Mixed Mucoadhesive Amphiphilic Polymeric Nanoparticles Cross a

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Biological and Medical Applications of Materials and Interfaces

Mixed mucoadhesive amphiphilic polymeric nanoparticles cross a model of nasal septum epithelium in vitro Inbar Schlachet, and Alejandro Sosnik ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b04766 • Publication Date (Web): 24 May 2019 Downloaded from http://pubs.acs.org on May 24, 2019

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ACS Applied Materials & Interfaces

Mixed Mucoadhesive Amphiphilic Polymeric Nanoparticles Cross A Model of Nasal Septum Epithelium In Vitro

Inbar Schlachet and Alejandro Sosnik*

Laboratory of Pharmaceutical Nanomaterials Science, Department of Materials Science and Engineering, Technion-Israel Institute of Technology, Haifa, Israel

*Corresponding author: Prof. Alejandro Sosnik Department of Materials Science and Engineering De-Jur Building, Office 607 Technion-Israel Institute of Technology Technion City 3200003 Haifa, Israel Phone #: +972-077-887-1971 Emails: [email protected], [email protected]

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ABSTRACT Intranasal administration of nano-drug delivery systems emerged as an appealing strategy to surpass the blood-brain barrier and thus, increase drug bioavailability in the central nervous system. However, a systematic study of the effect of structural properties of the nanoparticles on the nose-to-brain transport is missing. In this work, we synthesized and characterized mixed amphiphilic polymeric nanoparticles combining two mucoadhesive graft copolymers, namely chitosan-g-poly(methyl methacrylate) and poly(vinyl alcohol)-g-poly(methyl methacrylate) for the first time. Chitosan enables the physical stabilization of the nanoparticles by ionotropic crosslinking with tripolyphosphate and confers mucoadhesiveness, while poly(vinyl alcohol) is also mucoadhesive and, owing to its non-ionic nature, it improves nanoparticle compatibility in nasal epithelial cells by reducing the surface charge of the nanoparticles. After a thorough characterization of the mixed nanoparticles by dynamic light scattering and nanoparticle tracking analysis, we investigated the cell uptake by fluorescence light and confocal microscopy and imaging flow cytometry. Mixed nanoparticles were readily internalized at 37oC, while the uptake was inhibited almost completely at 4oC, indicating the involvement of energy-dependent mechanisms. Finally, we assessed the nanoparticle permeability across liquid-liquid and air-liquid monolayers of a nasal septum epithelial cell line and studied the effect of nanoparticle concentration and temperature on the apparent permeability. Overall, our findings demonstrate that these novel amphiphilic nanoparticles cross this in vitro model of intranasal epithelium mainly by a passive (paracellular) pathway involving the opening of epithelial tight junctions. KEYWORDS: Mucoadhesive amphiphilic polymeric nanoparticles; nano-drug delivery systems; intranasal (nose-to-brain) drug delivery; nasal epithelium monolayer; RPMI 2650 cell line. 2 ACS Paragon Plus Environment

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INTRODUCTION The nasal route is primarily utilized for the localized delivery of drugs in the treatment of local allergy.1–3 Owing to a large surface area, a highly permeable endothelial network and high total blood flow4–6, it has been also clinically implemented for the systemic administration of drugs that display significant hepatic first-pass metabolism after oral administration, that demand fast absorption or in case of patient unconsciousness.7-9 In more recent years, experimental evidence that there exists a direct nose-to-brain path that surpasses the blood-brain barrier (BBB), the anatomical barrier that controls the trafficking of drugs from the systemic circulation into the central nervous system (CNS), prompted the investigation of this administration route for the passive targeting of drugs to the brain in the therapy of cancer, infections and different neurological diseases.10,11 Other approaches to improve nose-to-brain delivery are by enhancing the interaction of the nanoparticle with the olfactory epithelium for which the nanoparticle surface can be modified with lectins or cell-penetrating peptides.12 Intriguingly, intranasal delivery appears to be more efficient for nanoparticles than soluble

matter.13

Thus,

different

nanoformulations

such

as

pure

drug

nanoparticles13,14, polymeric nanoparticles and micelles15–18, liposomes and solid lipid nanoparticles19,20 and nanoemulsions21,22 have been incipiently investigated to deliver drugs to the CNS by the intranasal route. Amphiphilic nanocarriers (e.g., polymeric micelles) are formed by the self-assembly of amphiphilic block or graft copolymers in aqueous medium and they have shown great potential for oral and other transmucosal drug delivery approaches.23–25 In this framework, we demonstrated the 3-fold increase of the brain bioavailability of the antiretroviral efavirenz with respect to plasma when the drug was nanoencapsulated within poly(ethylene oxide)-poly(propylene oxide) polymeric micelles (10-250 nm) and 3 ACS Paragon Plus Environment


