PolyesterâSolid Lipid Mixed Nanoparticles with Improved Stability in

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Polyester-solid lipid mixed nanoparticles with improved stability in gastro-intestinal tract facilitated oral delivery of larotaxel Jingxin Gou, Shuangshuang Feng, Yuheng Liang, Guihua Fang, Haotian Zhang, Tian Yin, Yu Zhang, Haibing He, Yanjiao Wang, and Xing Tang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00503 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 28, 2017

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Molecular Pharmaceutics

Polyester-solid lipid mixed nanoparticles with improved stability in gastro-intestinal tract facilitated oral delivery of larotaxel

Jingxin Gou1, Shuangshuang Feng1, Yuheng Liang1, Guihua Fang4, Haotian Zhang3, Tian Yin2, Yu Zhang1, Haibing He1, Yanjiao Wang1, Xing Tang1 1 Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University 2 Department of Wine, School of Functional Food and Wine, Shenyang Pharmaceutical University 3 Department of Pharmacology, School of Life Sciences and Bio-pharmaceuticals; 4 School of Pharmacy, Nantong University Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang, China, Postal code: 110016. Nantong University, No. 19 Qixiu Road, Nantong China, Postal code: 226001. Corresponding Authors: Prof. Xing Tang Email: [email protected] Tel: 86 24 23986343 Fax: 86 24 23911736 And Dr. Tian Yin Email: [email protected] Tel: 86 24 43520558

ACS Paragon Plus Environment


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Abstract:

The objective of this study was to investigate the role of core stability of

nanoparticles on their performances in oral drug delivery. Solid lipids (Geleol Mono and Diglycerides Nf) were incorporated into nanoparticles composed of mPEG-b-PCL by the dialysis method. The prepared solid lipid-loaded nanoparticles were found to be spherical nanoparticles with a core state and size distribution dependent on the amount of solid lipid incorporated. The critical aggregation concentrations of lipid-loaded nanoparticles were determined using pyrene fluorescence. Then, the stability of block copolymer in nanoparticles with different solid lipid contents was studied in simulated gastric fluid and simulated intestinal fluid. Solid lipids were found to stabilize nanoparticle cores by improving not only the thermodynamic stability (lowered CAC) of the nanoparticle but also the chemical stability of the block copolymer in the gastrointestinal environment. The stability of the loaded drug (larotaxel, LTX) in nanoparticles with different solid lipid contents was challenged by intestinal homogenate and rat liver microsome, and solid lipid-loaded nanoparticles showed superior drug-protecting capability. Solid lipid incorporation exhibited limited influence on the cytotoxicity and cellular uptake but improved the transcytosis of nanoparticles in Caco-2 monolayers. The results of pharmacokinetic study indicated that core stabilization was helpful in promoting oral larotaxel absorption as the absolute bioavailability of LTX delivered by solid lipid-loaded nanoparticles was found to be 13.17%, compared with that by the lipid-free nanoparticles (6.264%) and LTX solution (2.435%). Additionally, the results of biodistribution study indicated relatively higher particle integrity of solid lipid-loaded nanoparticles, shown by slower liver and spleen accumulation rate, compared with its lipid-free counterpart. Overall, incorporation of solid lipids made the nanoparticles more suitable for oral drug delivery.

Keywords: larotaxel, oral drug delivery, mixed nanoparticles, core stability


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1. Introduction. Taxane derivatives, such as paclitaxel and docetaxel, are effective chemotherapeutic agents widely applied in tumor treatment. Oral delivery of these drugs is an important step towards realizing the “therapy at home” model of treatment, which would improve patient compliance, as well as quality of life of patients in late stage cancer1, 2. However, oral absorption of taxane derivatives is usually limited due to poor drug solubility/permeability, drug efflux caused by P-glycoprotein (P-gp) that widely exist in the intestinal tract and enzymatic drug degradation after absorption2-4. Therefore, it is necessary to find approaches that can efficiently improve the oral bioavailability of certain anticancer drugs. Recently, the use of polyester or lipid-based nanoparticles as delivery systems have been shown to promote oral absorption of anticancer agents2, 5-8, and particularly, the combined use of these two adjuvants has achieved great success in promoting the oral availability of docetaxel under the assistance of enteric materials, which helped with nanoparticle stabilization in the stomach 9, 10. Structural integrity of the nanoparticles is another prerequisite for oral absorption of nanoparticles beside carrier functionalizations like bio-adhesion2, 5, 6 and P-gp inhibition11, 12. This is important as orally administered nanoparticles are challenged sequentially by acidic gastric juice and enzyme-rich intestinal juice, which cause disintegration of drug carriers composed of biodegradable building blocks. Additionally, drug leakage caused by nanoparticle disintegration lowers the overall drug absorption, as the leaked drugs can hardly be absorbed due to their poor membrane permeability. This has been demonstrated by a study that co-administered a drug solution with empty carriers, which resulted in a low bioavailability 9. To avoid carrier disintegration in the gastro-intestinal (GI) tract, enteric materials have previously been applied, and the resultant oral delivery systems with high bioavailability proved this to be a facile method for promoting oral absorption6, 9, 10, 13, confirming the necessity of nanoparticle stabilization in the GI tract. After nanoparticles absorption into the intestinal wall, oral bioavailability can still be limited by factors such as the mild acidic lumen of apical early endosomes (pH 5-6)14 that can destabilize the nanoparticles, and any leaked drugs will be effluxed by P-gp or metabolized by first pass



