption windows limited to the stomach or upper GIT. DLS and turbidity measurements are two common methods employed to investigate the interactions of mucoadhesive polymers, microparticles, and NPs with mucin. This is based on the assumption that such interactions can lead to the formation of large mucin-NP aggregates which can be detected by DLS and lead to an increase in solution turbidity [37, 46]. pD-coated NPs exhibited a marked change in their particle size distribution compared to uncoated NPs, attributed to their interactions with mucin. These interactions also corresponded to an increase in solution turbidity. These changes were only observed at relative high mucin/NP ratios, which might suggest that pD-coated NPs may not be as strongly mucoadhesive as would be desired. However, as seen from the mucin adsorption assay, pD-coated NPs were able to adsorb significantly higher amount of mucin even at very low mucin/NP ratios. Therefore, we hypothesize that the NPs start to interact with mucin at low mucin concentrations, but only start to form large aggregates at higher mucin concentrations. At this point, the surface of the NPs is saturated with adhered mucin, and the NPs start to lose their ability to adsorb additional mucin, which explains the plateau reached in Figure 5 at mucin/NP ratio of 2:1 and 4:1 w/w. Our results also indicate that at these high mucin concentrations, the adsorption ability of pD-coated NPs starts to resemble that of uncoated NPs due to nonspecific interactions. As for zeta potential measurements, the insignificant change in values for mucin/pDcoated NP mixtures strongly suggests that the mucin-NP interactions are not electrostatic in nature, due to the neutral surface charge of the NPs and the slightly negative charge of mucin at low pH. This distinguishes the mucoadhesive interactions of catechol-based coatings from those of other polymers such as chitosan, where electrostatic interactions play a significant role in its mucoadhesive properties [19, 38]. In the case of pD and other mussel-inspired coatings, their adhesive characteristics lie in the catechol moieties which can react with thiol- and aminecontaining molecules through Schiff base or Michael addition reactions [15, 16]. Note that mucin is a very large glycoprotein containing both primary amines and thiol groups capable of forming covalent bonds with pD-coated NPs, which is the main contributor to their mucoadhesive 14

properties. The in vitro mucoadhesion tests were further corroborated with an ex vivo wash-off test using freshly excised sheep stomach. pD-coated NPs were able to achieve higher % mucoadhesion upon initial contact with the stomach mucosa, and demonstrated excellent retention ability throughout the duration of the study. PEGylated NPs have been shown to exhibit limited interactions with mucin due to their near-neutral surface charge, which impedes their attachment to mucosal surfaces but improves their mucus penetration ability [47]. Our findings indicate that the pD coating enables the NPs to overcome these barriers, resulting in significantly improved mucoadhesion. RF release from pD-coated NPs revealed a typical release profile that was unaffected by the presence of the pD layer. Even though the NPs did not completely release the drug after several days, which is much longer than gastric residence time, it is expected that with the improved mucoadhesion, the NPs will be taken up by the gastric mucosa more readily, allowing for the drug to be released inside the cytoplasm of the endothelium. Alternatively, a copolymer with a smaller PCL MW can be employed to tune the drug release kinetics.

CONCLUSION We have demonstrated through a series of in vitro and ex vivo studies the mucoadhesive ability of pD-coated NPs. Our findings strongly confirm that mussel-inspired pD coatings on polymeric NPs can achieve strong interactions with mucosal membranes even at low pH such as that of the stomach, which supports their use for gastro-retentive drug delivery. There remain other physiological barriers to be overcome such as mucus turnover, which necessitates the optimization of drug release/NP uptake kinetics in order to maximize the therapeutic efficacy of the drug delivery system.

ACKNOWLEDGEMENTS This project was financially supported by the Deanship of Scientific Research and Graduate Studies at Al-Zaytoonah University of Jordan. The authors would like to thank Prof. Phillip Messersmith (University of California, Berkeley) and Prof. Haeshin Lee (Korea Advanced Institute of Technology) for insightful discussions. The authors also thank Dr. Caroline Sugnaux (University of California, Berkeley) for assistance with XPS analysis. 15

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Fig. 1. Preparation of mPEG-PCL NPs by nanoprecipitation followed by oxidative selfpolymerization of dopamine in Tris buffer (pH 8.5) to produce pD-coated NPs.