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administered intranasally.15 More recently, we introduced a novel platform of mucoadhesive nanogels produced by the self-assembly of amphiphilic chitosan (CS)26,27 and poly(vinyl alcohol) (PVA)28 graft copolymers synthesized by the hydrophobization of the side-chain of multifunctional polymers with different hydrophobic blocks such as oligo(N-isopropylacrylamide)26,28 and poly(methyl methacrylate) (PMMA)27 and the non-covalent crosslinking of the hydrophilic domains with sodium tripolyphosphate (TPP)26,27 or boric acid,28 respectively. We also demonstrated the ability of these nanoparticles to cross a co-culture in vitro model of the intestinal epithelium.29 Due to the self-assembly nature of these nanoparticles, different graft copolymers could be eventually combined in one single nanoparticle to adjust final properties such as size, surface charge and cell compatibility and uptake. Despite its great potential, intranasal delivery remains elusive to the clinical practice mainly due to the relatively small administrable volume per nostril.30 For instance, the clinical implementation of this route to treat diseases of the CNS has been very recent31,32 and this niche in the market of advanced drug delivery systems is steadily growing supported by the development of new medical devices that ease the intranasal deposition of the nanoformulation.33 To overcome this limitation, very concentrated formulations of highly potent drugs are usually required. The use of mucoadhesive nano-drug delivery systems emerges as a very appealing approach to prolong the residence of the drug in the nasal mucosa and hence, to increase the drug bioavailability in the brain.23,34–36 Prolongation of the residence time relies on the interaction of the nano-drug delivery system with the mucin layer that covers the nasal epithelium. Intriguingly, the cells and mechanisms behind the complex nose-to-brain transport have not been fully unraveled yet. Several reports suggested that axon terminals of olfactory neurons located at the olfactory bulb and exposed at the nasal 4 ACS Paragon Plus Environment

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mucosa are involved in the active uptake of the nanoparticles and their transport.37 In this regard, the need for initial nanoparticle permeability across the nasal epithelium that covers the whole cavity is unknown. Furthermore, the fundamental nanoparticle features (e.g., size, shape, surface chemistry) that govern the nose-to-CNS pathway remain to be elucidated.38 It is also unclear whether the transport mechanism of particles displaying different size ranges and/or surface characteristics are identical or not. In vitro models rely on the use of chamber systems where a donor and a receptor medium are separated by a semipermeable membrane or cell monolayer.39 Both primary cells and cell lines have been proposed over the years.40–44 In this work, we initially synthesized and characterized mixed amphiphilic polymeric nanoparticles combining two graft copolymers, namely CS-g-PMMA and PVA-gPMMA, for the first time. CS enables the physical stabilization of the nanoparticles by ionotropic crosslinking with TPP and confers mucoadhesiveness and the ability to open the epithelial tight junctions, while PVA improves their compatibility in primary nasal epithelial cells, a nasal septum epithelial cell line and human astrocytes in vitro by reducing the nanoparticle surface charge. Then, we conducted cell uptake assays and characterized the nanoparticle permeability across liquid-liquid (LL) and air-liquid (ALI) cell monolayers under different conditions to better understand the possible mechanisms involved in the transport. RESULTS AND DISCUSSION The rationale In this work, we aimed to investigate the interaction of mucoadhesive amphiphilic CSg-PMMA nanoparticles29 with a model of intranasal epithelium45 as a preamble to their application in the intranasal delivery of hydrophobic cargos to the CNS. CS nanoparticles have been extensively investigated in transmucosal drug delivery owing 5 ACS Paragon Plus Environment


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to their ability to transiently open tight junctions of different epithelia and increase paracellular drug transport.36,46–48 CS undergoes slow biodegradation in the biological milieu by human chitinases that belong to the glycoside hydrolase 18 family with an extent that decreases at higher degrees of deacetylation.49,50 In this context, given enough time, CS is expected to be excreted from the body. However, cell toxicity associated with the electrostatic interaction of the positively-charged surface of CS nanoparticles and the negatively-charged cell membrane has been also demonstrated in different cells in vitro26,51,52 such as a model of intestinal epithelium26 and adenocarcinomic human alveolar basal epithelial cells.51 Similar results were reported in human gastric carcinoma cell line MGC803 cells.53 The compatibility can improved upon partial neutralization of the positive charges by ionotropic crosslinking,26 or by decreasing the degree of deacetylation or chemically modifying or blending it with other polymers which reduce the availability of free primary amine groups of the sidechain of the polysaccharide on the nanoparticle surface.51,54,55 In this work, ionotropic crosslinking played a dual role of physically stabilizing the self-assembled nanoparticles to prevent disassembly upon dilution and reducing the positive surface charge to improve cell compatibility. Moreover, we blended it with a PVA-based nonionic amphiphile with higher cell compatibility than CS.28 In the current conceptual study, PMMA was chosen as hydrophobic block because it is approved by the Food and Drug Administration (FDA) as pharmaceutical excipient in formulations for oral administration56,57 and its use in a variety of chronic biomedical devices including intraocular lenses, and bone cement, screws and fillers.58-60 PMMA is biostable and the clearance of relatively short blocks from the body when covalently bound to hydrophilic backbones or residues of CS and PVA has to be investigated. In cases where biodegradation is required, PMMA could be replaced by other


