Transport Mechanisms of Butyrate Modified Nanoparticles: Insight into

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Transport mechanisms of butyrate modified nanoparticles: insight into “easy entry, hard transcytosis” of active targeting system in oral administration Lei Wu, Yuli Bai, Min Liu, Lian Li, Wei Shan, Zhirong Zhang, and Yuan Huang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00713 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 17, 2018

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Molecular Pharmaceutics

Transport mechanisms of butyrate modified nanoparticles: insight into “easy entry, hard transcytosis” of active targeting system in oral administration

Lei Wu, Yuli Bai, Min Liu, Lian Li, Wei Shan, Zhirong Zhang and Yuan Huang*

Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University. No. 17, Block 3, Southern Renmin Road, Chengdu 610041, P.R. China

Corresponding Author *E-mail: [email protected], Tel.: +86-28-85501617, Fax: +86-28-85501617.

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ACS Paragon Plus Environment

Molecular Pharmaceutics 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

Abstract The intestinal epithelium constitutes a major barrier for orally delivered nanoparticles (NPs). Although surface ligand modification can increase cellular uptake of NPs, the transepithelial transport of active targeting NPs is relatively limited. The phenomenon is described as “easy entry, hard transcytosis”. Yet, underlying mechanisms and potential solutions of this phenomenon are unclear. Here, butyrate modified polyethylene glycol coated NPs (Bu-PEG NPs) were chosen as the model active targeting NPs. Transport mechanism studies were performed to get a better understanding of intracellular trafficking and exocytosis fate. Results showed that after active binding to monocarboxylate transporter-1 (MCT-1), Bu-PEG NPs went through endolysosomal pathways, endoplasmic reticulum/Golgi recycling routes and microtubule-dependent shuttling within Caco-2 cells. Then, a larger proportion of Bu-PEG NPs was exocytosed from apical side. Notably, increasing the basal expression of MCT-1 by leptin facilitated basal exocytosis and transcytosis of Bu-PEG NPs, confirming that enhanced receptor recognition could promote “basal exit”. In addition to the effect of receptor recognition, surface properties also influenced the bidirectional exocytosis of Bu-PEG NPs. When surface hydrophobicity increased, Bu-PEG NPs were dominantly exocytosed from basal membrane. Hence, two strategies may help to overcome “hard transcytosis” of active targeting NPs. One is to enhance their affinity with basal membrane by reinforcing the receptor-ligand interaction; the other is to weaken apical exocytosis by optimizing surface hydrophobicity. Thereby, this study might provide important implications for the rational design of NPs to further increase transepithelial transport efficiency.

Keywords: Active targeting nanoparticles, “easy entry, hard transcytosis”, transport mechanism, bidirectional exocytosis, oral drug delivery, regulation of receptor distribution, surface hydrophobicity

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1. Introduction Nanoparticles (NPs) have been widely investigated in oral drug delivery due to their unique advantages including improved drug solubility and robust protection against enzymes1-2. Yet, the intestinal epithelium acts as a formidable absorption barrier, and severely limits the delivery of drugs into blood circulation3-4. For this reason, recent efforts have been made to increase the cellular internalization of NPs5-6. Among these strategies, ligand functionalization increased membrane affinity and enhanced cellular uptake via specific receptor recognition7.

Despite the merits of active targeting, it should be noted that current researches mainly focused on boosting the endocytosis process. Beyond endocytosis, the intracellular trafficking and exocytosis also determined the fate of NPs8-9. In view of the “polarization” of epithelia, the exocytosis from basolateral side is a key step for ultimate release of drugs into blood stream10. Unfortunately, limited migration from apical to basal side was frequently observed in many ligand-modified NPs, an event described

as

“easy

entry,

(NH2-C6-[cMPRLRGC]c-NH2)

hard

transcytosis”.

modified

For

nanoparticles

instance, (P22NPs)

peptide-22 targeted

low-density lipoprotein receptor (LDLR) and showed 2.5-fold increase in cellular uptake, whereas the transcytosis was only increased by 1.4-fold11. Likewise, folic acid functionalization elevated the cellular uptake of liposomes by ~3.3-fold, but the pharmacological availability was only increased by ~1.2-fold12. Collectively, even though ligands effectively ameliorated cellular internalization, the basolateral transport was still limited.

To our knowledge, the apical exocytosis exists and is unwanted, since it hampers the final transcytosis efficiency across the intestinal epithelium13. However, few in-depth mechanism investigations have been performed on “hard transcytosis” of ligand-functionalized NPs. Details in bidirectional exocytosis and the effect of receptor recognition on NP transportation remain unrevealed. A better understanding 3



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of how ligand-modification affects each stage of NP transcytosis would be beneficial to optimize the design of active targeting NPs in oral drug delivery.