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Fig. 2. A) TEM images of uncoated mPEG-PCL NPs and pD-coated NPs showing their spherical morphology is maintained after coating, scale bar = 100 nm. B) XPS survey scans of uncoated and pD-coated NPs. High resolution scans of the N1s region (inset) confirmed successful pD coating as indicated by the appearance of a peak at ~400 eV, which was absent in uncoated NPs.

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A

B

Mucin/NP _________________

4:1

Frequency

Frequency

2:1 1:1 1:2 1:4 No mucin 10

100

1000

10

100

1000

Diameter (nm)

Diameter (nm)

Fig. 3. Representative volume-weighted particle size distributions of mucin-NP mixtures after 1 h incubation in HCl (pH 2) at 37 °C. pD-coated NPs (A) exhibited a marked decrease in the frequency of the NP peak at mucin/NP ratio of 1:1 and higher, indicating their interaction with mucin. Particle size of uncoated NPs (B) remained unchanged even at high mucin/NP ratios, indicating limited interaction with mucin. Note that particle size distribution of mucin (1 mg/mL in HCl pH 2) after sonication for 30 min typically shows three main peaks around 10 nm, 170 nm, and 800 nm (data not shown).

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A

B

Fig. 4. A) Zeta potential measurements of mucin-NP mixtures. Surface charge of pD-coated NPs remained unchanged throughout the experiment and approached neutrality, indicating the limited role of electrostatic interactions in the mucoadhesive properties of the NPs. At mucin/NP ratio of 4:1 w/w, uncoated NPs exhibited a zeta potential value approaching that of pure mucin, which demonstrates their limited ability to interact with mucin and alter its surface charge. Results represent the mean ± SD of three independent experiments. * p < 0.05. B) Turbidity of mucinNP mixtures as measured by UV absorbance at 500 nm. Results are plotted as the difference between the absorbance (ΔA) of each mixture and that of pure mucin and pure NP dispersion at the same concentration found in each mixture. The observed increase in ΔA at mucin/NP ratios of 2:1 and 4:1 w/w signifies the formation of large mucin-pD-coated NP aggregates. Results represent the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01.

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Fig. 5. Adsorption of mucin by the NPs prepared in this study plotted as % of the initial amount of mucin added to each mixture. pD-coated NPs start to adsorb the majority of mucin present in the mixture at mucin/NP ratio of 1:4 w/w, reaching a plateau in their adsorption ability at higher ratios. Uncoated NPs exhibited some adsorption ability at mucin/NP ratios of 1:1 w/w and higher, likely due to nonspecific interactions. Results represent the mean ± SD of three independent experiments. ** p < 0.01, **** p < 0.0001.

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Fig. 6. Ex vivo mucoadhesion and retention of NR-loaded NPs using excised sheep stomach after 1 h of incubation under static conditions (T0), followed by a wash-off test up to 8 h. pD-coated NPs were able to achieve significantly greater initial adhesion and were efficiently retained on the mucosa throughout the period of the study. Results are plotted as % of NPs remaining attached to the mucosa (% mucoadhesion) and represent the mean ± SD of triplicate samples for each group. **** p < 0.0001.

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Cummulative RF released (%)

50

mPEG-PCL@pD mPEG-PCL

40 30 30

20 20

10

10

0

0

4

8

12

16

20

24

0 0

12

24

36

48

60

72

84

96

Time (h)

Fig. 7. In vitro release test of RF-loaded pD-coated and uncoated mPEG-PCL NPs in HCl (pH 2) up to 4 days plotted as cumulative % RF released. Inset: Cumulative % RF released within the first 24 h. Results represent the mean ± SD of triplicate samples for each group.

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Table 1. Particle size and zeta potential of the NPs prepared in this study in HCl (pH 2). Results represent the mean ± SD of at least three different batches of NPs.

NP

Particle size (nm)

Zeta potential (mV)

mPEG-PCL@pD

55.4 ± 3.7

-0.1 ± 0.6

mPEG-PCL

58.1 ± 3.2

0.8 ± 0.2

27