maleic anhydride)-Doped PEGâPLA

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Poly(vinyl methyl ether/maleic anhydride) doped PEG-PLA nanoparticles for oral paclitaxel delivery to improve bioadhesive efficiency Qian Wang, Chan Li, Tianyang Ren, Shizhu Chen, Xiaoxia Ye, Hongbo Guo, Haibing He, Yu Zhang, Tian Yin, Xing-Jie Liang, and Xing Tang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00612 • Publication Date (Web): 11 Sep 2017 Downloaded from http://pubs.acs.org on September 12, 2017

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Molecular Pharmaceutics

Poly(vinyl methyl ether/maleic anhydride) doped PEG-PLA nanoparticles for oral paclitaxel delivery to improve bioadhesive efficiency II, †, ‡, §

*, †, ‡, §

II

†, ‡, §

†, ‡, §, ●

Qian Wang, Chan Li, Tianyang Ren, Shizhu Chen, Xiaoxia Ye, †, ‡, § II II II *, †, ‡, § *, II Hongbo Guo, Haibing He, Yu Zhang, Tian Yin, Xing-Jie Liang, Xing Tang II †

School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, P. R. China

Chinese Academy of Sciences (CAS) Center for Excellence in Nanoscience, National Center for

Nanoscience and Technology, Beijing, 100190, P. R. China ‡

Laboratory of Controllable Nanopharmaceuticals, CAS Key Laboratory for Biomedical Effects of

Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, 100190, P. R. China §

University of Chinese Academy of Sciences, Beijing, 100049, P. R. China

●

Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, P. R.

China

* Corresponding authors at: School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, P. R. China, Tel: +86-024-23986343, Fax: +86-024-23911736, E-mail: [email protected] (Xing Tang). Laboratory of Controllable Nanopharmaceuticals, Chinese Academy of Sciences (CAS) Center for Excellence in Nanoscience; and CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology of China, No.11, First North Road, Zhongguancun, Beijing, 100190, P. R. China, Tel: +86-010-82545569, Fax: +86-010-62656765, E-mail: [email protected] (Chan Li), [email protected] (Xing-Jie Liang).

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Abbreviations PTX：Paclitaxel PVMMA：Poly (vinyl methyl ether/maleic anhydride) mPEG-b-PLA: Poly(ethylene glycol) methyl ether - b - poly ( D , L - lactic acid) m-NPs: various drugs loaded mixed polymer (mixture of Poly (vinyl methyl ether/maleic anhydride) and Poly(ethylene glycol) methyl ether - b - poly ( D , L lactic acid) ) nanoparticles p-NPs: various drugs loaded pure polymer (Poly(ethylene glycol) methyl ether - b poly ( D , L - lactic acid) ) nanoparticles C6: Coumarin 6 DIR: DiR iodide, 1,1‘-dioctadecyl-3,3,3’,3‘-tetramethylindotricarbocyanine iodide C6/DiR/PTX-m-NPs: C6/DiR/PTX loaded mixed polymer nanoparticles C6/DiR/PTX-p-NPs: C6/DiR/PTX loaded pure polymer nanoparticles Papp: apparent permeability coefficient AUC0−t: area under the concentration−time curve IC50: the half maximal inhibitory concentration MCT: Medium chain triglycerides TPGS 1000: D-alpha-Tocopheryl polyethylene glycol 1000 succinate blank-m-NPs: no drug loaded m-NPs blank-p-NPs: no drug loaded p-NPs HBSS: Hank's balanced salt solution


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Abstract Bioadhesive nanoparticles based on PVMMA (poly (vinyl methyl ether/maleic anhydride)) and mPEG-b-PLA (Poly (ethylene glycol) methyl ether - b - poly (D, L lactic acid)) were produced by the emulsification solvent evaporation method. Paclitaxel was utilized as the model drug, with an encapsulation efficiency of up to 90.2 ± 4.0%. The nanoparticles were uniform and spherical in shape, and exhibited a sustained drug release compared with Taxol®. M-NPs also exhibited favorable bioadhesive efficiency at the same time. Coumarin 6 or DiR-loaded nanoparticles with/without PVMMA (C6-m-NPs/DiR-m-NPs or C6-p-NPs/DiR-p-NPs) were used for cellular uptake and intestinal adhesion experiments, respectively. C6-m-NPs were shown to enhance cellular uptake, and caveolae/lipid raft mediated endocytosis was the primary route for the uptake of the nanoparticles. Favorable bioadhesive efficiency led to prolonged retention in the intestine reflected by the fluorescence in isolated intestines ex vivo. In a ligated intestinal loops model, C6-m-NPs showed a clear advantage for transporting NPs across the mucus layer over C6-p-NPs and free C6. The apparent permeability coefficient (Papp) of PTX-m-NPs through Caco-2/HT29 monolayers was 1.3 fold and 1.6 fold higher than PTX-p-NPs and Taxol® respectively which was consistent with the AUC0−t of different PTX formulations after oral administration in rats. PTX-m-NPs also exhibited a more effective anticancer efficacy, with an IC50 of 0.2 ± 1.4 µg/mL for A549 cell lines, further demonstrating the advantage of bioadhesive nanoparticles. The bioadhesive nanoparticles m-NPs demonstrated both mucus permeation and epithelial absorption, and thus this bioadhesive drug delivery system has the potential to improve the bioavailability of drugs that are insoluble in the gastrointestinal environment.

Keywords: bioadhesive nanoparticles; oral delivery; mucus permeation; epithelial absorption; anticancer efficacy.