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effect-related enzymes15. Therefore, stability of the drug-loaded nanoparticle during transportation in intestinal wall, especially the drug loading moiety, is also a key parameter determining the performance of oral drug delivery systems. Various types of mixed nanoparticles with appropriate compositions that provide improved physical stability or enhanced drug loading capability have been identified16-18. Among these, polymer-lipid mixed nanoparticles, which combine the functionalization capability of polymers with the advantages of lipid-based nanoparticles, have been demonstrated to be robust drug delivery systems that could potentially meet the delivery requirements of various bioactive agents19, 20 and have previously been employed in the construction of oral drug delivery systems5, 9, 21. By utilizing the stability improving property of mixed nanoparticles22, 23, the influence of the core stability of nanoparticles on their performance in oral drug delivery can be investigated. In this work, based on a mixed drug delivery system composed of poly (ethylene glycol)-b-polycaprolactone (PEG-b-PCL) and solid lipids, as well as larotaxel (LTX), a water insoluble and bio-membrane impermeable, semi-synthetic taxane derivative with higher cytotoxic effect and lower affinity to P-gp compared with paclitaxel and docetaxel24-26, were used as the nanoparticle system and model drug, respectively. The influence of solid lipid introduction was first evaluated by monitoring the changes in core crystallinity, core polarity, critical aggregation concentration (CAC), and drug release profile of the prepared nanoparticles with solid lipids (SL-NPs). The stabilizing effect of the solid lipids was studied by monitoring the changes in particle size of empty SL-NPs incubated in simulated gastric fluids, and the changes in LTX content of drug-loaded SL-NPs incubated in intestinal homogenate. Finally, the in vivo pharmacokinetic and biodistribution profiles of NP and SL-NP were studied.

2. Materials and methods. 2.1 Materials. mPEG5k-b-PCL14k block copolymer was synthesized by ring-opening polymerization according to the method reported previously17, 27 using mPEG5k-OH as the initiator. Dialysis membranes (MWCO: 3.5 kDa and 14 kDa) were purchased from Ruidahenghui Co., Ltd., Beijing, China. Larotaxel (LTX, purity >99%) was obtained from Shandong Target Drug


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Research Co. Ltd., Yantai, China. Solid lipids (Geleol Mono and Diglycerides Nf) were provided by Gattefosse (Lyon, France). Tween 80 was purchased from BASF (Ludwigshafen, Germany). 3,3’-dioctadecyloxacarbocyanine

perchlorate

(DiD),

rhodamine

B

isocyanate

and

nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Aladdin Agent (Shanghai, China), mixed liver microsome suspension obtained from male SD rats (20 mg/mL) was purchased from Research institute for liver disease Co., Ltd. (Shanghai, China).All other chemicals and reagents were of analytical grade or chromatographic grade. 2.2 Cell line and animals. Human breast cancer MCF-7 cell line and human colon carcinoma Caco-2 cell line were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). MCF-7 and Caco-2 cells were maintained in low and high glucose DMEM medium, respectively, supplemented with 10% FBS in a humidified atmosphere containing 5% CO2 at 37 ˚C. Sprague-Dawley rats (male, body weight: 200 g ± 20 g) and Kunming mice (male, body weight: 20 g ± 2g) were provided by Liaoning Changsheng Biotech Co., Ltd. (Benxi, China). All animal experiments were approved by the Animal Ethics Committee of Shenyang Pharmaceutical University. 2.3 Characterization of polycaprolactone/solid lipids blends. PCL/lipids blends containing solid lipids of 5%, 10%, 15% and 20% of weight of PCL were prepared by casting a dichloromethane solution of PCL containing different amounts of solid lipids onto glass slides, followed by evaporation of the solvent. The wax-like solid obtained was subjected to DSC analysis to determine the melting enthalpies of the blends. Samples (approximately 2 mg, accurately weighted) were placed in hermetically sealed aluminum pans with pin holes in the lids in case of moisture retention. The instrument conditions were as follows: the nitrogen was set at a flow rate of 80 mL/min, the samples were heated from 25 °C to 80 °C and kept for 5 min, then cooled to 30 °C at the same rate, and finally the samples were reheated to 80 °C at the rate of 1 °C/min. 2.4 Preparation of nanoparticles with/without solid lipids. Nanoparticles with and without solid lipids were prepared using the dialysis method. Nanoparticles with solid lipids (SL-NPs) were prepared as follows: block copolymer (20 mg) and solid lipids were dissolved in dimethyl formamide (DMF, 1 mL). The obtained solution



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was heated and maintained at 60 °C, and then purified water (2 mL) was added at a speed of 0.1 mL/min under stirring. After water addition, the obtained mixture was transferred to a pre-swollen dialysis membranes (MWCO: 3500 Da) and dialyzed for 24 h. After complete removal of DMF, the nanoparticle suspension was centrifuged at 12,000 rpm for 10 min to remove any precipitates. Solid lipids-free nanoparticles (NPs) were prepared in the same way, except that no solid lipids were added. 2.5 Characterization of nanoparticles The particle size of the prepared nanoparticles was determined by dynamic light scattering (DLS) using a NicompTM 380 particle sizer (Particle Sizing System, Santa Barbara, CA) at room temperature. The morphology of both SL-NPs and NPs were observed using a JEM-2100 transmission electronic microscope (JEOL) with 200 Kv acceleration voltage. Samples were stained with phosphotungustic acid prior to imaging. The critical aggregation concentration (CAC) of NPs and SL-NPs containing different amounts of solid lipids (5%, 10%, named as SL5%-NPs and SL10%-NPs, respectively) was determined using pyrene as a fluorescent probe. The pyrene concentration in each sample was fixed at 6×10-5 M. The excitation spectrum was recorded from 300 to 350 nm with the emission wavelength set at 390nm. The fluorescence intensity ratio at 335 and 337nm (I337/I335) versus the logarithm of the polymer concentration was plotted from the excitation spectra; the CAC of each sample was obtained from the inflection point of this plot. Core polarity of the nanoparticles was determined using pyrene fluorescence28. The emission spectrum of pyrene for each sample in the range from 360 to 430 nm was recorded using an F-7000 spectrophotometer (Hitachi) at an excitation wavelength of 335 nm. The ratio of the fluorescence intensity of the first peak to the third peak in the emission spectrum was used to reflect the core polarity. The drug loading content (DLC) and encapsulation efficiency (EE%) of the nanoparticles were determined by high performance chromatography (HPLC). 100 μL samples were transferred into 10 mL volumetric flasks and were diluted with acetonitrile. The resultant solution was filtered with a 0.45 μm filter, and the filtrate was subjected to HPLC analysis (equipment: Hitachi, L-2130 pump, L-2400 UV detector, L-2200 auto-sampler and L-2300 column oven; mobile phase: acetonitrile-water (65:35, v/v); flow rate: 1 mL/min; column