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hydrophobic blocks such as the aliphatic ester poly(epsilon-caprolactone) that displays higher relative hydrophobicity than PMMA and undergoes slow hydrolysis and clearance.61 In preliminary screening studies, we characterized the cell compatibility of 0.1% w/v CS-g-PMMA nanoparticles utilizing primary human nasal epithelial cells (HNEpC). This primary cell type is a good model to optimize cell compatibility of the nanoparticles before permeability studies due to its higher sensitivity than immortalized epithelial cell lines. Results indicated the relatively high cell toxicity of CS-based nanoparticles with viability losses of 27-33% and 56% after 4 and 24 h, respectively, as shown with other cells (Supplementary Figure S1). Viability losses were substantial, even upon nanoparticle ionotropic crosslinking which reduces the surface-charge and increases the cell compatibility.26,29 In this content, we hypothesized that owing to the selfassembly nature of our nanoparticles, the synthesis of mixed nanoparticles combining CS-g-PMMA with a similar graft copolymer where CS is replaced by a non-ionic multifunctional polymer that mixes well with CS and that shows very good cell compatibility as hydrophilic backbone such as PVA would enable the optimization of the cell compatibility due to a less positive nanoparticle surface charge;28 the cytotoxicity of CS is associated with the positively-charged amine groups in the sidechain.26,51-55 PVA is very biostable and it is slowly eliminated from the body by mechanisms that do not involve saturable transport.62 At the same time, the use of a CS-based copolymer would preserve their ionotropic crosslinkability of the nanoparticle and its capacity to cross epithelia by a paracellular pathway owing to the opening of tight junctions mediated by the amine moieties. In this framework, we synthesized mixed nanoparticles with a CS-g-PMMA copolymer containing 30% w/w of PMMA, namely



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CS-PMMA30, and growing weight ratios of a PVA-g-PMMA copolymer that contains 16% w/w of PMMA (Figure 1).


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Figure 1. Formation and most likely structure of mixed CS-g-PMMA:PVA-g-PMMA nanoparticles.



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Then, we characterized their properties and performance in vitro. The good molecular interaction between CS and PVA chains and the production of blends was reported elsewhere and favored the formation of mixed nanoparticles where blended CS and PVA hydrophilic domains are combined with hydrophobic PMMA ones.63 Based on previous investigations of the self-assembly of this type of copolymer and the size of the mixed nanoparticles measured by dynamic light scattering (DLS, see below), the nanostructure formed is most likely a multi-micellar aggregate.26,27 The synthesis of both copolymers was confirmed by proton-nuclear magnetic resonance (1H-NMR) (Supplementary Figure S2A) and Fourier-transform infrared spectroscopy (FTIR) analyses (Supplementary Figure S2B). Synthesis and characterization of mixed amphiphilic nanoparticles Mixed CS-g-PMMA:PVA-g-PMMA nanoparticles containing different weight ratios (3:1, 2:1 and 1:1) were produced by the solution casting method63 and their cell compatibility preliminarily assessed in primary HNEpC before and after crosslinking over 4 and 24 h. This method ensured the intimate mixing of both copolymers to render mixed nanoparticles. Results confirmed that the gradual incorporation of greater relative amounts of the non-ionic copolymer PVA-g-PMMA into the nanoparticles reduces their cytotoxicity with viability values ˃75% for a 1:1 combination, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Supplementary Figure S1). Based on these preliminary cell compatibility findings, in advance, we utilized CS-g-PMMA:PVA-g-PMMA (1:1) nanoparticles. The formation of self-assembled nanoparticles was initially assessed by 1H-NMR, where preformed nanoparticles were freeze-dried and re-dispersed in dimethyl sulfoxide-d6 (DMSO-d6) and deuterium oxide (D2O) and analyzed. DMSO is a good solvent for all the copolymer blocks in the mixed nanoparticles and thus, the