Introduction Paclitaxel (PTX) is a first-line anticancer drug, and is widely used in the treatment of



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cancer such as ovarian, non-small cell lung, breast, and others.1 Currently, the clinical PTX formulations of Taxol® and Abraxane®2 as well as regionally approved formulations such as paclitaxel liposomes3 and Genexol®-PM,

4, 5

are all designed for

intravenous administration. However, a study evaluating the most acceptable routes of drug administration in female patients with recurrent cancer showed that 78.7% of interviewed patients are reluctant to receive intravenous injections due to the inconvenience and problems with toxicity.6 Compared with intravenous injections, the oral administration is a more convenient and widely accepted pathway, as it is non-invasive and administration is painless, removing risks of contamination and injection-related discomforts.7 However, as a BCS (Biopharmaceutical Classification System) class IV drug, oral delivery of paclitaxel is limited not only by its low aqueous solubility and poor intestinal permeability, but also by the first pass hepatic metabolism and high level of P-glycoprotein (P-gp) efflux.8, 9 The use of nanotechnology is considered a potential strategy to overcome these problems and improve the oral bioavailability of paclitaxel by increasing paclitaxel solubility and decreasing P-gp induced drug efflux. Proposed strategies includes delivery systems such as polymer-drug conjugate nanoparticles,10 solid lipid nanoparticles,11 nanomicelles12 and nanoemulsions.13 However, thus far the improvement of bioavailability has been limited, and still falls short of the clinical treatment requirements. A prominent area of research for oral nano delivery-systems is in bioadhesive formulations, which have been investigated for improvement of oral delivery of paclitaxel.11,

14, 15

Bioadhesive nanoparticles are also widely used in localized

antimicrobial drug delivery, intraperitoneal (i.p.) administration, buccal delivery, oral delivery and other drug delivery systems, owing to their unique adhesion properties.16-19 For oral delivery, the ability for nanoparticles to adhere to the mucosa is mainly achieved through by electrostatic, hydrophobic and van der Waals interactions, as well as through interpenetration or tangling of the polymer chains within the mucus mesh structure. Mucoadhesive nanoparticles are capable of increasing oral bioavailability as a result of their prolonged residence time, ability to


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protect drugs from proteolytic enzymes and assistance of direct particle uptake by intestinal cells.20 In addition, nanoparticles coated with PEG2000 chains can be rapidly transported through physiological human mucus. The presence of PEG coatings have also been reported to stabilize nanoparticles in simulated gastrointestinal fluids.21, 22 For oral delivery of PTX, combining the advantages of both mucus permeation and epithelial absorption is a promising strategy for improving oral bioavailability. In the biomedical field, mPEG-b-PLA (Poly (ethylene glycol) methyl ether - b poly (D, L - lactic acid)) is considered a safe pharmaceutical excipient with a good biocompatibility. Additionally, an mPEG-b-PLA based polymeric micelles product, Genexol®-PM, has already been marketed for cancer treatment.23 PVMMA (poly (vinyl methyl ether/maleic anhydride)), which is a GRAS (generally recognized as safe) excipient, also has excellent biocompatibility.24 As well, PVMMA has been reported to show strong adhesive capability in the gastrointestinal tract due to the formation of carboxylic groups deriving from polyanhydride residues. J.M. Irache and collegues have previously reported studies of this material, demonstrating that nanoparticulate systems based on PVM/MA whether loaded with antigen or loaded with drug all had good bioadhesive property.25-27 Based on this, in this work, we prepared a PVMMA and mPEG-b-PLA-based nanoplatform through an emulsification method, to produce biodegradable polymeric nanoparticles that combine the desired features of mucoadhesion with mucus permeation for oral delivery of paclitaxel. This approach is suitable for medical applications, as no complex chemical reactions (which can potentially introduce impurities) are involved in the preparation process. Through

an

emulsification

solvent

evaporation

method,

paclitaxel-loaded

PVMMA/mPEG-b-PLA nanoparticles (PTX-m-NPs) with good stability, uniformity, high encapsulation efficiency and drug loading capability were obtained. Enhanced bioadhesion due to the incorporated PVMMA was confirmed by cellular uptake, in vitro adhesion, ex-vivo retention and in vivo permeability experiments using fluorescent probe (coumarin 6 or DiR) loaded PVMMA/mPEG-b-PLA nanoparticles. Further, pharmacokinetic analysis of the PTX-m-NPs demonstrated improved cytotoxicity and bioavailability, and thus the results suggest the developed



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bioadhesive carrier system is a promising tool for improvement of bioavailability of hydrophobic and low permeable drugs. Experimental section Materials Paclitaxel (PTX) was purchased from Shanghai Zhongxi Sunve Pharmaceutical Co., Ltd (Shanghai, China). Poly (ethylene glycol) methyl ether - b - poly (D, L - lactic acid) (mPEG-b-PLA) was supplied by Jinan Daigang Biomaterial Co., Ltd (Jinan, China). Poly (vinyl methyl ether/maleic anhydride) (PVMMA) was a gift from Ashland (Kentucky, USA). Medium chain triglycerides (MCT) were obtained from Beiya Industrial Pharmaceutical Oil Co., Ltd (Tieling, China). TPGS 1000 (D-alpha-Tocopheryl polyethylene glycol 1000 succinate) and DiR were obtained from Sigma–Aldrich (Missouri, America) and Dalian meilun biology technology Co., Ltd (Dalian, China) respectively. Coumarin-6 (C6), pepsin and trypsin all were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd (Shanghai, China). Ultra-purified water was supplied by Milli Q Plus system (Milli-Q, Merck Millipore, USA). Without further purification, all chemical reagents utilized in this study were analytical grade. Human colon carcinoma Caco-2, HT29 cells and A549 cells were purchased from American Type Culture Collection (ATCC). Cell culture mediums were from WisentInc (Quebec, Canada). Fetal bovine serum (FBS) and L-glutamine was purchased from Gibco (New York, USA). 0.25% Trypsin-EDTA, penicillin and streptomycin were purchased from Invitrogen (Carlsbad, USA). Non-essential amino acid was from Merck Millipore (Billerica, USA). Culture dishes and plates were from Corning (New York, USA). Fabrication of nanoparticles The emulsification solvent evaporation method was utilized for the fabrication of the nanoparticles. MPEG-b-PLA and PVMMA were used as the carrier materials and TPGS 1000 was used as the emulsifier, and the preparation process was carried out in an ice bath.