Hence, in the present study, butyrate modified polyethylene glycol coated NPs (Bu-PEG NPs) were chosen to be further investigated. In our previous research, active targeting Bu-PEG NPs showed enhanced cellular uptake compared with unmodified PEG NPs by binding to monocarboxylate transporter-1 (MCT-1)14. Yet, the effect of butyrate modification on intracellular trafficking and exocytosis need further evaluation. Herein, the following questions were investigated: (1) How would the ligand modification influence the intracellular trafficking pathways of NPs? (2) What is the effect of butyrate functionalization on the bidirectional exocytosis from both apical and basolateral sides? (3) Are the transporters of butyrate involved in the intracellular trafficking of Bu-PEG NPs? (4) What is the effect of receptor recognition on NP exocytosis? (5) How would the surface characteristics, such as surface hydrophobicity, influence the transport of ligand-modified NPs? The understanding of these key points may aid future design of active targeting NPs with improved transcytosis efficiency.

2. Materials and methods 2.1. Materials Poly (lactic-co-glycolic acid) (PLGA, 50/50, viscosity: 0.26-0.54 dL/g) with one methoxy

end

group

was

obtained

from

Lactel

absorbable

polymers.

1,2-Distearoyl-sn-glycero-3-phosphatidylethanolamine-polyethylene glycol (DSPE– PEG) was purchased from Avanti Polar Lipids. DSPE-PEG with a butyrate end group (DSPE-PEG-Bu) was provided by Ponsure (Shanghai, China). Recombinant leptin was purchased from Cloud-Clone Corp. (Houston, USA). Rabbit anti-human monocarboxylate transporter-1 (MCT-1) and rabbit anti-human CD147 were provided by Absin Bioscience Inc. (Shanghai, China). TRITC Red conjugated IgG, lysosome-tracker green and endoplasmic reticulum-tracker green were obtained from 4


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KeyGEN perchlorate

Biotech (DiO)

Corp. and

(Nanjing,

China).

3,3-dioctadecyloxacarbocyanine

1,1-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine

perchlorate (DiI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were bought form Sigma-Aldrich (St. Louis, MO, USA), Aladdin Chemistry Co., Ltd. (Shanghai, China) and Shanghai Chemical Reagents Co. (Shanghai, China) were used as received, unless otherwise stated.

2.2. Preparation of NPs with varying surface hydrophobicity Polyethylene glycol (PEG) chains coated PLGA NPs (PEG NPs) were prepared via nano-precipitation as previously reported14. Butyrate modified PEG NPs (Bu-PEG NPs) with varying surface hydrophobicity were obtained by modulating the weight ratio of DSPE-PEG-Bu and PLGA. The mass fraction of DSPE-PEG-Bu was set as 33%, 50%, 80%, and 90% in corresponding NPs named as 33%, 50%, 80% and 90% Bu-PEG NPs, respectively. The PLGA content was altered, and the concentration of DSPE-PEG-Bu kept constant in a series of NP suspensions. Hydrophobic dye DiI or DiO was loaded in NPs as the fluorescent indicator.

2.3. Stability study To investigate the stability of NPs, the Förster resonance energy transfer (FRET) assay and dye release study were carried out15. For FRET study, DiI and DiO were co-encapsulated in Bu-PEG NPs. DiI-loaded NPs and DiO-loaded NPs were prepared as control groups. Then, different NPs were dispersed in water at the same concentration of DiO/DiI. The fluorescence intensity was measured by a fluorescence spectrophotometer (Shimadzu RF-5301, Japan) with an excitation wavelength of 460 nm and the emission spectrum was recorded from 490 to 620 nm. Next, NPs were incubated in PBS (pH 5.0 or 7.4). At determined time points, the fluorescence intensity of dual-loaded and single-loaded Bu-PEG NPs was detected, respectively. The energy transfer efficiency (E) was calculated by the following formula. E = 1 − / 5



FDA and FD represented the fluorescence intensity of the donor alone (DiO-loaded Bu-PEG NPs) and with the presence of the acceptor (DiO/DiI-loaded Bu-PEG NPs), respectively.

Dual-loaded Bu-PEG NPs were co-incubated with cells for 2 h and 4 h, respectively. The fluorescence of DiI and DiO within cells was visualized by confocal fluorescence microscopy (Zeiss LSM 800). Meanwhile, cells exposed to NPs were imaged with an excitation wavelength of 488 nm and emission wavelengths from 510 to 650 nm at 35 nm intervals16.

For dye release evaluation, PBS (pH 5.0 or 7.4) were selected as release medium. In brief, 1 ml of NP suspension was sealed in a dialysis bag (MWCO: 100 kDa) and released against 40 ml of release medium for 24 h17. At determined time points, an aliquot of exterior medium (50 µL) was withdrawn and measured via Varioskan Flash Multimode Reader. The accumulative amount of released dye was then calculated.

