Entrapping of Nanoparticles in Yeast Cell Wall Microparticles for

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Entrapping of nanoparticles in yeast cell wall microparticles for macrophage-targeted oral delivery of cabazitaxel Tianyang Ren, Jingxin Gou, Wanxiao Sun, Xiaoguang Tao, Xinyi Tan, Puxiu Wang, Yu Zhang, Haibing He, Tian Yin, and Xing Tang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00357 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

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Molecular Pharmaceutics

Entrapping of nanoparticles in yeast cell wall microparticles for macrophage-targeted oral delivery of cabazitaxel Tianyang Ren1, Jingxin Gou1, Wanxiao Sun1, Xiaoguang Tao1, Xinyi Tan1, Puxiu Wang3, Yu Zhang1, Haibing He1, Tian Yin2, Xing Tang1, * 1

Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical

University, Shenyang 110016, Liaoning, PR China 2

School of Functional Food and Wine, Shenyang Pharmaceutical University,

Shenyang 110016, Liaoning, PR China 3

Department of Pharmacy, the First Affiliated Hospital of China Medical University,

Shenyang, Liaoning, PR China

*

Corresponding author: Professor Xing Tang, Department of Pharmaceutics

Science, Shenyang Pharmaceutical University. E-mail: [email protected] 103 Wenhua Road, Shenyang 110016, Liaoning, PR of China Tel: +8602423986343; Fax: +8602423911736;

1

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Abstract In this work, a nano-in-micro carrier was constructed by loading polymer-lipid hybrid nanoparticles (NPs) into porous and hollow yeast cell wall microparticles (YPs) for macrophage-targeted oral delivery of cabazitaxel (CTX). The YPs, primarily composed of natural β-1,3-D-glucan, can be recognized by the apical membrane receptor, dectin-1, which has a high expression on macrophages and intestinal M cells. By

combining

electrostatic

force-driven

self-deposition

with

solvent

hydration/lyophilization methods, the positively charged NPs loaded with CTX or fluorescence probes were efficiently packaged into YPs, as verified by scanning electron microscope (SEM), atomic force mircoscope (AFM) and confocal laser scanning microscopy (CLSM) images. NPs loaded YPs (NYPs) showed a slower in vitro drug release and higher drug stability compared with NPs in a simulated gastrointestinal environment. Biodistribution experiments confirmed a widespread distribution and extended retention time of NYPs in the intestinal tract after oral administration. Importantly, a large amount of NYPs were primarily accumulated and transported in the intestinal Peyer’s patches as visualized in distribution and absorption site studies, implying that NYPs were mainly absorbed through the lymphatic pathway. In vitro cell evaluation further demonstrated that NYPs were rapidly and efficiently taken up by macrophages via receptor dectin-1 mediated endocytosis using a mouse macrophage RAW 264.7 cell line. As expected, in the study of in vivo pharmacokinetics, the oral bioavailability of CTX was improved to 32.1 % when loaded in NYPs, which is approximately 5.7 times higher than CTX solution, indicating the NYPs are efficient for oral targeted delivery. Hence, this nano-in-micro carrier is believed to become a hopeful alternative strategy for increasing oral absorption of small molecule drugs.

Keywords: oral delivery, yeast cell wall microparticle, macrophage targeting, 2


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nanoparticle, small molecule drug

1. Introduction Oral route is the most common and favorable way of administration, attributing to its high safety, convenience, and patient compliance1. Oral delivery of anticancer agents has increasingly gained focus in recent developments for cancer therapy2. Cabazitaxel (CTX), a novel semisynthetic taxane approved by the U.S. Food and Drug Administration (FDA), exerts greater antitumor activity and lower affinity for multidrug resistance (MDR) proteins than docetaxel and paclitaxel3, 4. However, similar to other taxanes, oral delivery of CTX is still limited due to its low hydrophilcity, poor

penetration across epithelium and specific sensitivity to

P-glycoprotein, pre-systemic metabolic enzymes, and sophisticated gastrointestinal environment2, 5. Thus, it is a great challenge to develop an oral carrier for the delivery of CTX to overcome these significant problems. In the past decades, polymers and lipids etc. have been widely used to develop various nanocarriers to load lipophilic small molecule drugs, such as taxanes, for oral delivery6, 7. These nanocarriers have shown their advantages for improving oral bioavailability of anticancer components by increasing the drug loading, enhancing drug stability against complex gastrointestinal environments, and more importantly, overcrossing the oral absorptive barriers depending on their biophysicochemical properties, but they are still limited1, 6. Recently, yeast cell wall microparticles(YPs), derived from baker’s yeast Saccharomyces cerevisiae, have provided a potential system for oral drug delivery, and were first used as micron-sized oral vehicle by the group of Ostroff et al8. These natural carriers are porous microspheres with hollow cavities, by which chemicals and small particles can be entrapped9-11. YPs are mainly composed of β-1,3-D-glucan, which can be recognized by the apical membrane receptors on phagocytic cells10, 12. The primary membrane β-glucan receptor is dectin-1, which is a highly expressed lectin on the surface of phagocytic cells, such as macrophages, dendritic cells and specifically M cells13-15. β-glucan microparticles can be easily taken up by 3



macrophages via dectin-1 mediated endocytosis. Hence, cargo loaded YPs could be rapidly internalized by M cells and transported by macrophages to the circulation via the lymphatic system10. Therefore, YPs are ideal to be applied as a vehicle for targeting phagocytic cells, especially intestinal M cells. Until now, YPs have been primarily employed to encapsulate and orally deliver soluble macromolecules, e.g. DNA8, siRNA12,