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For the oil phase, 6.5 mg PTX, 60 mg mPEG-b-PLA and 40 mg PVMMA were dissolved together in 2 mL ethyl acetate containing 30% MCT. The oil phase was subsequently added to the water phase containing TPGS 1000 (0.2%, m/v) under high shear homogenization (modular homogenizer, Tianjin Hengao, China) to form an oil in water emulsion. In order to reduce particle size, the primary emulsion was sonicated by a probe sonicator (Scientz-IID, Ningbo Scientz, China) at 10% power for 5 min. The nanoemulsion was then solidified by removing the organic solvents by rotary evaporation (RV 10 digital V, IKA, German) for 20 min. Finally, the final nanoemulsion was obtained by filtration through a 0.45 µm syringe filter to remove free drug and large particles. PVMMA-free nanoparticles (PTX-p-NPs) and PTX-free nanoparticles (blank-m-NPs and blank-p-NPs) were prepared by the same method with the exception of no added PVMMA or PTX. Mean particle size and particle morphology The average particle size, zeta potential and polydispersity index (PDI) of nanoparticles were measured by dynamic light scattering (ZEN5000, Malvern, UK). 200 µL of each nanoemusion sample was diluted with ultra-purified water to 1 mL before measurement. Each sample was tested in triplicate. The morphology of PTX-m-NPs and PTX-p-NPs was observed by transmission electron microscopy (TEM) (HT-7700, Hitachi, Japan). A single drop of sample with a concentration less than 1 mg/mL was dropped onto a carbon grid and then negatively stained with 2% phosphotungstic acid for 2 min. The samples were observed after drying at room temperature. Measurements of PTX content The PTX content in the particles was measured by high performance liquid chromatography (LC-20A, Shimadzu, Japan). Briefly, 20 µL of the nanosuspension was diluted to 1 mL with organic solvent (acetonitrile/DMSO: 40/60), and manually vortexed to ensure the drug was fully dissolved. All solutions were filtered through a 0.22 µm syringe filter before measurement. The drug loading (DL) and encapsulation efficiency (EE) of PTX-m-NPs and PTX-p-NPs were calculated according to equations derived by the Dong Zhang group.24



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Physical state of PTX in nanoparticles The thermal behavior of lyophilized samples of PTX-m-NPs, PTX-p-NPs, blank-m-NPs, blank-p-NPs as well as crude drug PTX was used to analyze the physical state of PTX within nanoparticles by Differential Scanning Calorimetry (DSC) (Q2000, TA, America). Accurately weighed samples (4 mg) were sealed in an aluminum plate and heated from 30 °C to 260 °C with a 10 °C per minute heating rate. In vitro drug release The drug release profile was tested using the widely used dialysis method. 1 mL PTX-m-NPs, PTX-p-NPs and Taxol® (all contain nearly 100 µg PTX) were sealed in a dialysis membrane (molecular weight from 8000 to 14,000 Da). The release medium was 20 mL pH 6.8 PBS or pH 1.2 HCl solutions. In order to achieve sink conditions, 0.1% Tween-80 was used as an additional solubilizer for the drug. All samples were placed in a constant shaking water bath with a shaking rate of 100 rpm/min at 37 ± 0.5 °C. At predetermined time intervals, 2 mL samples were withdrawn and replaced with the same volume of fresh medium immediately. The drug content in each sample was analyzed by HPLC. Storage stability 4 mL of freshly prepared nanoparticles were diluted to 20 mL with pH 7.4 PBS and stored at 4 °C for seven days. An appropriate volume was removed and analyzed every day. Size, drug content change, and stability were the main methods of nanoparticle characterization. Cell culture Caco-2 and HT29 cells were maintained in Dulbecco Modified Eagle’s Minimal essential medium (DMEM, 4.5 g/L glucose) with 10% (v/v) fetal bovine serum and 1% (v/v) antibiotic solution, 1% (v/v) L-glutamine, and 1% (v/v) non-essential amino acids. A549 cells were maintained in Modified Eagle’s Minimal Essential Medium with 1% (v/v) antibiotic solution. All cells were cultured in an atmosphere with 90% relative humidity and 5% CO2 at 37 °C. Caco-2 cells were subcultured every 5 days in a 1:3 passage ratio, while HT29 cells and A549 cells were subcultured every 3 days


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with 1:6 and 1:3 passage ratios respectively. Evaluation of cell viability in vitro Caco-2 cells were used as an in vitro model for oral chemotherapy, to investigate the uptake mechanism in detail. The influence of the blank nanoparticles on Caco-2 cell viability was measured using an MTT assay. A549 cell viability was also measured in the same manner to determine the anticancer efficacy of drug-loaded nanoparticles.28 In brief, after the incubation of nanoparticles with the cells for a predetermined time, MTT solution (100 µL, 0.5 mg/mL in medium) was added to each well for another 2.5 h. Following the removal of MTT medium, cells were dissolved with DMSO and prepared for analysis. Samples were analyzed with a microplate reader (M200, Tecan, Switzerland) at an absorbance wavelength of 570 nm and 630 nm. Untreated cells were used as a control, and the cell viability was calculated using the following formula: Cell viability (%) = ODtest/ODcontrol × 100% Cellular uptake of C6-loaded nanoparticles Flow cytometry system (FCS) (Attune, ABI, USA) and laser scanning confocal microscopy (LSCM) (Zeiss 710, Zeiss, Germany) were utilized to investigate the cellular uptake of C6-loaded nanoparticles. Caco-2 cells were seeded in a sterile 12-well plate with a density of 1.5×105 cells per well. After proliferation for five days, caco-2 cells were incubated with free C6, C6-m-NPs and C6-p-NPs (with equal concentration of C6) for 0.5 h and 2 h. Cells were then digested with 0.25% Trypsin-EDTA to form single cell suspensions and washed by PBS. Flow cytometry system was performed on each sample and the mean intracellular fluorescence intensity was measured. For LSCM experiments, cells were seeded at the same density in specially designed dishes, and treated with complete medium containing C6-NPs or free C6. At predetermined time points, Caco-2 cells were rinsed with cold PBS and fixed in 4% paraformaldehyde (PFA) for subsequent nuclear staining with Hochest 33342. Z-stack LSCM analysis was performed to collect images of m-NP transmembrane transport. Transcellular investigation through the co-cultured Caco-2/HT29 cell monolayer