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temperature: 25 °C). DLC and EE% were calculated according to the following equations: ℎ × 100% ℎ ( + + ) ℎ EE (%) = × 100% ℎ $

DLC (%) =

In vitro drug release profiles of LTX-solution, NPs and SL10%-NPs were studied using simulated gastric fluids (SGF) and simulated intestinal fluid (SIF) containing 0.5% (w/v) Tween 80 as release media. LTX-loaded samples (1 mL containing approximately 100 μg LTX) were added into dialysis membranes (MWCO: 14 kDa). Then the dialysis membranes were placed in glass vials containing 10 mL of release medium. And the glass vials were placed in a water bath shaker maintained at 37 °C with a shaking speed of 100 rpm. At pre-determined time points, 4 mL release media were taken and centrifuged at 10,000 rpm for 10 min before HPLC analysis. The rest of the media was discarded and replaced with 10 mL fresh media. 2.6 Stability of NPs and SL-NPs. Colloidal stability of LTX-free NPs and SL10%-NPs in SGF and SIF was investigated. 1 mL sample was mixed with 5 mL SGF or SIF and the mixtures were incubated at 37 °C for 10 days. Every two days, 500 μL samples were withdrawn for DLS measurement. After 10 days of incubation, the samples were neutralized and lyophilized. The obtained powders were dissolved in DMF and then filtered for GPC analysis using pristine copolymer and macroinitiator (mPEG5k-OH) as the controls. The drug-protecting capability of LTX-loaded NPs and SL10%-NPs was evaluated in intestinal homogenate and mixed liver microsome obtained from male SD rats. For the intestinal homogenate study, changes in LTX content during incubation were monitored: 0.1 mL sample was mixed with 0.5 mL intestine homogenate and the mixture was incubated at 37 °C for 4 h. At pre-determined time points, 200 μL samples were taken and transferred into 10 mL volumetric flasks containing 2 mL DMSO. The mixture was diluted with acetonitrile and filtered, and then the LTX content was determined using HPLC. Intestinal homogenate was obtained according to the following method. Briefly, male SD rat was fasted for 24 h before being sacrificed, after which the small intestines (approximately 1 cm under stomach, 0.5 g in weight) were harvested, washed with normal saline and dried with filter paper. The intestinal segments were then cut into small pieces and homogenized with 1 mL normal



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saline. The primary homogenate was diluted with 4 mL normal saline before use. For the mixed liver microsome study, LTX-loaded NPs and SL10%-NPs were incubated with rat liver microsome and NADPH. The total volume of the reaction system was fixed at 0.5 mL, in which the concentrations of LTX, protein and NADPH were 80 μg/mL, 0.4 mg/mL and 1mmol/L, respectively. The whole system was incubated in 37 °C water bath. After 4 h incubation, the mixture was cooled in ice bath to terminate the reaction. Then, acetonitrile was added to precipitate the proteins and dilute the mixture to a final volume of 5 mL. LTX content in the final mixture was determined by HPLC. Rat liver microsome-free mixtures were used as negative control. The percentage of LTX remained was calculated by the following equation: LTX remained (%) =

. /

where LTXa indicates LTX recovered from microsome-containing mixtures and LTXb indicates LTX recovered from microsome-free mixtures . 2.7 Cell studies. The cytotoxicity of empty nanoparticles and LTX-loaded nanoparticles were studied in MCF-7 cells and Caco-2 cells using MTT assay. Cell density in each well was 5000 cells per well, and the concentrations of LTX-free samples were 0.1, 2, 5, 10, 50 and 100 μg/mL, and the concentrations of LTX-loaded samples were 0.003, 0.03, 0.3, 3 and 30μg/mL. The incubation time was 72 h and the absorbance of the formed formazan crystals was measured at 570 nm using a microplate reader (Biotek SynergyTM HT Reader). Cell uptake of NPs and SL10%-NPs was studied using coumarin 6 as a fluorescence probe. Cells density in each well was 1.5×104, and the incubation times were set to 1 and 4 h for each sample. Cell nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI). Images and intracellular fluorescence intensity were obtained using an Image Xpress® Micro instrument (Molecular Devices, Metaxpress™). Transcytosis of NPs and SL10%-NPs was studied using Caco-2 monolayers. Caco-2 cells were seeded on transwell inserts (6-well plates) at a cell density of 7.5×105 cells per well and the culture medium was replaced every 2 days. The cells were incubated for 21 days to allow cell differentiation. Then the integrity of cell monolayers was checked using sodium