16

, vaccines17-19 and therapeutic proteins20-22. However, the

employment of YPs for oral delivery of active small molecules is quite limited by YPs, as most major small molecule drugs are small in size, neutral in charge and insoluble in water, and are thus rarely steadily entrapped in YPs11, 23, 24. To solve this problem, a nano-in-micro method, that is, entrapping drug-loaded nanocarriers in the YPs, can be applied in development of new oral delivery systems. Nanoparticles (NPs), with different physicochemical properties, can be selected to realize orally targeted delivery of small molecule drugs, such as CTX, by packaging into YPs. In this study, a nano-in-micro carrier was developed to orally deliver CTX by targeting intestinal M cells (Scheme 1). Initially, CTX was loaded in the charged polymer-lipid matrix hybrid NPs, which have been shown to be useful in improving drug loading and sustained release in our previous study25. After this, the CTX loaded NPs (CTX-NPs) were entrapped in the hollow, porous YPs by combining electrostatic force-driven self-deposition with a solvent hydration/lyophilization method. The characterizations of CTX-NPs loaded YPs (CTX-NYPs) were generally performed. To investigate the intestinal absorption process of NYPs, fluorescence labeled NYPs were used to visualize the biodistribution in mouse intestinal tracts after oral administration, as well as the absorption site in the intestinal segments by directly intestinal lumen injection. In vitro cell evaluation was implemented to study the rapid internalization and uptake mechanism of NYPs by phagocytic cells using a mouse macrophage cell line, Raw 264.7 cell. Finally, the bioavailability of CTX-NYPs orally administrated to rats was measured to verify the efficiency of the nano-in-micro carriers for macrophage-targeted delivery of anticancer agents.

4


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Scheme 1. Schematic illustration of the preparation of CTX-loaded nano-in-micro carriers(A) and their intestinal absorptive pathway by targeting M cells after oral administration(B). 2. Materials and methods 2.1 Materials and animals CTX (> 99%) was obtained from Chengdu D-innovation Pharmaceutical Co., Ltd. (Sichuan, China). Larotaxel (LTX, > 99%) was provided by Shandong Target Drug

Research

Co. Ltd.

(Yantai,

China).

Baker’s

yeast

(Anqi,

China).

Poly(ε-caprolactone) (PCL, molecular weight: 5000 Da) (Jinan Daigang Biomaterial Co., Ltd, Shandong, China). Medium chain triglyceride (MCT) and soyabean lecithin (Lipoid S75) (Lipoid KG, Ludwigshafen, Germany). 1,1′-Dioctadecyltetramethyl indotricarbocyanine iodide (DiR), coumarin-6, hexadecyl trimethyl ammonium chloride (CTAC) and rhodamine B isothiocyanate (RBITC) were purchased from Sigma (St. Louis, MO, USA). 4’, 6-Diamidino-2-phenylindole (DAPI) was obtained 5



from Gen-view Scientific Inc. (USA). Penicillin, streptomycin and 0.25% trypsin-EDTA were all bought from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA) and culture medium was bought from Wisent Inc. (St Bruno, Quebec, Canada). The deionized water was used, and the other solvents and chemicals were analytical or HPLC grade. CTX solution (CTX-Sol) was prepared with Tween 80, ethanol and saline as the formulation of JEVTANA®. The animals (SD rats and KM mice) were provided from Changsheng Biotechnology Co., Ltd. (Liaoning, China). The animal experiments performed were in accordance with the approval and oversight of the ethical committee of Shenyang Pharmaceutical University. 2.2 Preparation and characterization 2.2.1 Preparation of CTX-NPs The CTX-NPs was prepared by the emulsion-solvent evaporation method. Firstly, the organic phase was formed by dissolving CTX, PCL, MCT and soybean lecithin in ethyl acetate and CTAC was separately dissolved in deionized water to form the aqueous phase. And then, the organic phase was transferred dropwise to the aqueous phase with 400 rpm stirring. After the formation of primary emulsion, this suspension was under ultrasonication process with a probe sonicator at 200 w for 2 min. Finally, the organic solvent was completely removed from the obtained emulsion under a reduced pressure condition by use of the rotary evaporator. 2.2.2 Preparation of YPs YPs was prepared from baker’s yeast Saccharomyces cerevisiae according to alkali and acid treatments as previously reported with some modifications8. Briefly, 50 g baker’s yeast was dispersed in 1 L NaOH solution (1 M), and the suspension was stirred continuously for 1 h at 80 °C. After centrifugation for 5 min at 3,000 rpm, the obtained precipitate was rinsed twice with deionized water and re-suspended in 1 L HCL solution (pH 4.0), and stirred at 60 °C for 1 h. The YPs were collected by centrifugation and rinsed twice with deionized water. Afterwards, the YPs were rinsed 6