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characteristic peaks of CS, PVA and PMMA could be detected in the spectrum at 2.8 (H-C-NH2), 1.3 (-CH2- backbone) and 0.8-1.0 ppm (-CH3-C), respectively (Figure 2). Conversely, in D2O, the disappearance of PMMA peaks at 0.8-1.0 ppm was consistent with the formation of self-assembled nanoparticles displaying outer hydrophilic CS and PVA and inner hydrophobic PMMA domains, as previously shown for CS-g-PMMA counterparts.27 DO 2

2.8 ppm

1.3 ppm 0.8-1 ppm

CS-PMMA30:PVA-PMMA16 (1:1) nanoparticles in DMSO-d6

CS-PMMA30:PVA-PMMA16 (1:1) nanoparticles in D2O

DMSO-d6

*

Figure 2. 1H-NMR spectra of mixed CS-PMMA30:PVA-PMMA16 (1:1) nanoparticles in DMSO-d6 (complete dissolution) and D2O (formation of amphiphilic nanoparticles). The peak of residual DMSO in the lower spectrum is indicated with an asterisk (*). The formation of multi-micellar nanoparticles by CS-g-PMMA copolymers where PMMA blocks are surrounded by hydrophilic CS domains was reported elsewhere.27 Owing to the good miscibility of CS and PVA62 and the use of hydrophobic blocks of identical relative hydrophobicity, mixed nanoparticles are anticipated to display a similar aggregation pattern. In addition, the characteristic peak of DMSO at 2.5 ppm in D2O samples was negligible, confirming the efficient elimination of this solvent during the preparation of the nanoparticles.



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The size (hydrodynamic diameter, Dh), size distribution (polydispersity index, PDI) and zeta-potential (Z-potential) of 0.1% w/v non-crosslinked and crosslinked CS-gPMMA:PVA-g-PMMA (1:1) nanoparticles was analyzed by DLS and compared to pure nanoparticles produced with each one of the copolymers, at 25oC. All the samples showed a monomodal size distribution, while the PDI depended on the composition and the crosslinking (Table 1). For example, CS-PMM30 nanoparticles showed Dh of 184 and 332 nm before and after crosslinking with 5 L/mL of suspension of 1% w/v TPP solution and accompanied with an increase of the PDI from 0.20 to 0.34 and a decrease of the Z-potential from +39 to +28 mV (Table 1). PVA-PMMA16 nanoparticles showed smaller size (92 nm) and negatively-charged surface (Zpotential = -16 mV).28 When non-crosslinked mixed CS-g-PMMA:PVA-g-PMMA (1:1) nanoparticles were produced, the size was similar to that of pure CS-PMMA nanoparticles before crosslinking with an enlargement to 249 nm upon crosslinking with 2.5 L/mL of suspension and a relatively small PDI of 0.259. When a higher amount of crosslinker was used (5 L/mL of suspension), the size increased substantially to 520 nm and the PDI to 0.44, indicating the formation of inter-micellar bridges. Based on these results, in advance, all the mixed nanoparticles used in cell experiments were crosslinked with 2.5 L/mL of suspension. As mentioned above, the size distribution was monomodal, in good agreement with the formation of mixed nanoparticles. As expected, mixed nanoparticles showed a less positive Z-potential (+30 mV) than pure CS-PMMA30 counterparts (+39 mV), this surface-charged being further decreased to +10 mV upon ionotropic crosslinking. It is worth stressing that Zpotential values were similar when nanoparticles were dispersed in Hank’s Balanced Salt Solution (HBSS) of pH 7.2, the medium used in permeability assays; e.g., mixed


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nanoparticles showed values of +25 and +7 mV, before and after ionotropic crosslinking, respectively. The size and concentration of the nanoparticles was also measured by nanoparticle tracking analysis (NTA) under scattering mode. For this, samples were diluted 10-500 times in the same medium used to prepare the nanoparticles, namely acetic acid solution in water of pH 5.5 to fit the concentration range of the instrument (107-109 particles/mL),

measured

immediately

after

dilution

to

prevent

nanoparticle

disassembly (in the case of non-crosslinked specimens), and the concentration corrected by the corresponding dilution factor. Sizes were in good agreement with DLS, considering that this instrument utilizes a different measurement method based on the visualization and tracking of the nanoparticle Brownian motion in suspension (Supplementary Videos 1). After ionotropic crosslinking, a procedure carried out before the dilution of the nanoparticles for NTA, the nanoparticle concentration increased, strongly suggesting that the aggregates were more physically stable under the conditions of the analysis. Overall, the size of mixed CS-PMMA30:PVA-PMMA16 (1:1) nanoparticles crosslinked with the smallest TPP volume fits very well the size range required to cross nasal epithelium monolayers.64,65 In this framework, these nanoparticles appear as good candidates for the nanoencapsulation of poorly watersoluble drugs, as shown elsewhere for graft copolymers made of CS and other hydrophobic blocks and drugs such as efavirenz,26 rifampicin61 and indinavir free base.66 In addition, the slightly positive surface charge is anticipated to facilitate the opening of epithelial tight junctions, while reducing cell toxicity with respect to pure CS nanoparticles.