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fluorescein solution. Before sample addition, the monolayers were equilibrated with pre-warmed Hank’s balanced salt solution (HBSS) for 15 min at 37 °C. After equilibration, the HBSS in the apical side was discarded and replaced with 0.5 mL HBSS containing LTX-loaded NPs or SL10%-NPs (final LTX concentration: approx. 100 μg/mL, accurately determined) and the monolayers were further incubated for 4 h at 37 °C. After incubation, the liquids in the apical side were collected, and the monolayer was washed three times with 0.2 mL HBSS. HBSS used in cell wash was combined with the liquids in the apical side. Then, the cells were trypsinized and suspended in 0.5 mL PBS. This cell suspension and the liquids in the basolateral side were collected separately. LTX content in the apical/basolateral liquid and in cells was determined using HPLC. 2.8 Intestinal uptake study. Uptake of NPs and SL10%-NPs into the intestine was studied using rhodamine B isocyanate (RBIC) as a fluorescent marker. RBIC was conjugated to the terminal hydroxyl group of the block copolymers through an esterification reaction using EDC·HCl as a dehydrating agent and DMAP as a catalyst according to the following procedures. RBIC (56 mg, 0.1053 mmol), EDC·HCl (101 mg, 0.5263 mmol) and DMAP (64 mg, 0.5263 mmol) were mixed and dissolved in 10 mL dry DCM, and the mixture was stirred in dark for 1 h to activate the RBIC. Block copolymer (1 g, 0.05263 mmol) dissolved in 20 mL DCM was then added and the mixture was allowed to react for 3 days. At the completion of reaction, the mixture was filtered and concentrated, and then washed three times with water and dried over MgSO4. The product was then precipitated from cold ether, collected by filtration and dried under vacuum. The dried product was then dissolved in THF and dialyzed against water for 48 h before lyophilization. The final product was a purple red powder (0.472 g). Sprague-Dawley rats (SD rats) were fasted overnight before oral administration with NPs and SL10%-NPs labeled with RBIC (NPs/RBIC and SL10%-NPs/RBIC) at a RBIC dose of 5 mg/kg. 45 min after administration, the rats were sacrificed to collect the duodenum. The collected intestinal segments were cryo-sectioned into slices 20 μm in thickness (SLEE MEV, Germany). Cell nuclei in the slices were stained with DAPI before being observed by CLSM (Nikon C2S, Japan). 2.9 In vivo pharmacokinetic study.



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24 male SD rats were randomly divided into four groups (6 rats per group). The rats were administered with LTX solution, NPs or SL10%-NPs orally at a dose of 10 mg/kg or injected intravenously with LTX solution at a dose of 4 mg/kg. Blood samples (0.5 mL) were taken from the orbital vein at pre-determined time points (oral: 0.25, 0.5, 1, 2, 3, 4, 6, 8 and 10 h; i.v.: 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, and 10 h) after administration. Plasma was obtained by centrifuging the blood samples at 6000 rpm for 5 min. The plasma samples were then treated as follows: plasma (100 μL) was mixed with 10 μL internal standard solutions (docetaxel in methanol, 500 ng/mL) and the drugs were extracted with 2 mL tert- butyl methyl ether by vortexing for 10 min. The mixture was then centrifuged for 10 min at 4000 rpm, and 1.6 mL of the supernatant was collected and dried under nitrogen flow at 35 °C. The residue was reconstituted with 100 μL of acetonitrile -water (9:1, v/v) and centrifuged at 12,000 rpm for 10 min, and an aliquot of 5 μL was used for analysis. The method of UPLC/MS/MS used in this study was as reported previously29. The absolute bioavailability (Fabs) of the orally administered formulations was calculated according to the following equation: F123 (%) =

AUC6718 × D9: × 100% AUC9: × D6718

where AUCoral and AUCiv are the areas under the curve of the oral formulation and LTX solution given intravenously, respectively; Doral and Div are the doses of oral formulation and LTX solution given intravenously, respectively. 2.9 In vivo biodistribution. DiD ethanol solution, DiD-loaded NPs and SL10%-NPs were prepared and administered to mice orally at a dose of 12 μg/kg. The mice were then sacrificed by cervical dislocation to collect major organs (brain, heart, lung, liver, spleen and kidneys) at 1 or 4 h post-administration. The collected organs were then visualized under a vivo imaging system (Carestream FX Pro, USA) using an excitation wavelength of 720 nm and an emission wavelength of 750 nm. 2.10 Statistical analysis. The pharmacokinetic parameters of CTX in rats were calculated using DAS 2.1 supplied by the Pharmacological Society of China (Beijing, China). All data are presented as mean ± SD


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and analyzed using Student’s t-test. Statistical significance was determined as a P value of < 0.05 unless stated otherwise.

3. Results. 3.1 Characterization of PCL/solid lipid blends. PCL, solid lipid (SL) and PCL-SL blends at different weight ratios were subjected to DSC analysis. As shown in Figure 1, the blends at all ratios (5%, 10%, 15% and 20%) were found to be partially mixed systems due to the existence of individual endothermic peaks but lowered melting points of both components, indicating the co-existence of a PCL phase and a SL phase at all blending ratios. By calculating the endothermic peak areas of PCL in blends with different ratios, the effect of solid lipids incorporation on the crystallinity of PCL was investigated. A low amount of solid lipid incorporated (5%) could effectively decrease the crystallinity of PCL in the mixture, however, when further increasing the lipid content, no further inhibition of crystallization was observed (Table 1), which indicated a limited mixing between SL and PCL.

Figure 1. DSC curves of PCL, Solid lipid and lipid-PCL blends at different weight ratios. Table 1. DSC characterization of PCL blended with different amounts of solid lipids. Lipid content (%)

ΔHm (J/g)

Crystallinity (%)*

0 5 10 15 20

67.94 26.86 26.59 24.31 25.20

48.06 19.25 19.07 17.43 18.06

* Crystallinity=ΔHm, sample/ΔHm, PCL× 100%, ΔHm, PCL=139.5 J/g30



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3.2 Characterization of nanoparticles with/without solid lipids. In this study, solid lipid-free NPs and SL-NPs were prepared by the classic dialysis method which has been widely used in the preparation of self-assemblies composed of amphiphilic copolymers with low water solubility, and the size of the self-assemblies have been found controllable by varying the organic solvents used or the composition of the copolymers31, 32. The mean particle size of the nanoparticles increased gradually with increased amount of incorporated solid lipid (Figure 2 A). And SL-NPs with a solid lipid content of 5% and 10% (SL5%-NPs and SL10%-NP) exhibited narrow size distribution, as indicated by the low PDI (less than 0.15). The formation of SL-NPs was similar with the formation process of polymeric micelles loading water insoluble drugs. So, when further increasing the amount of incorporated solid lipid, the size distribution increased drastically (15%: 0.216 ± 0.017; 20%: 0.543 ± 0.029) (Figure 2 A) due to the leakage of the over-loaded solid lipid that exceeded the loading capacity of the block copolymer.