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four times with 100 mL isopropanol and twice with 100 mL acetone, and then dried at room temperature. 2.2.3 Preparation of NYPs To prepare the NYPs successfully, a novel method was developed by combining electrostatic force-driven self-deposition with solvent hydration/lyophilization, as reported by Zhou et al.10 and Soto et al.24, respectively. Briefly, dry YPs were first incubated with sodium carbonate buffer solution (0.1 M, pH 9.2) at 37 °C for 0.5 h, then the NP suspension was added dropwise. After incubation for another 1 h, the buffer solution was replaced by deionized water by centrifugation at 4,000 rpm for 10 min, and the suspension was followed by a lyophilization-hydration cycle. The lyophilization-hydration procedure was repeated twice by utilizing water to push the NPs loaded in the holes or attached at the surface into the core of YPs by capillary action. After the second hydration, the NYPs were rinsed thoroughly with water to dispose of free NPs, followed by a final lyophilization. 2.2.4 Fluorescence labeling of YPs RBITC was used to label YPs as previously reported22, 26. Briefly, 100 mg YPs were incubated in 10 mL sodium carbonate buffer solution (0.1 M, pH 9.2) at 37 °C for 0.5 h, and then RBITC solution (1 mg/mL, dissolved in DMSO) was added dropwise and stirred overnight in the dark. The unreacted fluorescence probe was quenched in Tris buffer (1 M, pH 8.3) for 0.5 h. Then, the fluorescence labeled YPs were well washed with sterile water, rinsed with ethanol and acetone to remove the water, and dried at room temperature in the dark. 2.2.5 Size, zeta potential and particle morphology The average particle size, size distribution and zeta potential of particles was detected on a Malvern Zetasizer Nano ZS instrument at 25 °C. The morphology of NPs was visualized under transmission electron microscopy (TEM) (JEM-2100, Japan). The diluted NP suspension was transferred onto a copper grid appropriately, and then stained with 2 % phosphotungstic acid. The samples were dried in air at room temperature before observation. 7



The morphology of YPs and NYPs were observed under scanning electron microscopy (SEM) (S3400, Japan) and atomic force microscopy (AFM) (Cypher ES, UK). The dry samples of YPs and NYPs were sputter coated with gold under a high vacuum before SEM imaging. Topography images of the surface of YPs and NYPs were obtained in AFM AC mode using a medium-soft silicon cantilever (AC240TS-R3, Olympus). Fluorescence observation was performed on a laser scanning confocal microscope (CLSM) (Zeiss, Germany). 2.2.6 Drug loading (DL) and encapsulation efficiency (EE) The HPLC was employed to determine the DL and EE of CTX in lyophilized NYPs at 230 nm in accordance with the following formulas:

DL% = EE% =

× 100% × 100%

2.3 In vitro drug release study The CTX release profiles from different formulations were assessed in the mediums of pH 6.8 PBS and pH 1.2 HCl solution with addition of Tween 80 (0.5 %) by the use of a dialysis method25. In details, 1 mL of the samples were poured into the dialysis bag with a molecular weight cut off of 14 KDa and immersed in the vials with 10 mL medium. Then, the vials were gently placed in a shaking bath with constant temperature (37 °C) and shaking (100 rpm). The medium containing the released CTX was taken totally at predetermined time points and replenished with fresh medium. The drug concentrations of the obtained samples were measured using HPLC method at 230 nm and the accumulated drug release was performed. 2.4 Stability in simulated gastric fluid (SGF) and intestinal fluid (SIF) To investigate the stability of drug encapsulated in NPs and NYPs in the gastrointestinal tract, CTX loaded NPs and NYPs was mixed with SGF or SIF, and then incubated in a shaking bath with constant temperature (37 °C) and shaking (100 rpm). At pre-determined time points, 200 µL samples were removed, dissolved with appropriate acetonitrile, and then the amount of CTX was measured under HPLC. 2.5 Biodistribution of NYPs in intestine 8


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The fluorescently-labeled NYPs were used to study the biodistribution in rats or mice after oral administration by CLSM (Zeiss, Germany) and a living imaging system (Carestream FX Pro, USA). Coumarin-6 was selected as a probe to prepare fluorescently labeled NPs and NYPs. Mice were fasted for 12 h, and then dosed with either NP or NYP suspension orally at a dose of 4 mg/kg coumarin-6, meanwhile coumarin-6 dissolved in ethanol was administrated as the control. After 1h, mice were sacrificed, and intestinal tissues were carefully taken and rinsed with normal saline. The intestinal sections were prepared according to the process reported in our previous study25. The images of intestinal sections were obtained by CLSM. Similarly, the NPs and NYPs were labeled with DiR and orally administrated to rats at 4 mg/kg (DiR). After 1 h and 4 h, the rats were sacrificed, and intestinal tissue from stomach to ileum were removed and visualized under a living imaging system (excitation at 748 nm and emission at 780 nm). 2.6 Absorption site of NYPs in the intestine To observe the absorption site of NYPs in the intestine, a DiR loaded NYP suspension was locally injected into the intestinal lumen of rats under abdominal surgery. Briefly, the rats were fasted overnight, and an incision was done in the middle of the abdomen after anesthetization by injecting pentobarbital sodium solution intraperitoneally at 30 mg/kg. The body temperature of rats was kept normally using an infrared lamp. Subsequently, one segment (approximately 5 cm) in the ileum, which contains 1~3 Peyer’s patches, was ligated at both ends. After injection of 0.5 mL DiR loaded NYP suspension, the intestinal tissues were replaced into the abdominal cavity and a wound treatment was applied. After 1 h, the ileum tissue containing Peyer’s patches was collected, and ex vivo imaging was conducted under a living imaging system. DiR loaded NPs were prepared and used as a control formulation. To further investigate the effect of laminarin on absorption of NYPs, a pre-treatment of local injection of 0.5 mL laminarin solution (1 mg/ml) into the lumen of the ligated ileum was done 1 h before NYP suspension injection. 2.7 Cell evaluation 9