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Table 1. Hydrodynamic diameter (Dh), size distribution (PDI), Z-potential and concentration of CS-PMMA30, PVA-PMMA16 and CSPMMA30:PVA-PMMA16 (1:1) nanoparticles (total copolymer concentration of 0.1% w/v), as determined by DLS and NTA. Nanoparticle crosslinking was carried out with 1% w/v TPP. Nanoparticle

CS-PMMA30 PVA-PMMA16 CSPMMA30:PVAPMMA16 (1:1) a Volume

Dh – Intensity (nm) (± S.D.)

PDI (± S.D.)

Z-potential (mV)

Dh (nm) (± S.D.)

NTA Concentration (x 109 particles/mL) (± S.D.)

-

184 (4)

0.20 (0.01)

+39 (1)

107 (4)

113 (5)

5

332 (54)

0.33 (0.03)

+28 (2)

165 (1)

165 (4)

-

92 (4)

0.14 (0.01)

-17 (1)

86 (2)

142 (5)

-

193 (62)

0.23 (0.06)

+30 (1)

155 (3)

221 (37)

2.5

249 (26)

0.26 (0.01)

+10 (1)

142 (8)

541 (27)

5

520 (197)

0.44 (0.15)

+8 (0)

165 (1)

503 (40)

TPP volume (µL) a

DLS

of 1% w/v TPP solution per mL of nanoparticle dispersion.


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Compatibility in RPMI 2650 cell line and human astrocytes RPMI 2650 is the only human nasal epithelial cell line obtained from a spontaneous tumor, namely an anaplastic nasal septum tumor, and it recapitulates well the properties of the normal human nasal epithelium.65 Thus, it has been extensively used to study the permeability of different types of nanoparticles.65 Ensuring low cell toxicity was crucial to assess the permeability of the mixed nanoparticles without affecting the integrity of the cell monolayer due to cell death. As shown above, mixed nanoparticles showed very good compatibility in primary HNEpC cells that are more sensitive than cell lines (Supplementary Figure S1). Results confirmed the very good cell compatibility of non-crosslinked and crosslinked CSPMMA30:PVA-PMMA16 (1:1) nanoparticles in RPMI 2650, viability values after 4 and 24 h of exposure being >95% and >75%, respectively (Figure 3). These values comply with the ISO 10993-5 (Biological evaluation of medical devices Part 5: Tests for in Vitro Cytotoxicity).67 120

RPMI 2650 cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4h

24 h

100 80 60 40 20 0

0.1% w/v

sslinked

cro 0.1% w/v

CS-PMMA30/PVA-PMMA16 (1:1) concentration

Figure 3. RPMI 2650 cell viability upon exposure to 0.1 % w/v non-crosslinked and crosslinked CS-PMMA30:PVA-PMMA16 (1:1) nanoparticles at 37oC, as estimated by the MTT assay. Nanoparticle crosslinking was carried out with 1% w/v TPP (2.5 L/mL of nanoparticle suspension). 15 ACS Paragon Plus Environment


Cell compatibility was also assessed with rhodamine B isothiocyanate (RITC, red fluorescence)-labeled mixed crosslinked nanoparticles at two working concentrations (0.01% and 0.05% w/v) and temperatures (4 and 37oC) for 4 h; this derivative is used in the uptake and permeability experiments. Under these conditions, the viability was always ˃75% (data not shown). It is also important to remark that permeability studies in vitro are conducted at lower nanoparticle concentrations (0.01% and 0.05% w/v) for only 4 h. Thus, the cell compatibility of our nanoparticles is optimal for these in vitro studies. Since these nanoparticles are envisioned to target the CNS by bypassing the BBB after the intranasal administration, their compatibility was also assessed in human astrocytes as a model of CNS cell type. After 4 h, viability values were >80%, regardless of the crosslinking or not (Figure 4). After 24 h, a decrease in viability was recorded, values being 68% and 78% for non-crosslinked and crosslinked nanoparticles, respectively. 120

Human astrocyte viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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4h