Figure 2. Characterization of SL-NPs. (A). Average particle size and polydispersity index (PDI) of nanoparticles with different solid lipid contents; (B). TEM images of NPs (B1), SL5%-NPs (B2),


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SL10%-NPs (B3) and SL20%-NPs (B4). Scale bars: B1, B3 and B4, 100 nm; B2, 200 nm; (C). Changes in CAC (upper) and nanoparticle core polarity (lower) upon solid lipid incorporation; (D). In vitro drug release profiles of LTX solution, LTX-loaded NPs and SL10%-NPs in simulated gastric fluid (upper) and simulated intestinal fluid (lower) (mean ± SD, n=3).

TEM was used to observe the morphology of NPs and SL-NPs with solid lipid contents of 5%, 10% and 20%. NPs and SL5%-NPs exhibited a similar morphology, with typical spherical shapes and uniform size distributions (Figure 2 B1 and B2). SL10%-NPs were also spherical in shape, but with non-homogeneous cores (Figure 2 B3, marked by arrows). Similar core structures were also observed in the TEM image of SL20%-NPs (Figure 2 B4). The formation of the non-homogeneous cores was likely caused by phase separation, as was demonstrated in the DSC analysis. Due to the differences in the dimensions of the separated SL phase, it appears that the SL5%-NPs possess homogeneous cores. In the TEM image of SL20%-NPs, the amount of SL exceeded the loading capacity of the nanoparticles, and thus precipitated to form aggregates with irregular shapes which were considered to be the reason for the wide size distribution of the SL20%-NPs (Figure 2 A). The influence of solid lipid content (5% and 10%) on the thermodynamic stability of nanoparticles was studied by determining the critical aggregation concentration (CAC) of nanoparticles using pyrene fluorescence. In the solid lipid content range of 0%-10%, the CAC of nanoparticles decreased with increasing amount of incorporated solid lipid (Figure 2 C, upper), indicating that there was an improvement on thermodynamic stability upon addition of solid lipid. Similarly, the core polarity of SL-NPs also decreased with improved solid lipid content (Figure 2 C, lower), indicating there was an improved hydrophobicity of the SL-NPs cores. LTX-loaded NPs and SL10%-NPs were also prepared by the dialysis method, and the polydispersity index, drug loading content (DLC) and encapsulation efficiency (EE) of the LTX-loaded nanoparticles were determined (Table 2). Both of the drug-loaded nanoparticles exhibited a narrow size distribution and increased particle sizes due to the core expansion effect of loaded LTX. SL10%-NPs had an improved DLC over their lipid-free counterpart, which can be attributed to the core crystallization inhibition effect of the solid lipid incorporation as discussed (Table 1)17. Table 2. Particle size, size distribution and drug loading parameters of NPs and SL-NPs (mean ±



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SD, n=3). Sample

Particle size (nm)

PDI

DLC (%)

EE (%)

NPs SL10%-NPs

62.0 ± 4.0 76.8 ± 6.4

0.073 ± 0.007 0.083 ± 0.005

11.04 ± 0.84 12.90 ± 0.29

79.2 ± 1.3 81.5 ± 0.9

The drug release profiles of LTX solution, NPs and SL10%-NPs in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were studied (Figure 2 D). Considering the low stomach residence time of the nanoparticles, drug release profiles within 4 h were studied. Compared with the fast release of LTX solution, both the nanoparticles released approximately 5% of the loaded drug in SGF (Figure 2 D, upper), indicating negligible drug leakage in stomach. From the release curve of the LTX solution it could be confirmed that LTX adsorption by the dialysis membrane itself was negligible, as the amount of LTX release reached 96.3 % after 48 h. For the NPs and SL10%-NPs, the amount of drug released reached 60.5 % and 50.1 %, respectively, after 48 h, and it took the nanoparticles 7 days to achieve complete release (Figure 2 D, lower). These results indicated a sustained drug release behavior for both the nanoparticles. As seen in previous literature where the liquid lipids within mixed nanoparticles had an effect on drug release 17, incorporation of the solid lipids in this case could also sustain the release of LTX from the nanoparticles. The likely mechanism for this sustained release is the decreased core polarity and extended drug diffusion path. 3.3 Stability of NPs and SL-NPs As the mPEG-b-PCL is degradable in acidic environment, the colloidal stability of NPs and SL10%-NPs in SGF was studied using DLS. Both of the nanoparticles showed a relatively good stability in SGF during an initial 24 h period, and so the incubation time was extended out to 10 days to ensure observable changes in particle sizes. As shown in Figure 3 A, the particle size of NPs and SL10%-NPs increased from 51.8 ± 16.5 nm to 312.4 ± 147.8 nm, and 57.1 ± 14.2 nm to 102.8 ± 63.2 nm, respectively. From the changes in particle size of NPs and SL10%-NPs, it could be concluded that incorporation of solid lipids provided stabilization of the nanoparticle in the acidic environment. After 10 days, the block copolymers were isolated by lyophilization for GPC analysis in order to investigate the mechanism of particle size change. Comparing the elution curves of the pristine block copolymers, mPEG5k-OH (the macroinitiator) and block copolymers obtained from NPs and SL10%-NPs after the 10-day


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incubation in SGF, it was found that mPEG was cleaved from both the nanoparticles due to hydrolysis at the core-shell interface (Figure 3 B). The elution curve of the block copolymers from NPs showed a new peak at retention time around 19.5 min, which was deduced to be PCL fragments as a result of PCL core hydrolysis. Very little PCL hydrolysis could be observed in the block copolymers from SL10%-NPs, indicating improved core stability in this sample. Additionally, the GPC analysis was able to provide more information on the changes in particle size of NPs and SL10%-NPs: in NPs, the 6-fold increase in particle size was likely the consequence of uncontrollable PCL fragment aggregation, while the 2-fold increase in particle size of SL10%-NPs was likely induced by inter-particle aggregation due to decreased PEG coverage. Subsequently, the colloidal stability of NPs and SL10%-NPs in SIF was also studied. Due to the more neutral pH environment of SIF (pH 6.8), both the nanoparticles showed less changes in particle size compared with those incubated in SGF (Figure 3 A). And according to the results of GPC analysis (Figure 3 C), block copolymers obtained from both the nanoparticles were found with less degradation. Overall, the degradation studies found that SL10%-NPs had a better colloidal and chemical stability than NPs in environments like SIF and SGF.