2.7.1 Raw 264.7 cell culture The mouse macrophage cell line, Raw 264.7 cells, purchased from American Type Culture Collection (Manassas, VA), were cultured in RPMI 1640 medium containing 10 % (v/v) FBS, 1 % (v/v) penicillin and 1 % (v/v) streptomycin. The culture was proceeded at 37 oC in a 5 % CO2 and 90 % humidified environment. 2.7.2 Cytotoxicity assays The in vitro cytotoxicity of blank particles was measured by an MTT assay against Raw 264.7 cells22. The cells were seeded in a 96-well sterile plate with a concentration of 1×105 cells/mL and cultured for 24 h. After treatments with different concentrations of blank YP or NYP suspensions (5~200 µg/mL) for 24h, 10 µL of MTT (5 mg/mL) was added in each well for a further 4 h at 37 ℃. The supernatant was completely drained and then 100µL of DMSO was used to dissolve the formazan. The absorbance was then measured at 570 nm on a microplate reader. 2.7.3 Cellular uptake investigation The cellular uptake of double fluorescence-labeled NYPs was performed on Raw 264.7 cells20, 22, 27. RBITC was selected to label the YP shells, and hydrophobic probe coumarin-6 was loaded into the core of particles. The 12-well sterile plate was used to culture the cells with a concentration of 1×105 cells/mL for 24 h. Raw 264.7 cells were first incubated with NYP suspensions at different concentrations of 1, 10, 20, 50 µg/mL for 4 h. To confirm a time-dependent behavior of cellular uptake of NYPs, the cells were incubated with NYPs (20µg/mL) for 2 h, 4 h and 8 h. At these time points, the cells were rinsed with PBS and fixed in 4 % paraformaldehyde solution. After staining with DAPI, the cells were observed by CLSM. To quantify the extent of NYPs internalization, the cells incubated with NYPs were washed, digested, collected by centrifugation and re-suspended in PBS. Finally, the cell suspension was analyzed by flow cytometry system (FCS). 2.7.4 Uptake mechanism study Laminarin is a soluble β-1,3-glucan which could also be recognized by the dectin-1 receptor expressed on macrophage cells. In this study, laminarin was used as 10


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an inhibitor to study the uptake mechanisms of NYPs10, 20. The cells were seeded in a 12-well sterile plate and incubated with NYPs suspensions with or without added laminarin. The concentration of laminarin was set at 0.1 mg/mL and 1 mg/mL. After incubation for specified time periods, the uptake by the cells was verified by CLSM and FCS. 2.8 In vivo pharmacokinetics Twenty-four male SD rats were randomly divided into four groups. After fasting overnight, the rats were orally administrated with CTX-Sol, CTX-NP and CTX-NYP at a dose of 10 mg/kg. And the fourth group was intravenously injected with CTX-Sol at 4 mg/kg. At predetermined time points, blood samples were withdrawn from the orbital cavity. The plasma was obtained by centrifugation at 8,000rpm for 10 min and the drug concentrations were measured by UPLC−MS/MS method as previously reported25. DAS 2.0 software was used to analyze the pharmacokinetic data. The relative bioavailability (Frb) and absolute bioavailability (Fab) were obtained as the following formulas: !"#$ ∙ &'()*+, × 100% !"#*+, ∙ &'()$ !"#.+ ∙ &'()/0 - % = × 100% !"#/0 ∙ &'().+ % =

2.9 Statistical analysis The significant difference was evaluated using student’s t test. And, P < 0.05 was considered statistically significant and P < 0.01 was considered very statistically significant. All the Data was presented as the mean ± standard deviation (SD). 3. Results and discussion 3.1 Preparation and characterization The aim of this study was to develop a nano-in-micro carrier for oral delivery of CTX with targeting to intestinal M cells. YP, as a desirable micro carrier, not only possesses macrophage targeting properties by recognizing cell-surface receptors, but is also able to package a variety of nanoparticulate cargoes. The YPs were easily obtained after processing with alkali, acid, isopropanol and acetone to remove the proteins, nucleic acids and other components8. The retained components were 11



primarily β-glucan, which provided the hollow and porous structure of YPs for cargo loading. The size of the blank YPs was 2~4 µm in diameter, as shown in Figure 1A, and visualized by SEM (Figure 1B) and AFM (Figure 1C), which is in accordance with previous reports12, 28. The SEM image (Figure 1B) showed the bumpy and porous surface morphology of the yeast cell wall in the absolute dry state, and the AFM image (Figure 1C) further indicated that the porous channels found in the glucan shell were nonuniform in size, suggesting that the YPs are suited for loading cargoes with a broad size distribution. To confirm the hollow architecture of the empty YPs, a fluorescence dye, calcofluor white, was used to selectively bind to the glucan wall for CLSM imaging10. As expected, a typical capsular structure was observed, as shown in Figure 1D. To achieve the high loading of cargoes within YPs, the nano-carrier was expected to have a high loading of CTX and physiochemical properties suited for entrapping. In our previous study25, a polymer and lipid hybrid nano-carrier was optimized for drug loading and sustained release in oral delivery. Similarly, in this study, PCL and MCT were also selected as the loading core, and soybean lecithin and CTAC were used as emulsifying agents to prepare the NPs by the emulsion-solvent evaporation. The ratio of PCL: MCT: soybean lecithin: CTAC in weight was optimized as 9: 1: 8: 2. The CTX-NPs had a uniform distribution with a mean hydrodynamic diameter of 67.8 nm (Figure 1A), and a positively charged surface with a mean zeta potential of +62.0 mV (Figure 1E). The CTAC provided the NPs with a positively charged surface, which is requisite in the entrapping process, as previously reported10. The TEM images showed a uniformly spherical appearance (Figure 1F), which is consistent with the results determined by the Malvern Zetasizer (Table S1). The drug loading reached a maximum of 19.7 %, likely due to the improved loading capacity by incorporating MCT in the PCL matrix25. It was probably attributed to the reduced core crystallinity by the incorporated MCT, which was able to provide more amorphous areas in the core suited for drug loading, as verified in our previous work29. 12