2.4. Intracellular trafficking of NPs Firstly, NPs were incubated with Caco-2 cells for 2 h. Then, NPs were removed followed by washing cells thrice with fresh PBS. Specific inhibitors (i.e., brefeldin A, monensin, LY294002 and nocodazole) and cells were co-incubated for another 2 h18. The amount of intracellular NP was detected via flow cytometric analysis as previously reported11. The concentrations of inhibitors were listed as follows. Brefeldin A was 25 µg/ml, monensin was 33 µg/ml, LY294002 was 1 mM and nocodazole was 10 µM.

For colocalization analysis, cells were treated with trackers of subcellular organelles after co-incubation with NPs for 2 h. Endoplasmic reticulum (ER)-tracker green and lysosome-tracker green were used according to the instruction11. Anti-β-tubulin (1:1000) was applied to stain the microtubules. The immunofluorescence protocols 6


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were performed as previously reported19. 4′, 6-diamidino-2-phenylindole (DAPI) (5 mg/mL) was applied to stain the nucleus for 3 min before imaging. The images were obtained via confocal fluorescence microscope. Pearson’s correlation coefficient (Rr) was calculated and line profile analysis was performed via Image Pro Plus.

2.5. Exocytosis from apical and basolateral membrane Briefly, NP suspension was added in the apical chamber and fresh medium was in the basolateral side. NPs were allowed to transport across the Caco-2 cell monolayer. After 2 h, NP suspension (apical side) and fresh medium (basolateral side) were both replaced by medium containing specific inhibitors. The concentrations of inhibitors were same as those in intracellular trafficking assay. The exocytosis of NPs in two directions was detected. At determined time points (15, 30, 60, 120, 240 and 480 min), an aliquot of apical and basolateral medium (50 µL) was withdrawn, respectively. 50 µL of fresh medium containing inhibitors was replenished. Then, 50 µL of DMSO was added to destroy NPs and the fluorescence intensity of each well was measured via Varioskan Flash Multimode Reader. The exocytosis rate was calculated as follows: where dQ/dt is the transport amount of Dil into apical (or basal chamber) per second. Exocytosis rate =

2.6. Immunofluorescence microscopy To investigate the influence of leptin on protein expression, leptin (100 nM) was incubated with Caco-2 cells for 18 h and then removed. Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, then permeabilized with 0.1% Triton-100 for 15 min and blocked with 5% goat serum for 1 h at 37 °C. Coverslips were immunostained with rabbit anti-human MCT-1 for 2 h at 37 °C. Cells were washed and incubated in the dark for 2 h with goat anti-rabbit TRITC-conjugated IgG. Finally, coverslips were stained with DAPI (5 µg/ml) for 5 min, mounted with anti-fade reagent and visualized using confocal fluorescence microscopy (Zeiss LSM 800). For staining CD147, the rabbit anti-human MCT-1 was 7



replaced by rabbit anti-human CD147 and other procedures were same as described above.

2.7. Surface hydrophobicity assay NP dispersions of increasing concentrations were incubated with Rose Bengal (RB) solution at fixed concentration (20 µg/ml) in a shaker (37 ℃, 60 rpm) for 3 h20. Next, particles were removed by centrifugation. Amount of free RB, which was not bound to particle surface, was measured by fluorescent spectrometry. Binding constant (K) was calculated according to the following formula. = − N is the maximal amount of bound RB, r represents concentration of bound RB and a is the equilibrium concentration.

2.8. Cellular uptake of NPs with varying surface hydrophobicity After exposed to NPs for 4 h, Caco-2 cells were washed thrice with PBS. Then, cells were harvested and analyzed by flow cytometry as previously reported21-22. Mean fluorescence intensity was detected for 1 × 104 cells in each sample. The fluorescence intensity indicated relative amount of cellular uptake.

3. Results and discussions 3.1. The stability of NPs during the trafficking within Caco-2 cells Bu-PEG NPs and PEG NPs were prepared via self-assembly nanoprecipitation according to our previous study14. The diameters of both NPs were around 90 nm. The zeta potential declined from -6 mV to -16 mV after butyrate-functionalization. Hydrophobic dye DiI was loaded to track NP trafficking within Caco-2 cells15. As shown in Fig. 1A, around 95% of DiI remained in NPs after incubation at pH 7.4 within 5 h. Similar results were also observed in Fig. 1B that over 97% of DiI remained in Bu-PEG NPs under acidic environment, mimicking lysosomal condition23. Therefore, both NPs exhibited good stability at pharmacological and acidic pH. 8