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Caco-2 and HT29 cells were seeded on Transwell inserts (12 well plates, Corning, USA) at a ratio of 90:10 and a total cell density of 4.5×105 cells per well. Culture medium was replaced every 1-2 days. After 21-days of cell differentiation, sodium fluorescein was used to check the integrity of the cell monolayer and TEM was used to observe the morphology of the cell monolayer. Transepithelial transport experiments were completed as described by Liu group with some minor changes.29 Prior to the transport studies, culture medium was replaced by pre-warmed HBSS (0.5 mL in apical side and 1.5 mL in basolateral side) to equilibrate the cell monolayer at 37 °C for 15 min. The HBSS solution in the apical side was then discarded and 0.5 mL PTX-m-NPs, PTX-p-NPs HBSS suspension and Taxol® (20 µg/mL of PTX equivalent) were added to the upper side. 200 µL samples were collected from the basolateral chamber and the equivalent fresh HBSS solution was supplemented immediately at predetermined time intervals (0.25, 0.5, 1, 2 and 4 h). Before HPLC analysis, 1 mL of organic solvent (acetonitrile/DMSO: 40/60) was added to the sample to completely dissolve the drug. The apparent permeability coefficient (Papp) was calculated as follows for each treatment group. Papp (cm/s) = dQ/ (dt ×A×C0) Where dQ/dt was the amount of permeated PTX (µg) every second, A was 1.12 cm2, C0 was initial concentration of PTX (20 µg/mL). Mechanism of cellular uptake Different endocytic inhibitors were used to elucidate the uptake mechanisms of C6-m-NPs into cells. In this experiment, Caco-2 cells were divided into six different groups. The control group received no treatment, and the other groups of Caco-2 cells were pre-treated with 2 mg/mL sodium azide, 10 µg/mL Chlorpromazine, 18 µM Nystatin, 45 µM cytochalasin D or 15 mM beta-Cyclodextrin methyl ethers respectively for 1 h at 37 °C followed by incubation with C6-m-NPs suspension for a further 0.5 h. After this, the evaluation of cellular uptake was as above. Adhesion, Retention and Permeability (ARP effect) Adhesion effect in vitro In order to evaluate the adhesion effect of PTX-m-NPs on protein-rich mucosa, C6


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loaded nanoparticles were incubated with lysine-coated 96 well plates as previously described by the Saltzman group.17 Briefly, nano-suspensions of equal concentrations were incubated with lysine-coated 96 well plates for 30 min with gentle shaking. The excess nano-suspension was then removed and the plate washed with PBS. Acetonitrile/DMSO (40/60) was added to dissolve the nanoparticles that had adsorbed on the plate. The fluorescence in each well was then measured using an Infinite M200 microplate reader. Additionally, the interaction between mucin and nanoparticles also was analyzed. In short, mucin and nanoparticles were incubated together with shaking at 37 °C. After high-speed centrifugation, free mucin in the supernatant was measured using a coomassie blue staining method. The amount of mucin associated with the nanoparticles was calculated by subtracting the supernatant protein content from the total protein present Retention effect ex vivo All animal studies were conducted under the instruction of the local Institutional Animal Ethical Care Committee (IAEC). Male Kunming mice (Vital River Company, Beijing, China) of 18-22 g were fasted but had free access to water for 24 h prior to experiments. Three formulations (Free DiR, DiR-m-NPs, DiR-p-NPs) were given to the mice by intragastric administration with a DiR dosage of 2.5 mg/kg. After 0.5 h, 5 h and 12 h, the mice were sacrificed and the intestines were removed. The retention time of the nanoparticles in the intestine were reflected by the fluorescence signal using an in vivo fluorescence imaging system (Maestro 2, CRI, USA). The total amount of fluorescence in the intact intestine, duodenum, jejunum and ileum was measured by fluorescence spectrometer (F7000, Hitachi, Japan).30 Permeability effect in vivo In order to evaluate the permeability of the nanoparticles after their adhesion on the surface of intestines, the ligated intestinal loops model was applied.31, 32 Coumarin 6 (C6) dye was incorporated in the nanoparticles to track their position. After incubating free C6, C6-m-NPs or C6-p-NPs with the ligated jejunum loops for 2 h, SD rats were sacrificed and each loop was removed. The intestinal loops were washed with PBS



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and fixed in 4% paraformaldehyde for 24 h. The samples were dehydrated, and then the intestinal tissues were embedded in paraffin and sliced. The small intestinal tissue-sections were mounted onto polylysine-coated glass slides. Subsequently, the intestinal epithelial cells were stained with Hochest 33342 to label the nucleus, and finally, the sections were observed by LSCM. The fluorescence intensity of the intestinal epithelial cells was quantified with ImageJ and compared between treatment groups. In vivo pharmacokinetic studies Eighteen male SD rats (fasted for 12 h but allow free access to water) were divided randomly into three groups. The three groups were treated with Taxol®, PTX-m-NPs or PTX-p-NPs by oral gavage at 10 mg/kg (n = 6). At predetermined times (15 and 30 min, 1, 2, 4, 6, 8, 12 h), whole blood samples were withdrawn from the rats and collected into heparinized tubes. After centrifugation at 8,000 rpm for 10 min, the plasma was obtained and stored at -20 °C for UPLC-MS/MS analysis. The UPLC-MS/MS method used was as reported in previous studies.33 Statistics All data was expressed as mean ± SD, two groups was compared by one-way ANOVA analysis using statistical software (SPSS 17.0). *P < 0.05 and **p < 0.01 represented general difference and high difference respectively. Results Preparation, optimization and characterization of PTX-m-NPs and PTX-p-NPs The final PTX-m-NPs were comprised of 6.5 mg PTX, 60 mg mPEG-b-PLA, 40 mg PVMMA and 30 mg medium chain triglycerides (MCT), which gave the optimum encapsulation efficiency and uniform particle size of the nanoparticles. The addition of MCT improved the stability of the NPs in the dispersion and reduced drug leakage. Figure 1 shows a schematic illustration of the fabrication process of the PTX loaded PVMMA/mPEG-b-PLA nanoparticles (PTX-m-NPs). In order to confirm the necessity of a mucoadhesion effect provided by PVMMA for oral delivery, PTX loaded mPEG-b-PLA nanoparticles without PVMMA (PTX-p-NPs) were prepared by the same method. The physical appearance of the final nanoparticles suspension is