24 h

100 80 60 40 20 0

0.1%w/v

0.1% w/v

ed

crosslink

CS-PMMA30:PVA-PMMA16 (1:1) concentration

Figure 4. Human astrocyte viability upon exposure to 0.1 % w/v non-crosslinked and crosslinked CS-PMMA30:PVA-PMMA16 (1:1) nanoparticles at 37oC, as estimated by the MTT assay. Nanoparticle crosslinking was carried out with 1% w/v TPP (2.5 L/mL of nanoparticle suspension). 16 ACS Paragon Plus Environment

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These results confirmed the contribution of ionotropic crosslinking and partial positivecharge neutralization to the cytocompatibility of the nanoparticles. Characterization of cell compatibility with other cells of the CNS was beyond the scope of the current work. Nanoparticle uptake in vitro Nanoparticles can exploit two different pathways to cross epithelial barriers, namely transcellular and paracellular.68 The former is usually energy-dependent, while the latter is energy-independent. Aiming to improve the understanding of the interaction between our mixed mucoadhesive amphiphilic nanoparticles and RPMI 2650 cells, we characterized nanoparticle uptake at 4 and 37oC and initially visualized them by fluorescent microscopy. In general, RPMI2650 cells showed good nanoparticle internalization at 37°C after 4 and 24 h (Figure 5). At 4°C, nanoparticle uptake was substantially diminished; note the decrease in fluorescence (Figure 5C). These results suggested the involvement of mainly energy-dependent pathways. On the other hand, it is important to stress that conventional microscopy is not conclusive because nanoparticles could undergo both non-specific adsorption on the cell membrane and internalization and this technique does not discern between them.



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Figure 5. Light and fluorescent microscopy micrographs of RPMI 2650 cells exposed to RITC-labeled non-crosslinked and crosslinked 0.1% w/v mixed CS-PMMA30 PVAPMMA16 nanoparticles (1:1). (A) 4 h at 37oC, (B) 24 h at 37oC and (C) 4 h at 4oC. Nanoparticle crosslinking was carried out with 1% w/v TPP (2.5 L/mL of nanoparticle suspension). In

this

framework,

we

also

visualized

nanoparticle

uptake

by

confocal

laser scanning fluorescence microscopy (CLSFM). Since cellular f-actin was stained in red with phalloidin-Atto647N, nanoparticles were fluorescently labeled with fluorescein thioisocyanate (FITC, green fluorescence). At 37oC, cells showed nanoparticle uptake even after 4 h (Figure 6A). Conversely, at 4oC, no uptake could be visualized (Figure 6C). These results were confirmed by Z-stack analysis and indicated that the uptake mechanism is mainly energy-dependent (Supplementary Figure S2). It is worth stressing that the morphology of RPMI 2650 cells incubated at 4oC for 4 h remained unaltered (phase contrast micrographs, Figure 5C). In addition, we conducted the MTT assay with RITC-labeled nanoparticles and the cell viability under these conditions always remained ˃75% (data not shown). These results together with imaging fluorescent-activated cell sorting (FACS) assays (see below)


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indicated that RPMI 2650 cells withstand these incubation conditions and thus, we conducted uptake and permeability assays also at 4oC.

Figure 6. Fluorescent confocal micrographs of the RPMI 2650 cells exposed to FITClabeled non-crosslinked and crosslinked 0.1% w/v mixed CS-PMMA3: PVA-PMMA16 nanoparticles (1:1). (A) non-crosslinked, 4 h at 37oC (B) crosslinked, 4 h at 37oC and (C) crosslinked 4 h at 4oC. (A1,B1,C1) Nuclear staining with DAPI (blue), (A2,B2,C2) f-actin staining with phalloidin-Atto647N (red), (A3,B3,C3) FITC-labeled nanoparticles and (A4,B4,C4) merge. Nanoparticle crosslinking was carried out with 1% w/v TPP (2.5 L/mL of nanoparticle suspension). To quantify the uptake extent, cells exposed to RITC-labeled nanoparticles at 37 (4 and 24 h) and 4oC (4 h) were harvested and analyzed by FACS. The uptake of both non-crosslinked and crosslinked mixed nanoparticles at 37oC was confirmed, as exemplified in Figure 7.