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Next, fluorescence resonance energy transfer (FRET) analysis was performed to monitor the particle integrity15, 18. A pair of FRET dyes, DiO and DiI, recognized as the donor and acceptor, was encapsulated into Bu-PEG NPs. FRET signal was indicated by the quenching of emission at 510 nm and increase in emission at 560 nm in DiO/DiI-loaded NPs (Fig. 1C), which demonstrated that the distance between DiO and DiI was less than 10 nm24. After incubation in both neutral and slightly acidic medium, the FRET phenomenon still existed (Fig. S1), indicating the integrity of nano-structure. Further, the stability of NPs after endocytosis was evaluated. Dual FRET dyes-loaded NPs were exposed to Caco-2 cells for 2 and 4 h, followed by CLSM imaging. Fig. 1D showed the colocalization of DiI and DiO within cells, indicating that a large proportion of NPs remained intact during the intracellular trafficking. Moreover, when excited at 488 nm, fluorescence intensity could be detected around 560 nm (Fig. 1E), further demonstrating the integrity of NPs within cells16. Therefore, it was feasible to monitor the behaviors of NPs by dye tracking in the following studies.

Fig.1 The percentage of DiI remained in NPs at pH 7.4 (A) and pH 5.0 (B). Error bars represent SD (n=3). (C) Emission spectrum of DiO-loaded NPs, DiI-loaded NPs and DiO/DiI-loaded NPs excited at 460 nm. (D) CLSM images of cellular uptake of Bu-PEG NPs at 2 or 4 h. DiO (green) and DiI (red) were loaded in NPs. DAPI (blue) 9



represented the nucleus. Scale bar: 20 µm. (E) Cells exposed to Bu-PEG NPs suspensions for 4 h were imaged by CLSM with an excitation wavelength of 488 nm and emission wavelengths from 510 to 615 nm at 35 nm intervals. FRET images at different emission wavelength and fluorescence spectra of cells after incubation with DiI/DiO-labeled Bu-PEG NPs. Scale bar: 20 µm.

3.2. Butyrate modification did not alter the intracellular trafficking pathways of NPs Our previous research demonstrated that PEG NPs and Bu-PEG NPs were both internalized into cells via multiple routes, including clathrin-mediated and caveolin-mediated endocytosis, while micropinocytosis was only observed in unmodified PEG NPs (Scheme 1)14. In this section, intracellular trafficking pathways of both NPs were investigated. Firstly, to investigate whether NPs were trafficked to lysosomes, LY294002 (PI3-kinases inhibitor) was added to block the lysosomal pathway. The apical exocytosis of both NPs was inhibited by LY294002, reflected by the enhanced retention within cells (Fig. 2A), demonstrating the involvement of lysosomal pathway in intracellular trafficking. To visualize the colocalization of lysosome with NPs, we stained cells with LysoTracker Green after NP incubation25. CLSM images and line profiles in Fig. 2B and S2 revealed the colocalization of NPs and lysosomes (Rr>0.5), further confirming the endolysosomal trafficking of both NPs.

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Scheme 1 The scheme of transport pathways of Bu-PEG NPs and several factors that influenced each stage of transcytosis.

Next, brefeldin A and monensin were used to inhibit endoplasmic reticulum (ER)/Golgi and Golgi/plasma membrane (PM) pathway, respectively9, 26. Fig. 2C showed that both Bu-PEG NPs and PEG NPs were transported to ER and Golgi apparatus. Colocalization of NPs with ER (Rr>0.5) was also observed via CLSM (Fig. 2D and S2). Both clathrin- and caveolae-mediated routes would exchange their cargos with ER and Golgi apparatus, thus avoiding the lysosomal degradation to some extents9. If NPs could take the advantages of ER and Golgi routes, they might further increase the drug stability and transcytosis efficiency.

Moreover, the colocalization of both NPs with microtubules were confirmed by immunostaining (Fig. 2F and S2). The colocalization value (Rr) observed with Bu-PEG NPs and PEG NPs was 0.78 and 0.63, respectively. It suggested the microtubule-dependent active movement of both NPs. Notably, the inhibitor 11



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nocodazole, as a microtubule disruptor, significantly blocked the intracellular transport of Bu-PEG NPs, but barely affected the trafficking of PEG NPs (Fig. 2E). Considering the complexity of intracellular trafficking, the distinct trafficking rates may result in the different sensitivity to nocodazole-treatment. Collectively, butyrate functionalization did not alter the intracellular fate of PEG NPs. Both NPs were transported

via

endolysosomal

routes,

ER/Golgi

recycling

pathways

and

microtubule-dependent shuttling.

Fig.2 Intracellular trafficking of NPs in different cell organelles based on colocalization of NPs with cell organelles. (A, C, E) The influence of different transport inhibitors on intracellular trafficking and exocytosis of NPs. The amount of NP retention within cells after exocytosis of 2 h was measured. *p

Transport Mechanisms of Butyrate Modified Nanoparticles: Insight into

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