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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Polydopamine Coating Enhances Mucopenetration and Cell Uptake of Nanoparticles Barbara Poinard,† Syafiqah Kamaluddin,‡ Angeline Qiao Qi Tan,§ Koon Gee Neoh,†,∥ and James Chen Yong Kah*,†,‡ †
NUS Graduate School of Integrative Sciences and Engineering, National University of Singapore, 117456 Singapore Department of Biomedical Engineering, National University of Singapore, 117583 Singapore § School of Life Sciences & Chemical Technology, Ngee Ann Polytechnic, 599489 Singapore ∥ Department of Chemical and Biomolecular Engineering, National University of Singapore, 117585 Singapore
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ABSTRACT: Mucus is an endogenous viscoelastic biopolymer barrier that limits the entry of foreign pathogens and therapeutic carriers to the underlying mucosal cells. This could be overcome with a hydrophilic and nonpositively charged carrier surface that minimizes interactions with the mucin glycoprotein fibers. Although PEGylation remains an attractive surface strategy to enhance mucopenetration, cell uptake of PEGylated nanoparticles (NPs) often remains poor. Here, we demonstrated polydopamine (PDA) coating to enhance both mucopenetration and cell uptake of NPs. PDA was polymerized on carboxylated polystyrene (PS) NPs to form a PDA coating, and the resulting PS-PDA achieved a similar level of mucopenetration as our PEGylated PS (PS-PEG) positive control in three separate studies: NP−mucin interaction test, transwell assay, and multiple particle tracking. Compared to water, the diffusions of PS-PDA and PS-PEG in reconstituted mucus solution were only 3.5 and 2.4 times slower, respectively, whereas the diffusion of bare PS was slowed by up to 250 times. However, the uptake of PS-PDA (61.2 ± 6.1%) was almost three times higher than PS-PEG (24.6 ± 5.4%) in T24 cells, which were used as a model for underlying mucosal cells. Our results showed a novel unreported functionality of PDA coating in enhancing both mucopenetration and cell uptake of NPs for mucosal drug delivery applications, not possible with conventional PEGylation strategies. KEYWORDS: polydopamine, nanoparticles, mucopenetration, particle tracking, cell uptake
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INTRODUCTION One major clinical challenge that hinders the use of nanoparticles (NPs) for mucosal delivery is their ability to penetrate quickly through mucus and present themselves with high uptake in underlying cells. The mucus layer that lines the major organs of the body such as the eyes, lungs, gut, and urinary tract presents a biological barrier that protects underlying tissues by efficiently trapping bacteria and foreign particulates1−3 before removing them as it sheds. This porous yet dense viscoelastic biopolymer matrix comprises mostly mucin glycoproteins densely coated with short glycans, most of which are negatively charged (carboxyl or sulfate groups) and separated by cysteine-rich hydrophobic domains. The matrix also presents a mesh pore size of ∼300 nm4 and viscosity of 100−10 000 times higher than water,5−7 which collectively allows the same mucus layer to also limit the penetration of therapeutics into target tissues for delivery to underlying aberrant cells.8−12 Several viruses have adapted abilities to minimize mucointeraction and diffuse efficiently through the mucus © XXXX American Chemical Society
barrier by having a combination of small size ( 100 for statistical significance). The hydrodynamic diameter (DH), expressed in terms of the intensity average, and ζ potential of the NPs were determined by dynamic light scattering (DLS) and laser Doppler anemometry using a Nanosizer ZS90 (Malvern Instruments, Southborough, MA) at 25 °C. The fluorescence spectra of the NPs were acquired with a microplate reader with λex/λem = 400/605 nm (Tecan, Switzerland). Fourier transform infrared (FTIR) spectroscopy was used to verify successful surface modifications of PS NPs with PDA by checking for the presence of specific functional groups, including bands at 3000 and 1500 cm−1 characteristic of aromatic C−H groups and COO− groups, respectively, for PS and a band at 3500 cm−1 characteristic of OH functional groups for PDA. Hydrophobicity of NPs. The surface hydrophobicity of NPs was determined using Rose Bengal (RB) adsorption assay.43,44 The PS NPs with different surface modifications were mixed with an aqueous solution of 11.23 μg/mL RB and incubated overnight to allow for the adsorption of the hydrophobic dye molecules on the NPs. After incubation, NPs were removed from the mixture by centrifugation at 21 100g for 30 min, and the optical density (OD) of the supernatant was measured at 550 nm to determine the amount of unadsorbed RB molecules. The surface hydrophobicity of NPs was determined from the decrease in OD due to hydrophobic interaction between the RB dye and the surface of PS NPs. Mucointeraction Measurement. Reconstituted mucus solution was prepared by dissolving 5.0 mg/mL unpurified porcine-stomach type III mucin powder (Sigma) in ultrapure water adjusted to pH 4.5. The solution was vortexed for 10 s and subsequently incubated overnight at 37 °C. After incubation, the solution was sonicated at 37