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To load the NPs into YPs successfully, the electrostatic force-driven self-deposition method was done, followed by a solvent hydration/lyophilization process10, 24. The empty YPs have a zeta potential of -8.7mV after hydration (Figure 1E) due to the residual carboxyl groups on the surface of the cell wall19, and positively charged NPs were easily captured and packaged into the YPs by electrostatic force during incubation. The following lyophilization-hydration procedure improved the loading efficiency by utilizing water to push the NPs loaded in the holes or attached at the surface into the core of YPs by capillary action24. After loading, the mean hydrodynamic diameter of NYPs was 3.686 µm as shown in Figure 1A, with no apparent change compared to blank YPs (3.777 µm). Figure 1E indicated that the zeta potential of NYPs varied from -8.7 mV (blank YPs) to +1.5 mV, probably due to a small amount of NPs retained on the surface of NYPs during the loading process. The SEM image of NYPs (Figure 1G) obtained demonstrated a smooth surface morphology compared with blank YPs (Figure 1B), suggesting that the deep holes present in the yeast cell wall were filled with the NPs after loading. Similar results were obtained by AFM, where the number of deep holes in the NYPs (Figure 1H) was significantly reduced compared to blank YPs (Figure 1C). In order to validate the cargo loading visually, fluorescently labeled NPs were prepared with coumarin-6 instead of the CTX, and used for loading and imaging by CLSM. As shown in Figure 1D, fluorescence signals (green) were primarily concentrated in the core of the yeast cell wall, indicating that the NPs were efficiently and successfully packaged into the YPs. As determined by HPLC at 230 nm, the CTX loading content in the NYPs was 1.8 %. The particle size, zeta potential, DL and EE of the particles are summarized in detail in Table S1.

13



Figure 1. Characterization of nanoparticles (NPs), yeast cell wall microparticles (YPs) and NP loaded YPs (NYPs). (A) Particle size distribution of CTX-NPs, blank YPs and CTX-NYPs. (B) SEM image of blank YPs. Scale bar: 1 µm. (C) AFM images of blank YPs. Scale bar: 3 µm (left) and 350 nm (right). (D) CLSM images of coumarin-6 loaded NYPs (green) with calcofluor white (blue) labeling the yeast cell wall. Scale bar: 5 µm. (E) Zeta potential of CTX-NPs, blank YPs and CTX-NYPs. (n=3). (F) TEM image of CTX loaded NPs (CTX-NPs). Scale bar: 100 nm. (G) SEM image of CTX-NYPs. Scale bar: 1 µm. (H) AFM images of CTX-NYPs. Scale bar: 3 µm (left) and 350 nm (right).

3.2 In vitro drug release and stability of drug in NPs and NYPs 14


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The drug release properties of CTX-NPs and CTX-NYPs were studied in the release medium of pH 1.2 over 4 h and pH 6.8 over 72 h. As shown in Figure 2A and 2B, the NPs released approximately 17 % of the loaded CTX in pH 1.2 medium within 4 h and 77 % in pH 6.8 medium within 72 h. Compared with the NPs, the NYPs showed a slower drug release in both release medium, indicating that entrapping the NPs into the YPs was able to slow the drug release to a certain extent. One possible reason is that a mass of NPs coagulating in the holes of the yeast cell wall and the inner cavity of YPs lead to a reduced and delayed contact with release medium. The stability of CTX loaded in NPs and NYPs in the gastrointestinal fluid after oral gavage was evaluated using SGF and SIF. According to Figure 2C, the percentage of CTX retained in NPs and NYPs were both more than 90 % after incubation in SIF for 8 h, indicating that the CTX loaded in NPs and NYPs was stable, with only slight degradation in SIF. In contrast, the retained CTX in NPs and NYPs was clearly lower in SGF than SIF after incubation for 8 h, probably due to the high sensitivity of CTX to gastric juices30, 31. It could be concluded that the CTX leaked from the carriers would be rapidly degraded in gastric fluid. Furthermore, the CTX-NYPs exerted better stability in SGF in comparison with CTX-NPs, which could be attributed to the slow drug release from NYPs. In total, the NYPs have shown good drug-protecting capability in the gastrointestinal environment.