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shown in Figure S1. The diameter of the hydrated PTX-m-NPs and PTX-p-NPs in solution were 120 nm and 90 nm (Figure 2A and 2C) respectively. TEM images (Figure 2B and 2D) confirmed that the PTX-m-NPs and PTX-p-NPs were both spherical in shape and uniform in size distribution. The properties of PTX-m-NPs and PTX-p-NPs including size, zeta potential, drug loading and drug encapsulation efficiency are summarized in Table 1. Particle size and surface charge played an important role in the cellular uptake of NPs.34 There are reports on the influence of nanostructured lipid carriers (NLCs) with three different particle sizes (100 nm, 200 nm and 300 nm) on cell permeability within Caco-2 monolayers, and the area under the concentration-time curve (AUC) after oral drug delivery, where NLCs-100nm showed a clear advantage.35 Studies have also shown that nanoparticles with negatively charged surfaces tended to bind to the biomolecules in the intestine and then be absorbed by the small intestinal epithelial cells.36 The zeta potential of blank-m-NPs (-20.03 ± 0.12 mV) was much lower than that of blank-p-NPs (-1.59 ± 0.10 mV). The difference in zeta potential suggested the successful preparation of PVMMA doped nanoparticles, as the change is attributed to the hydrolysis of anhydride linkages of the PVMMA on the surface of the nanoparticles.27 Compared with PTX-p-NPs, the presence of PVMMA also resulted in a more negative zeta potential for the PTX-m-NPs. This means that PTX-m-NPs are more stable than PTX-p-NPs, due to increased repulsion between the nanoparticles.37 PTX-m-NPs showed comparable encapsulation efficiency and drug loading content to PTX-p-NPs, indicating that the addition of PVMMA had no obvious effect on the encapsulation and drug loading ability of mPEG-b-PLA. Storage stability tests concluded that the shelf life of samples was stable within 7 days (Figure S2).



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Figure 1. Schematic illustration of the preparation and composition of nanoparticles. Differential Scanning Calorimetry (DSC) was utilized to characterize the physical state of paclitaxel in the NPs (Figure 2E). For pure paclitaxel, a melting endothermic peak appeared at 214 °C, consistent with previous reports, 38 showing that paclitaxel alone was in a crystalline form. However, the melting peak disappeared in both the PTX-m-NPs and PTX-p-NPs formulations, indicating that the paclitaxel encapsulated within nanoparticles was mostly in an amorphous or molecular state.


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Figure 2. Dynamic light scattering (DLS) of PTX-m-NPs (A) and PTX-p-NPs (C), and TEM micrographs of PTX-m-NPs (B) and PTX-p-NPs (D), Scale bar = 100 nm. (E) DSC profiles of PTX, blank carriers and PTX-loaded nanoparticles. In vitro drug release In order to illustrate the necessity of the addition of PVMMA for the oral nano-formulation design, PTX-p-NPs served as a control group in the drug release tests. The drug release profiles of paclitaxel from the different formulations in vitro were shown in Figure 3. As polymeric nanoparticles tend to pass through the stomach quickly, the influence of the acidic environment of the stomach has often been overlooked in previous reports.39 Figure 3A shows the release profile of PTX from nanoparticles in a stimulated gastric juice environment (pH 1.2). The cumulative release of PTX-m-NPs was lower than 10% in 2 h, indicating that PTX-m-NPs



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possess a higher stability than PTX-p-NPs and Taxol®. In simulated intestinal fluid (pH 6.8), Taxol® exhibited quick release, while PTX-m-NPs and PTX-p-NPs showed sustained release profiles (Figure 3B). For Taxol®, up to 80% of PTX was released by 6 h; however it took nearly 24 h for PTX-m-NPs to reach a comparable cumulative release. Within the first four hours, PTX-m-NPs and PTX-p-NPs showed similar release profiles, but at later time points PTX-p-NPs displayed a faster release rate. These results imply that PTX-m-NPs possess a high stability within the gastric juice environment, and sustained release in an intestinal environment, which are both necessary characteristics for oral drug delivery.