hypersensitivity,23,24 which collectively points to an existing gap in having an appropriate NP surface capable of simultaneously enhancing mucopenetration and uptake in underlying cells. By taking inspiration from the surface properties of mucopenetrating pathogens, we showed that the hydrophilic and negative surface coating of polydopamine (PDA), a biopolymer polymerized from dopamine,25,26 could also enhance mucopenetration by minimizing interaction with the negatively charged and hydrophobic pockets in mucus, while also promoting interaction with positively charged choline groups on the lipid membrane to enhance cellular uptake of NPs. This was possible due to the hydroxyl groups present on PDA, which confer hydrophilicity, as well as the presence of other multiple functional groups (e.g., amino, phenol), which confer zwitterionic properties (isoelectric pH 4−4.5).27,28 Hence, at physiological pH, the phenolic groups deprotonate to a negative surface charge. Although PDA has been widely used for coating medical implants29−32 and NPs to enable multifunctional surface chemistries,25,33,34 as well as a nanocarrier for drug loading35,36 with known biocompatibility37 and minimal immune response,38 their ability to simultaneously facilitate mucopenetration and enhance cell uptake has not been reported to our knowledge. Here, we demonstrate PDA as a coating on carboxylated fluorescent polystyrene (PS) NPs capable of enhancing their diffusion in reconstituted mucus at a comparable level to our positive control of PEGylated PS (PS-PEG), yet being able to achieve a much higher uptake than PS-PEG in T24 cells, which were used as a model for underlying tumoral urothelium in bladder cancer. Our results provide the first evidence of PDA surface modification to facilitate mucopenetration of NPs for potential enhanced mucosal delivery of therapeutics with PDAcoated nanocarriers.
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MATERIALS AND METHODS
Preparation and Characterization of NPs with Different Surface Modifications. Fluorescent carboxylated 200 nm PS NPs (initial concentration of 4.55 × 1012 NPs/mL, Life Technologies, MA) were used as our model NP for fluorescence tracking of their trajectory in reconstituted mucus. The PS NPs were surface functionalized with PDA, PEG (our mucopenetrative positive control), and poly(diallyldimethylammonium chloride) (PDADMAC) (cationic polyelectrolyte as our mucopenetrative negative control) to form fluorescent PS NPs with different surface modifications. The PDA coating on PS was formed by resuspending 10 μL of 7.5 nM PS NPs in an alkaline 0.3 mg/mL dopamine solution in 10 mM Tris buffer (pH 10.5) for 2 h. The thickness of the PDA B
DOI: 10.1021/acsami.8b18107 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces Hz for 30 min and washed once via centrifugation at 1500g for 30 min. The pellets were discarded and the supernatant collected and stored at 4 °C until further use. Using the prepared reconstituted mucus, we probed the interaction between PS NPs with different surface modifications and mucin, one of the major components of the mucus barrier, using DLS (Nanosizer ZS90, Malvern Instruments, Southborough, MA) to examine for any increase in DH of the NPs due to their interaction with the submicrometer-sized mucin particles.45,46 Mucopenetration Measurement. Transwell inserts (polyethylene 1 μm pores, 6.5 mm diameter to give a surface area of 0.33 cm2 membrane filter, Corning) were used to examine the mucopenetration of PS NPs with different surface modifications. Briefly, 900 μL of ultrapure water was added to the bottom of 24-well plates, and 250 μL of NP colloid solution containing 9 × 109 NPs at known concentrations of 60 pM was deposited gently in the transwell insert precoated with 20 μL of reconstituted mucus, which has been previously left to settle on the transwell insert for 30 min to form a ∼600 μm mucin layer based on calculation, that is, 20 nm3/0.33 cm2. We noted that the mucin layer used in this transwell assay was much thicker than the physiologically relevant mucus thickness of ∼10 μm. Unfortunately, we were unable to create this physiologically relevant thickness in the transwell assay as this would require a nonfeasible volume of 0.33 μL of mucin solution to be added to the transwell insert. Nonetheless, a thicker mucin layer would still allow us to assess the mucopenetration of the NPs. The NPs were left to incubate with the reconstituted mucus for 24 h at 37 °C to allow for migration of NPs through the mucin layer (Figure 1). The percentage of NPs that crossed the mucin layer in the mucincoated transwell cassette was normalized against the amount that passed through the uncoated transwell cassette, as measured by their fluorescence45,47,48 and using the following formula
obtained from the gradient of the ln⟨MSD⟩ versus ln(τ) plot, whereas D could be obtained from the y-intercept of the same plot. Computational Estimate of Mucopenetration across a Specific Mucus Thickness. From the particle diffusion coefficient D obtained earlier, we also determined the theoretical mucopenetration profile of NPs over time after diffusing through a chosen mucus thickness as modeled by an initial constant concentration of NPs on the surface (Cx=0, t) and no NPs in the mucus layer at t = 0 (C0