15



Figure 2. In vitro drug release and stability of drug in NPs and NYPs. (A) drug release in pH 1.2 HCl solution, (B) drug release in pH 6.8 PBS and (C) the stability of drug in NPs and NYPs after incubation in simulated gastrointestinal fluid. (n=3)

3.3 Biodistribution of NYPs in intestine The distribution of fluorescently-labeled particles in the duodemum, jejumum, ileum and Peyer's patches of the mice was observed under CLSM. As shown in Figures 3A-C, stronger fluorescence signals from NPs and NYPs could be visualized in the entire intestinal tract in comparison with free coumurin-6 solution, suggesting that the intestinal distribution could be efficiently enhanced by loading coumurin-6 in particles. The coumurin-6 in ethanol solution was quickly cleared after oral gavage, and only a small proportion reached the ileum after 1 h. A mass of coumurin-6 loaded NPs arrived at the jejumum and ileum within 1 h due to the intact nano-sized 16


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construction, and they not only existed on the villi surface, but also partly permeated into the inner of the villi, indicating the NPs could be rapidly transported across the intestinal epithelium. This is attributed to the nano-size and positively charged surface of NPs, which have a high affinity to enterocytes32. After successfully entrapping the NPs into the YPs, coumurin-6 was distributed in a microparticle state throughout the intestinal tract, particularly in the ileum. As well, they easily spread to the root of the intestinal villi, notably providing enough chances to contact with intestinal epithelium, to then be rapidly taken up by M cells. Figure 3D demonstrates that the distribution of fluorescence signals in the Peyer's patches, which mostly existed in the ileum32. As observed, coumurin-6 solution could only minimal arrive in the Peyer's patch, due to its short residence time in the intestine, while strong fluorescence signals from NPs were found in the tubular structures, indicating the NPs were partly absorbed in Payer's patches. Further, large amounts of NYPs were accumulated in the Peyer's patch, suggesting an absorptive pathway. The CLSM image further demonstrated that the NYPs permeated into the intestinal epithelium and reached the subepithelial dome, as shown in the Figure 3D enlarged area marked with a white arrow. It can be inferred that the NYPs were absorbed by M cells located in the follicle-associated epithelium (FAE) via the transcellular route. Similar results were obtained by Rebecca De Smet, who demonstrated that ovalbumin loaded glucan particle uptake occurred via the transcellular pathway in M cells in in vivo ligated loop experiments17. In addition, coumurin-6 signals from NYPs also were found in the tubular structures of Peyer's patches, probably due to coumurin-6 release from NYPs after absorption and transportation in the Peyer's patches. To study the biodistribution at different time points in the whole intestine, a living imaging system was used after oral administration of DiR labeled NPs and NYPs. As shown in Figure 4, the fluorescence signals from NYPs were observed in the whole gastrointestinal tract at 1 h, while the fluorescence signals from NPs were primarily distributed in the jejunum and ileum, which were consistent with the previous results from Figure 3. These demonstrated that the NYPs exerted a longer retention time in 17



the intestine than the NPs, probably due to the micro size of NYPs. Consistently, at 4 h, more NYPs were still retained in the intestine compared with NPs. It is advantageous that the NYPs could be fully absorbed and transported in the intestinal tract during long residence times.

Figure 3. The CLSM images of duodenum (A), jejunum (B), ileum (C) and Peyer's patches (D) at 1 h after oral gavage of coumarin-6 (green) labeled Sol, NPs and NYPs. Cell nuclei were stained by DAPI (blue).

18


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Figure 4. Fluorescent images of the intact intestine tract ex vivo at 1h and 4 h after oral gavage of DiR-labeled NYPs and NPs.

3.4 Absorption sites of NYPs in the intestine To observe the absorption sites of NYPs in the intestinal segments, a DiR labeled NYP suspension was locally injected into the lumen of intestinal segments containing Peyer’s patches, with DiR loaded NPs used as the control. As shown in Figure 5, after treatment with NPs and NYPs for 1 h, the intestinal segments were completely filled with fluorescence signals, and more importantly, stronger fluorescence was largely concentrated in the Peyer’s patches marked with white arrows compared to other locations for the NYP group, but this effect was not apparent in the NP group. This indicated that the NYPs were prone to be absorbed in the Peyer’s patches, which was in line with the intestinal distribution study. When pretreated with laminarin, the NYPs were mainly retained in the lumen with much less accumulation at the Peyer’s patches. These results were consistent with those obtained by Zhou et al., who validated that the absorption of quantum dot loaded YPs primarily occurred in 19



intestinal Payer’s patches after local injection into intestinal lumen, and this phenomenon was easily reversed by pretreatment with laminarin10, 11. The above results further confirmed that the NYPs were primarily taken up and transported by M cells in the Peyer’s patches, and subsequently endocytosed by other macrophages and absorbed through lymphatic transport.

Figure 5. Fluorescent images of the ileum segments ex vivo containing Peyer’s patches (marked with white arrows) at 1 h after local injection of DiR-labeled NP suspension, DiR-labeled NYP suspension with or without pretreatment with laminarin (1mg/mL) in the intestinal lumen.

3.5 Cell culture and evaluation 3.5.1 Cytotoxicity assays Cytotoxicity assays were used to assess the safety of blank YPs and NYPs on RAW 264.7 cells. Figure 6 shows that the RAW 264.7 cells retained nearly 100 % viability when treated with blank YPs at concentration of 5~200µg/ml for 24h, suggesting that YPs are non-cytotoxic against macrophages. As for blank NYPs, although there was a slight decrease in cell viability with the concentration increasing, the cell viability remained above 80 %, which is considered to be no effect in the cellular uptake studies. The slight cytotoxicity of blank NYPs against RAW 264.7 cells was likely caused by the positively charged NPs retained on the surface of YPs during the 20


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loading process. It was reported that positively charged particles were commonly toxic against cells due to their cationic nature33. In this study, the NPs were positively charged to achieve effective adsorption and loading in the YPs, which lead to a slight amount remaining on the NYPs after loading. However, the blank NYPs were considered safe to RAW 264.7 cells for further cell evaluation.