Table 1. Summary of basic properties of blank carriers and PTX-loaded nanoparticles. Zeta a

Size ( nm )

potential

PDI

DL(%)

EE(%)

( mV ) PTX-m-NPs

120.3 ±1.1

-20.3 ± 0.2

0.2 ± 0.0

4.69 ± 0.18

90.2 ± 4.0

PTX-p-NPs

90.4 ± 2.5

-1.0 ± 0.0

0.3 ± 0.0

4.3 ± 0.2

90.9 ± 5.4

blank-m-NPs

117.6 ± 0.8

-20.03 ± 0.12

0.2 ± 0.0

-

-

blank-p-NPs

94.9 ± 1.8

-1.59 ± 0.10

0.3 ± 0.0

-

-

a

Measured by intensity


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Figure 3. Cumulative release of PTX from PTX-m-NPs, PTX-p-NPs and Taxol® in HCl solution (pH 1.2) containing 0.1% Tween 80 (A) and PBS (pH 6.8) containing 0.1% Tween 80 (B). Cellular uptake of C6-loaded nanoparticles Before cellular uptake experiments were conducted, the cytotoxicity of the blank carriers was evaluated using an MTT assay (Figure S3). The results showed that the concentration of nanoparticles should be maintained at less than 1 mg/mL in order to reduce the impact of the materials on the activity of cells in vitro. Coumarin 6 (C6), a commonly used hydrophobic fluorescent dye, was encapsulated in the nanoparticles and used for imaging the cellular uptake of the nanoparticles.40-42 As shown in Figure 4, the uptake of C6-m-NPs and C6-p-NPs by the Caco-2 cell monolayer was much higher than that of free C6 after 0.5 h and 2 h incubation.

Figure 4. The uptake of coumarin 6 loaded nanoparticles by Caco-2 cells monolayer at different timepoints. The Z-stack LSCM analysis is shown for 0.5 h (A) and 2 h (B), the yellow arrows indicate from the bottom to the top of the dish, the blue color represents Hochest and green color represents coumarin 6. Scale bar = 10 µm.



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Quantitative analysis and mean fluorescence intensity by flow cytometry at 0.5 h (C, D) and 2 h (E, F). Mechanisms of cellular uptake The mechanisms of endocytosis of the nanoparticles were investigated in detail in the presence of different endocytic inhibitors. As shown by the LSCM images (Figure 5A), compared with the control group, the fluorescence intensity of C6-m-NPs in Caco-2 cell monolayers that were pre-treated with endocytic inhibitors was decreased significantly. Quantitative analysis was also carried out by FCS, and is shown in Figure 5B and 5C. After treating the cells with sodium azide and 4 °C, the uptake of C6-m-NPs was reduced to 25.2 ± 3.2% and 23.5 ± 3.1% (p < 0.01) respectively, indicating that the uptake of particles was an energy-dependent process. Nystatin and methyl-β-cyclodextrine showed the most significant inhibitory effects. Therefore, it can be inferred that the PTX-m-NPs were internalized into the cells by caveolae/lipid raft mediated endocytosis. This result is exciting as it suggests that PTX-m-NPs can be transported to the endoplasmic reticulum, and escape the usual fate of lysosomal degradation.24, 42


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Figure 5. Uptake mechanism of C6-m-NPs by Caco-2 cell monolayer. (A) Confocal images of cells treated with C6-m-NPs and different endocytotic inhibitors. (B) Quantitative analysis by flow cytometry. (C) Mean fluorescence intensity by flow cytometry. (Chl: Chloropromazine hydrochloride, Nys: Nystatin, Cyt-D: Cytochalasin D, M-β-CD: Methyl-beta-cyclodextrin). Adhesion, Retention and Permeability (ARP effect) Adhesion in vitro The potential for bioadhesion of the particles as a result of chemical interactions between NP acid anhydride groups and intestinal proteins was investigated. The adhesion ability of C6-m-NPs, C6-p-NPs and free C6 was assessed by determining the NP binding to the surface of a lysine-coated 96 well plate, and the results are presented in Fig. 6A. Compared with C6-p-NPs, the presence of PVMMA in



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C6-m-NPs increased their adhesion efficiency by 2-fold. The adhesion of free C6 was particularly low. The results indicated that C6-m-NPs showed the greatest ability for attachment to the lysine coating. The interaction between the carboxylic groups deriving from polyanhydride residues and amino groups in lysine is beneficial to immobilize the NPs on the surface of lysine-coated 96 well plates 25. The formation of numerous hydrogen bonds between the hydrophilic functional groups (-COOH) of the carriers and the -OH groups of the mucus glycoproteins also contributed to the observed bioadhesion.43 Following the hydrolysis of the acid anhydride bonds of PVMMA on the NPs, adsorption of mucin to the PTX-m-NPs surface was favorable, as shown in Fig. 6B. The amount of mucin adsorbed by PTX-m-NPs was 68.4 ± 4.4%, in comparison to 32.9 ± 5.0% for PTX-p-NPs (p < 0.01). Although it has been reported that larger sizes and lower zeta potentials may prevent the absorption of mucin to nanoparticles,32 we postulate that the acid anhydride bonds in PVMMA play a definitive role in the adhesive effects found for PTX-m-NPs. Retention ex-vivo It is well known that the intestinal epithelium is covered by mucus glycoproteins, in order to prevent pathogens and foreign bodies from entering the systemic circulation. To further investigate the retention effects of the nanoparticles in the intestine, DiR-loaded PVMMA/mPEG-b-PLA nanoparticles (DiR-m-NPs) and DiR-loaded mPEG-b-PLA nanoparticles (DiR-p-NPs) were prepared. Ex vivo mouse intestine samples were collected after oral dosing of the samples over a series of pre-determined time points. As shown in Figure 6C, fluorescence in the free DiR group disappeared rapidly after dosing, and in the DiR-p-NPs group became very weak by 12 h, however the fluorescence in the DiR-m-NPs group was still strong. The overall total fluorescence in the intestines also confirmed the longer retention properties of the DiR-m-NPs (Figure 6D). The fluorescence intensity in different intestine segments demonstrated the preferred residence of DiR-m-NPs as within the jejunum, which is characterized by a thicker mucus layer and larger population of goblet cells (Figure 7E).


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Figure 6. Adhesion, retention and permeability effect (ARP effect). (A) Adhesion of C6-m-NPs on (poly-L-lysine) - coated plates compared with C6-p-NPs and free C6. (B) In vitro interaction between nanoparticles and mucin. (C) Ex-vivo images of the mouse intestinal tract. (D) Total fluorescence intensity in intestine analyzed by ImageJ.