Figure 3. Stability of NPs and SL10%-NPs. (A). Changes in particle size of NPs and SL10%-NPs in



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SGF and SIF during 10-day incubation; (B). GPC elution curves of block copolymers from NPs and SL10%-NPs incubated in SGF for 10 days; (C). GPC elution curves of block copolymers from NPs and SL10%-NPs incubated in SIF for 10 day; (D). Changes in LTX contents in NPs and SL10%-NPs incubated in intestinal homogenate for 4 h; (E). Changes in LTX contents in NPs and SL10%-NPs incubated in rat liver microsome for 4 h (mean ± SD, n=3; *p＜0.05).

The drug-protecting capability of the nanoparticles was first studied by measuring the total LTX content in mixtures of nanoparticles (LTX-loaded NPs and SL10%-NPs) and intestinal homogenate after incubation in a 37 °C water bath. As shown in Figure 3 D, SL10%-NPs showed superior drug-protecting capability than NPs in the intestinal homogenate, with more than 80% of the loaded drug still remaining after 4 h incubation. In contrast, the percentage of LTX remaining in NPs dropped sharply to approximately 50% during the first 15 min of incubation, followed by a continued decline at a relatively lower degradation rate (which was considered to be enzyme saturation over longer incubation time). It could therefore be concluded that the loaded LTX would be leaked from the vehicles and the leaked LTX was sensitive to the enzymes expressed in the small intestine. With these data in hand, the differences in drug-protecting capability of the nanoparticles against drug-metabolism-related enzymes were further studied using rat liver microsome (RLM) as enzyme source. Compared with the corresponding RLM-free negative controls, LTX in SL10%-NPs remained nearly unchanged after 4 h incubation, while a nearly 10% of LTX loss was observed in NPs group (Figure 3 E). These results again demonstrated the better drug protecting capability of SL10%-NPs. The lower percentage of LTX degradation could be attributed to the lower complexity in the RLM system compared with intestine homogenate, which shifted the manner of LTX degradation to “enzyme attack”-dependent in RLM (Scheme 1 A) instead of drug leakage-dependent in intestinal homogenate.


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Scheme 1. (A). Schematic illustration of stabilizing effect brought by solid lipid incorporation; (B). Enzymatic metabolization site of taxane derivatives.

3.4 In vitro cell viability, cellular uptake and transcytosis studies. The influence of solid lipid incorporation on the performance of SL10%-NPs at the cellular level was studied in MCF-7 breast cancer cells and Caco-2 human colon carcinoma cells. By comparing the cell viability curves of empty SL10%-NPs with NPs, it could be found that under all concentrations tested, incorporation of solid lipids did not induce any significant cytotoxicity in both cell lines (Figure 4 A), confirming the biocompatibility of SL10%-NPs. The cytotoxicity of LTX-loaded NPs and SL10%-NPs was then compared, and SL10%-NPs were found to have a slightly lower cytotoxicity compared with LTX-loaded NPs in both cell lines (Figure 4 B). Besides, both LTX-loaded NPs and SL10%-NPs showed lower cytotoxicity in Caco-2 cells compared with MCF-7 cells (Figure 4 B), and this insensitivity of Caco-2 cells to LTX could be attributed to their slower growth rate than MCF-7 cells. Cellular uptake of NPs and SL10%-NPs during 1 or 4 h incubation was investigated using coumarin 6 as a fluorescence probe. The fluorescence intensity of the coumarin 6 in cells treated with SL10%-NPs was comparable with that of the cells treated with NPs at both time points (Figure 4 C and D), indicating similar cell internalization behavior. This was expected as both SL10%-NPs and NPs demonstrated identical surface properties (PEG coverage), shape (spherical) and comparable particle sizes (64.5 ± 4.6 nm versus 52.3 ± 5.4 nm), which are key factors that determine the cellular uptake of nanoparticles33. Combining the results of the cell uptake studies, it was shown that incorporation of solid lipids could only have limited influence on the performance of nanoparticles at the cellular level.



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Figure 4. Results of cell studies. (A). Cell viability of blank nanoparticles; (B). Cell viability of LTX-loaded nanoparticles; (C). Intracellular fluorescence intensity after 1 or 4 h incubation with coumarin 6-loaded NPs and SL10%-NPs; (D). Fluorescence images of DAPI-stained MCF-7 cells incubated with coumarin 6-loaded NPs for 1 or 4 h; (E). LTX contents in cells and basolateral liquids after transcytosis study (mean ± SD, n=3; *p＜0.05).

In the transcytosis experiment, LTX contents in the upper apical liquids (NPs: 103.8±1.579 μg; SL10%-NPs: 102.8±3.040 μg), middle cellular layer and lower basolateral liquids were determined (Figure 4 E). The 1.65-fold higher intracellular LTX content of the SL10%-NPs group was considered to be the consequence of its better drug-protecting capability, since the two nanoparticles showed comparable cellular uptake (Figure 4 C) and there are also drug metabolizing enzymes expressed in Caco-2 cells34. So, the higher LTX content observed in the lower basolateral liquids of SL10%-NPs group indicated its better transcytosis capability realized by superior drug-protecting capability.