Figure 6. In vitro cell evaluation. (A) Cell viability of RAW 264.7 cells after treatment with blank YPs and blank NYPs at different concentrations for 24 h by MTT assay. (B) The FCS results of uptake of double fluorescence-labeled NYPs by RAW 264.7 cells after incubation for 4 h at 1, 10, 20 and 50 µg/mL of NYP suspension. (C) The FCS results of uptake of double fluorescence-labeled NYPs by RAW 264.7 cells after incubation for 2 h, 4 h and 8 h at 20 µg/mL of NYP suspension. (D) The effect of laminarin (0.1 or 1 mg/mL) on uptake behavior of double fluorescence-labeled NYPs after incubation with RAW 264.7 cells for 4 h at 20 µg/mL of NYP suspension. (n=3, **P < 0.01, *P < 0.05, compared with control).

3.5.2 Cellular uptake investigation 21



According to previous studies, β-glucans, primary constituents of YPs, obviously increase the uptake of YPs by macrophages due to their high specificity and selectivity to the membrane receptor dectin-110, 21. To certify that NYPs still possess the high affinity to macrophages, double fluorescence-labeled NYPs (Figure S1) were used to investigate in vitro cellular uptake by RAW 264.7 cells. First, the RAW 264.7 cells were incubated with different concentrations of NYP suspension (1, 10, 20 and 50 µg/mL) for 4 h, and the CLSM images are shown in Figure 7. It was clear that NYPs were internalized in their whole state by RAW 264.7 cells, even at the lowest concentration of 1 µg/mL. With increasing concentration, the NYPs being absorbed into the cytoplasm of a single cell increased as well. When the concentration reached the highest 50 µg/mL, nearly ten NYPs could be phagocytosed by only one RAW 264.7 cell, indicating that the macrophages had a strong capacity to absorb the NYPs. These results implied that the NPs loaded in the YPs had no effect on the phagocytic behaviors of the macrophages, and that the surface components of the NYPs facilitated recognition by glucan receptors expressed on the macrophages34. Furthermore, colocalization of both the RBITC (red) and coumarin-6 (green) fluorescence signals further demonstrated that the NPs were successfully entrapped in the YPs, and that internalization of fluorescence labeled NPs in cells could be attributed to the intact micro-size carriers. To determine quantitative cellular uptake, flow cytometry experiments were done, and the results are shown in Figure 6B. Similar to the CLSM images, the mean fluorescence intensity of uptake was concentration-dependent and the signals of coumarin-6 and RBITC were consistent, indicating that coumarin-6 entered the cells via endocytosis of NYPs. To confirm a time-dependent manner in cellular uptake of NYPs, RAW 264.7 cells were incubated with NYPs (20µg/mL) for 2, 4 and 8 h, and then analyzed by CLSM and FCS. As shown in Figure 8 and Figure 6C, the cell uptake efficiency of NYPs rapidly increased from 2 h to 8 h, demonstrating time-dependent uptake behavior. When the incubation continued to 8 h, the coumarin-6 signals were primarily not co-localized with the RBTIC signals anymore. It was likely that the coumarin-6 22


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loaded in the NYPs was partly released from the carriers after internalization by macrophages after 8h. This suggested that the absorption of small molecular drugs could be enhanced by loading in NYPs to increase the cellular internalization in micro-size carrier form, with subsequent release and transportation.

Figure 7. The CLSM images of uptake behavior of double fluorescence-labeled NYPs after incubation with RAW 264.7 cells for 4 h at 1, 10, 20 and 50 µg/mL of NYP suspension.

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Figure 8. The CLSM images of uptake behavior of double fluorescence-labeled NYPs after incubation with RAW 264.7 cells for 2 h, 4 h and 8 h at 20 µg/mL of NYP suspension.

3.5.3 Uptake mechanism study As reported, β-glucans are rapidly recognized by the membrane receptor dectin-1, which has a high expression on macrophages13-15. To verify the importance of dectin-1 in NYP uptake by macrophages, laminarin, a ligand of dectin-1, was added to the NYP suspension (20 µg/mL) at concentration of 0.1 or 1 mg/mL, to co-incubate with RAW 264.7 cells. The CLSM images (Figure 9) showed that the addition of laminarin led to a significant decrease in uptake of NYPs, and a stronger inhibiting effect was observed at the high concentration of laminarin. The quantitative FCS results (Figure 6D) indicated that the NYP uptake was reduced to approximately 67.1 % (coumarin-6) and 75.0 % (RBITC) at 0.1 mg/mL laminarin addition compared with the control. When the concentration of laminarin was increased to 1 mg/mL, the amount of cell 24


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uptake further decreased to 31.3 % (coumarin-6) and 33.4 % (RBITC). These results suggest that phagocytosis of NYPs by macrophages is primarily attributed to the recognition of β-glucan by dectin-1, which is in line with previous reports10, 20. As well, it is clear that laminarin is a competitive inhibitor of dectin-1 for β-glucan, as the stronger inhibiting effect was obtained by increasing the amount of added laminarin. Similarly, when the concentration of NYP suspension was set to 50 µg/mL, the uptake inhibition was weakened accordingly at 0.1 or 1 mg/mL laminarin, as shown in Figure S2-3. In summary, the in vitro cell studies demonstrated that NYPs are useful carriers to efficiently deliver small molecular drugs to phagocytic cells. The strong phagocytic capacity of macrophages provides the possibility for NYPs to be efficiently internalized and transported by phagocytic cells. It is quite clear that the specific targeting of the NYPs to phagocytes is mainly mediated by the recognition of surface β-glucan by specific receptors on the surface of phagocytic cells.