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A1 Brightfield

RITC

RITC/Brightfield A2 Brightfield

RITC

RITC/Brightfield

B1 Brightfield

RITC

RITC/Brightfield B2 Brightfield

RITC

RITC/Brightfield

Figure 7. Imaging FACS analysis of RPMI 2650 cells exposed to RITC-labeled 0.1% w/v CS-PMMA30:PVA-PMMA16 for (A1,B1) 4 and (A2,B2) 24 h, at 37oC (A) before and (B) after crosslinking. Nanoparticle crosslinking was carried out with 1% w/v TPP (2.5 L/mL of nanoparticle suspension). Regardless of the exposure time, at 37oC, the percentage of stained cells was ˃98% (Table 2); approximately 13-15% of the nanoparticles did not undergo complete uptake and were detected on the cell membrane, while the rest was visualized in the cytosol. We also investigated the fluorescence intensity ratio between non-crosslinked and crosslinked nanoparticles within the cells at different time points. In general, crosslinking was not detrimental of nanoparticle uptake and the intensity ratio was ~1 (Table 1). In addition, the relative fluorescence intensity increased between 4 and 24 h by approximately 3-fold, regardless of the crosslinking. FACS results also suggest that upon internalization, nanoparticles are released to the cytosol as, otherwise, their localization in cell organelles should have been observed. Studies to reveal the intracellular nanoparticle trafficking were beyond the scope of this work. Finally, incubation at 4oC for 4 h (longer times resulted in massive cell death and FACS measurements were not possible) led to a significant 10- to 13-fold decrease of the uptake, in good agreement with confocal microscopy data.


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Table 2. Quantification of nanoparticle uptake by RPMI 2650 exposed to 0.1% w/v non-crosslinked and crosslinked RITC-labeled CS-PMMA30:PVA-PMMA16 (1:1) 0.1% w/v, as determined by imaging FACS analysis. Crosslinking was carried out with 1% w/v TPP (2.5 L/mL of nanoparticle suspension). Stained cytosol (%)

Temperature (°C)

Incubation time (h)

TPP crosslinkinga

Stained cells (%)

Stained membrane (%)

37

4

No Yes No

98.8 ± 0.8 98.5 ± 1.2 99.7 ± 0.3

14.0 ± 1.0 14.3 ± 1.5 15.0 ± 2.4

86.0 ± 1.0 85.7 ± 1.5 85.0 ± 2.4

Yes No Yes

99.8 ± 0.3 91.5 ± 5.7 93.4 ± 1.1

13.0 ± 1.7 19.2 ± 0.9 19.7 ± 0.5

87.0 ± 1.7 80.8 ± 0.9 80.3 ± 0.5

24 4

4 a

Total intensity ratio noncrosslinked/crosslinked nanoparticles

Intensity ratio 24 h/4 h

Intensity ratio 37°C/4°C 4h

1.06 ± 0.04

-

-

1.03 ± 0.04

2.85 ± 0.80 3.17 ± 0.83

0.86 ± 0.05

10 ± 2 13 ± 2

Nanoparticle crosslinking was carried out with 2.5 L of 1% w/v TPP per mL of 0.1% w/v nanoparticle dispersion.



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It is important to stress that some differences between FACS and CLSFM analyses may stem from the different sample preparation methods used (see experimental section for details). For instance, in FACS analysis cells were trypsinized and harvested before analysis, while in CLSFM they were initially fixed and washed several times between every staining stage. Permeability in vitro The permeability assay was evaluated in two comparable RPMI 2650 models with different interface, namely ALI and LL, by measuring the apparent permeability (Papp) of the nanoparticles. The comparison is based mainly on the physiological fact that the human nasal epithelium is not covered by liquid and, conversely, it is exposed to air what usually leads to higher formation of tight junctions.44,69 It is worth mentioning that the ability of RPMI 2650 cells to produce mucin has not been established.64 On the other hand, CS transiently opens these junctions and thus, facilitates paracellular permeability. Since after adjusting the conditions (TPP volume), nanoparticle crosslinking did not markedly change the interaction of the nanoparticles with nasal epithelial cells and these nanoparticles are physically stable and not affected by dilution, for these experiments, we utilized only 0.01% and 0.05% w/v crosslinked mixed nanoparticles. The assessment of two different nanoparticles concentrations relies on a recent work done with an in vitro model of intestinal epithelium where we demonstrated that the Papp is concentration-dependent.29 In addition, since transcellular (energy-dependent) and paracellular (energy-independent) mechanisms could be involved in the permeability, we conducted these experiments at both 4 and 37oC. To estimate the integrity of the cell monolayers, transepithelial electrical resistance (TEER) values were measured over 14-21 days until a constant value between 140


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and 170 Ω·cm2 was measured for LL and ALI conditions, respectively.58–60 It is important to stress that the TEER increase of 25-30 Ω·cm2 under ALI incubation conditions on the same experiment day was consistent with the formation of tighter junctions.69-71 The Papp was calculated according to Equation 1