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3.5 Intestinal uptake study. Intestinal slices were imaged using CLSM to determine the location of RBIC-labeled nanoparticles within the intestinal wall. Both of the nanoparticles could be found with a high distribution in the villi (Figure 5), as they are easily accessible by nanoparticles due to their stretched state in the intestinal lumen. At the basolateral side, red fluorescence could be also observed in the CLSM images of both samples, indicating the permeation of the two nanoparticles into the deep area of intestine. Moreover, by inspecting the areas with red fluorescence in the basolateral side (Figure 5, white circles), a stronger and wider distributed red fluorescence was observed in the CLSM image of SL10%-NPs group, indicating its stronger intestinal penetrative capability.

Figure 5. CLSM images of intestinal villi, and intestinal wall from SD rats administered with NPs/RBIC and SL10%-NPs/RBIC. 3.6 In vivo pharmacokinetic study.

The oral availability of LTX solution, LTX-loaded NPs and SL10%-NPs was studied in SD rats using intravenously injected LTX solution as a reference formulation. As a BCS IV drug24,



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LTX solution exhibited poor oral bioavailability, with an AUC0-t of 101.5 ± 21.56 μg/L*h (Figure 6 and Table 3). However, the AUC0-t of LTX-loaded NPs was 2.57-fold of that of the LTX solution, indicating that drug encapsulation within nanoparticles could enhance the absorption. More importantly, incorporation of solid lipids was able to further improve the AUC0-t of LTX by more than 4-fold above the LTX solution (Table 3). The AUC0-t of LTX solution administered through intravenous injection was determined to calculate the absolute bioavailability (Fabs), which gave the Fabss of LTX delivered by solution, NPs and SL10%-NPs as 2.435%, 6.264% and 13.17%, respectively. These results confirmed that the oral availability of LTX could be improved by nanoparticles stabilized using solid lipid.

Figure 6. Pharmacokinetic profiles of LTX solution, NPs and SL10%-NPs administered orally (10 mg/kg); inset: PK profile of LTX solution administered via i.v. injection (4 mg/kg) (mean ± SD, n=6). Table 3. Pharmacokinetic parameters of oral formulations (LTX solution, NPs and SL10%-NPs) and i.v. formulation (LTX solution) (mean ± SD, n=6). Parameters

LTX solution (i.v.)

LTX solution (oral)

NPs

SL10%-NPs

AUC0-t (μg/L*h)

1667.3 ± 103.7

101.5 ± 21.56

261.1 ± 111.2

548.9 ± 71.37

t1/2z (h)

3.833 ± 3.302

2.399 ± 1.972

2.163 ± 1.664

2.518 ± 1.172

tmax (h)

0.08333

0.75 ± 0.274

0.667 ± 0.258

1.167 ± 0.683

Cmax (ng/mL)

2268.8 ± 665.7

55.46 ± 8.863

128.0 ± 29.29

285.2 ± 22.85

CLz/F (L/h/kg)

2.319 ± 0.085

77.90 ±41.62

39.45 ± 28.79

17.77 ± 1.784

Vz/F (L/kg)

12.77 ± 10.95

177.9 ± 50.18

78.08 ± 52.62

43.91 ± 32.89


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3.6 Biodistribution study. Accumulation of orally administered nanoparticles in the major organs (brain, heart, liver, spleen, lung and kidneys) was studied using DiD-labeled NPs and SL10%-NPs (NPs/DiD and SL10%-NPs/DiD). As shown in the ex vivo images of the major organs collected from mice treated with NPs/DiD and SL10%-NPs/DiD (Figure 7), the two nanoparticles exhibited different organ accumulation profiles, particularly in reticuloendothelial system-rich organs such as the liver and spleen. NPs/DiD were found to have a massive accumulation in the liver and spleen by 1 h after oral administration, while it took SL10%-NPs/DiD 4 h to reach comparable accumulation levels in these two organs. To determine whether DiD accumulation in these organs was achieved by nanoparticle delivery or by the physicochemical property-determined spontaneous distribution of the probe molecule, DiD ethanol solution was administered orally as a reference. It was found that, when administered in solution form, DiD exhibited very limited accumulation in spleen, lung and brain at both time points, and nearly no accumulation in liver, heart and kidneys (Figure 7). These results indicated that i) DiD-loaded nanoparticles were absorbed as a whole to achieve high organ accumulation of DiD, and ii) NPs/Did and SL10%-NPs/DiD exhibited different blood retention behaviors after intestinal absorption.

Figure 7. Ex vivo images of major organs 1 and 4 h after oral administration of DiD solution, NPs/DiD and SL10%-NPs/DiD.

4. Discussion. Encapsulation of drugs within nanoparticles is a promising solution to promote oral



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absorption of BCS IV drugs6, 9, 35. However, effective oral drug delivery by nanoparticles composed of biocompatible polymers is usually challenged by the harsh environment in the gastro-intestinal tract (acidic gastric juice, enzymes in gastric/intestinal contents and intestinal wall). Recently, dispersion of nanoparticles in enteric materials has been reported to be a successful method to improve oral bioavailability6, 9, indicating the importance of protecting the nanoparticle in the acidic gastric juice. This is of particular importance for the oral delivery of anticancer drugs, as the integrity of the nanoparticles together with the encapsulated state of the cargo become more important, as the toxic cargos are to be delivered to tumor tissues through the enhanced permeation and retention effect (EPR effect) which facilitates selective tumor accumulation of nanoparticles in the size range of 60-100 nm 36, 37. In this work, the influence of incorporating solid lipids (SLs) on the core stability of mPEG-b-PCL nanoparticles was studied. SLs, including tripalmitin, cetyl palmitate, glyceryl mono/di-stearate and phospholipids, are lipids that could maintain solid state under body temperature that have been widely applied in the construction of solid lipid nanoparticles38. One unique property of SLs in the construction of polyester-lipid mixed nanoparticles is their CAC reduction capability as observed by us (Figure 2 C) and other22, while the CAC of mixed nanoparticles increased upon incorporation of certain amounts of medium triglycerides, a liquid lipid17. Stability of nanoparticles, as reflected by CAC, is determined by factors including core crystallinity, glass transition temperature (Tg) of the core-forming block and core hydrophobicity31, 39. Incorporation of SL was found to result in a reduction of core crystallinity (Table 1) and an improvement in core hydrophobicity (Figure 2 C) simultaneously. It was found that these two changes in core characteristics acted against each other in nanoparticle stabilization17, however, according to the decreased CACs of nanoparticles with increased SL contents (Figure 2 C), the destabilizing effect of crystallinity reduction was satisfactorily compensated by the improved core hydrophobicity. Phase separation is a common phenomenon that occurs when different non-miscible substances are blended. According to our previous studies17,