Figure 9. The effect of laminarin (0.1 or 1 mg/mL) on cellular uptake of double 25



fluorescence-labeled NYPs by RAW 264.7 cells after incubation for 4 h at 20 µg/mL of NYP suspension under CLSM.

3.6 In vivo pharmacokinetic study The aim of this work was to enhance the oral absorption of CTX using the nano-in-micro carrier targeted to intestinal M cells. To prove the efficiency of NYPs in oral delivery, the plasma concentration of CTX was measured after oral gavage of CTX-NPs, CTX-NYPs and CTX-Sol in rats. The mean concentration-time curves are illustrated in Figure 10, and the major pharmacokinetic parameters are concluded in Table 1. The Cmax and AUC (0-∞) of CTX in NPs increased to 108.62±85.06 µg/L and 325.25±162.49 µg/L·h, which is 2-fold and 4.5-fold higher than CTX-Sol, respectively. These results indicate that the NPs are superior to the control Sol for enhancing oral drug delivery. It can be explained that the PCL and MCT mixed core of the NPs are suited for loading drugs by decreasing the core crystallinity, increasing drug stability in the gastrointestinal tract, releasing drugs in a sustained manner etc, which has been previously demonstrated in our work25, 29. In addition, the NPs can be absorbed in different pathways, including intestinal epithelial cell uptake and M cell transport in Payer's patches, as visualized in Figure 3 in the biodistribution study. Compared to CTX-NPs, CTX-NYPs further improved the Cmax and AUC (0-∞) of CTX to 190.13±67.58 µg/L and 393.38±103.16 µg/L·h respectively, indicating that entrapping NPs into YPs indeed enhanced the oral delivery of CTX. It is apparent that the NYPs combine the superiority of NPs for drug loading, with the macrophage targeting effect conferred by the YPs. However, it is still difficult to make a large breakthrough in oral delivery of CTX by NYPs. One possible reason is the extremely low distribution of M cells in the intestine. As reported, the gut-associated lymphoid tissue (GALT) comprises less than 10% of the intestinal surface, and only ~10% of the epithelial cells in GALT are M cells35. This brought up a barrier for NYPs in intestinal transportation by M cell, although the M cells have a high transcytotic activity for particles35. As well, the Tmax of CTX was prolonged to 0.92±0.20 h by 26


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NYPs, approximately 2-fold to NPs, and the t1/2 of CTX was larger in NYPs than NPs. On one hand, the NYPs achieved a longer retention time in the gastrointestinal tract than NPs, providing enough time for drug transport and absorption. On the other hand, the NYPs were primarily absorbed and transported by M cells and other phagocytic cells, and entered the blood circulation through the lymphatic transport, which led to a delayed peak reaching. Finally, utilizing the characteristics of the nano-in-micro carrier, the oral absolute bioavailability (Fab) of CTX was significantly improved from 5.7 % (Sol) to 32.1 % by NYPs, which is approximately 5.7-fold to Sol. In addition, in order to evaluate the intestinal biocompatibility of NYPs, a histological examination was performed by use of hematoxylin-eosin (H&E) staining at 3 h and 6 h after oral administration of NYPs at a dose of 400 or 800 mg/Kg. As shown in Figure S4, no obvious mucosal erosions and disruption was observed in histological sections throughout the intestinal tract at a dose of either 400 or 800 mg/Kg, indicating that the NYPs have a good biocompatibility and safety for oral drug delivery. In a word, this is a feasible and useful method to improve the oral absorption of insoluble small molecule drugs by loading them into the nano-in-micro carriers for phagocytic cell targeted delivery.

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Figure 10. The mean plasma concentration time curves of CTX after intravenous injection of CTX-Sol (A) at a dose of 4 mg/kg and oral gavage of CTX-NPs, CTX-NYPs and CTX-Sol (B) at a dose of 10 mg/kg. (n=6).

Table 1. The major pharmacokinetic parameters after intravenous or oral administration of different CTX formulations. (n=6). Parameter Dose(mg/kg)

CTX-Sol (iv) 4

CTX-Sol (po) 10

CTX-NPs (po) 10

CTX-NYPs (po) 10

Cmax(μg/L)

486.07±96.78

53.53±34.97

108.62±85.06*

190.13±67.58**#

Tmax(h) t1/2(h)

0.08 3.84±1.76

0.42±0.30 3.02±0.41

0.46±0.29 4.63±2.72

0.92±0.20 5.13±2.45

AUC(0-t)(µg/L·h)

476.04±169.92

67.48±48.43

301.55±145.97*

382.01±102.66**#

AUC(0-∞) (µg/L·h)

485.13±176.81

68.21±48.14

325.25±162.49*

393.38±103.16**#

Frb(%)

-

-

446.9

566.1

Fab(%)

100

5.7

25.3**

32.1**#

* P

Entrapping of Nanoparticles in Yeast Cell Wall Microparticles for

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