𝑃𝑎𝑝𝑝 =

𝑑𝑐 1 [𝑐𝑚 ⋅ 𝑠 ―1] ⋅ 𝑑𝑡 𝐴 ⋅ 𝐶0

(1)

where 𝑑𝑐/𝑑𝑡 is the permeability rate (μg/s) across the monolayer, 𝐶0 is the initial concentration in the donor compartment (μg/cm3), and 𝐴 is the surface area of the membrane (cm2). In general, Papp values were in good agreement with previous studies conducted with CS-based amphiphilic nanoparticles and utilizing other in vitro models of epithelium (Figure 8).72

Figure 8. Apparent permeability coefficient (Papp) of 0.01% and 0.05% w/v crosslinked CS-PMMA30:PVA-PMMA16 (1:1) nanoparticles at 37 and 4oC in RPMI2650 monolayers under ALI and LL conditions. Nanoparticle crosslinking was carried out 23 ACS Paragon Plus Environment


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with 1% w/v TPP (2.5 L/mL of nanoparticle suspension). *Statistically significant difference (P 130 Ω·cm−2 were used. Nanoparticles for these experiments were prepared as described above for cell uptake assays and 1% w/v RITC-labeled samples were diluted to a final concentration of 0.1% w/v in HBSS (Sigma-Aldrich) buffered to pH 7.2 with 25 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES, Sigma-Aldrich) that was used as transport medium. Next, the crosslinking solution (1% w/v TPP in HBSS) was added, and nanoparticles incubated at 25°C overnight. Finally, the crosslinked samples were diluted to a final concentration of 0.05% or 0.01% w/v. At the beginning of the experiment, the medium in the apical and basolateral was replaced with transport medium (HBSS) and cells incubated for 15 min at 37°C in a humidified 5% CO2 atmosphere to enable their adjustment to the new medium. Then, transport medium in the donor (apical) compartment was replaced by the corresponding sample (0.4 mL) and in the acceptor compartment (basolateral) by fresh transport medium (1.2 mL). After 5, 10, 15, 30, 45, 60, 90, 120, 180, and 240 min, 600 μL of medium was extracted from the basolateral compartment and replaced


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by the same volume of fresh transport medium to maintain the total volume in the chamber constant. The extracted medium was used for quantification of the transported copolymers by fluorescence spectrophotometry (Fluoroskan Ascent Plate Reader, Thermo Fisher Scientific Oy) using black 96-well flat bottom plates (Greiner Bio-One, Kremsmünster, Austria) at wavelengths of 485 nm for excitation and 635 nm for emission. The red fluorescence in the acceptor chamber due to the permeability of RITC-labeled nanoparticles was measured and interpolated in a calibration curve built with different nanoparticle concentrations (in the 0.0001–0.1% w/v range) containing 20% w/w of RITC-labeled PVA-g-PMMA copolymer (R2 = 0.995). In addition, an experiment with cell-free inserts was conducted to calculate PE values. Results are expressed as mean ± S.D. of at least three experiments. In addition, at the end of each permeability experiment, we measured the fluorescence in both the donor and the acceptor chamber to calculate the mass balance and the %Nanoparticles retained by cell monolayer. Statistical analysis Statistical analysis of permeability experiments was performed by t-test on raw data (Excel, Microsoft Office 2013, Microsoft Corporation). The analysis was performed for Papp obtained from the different conditions and concentrations. P-values were compared between different concentrations (0.05% and 0.01% w/v) at the same temperature and between different temperatures at the same concentration. In addition, a further analysis was performed, and p-value was calculated between the ALI and LL conditions. Supporting Information The Supporting Information is available free of charge on the ACS Publications Web site. HNEpC cell viability upon exposure to different weight ratios of CS-PMMA30:PVAPMMA16 before and after crosslinking, 1H-NMR and FTIR spectra of pure CS, pristine 35 ACS Paragon Plus Environment


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PMMA, pure PVA, CS-PMMA30, PVA-PMMA16 and mixed CS-g-PMMA:PVA-gPMMA nanoparticles and epithelial permeability of crosslinked CS-PMMA30:PVAPMMA16 (1:1) nanoparticles at 37 and 4oC in RPMI2650 monolayers under ALI and LL conditions. ACKNOWLEDGMENTS. This research was funded by the Israel Science Foundation (ISF Grant #269/15) and the Teva National Network of Excellence in Neuroscience Research Grant. Partial support of the Russell Berrie Nanotechnology Institute (Technion) is also acknowledged.

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Graph for Table of Contents

Brain

LL

ALI

TJ

Olfactory area

T J

Nasal cavity

TPP

....

+

NH3

CS-PMMA30:PVA-PMMA16 nanoparticle

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Mixed Mucoadhesive Amphiphilic Polymeric Nanoparticles Cross a

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