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(E) Fluorescence intensity of the contents in the duodenum, jejunum, and ileum segments. (F) Localization of C6 loaded NPs in villi of jejunum at 2 h using LSCM, blue color represents nuclei and green color represents C6, red arrow represents deep permeation. Scale bar = 100 µm. (G) Semi-quantitative fluorescence analyzed by ImageJ. Permeability in vivo Visualization of the permeation of C6-m-NPs, C6-p-NPs and free C6 into the villi of the jejunum was qualitatively observed by LSCM. Figure 6F illustrates the penetrative ability of the C6-m-NPs, with deep permeation of the particles into the villi, as indicated by the red arrow. Through quantitative analysis of the images by ImageJ (Figure 6G), both C6-loaded nanoparticles showed enhanced permeability compared with free C6, and the permeability of C6-m-NPs in the villi was approximately 1.5 fold higher than for C6-p-NPs. It can be concluded that the enhanced permeability ability of C6-m-NPs is related to its high absorption ability, which is derived from the presence of PVMMA. Transcytosis A mucus-secreting Caco-2/HT29 co-culture cell model was established to further evaluate the transport of nanoparticles across the epithelial cells. Sodium fluorescein exhibited a low Papp of 3.98 ± 0.15×10-7 cm/s after 4 h, suggesting the formation of an intact cell monolayer.44 As well, both microvilli and tight junctions could be observed clearly by TEM (Figure S4). Fig 7A depicts the cumulative amount of transported PTX at the predetermined time points. For PTX-m-NPs and PTX-p-NPs, as well as Taxol®, the cumulative amount of transported PTX showed a time-dependent behavior. PTX-m-NPs demonstrated the best transmembrane transport properties, and these results are consistent with the cellular uptake demonstrated by both the Z-stack of LSCM and FCS analysis. The Papp of PTX-m-NPs through the co-cultured cell monolayer was 1.6-fold of that for Taxol® and 1.3-fold of that for PTX-p-NPs after 4 h (Figure 7B). Significant differences were observed at the same time points.


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Figure 7. (A) Cumulative amount of transported PTX through Caco-2/HT29 monolayers at different time. (B) Papp (apparent permeability coefficient) of PTX from different formulations at 4 h. In vitro cytotoxicity Cytotoxicity of the particles was evaluated by culturing A549 cells with PTX formulated in the PTX-m-NPs, PTX-p-NPs or Taxol® at the same PTX doses (0.03, 0.3, 3, 30 and 300 µg/mL, n = 3) for 48 h. The profile of cell viability was measured and the results are depicted in Figure S5 and Table 2. The IC50 values of Taxol®, PTX-m-NPs and PTX-p-NPs were 6.1 ± 1.5 µg/mL, 0.2 ± 1.4 µg/mL and 1.2 ± 1.4 µg/mL respectively. The anti-cancer efficiency was most significant for PTX-m-NPs, showing an almost 30 and 6-fold greater cytotoxicity compared with Taxol® and PTX-p-NPs respectively, after the 48 h treatment. Table 2. IC of A549 cells after 48 h incubation with PTX formulated in the Taxol®, 50 PTX-m-NPs and PTX-p-NPs.

IC50 (µg/mL)

Taxol®

PTX-m-NPs

PTX-p-NPs

6.1 ± 1.5

0.2 ± 1.4

1.2 ± 1.4

In vivo pharmacokinetics All in vitro and ex-vivo results showed that the PTX-m-NPs formulation had an advantage over PTX-p-NPs and Taxol®, owning to the bioadhesive effect of the particles. For further validation of this phenomenon, a pharmacokinetic experiment was performed. The mean concentration-time curve and pharmacokinetic parameters



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of Taxol®, PTX-m-NPs, and PTX-p-NPs after oral administration to rats is shown in Figure 8 and Table S1. The peak concentrations (Cmax) of PTX were observed at approximately 1 h for the three formulations. Similar to the in vitro cytotoxicity study, the two nanoparticle formulations showed a certain degree of increase in Cmax, however the rate of increase was more significantly for PTX-m-NPs. The AUC0-t (ng·h/mL) for PTX-m-NPs, PTX-p-NPs and Taxol® was 62.2 ± 10.4, 30.8 ± 5.5 and 29.3 ± 7.8 respectively, and thus the PTX-m-NPs showed a higher bioavailability as determined by AUC0-t. PTX-p-NPs and solutions exhibited a similar AUC0-t. The complex physiological environment of the gastrointestinal tract (GI) makes it difficult to pinpoint the exact reason for this, however it can be theorized that the faster release of PTX-p-NPs in pH 1.2 and pH 6.8 medium may be responsible for the low bioavailability of PTX, as the released drugs from this formulation was comparable to the free PTX solution.

Figure 8. Mean concentration-time curve of PTX after oral administration of different formulations. Discussion In this work, bioadhesive nanoparticles loaded with paclitaxel were successfully produced, with encapsulation efficiency up to 90.2 ± 4.0%. The sustained drug release of PTX from PTX-m-NPs compared with Taxol® and PTX-p-NPs were reflected by


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the release profiles. By the Z-stack analysis for the cellular uptake, the phenomenon that C6-m-NPs were tended to be transported from the apical to basolateral sides of the cell monolayer was observed and it meant C6-m-NPs could overcome the barrier of the cell monolayer and be transported into the circulatory system and this phenomenon pushed us to carry out the experiment of transcytosis on another hand. The Caco-2 cell line is a classic in vitro model of the GI barrier used to predict drug absorption via oral delivery，as they can be used to form a similar structure to the small intestinal epithelium.45,

46

However, because of the lack of mucus, the barrier effect of the

mucus layer has often been ignored.47 Therefore, the establishment of a mucus-secreting Caco-2/HT29 co-culture cell model was necessary to further evaluate the transport of nanoparticles across the epithelial cells. The ratio of 90:10 between Caco-2 and HT29 cell was the optimal in vitro co-culture model for transmembrane transport studies as previously reported,