27

, lipids with shorter

hydrocarbon chains are more compatible with PCL than the SL used in this study, and thus a higher lipid ratio needs to be reached before phase separation occurs. In this case, due to the


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stronger intermolecular forces between the long hydrocarbon chains of SL molecules, the SL was found to induce phase separation at relatively lower SL-PCL ratios (Figure 1, Figure 2 B3). Thus, the amount of SL incorporated should be precisely controlled in order to prepare nanoparticles with uniform size distributions, like SL5%-NPs and SL10%-NPs (Figure 2 A). It has previously been reported that block copolymers in lipid-PCL mixed nanoparticles with non-homogeneous cores exhibit better chemical stability in aqueous medium27. In this study, incorporation of SL also exhibited a block copolymer stabilizing effect, and the reason for this stabilizing effect was attributed to a decreased core polarity induced by incorporation of SL. As shown in Scheme 1 A, there is less accessibility of H+ or enzymes into the deeper regions of the core in SL10%-NPs, whereas the chance of H+ or enzyme infiltration into the core of NPs is higher due to their higher core polarity. Therefore, although mPEG shedding was observed in the GPC trace of block copolymers obtained from SL10%-NPs incubated in SGF and SIF, negligible degradation in the PCL block was observed (Figure 3 B and C). Enzymes, including CYP3A enzymes, expressed in the small intestine are partly responsible for the first-pass effect of absorbed drugs15, so an improved oral docetaxel availability has been observed in cyp3a-knockout mice40. Additionally, taxane derivatives like paclitaxel, docetaxel and cabazitaxel are reported to be substrates of CYP3A enzymes41, 42. Marre et al.43 reported that docetaxel was metabolized by CYP3A enzymes through oxidation at the tert-butyl group, which is also a characteristic group of other taxane derivatives like LTX, cabazitaxel and TM-2 (Scheme 1 B). This indicates that the taxane derivatives would all potentially be substrates of CYP3A enzymes, and that a proportion of LTX could be metabolized during intestinal absorption as demonstrated by the different intracellular LTX contents in transcytosis study (Figure 4 E). Moreover, attentions should be paid to drug protection during absorption especially when designing oral drug delivery systems for human uses since the expression of CYP3A4 in human jejunum has been found 871-fold higher than that in Caco-2 cells34. Nanoparticles protect loaded drugs by isolating the drugs spatially from drug-decomposing factors44, but only when the nanoparticles maintain their own integrity. As indicated by the results of the intestinal homogenate stability test (Figure 3 D), the difference in the amount of LTX remained indicated a higher intestinal stability of SL10%-NPs over NPs. And this higher intestinal stability was then able to facilitate the intestinal



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absorption of SL10%-NPs was demonstrated by the higher accumulation in the basolateral side (Figure 5) in CLSM studies and a higher Cmax as well as AUC0-t in the pharmacokinetic studies (Figure 6, Table 3). The liver and spleen are responsible for the clearance of foreign particles in the bloodstream, including nanoparticles intravenously (i.v.) injected or orally absorbed. Surface PEGylation is a common method employed to reduce nanoparticle clearance, and the effectiveness of this approach is determined by factors such as the molecular weight of PEG, surface PEG density and PEG chain conformation45. Beside surface PEGylation, particle size is another key factor determining blood clearance rate and tumor penetration capability: for two nanoparticles (no larger than 200 nm) sharing a similar surface PEG density but having different particle sizes, the smaller one would be characterized with faster blood clearance but wider intratumoral distribution37, 46. NPs and SL10%-NPs had a similar surface PEG density and PEG chain conformation as they were prepared with same block copolymer and had comparable particle sizes (Figure 2 A), so they exhibited similar pharmacokinetic profiles after i.v. injection (Figure S1, Table S1). Since NPs and SL10%-NPs showed comparable pharmacokinetic profiles, particle size and surface property, it follows that they should also show similar biodistribution profiles47. Therefore, the observed differences in the biodistribution profiles between orally administered NPs and SL10%-NPs (Figure 7) could be attributed to the changes in nanoparticle structure during absorption, including possible shedding of the PEG chains from NPs surface, making them more easily arrested by the liver and spleen.

5. Conclusion. In conclusion, solid lipids (Geleol Mono and Diglycerides Nf) have been demonstrated to be a core stabilizing agent for the improvement of thermodynamic stability of nanoparticles and the chemical stability of both block copolymer as well as the loaded drug without affecting the performances of nanoparticles in other aspects. By overcoming the harsh environment in the GI tract and drug-metabolizing enzymes in the intestinal wall, the absolute bioavailability of larotaxel was promoted by solid lipid incorporation by one fold compared with the polymeric nanoparticle (13.17% versus 6.264%). It has been shown that


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core stability, including the colloidal stability and chemical stability, is another key parameter determining the oral delivery efficiency of nanoparticles that could be feasibly improved by solid lipids incorporation. This is a method that affects only the hydrophobic cores of nanoparticles, and thus it could be readily transferred into other drug delivery systems that are designed to face harsh biological environments. Supporting information In vivo pharmacokinetics of NPs and SL10%-NPs administered via intravenous injection. This material is available free of charge via the Internet at http://pubs.acs.org Acknowledgements This work is supported by the National Basic Research Program of China (973 Program, No. 2015CB932103), National Natural Science Foundation of China (No. 81673378) and China Postdoctoral Science Foundation (2016M600216). The authors appreciated Dr. Amanda Pierce for her help in polishing the manuscript.



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