48, 49

and Woitiski’s group found that the

permeability of insulin across the Caco-2/HT29 (90:10) co-culture cell model was similar to the animal-model intestinal membrane. In our results, PTX-m-NPs showed great potential for transcytosis, which highlights the benefit of the bioadhesive effect. Clinically, a paclitaxel injection combined with cisplatin is the standard first-line treatment for non-small cell lung cancer, and thus for this work a non-small cell lung cancer cell line A549 was chosen as the model cell line to evaluate the cytotoxicity of different PTX formulations. In the literature, the process of endocytosis of nanoparticles has been shown to be defined by two sequential stages. First, there is surface binding followed by vesicle internalization, which demonstrates the importance of bioadhesion for facilitating the endocytic process.42 Based on this, we believe that the adhesive properties of PTX-m-NPs increased the probability of surface binding and thus enhanced the internalization of NPs, as more binding leads to more internalization. The increase in the cytotoxicity of PTX-m-NPs is likely due to the increase in cellular uptake, which is in agreement with the transcellular investigation. Obvious difference of cellular uptake between LSCM and FCS experiments was



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observed and we ascribed the difference to tight junctions. Tight junctions of the Caco-2/HT29 monolayers could be observed clearly (Figure S4). Through the analysis of cellular uptake, it is interesting to find that the cellular uptake of the nanoparticles as analyzed by LSCM decreased in the following order: PTX-m-NPs > PTX-p-NPs > free C6 (Figure 4A, 4B), while FCS analysis showed no difference between C6-m-NPs and C6-p-NPs, although uptake was significantly higher for the free C6 control group (Figure 4C-F). In contrast with the analysis by FCS for single cell suspension, the imaging of LSCM was carried out with an intact cell monolayer, thus the presence of the tight junctions played an important role in the difference between the LSCM and FCS experiments. It has been reported that the chelation of Ca2+ can enhance the endocytosis of NPs in Caco-2 cell monolayers by increasing the paracellular permeability,

50

and thus would suggest that changing tight junctions

would have an influence on endocytosis. Compared with the control C6-p-NPs, the addition of PVMMA decreased the zeta potential value from -1.59 ± 0.10 mV to -20.03 ± 0.12 mV; the more negative zeta potential likely improved the calcium depletion and enhanced the penetration of C6-m-NPs through the tight junctions. The results of the in vitro experiments can be summarized as follows: bioadhesive nanoparticles m-NPs had a stronger ability to cross the barrier of the mimic small intestine, and further showed enhanced uptake by cancer cells to exhibit anti-tumor activity. Adhesion, retention and permeability are referred to as the “ARP effect” and for this work were adapted to evaluate the bioadhesion effect in vitro as well as its advantages ex vivo and in vivo. In summary, the addition of PVMMA led to strong adhesion between mucus and m-NPs which was beneficial to increase the retention time and further improve the permeability. DiR-m-NPs exhibited long time retention in the intestine and C6-m-NPs had an obvious increase in intestinal absorption compared to the C6-p-NPs and free drugs, confirming the necessity of bioadhesive properties for facilitating NPs transportation across the mucus layer. By using the ligated intestinal loop model, the fluorescence intensity of the different C6 formulations could be easily characterized. It should be noted that although there was


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an obvious bioadhesive effect of the m-NPs observed both in vivo and in vitro analysis, the improvement of the bioavailability for the PTX-m-NPs only was approximately 2 fold higher than PTX-p-NPs and Taxol®. In other words, the bioavailability was only slightly increased when only the bioadhesion of the nanoparticles was increased. Based on this, further work is required to further increase the bioavailability of the particles. At first, the formulation m-NPs was proved to have excellent adhesion effect with protein-rich mucosa by the experiment of adhesion effect in vitro. Second, the advantage of bioadhesive efficiency was further illustrated through cell and animal experiments. By combining the cellular uptake results of the nanoparticles with “ARP effect”, the improvement of bioavailability could be attributed as followed that prolonged nanoparticle retention in the intestinal tract facilitated internalization of PTX-m-NPs into cells mainly through caveolae/lipid raft mediated endocytosis. In addition, the negative zeta potential of PTX-m-NPs resulted in an improvement of calcium depletion in the intestines, which can further increase the paracellular permeability to some extent. Conclusion In this work, nanoparticles with PVMMA (m-NPs) that exhibited improved bioadhesion were produced, and revealed the importance of a bioadhesion effect for intestinal absorption. Nanoparticles without PVMMA (p-NPs) and free drug were used as control groups to confirm the bioadhesive properties. The enhanced bioadhesion due to the presence of PVMMA was confirmed by cellular uptake, in vitro adhesion, ex-vivo retention and in vivo permeability experiments using fluorescent molecules (coumarin 6 or DiR) loaded nanoparticles. As a drug delivery system, PTX-m-NPs showed improved cytotoxicity and bioavailability as determined by in vitro cytotoxicity and in vivo pharmacokinetic analysis respectively, compared with free drug and PTX-p-NPs. Therefore, the developed bioadhesive drug delivery system is a promising tool for improving the bioavailability of drugs. This work focused on the effect of bioadhesion alone for gastrointestinal absorption of the nanoparticles, and in future should contribute to further research towards the oral



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delivery of PTX. Acknowledgements This work was supported by the National Natural Science Foundation of China (41310262), National major scientific research program (973 program, No. 2015CB932103), National Natural Science Foundation of China (Grant No. 31600810), Beijing Natural Science Foundation (Grant No. 7174332), Natural Science Foundation key project (31630027 and 31430031), and National Distinguished Young Scholars grant (31225009). The authors also appreciate the support by the Strategic Priority Research Program (XDA09030301). We are grateful to Antoinette Nelson from the Department of Biomedical Engineering at Rutgers University for her help in editing this manuscript. Supporting information Details about preparation process and prescription exploration; storage stability, cell viability under different conditions; confirmation of structure of Caco-2/HT29 monolayer and the pharmacokinetic results. References 1.

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