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Gastric environment-stable oral nanocarriers for in situ colorectal cancer therapy
International Journal of Biological Macromolecules 139 (2019) 1035–1045
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Gastric environment-stable oral nanocarriers for in situ colorectal cancer therapy Ting Wang a, Fangqin Wang a, Mengjie Sun a, Meiping Tian a, Yuzhi Mu a, Xiguang Chen a,b, Ya Liu a,⁎ a b
College of Marine Life Science, Ocean University of China, Qingdao 266003, PR China Qingdao National Laboratory for Marine Science and Technology, Qingdao 266000, PR China
a r t i c l e
i n f o
Article history: Received 11 April 2019 Received in revised form 3 August 2019 Accepted 10 August 2019 Available online 11 August 2019 Keywords: Gastric environment Stable Nanocarriers Oral delivery Colorectal cancer
a b s t r a c t Colorectal cancer (CRC) is a prevalent and fatal cancer. Oral administration provided the potential for in situ treatment of the colorectal cancer. However, drugs couldn't be well-absorbed mainly due to its degradation in the gastric area and poor intestinal permeability. In this study, we synthesized deoxycholic acid and hydroxybutyl decorated chitosan nanoparticles (DAHBC NPs) as oral curcumin (CUR) delivery system for colorectal cancer treatment. DAHBC with lower critical solution temperature (LCST) below 37 °C (27–33 °C) was obtained. DAHBC NPs were correspondingly stable in simulated gastric conditions (pH 1.2, 37 °C), due to the offset of size change between pH-responsive expansion and thermo-responsive shrinkage. In simulated intestinal tract (pH 7.0–7.4, 37 °C), DAHBC NPs exhibited burst release of CUR owing to the onefold effect of thermoresponsive shrinkage. DAHBC27 NPs showed the minimum CUR leakage (~10%) in simulated gastric conditions, because a furthest temperature-sensitive shrinkage caused by the lowest LCST offset the expansion in acid environment. DAHBC27 NPs induced ~10-fold increased (P b 0.05) CUR absorption by paracellular transport pathway, compared to the free CUR. Thus, DAHBC NPs stabilized in the gastric environment may be a promising oral drugs delivery system for effective in situ colorectal cancer therapy. © 2019 Published by Elsevier B.V.
1. Introduction Colorectal cancer (CRC) is among the most commonly cancer and a major source of cancer-related deaths worldwide [1]. Most chemotherapy regimens administered through intravenous injection (i.v.), leading to the side effects and limited therapeutic efficacy [2]. Oral administration is a convenient and safe route that make drug acted directly on the intestinal tract, avoiding the cytotoxicity on normal tissues to consequently minimize the side effects [3,4]. Curcumin (CUR), a natural plant-derived therapeutic agent, demonstrated its antitumor activity in various cancer cell models [5–8]. However, the bioavailability of CUR is restricted by the instability and poor intestinal permeability in the gastrointestinal (GI) tract [9–11]. Nano-engineered drug delivery systems had gained sustaining attention for improving oral bioavailability of anticancer drugs because they provided the possibility of loading hydrophobic compounds [12–14]. To enhance the performance of the oral drug nanocarriers, the protection of drug in gastric environment and the drug release in intestinal conditions were essential [15,16]. Therefore, nanoparticles
⁎ Corresponding author at: College of Marine Life Science, Ocean University of China, 5# Yushan Road, Qingdao 266003, PR China. E-mail address: [email protected] (Y. Liu).
https://doi.org/10.1016/j.ijbiomac.2019.08.088 0141-8130/© 2019 Published by Elsevier B.V.
(NPs) stabilized in gastric environment were desired as oral drug delivery platforms for colorectal cancer therapy. Chitosan (CS) had been commonly used as a material for oral drugs delivery systems [17–19]. Its amino could be protonated under acidic conditions to carry a positive charge in response to pH stimulation, led to morphology expansion of NPs [20,21]. In addition, CS could open the tight junctions between intestinal epithelium and enhance the membrane permeability of drug rely on a translocation of tight junction proteins from the membrane to the cytoskeleton [22]. In our previous research, we prepared thermo-responsive CS nanoparticles, which could shrink at a temperature higher than the lower critical solution temperature (LCST), due to heightened intermolecular hydrogen bonding interactions and enhanced hydrophobic effect, finally resulting in the temperature-sensitive drug release [23,24]. Based on the opposite effects of pH-responsive expansion and thermo-responsive shrinkage in particle size, we hypothesized that thermo and pH dual-responsive nanoparticles with appropriate LCST would be efficient for the protection and the stable existence of drugs in gastric environment and on-demand release in the intestinal tract. Herein, hydrophobic deoxycholic acid (DOCA) and hydrophilic hydroxybutyl decorated chitosan nanoparticles (DAHBC NPs) were prepared as oral CUR delivery system for the in situ treatment of colorectal cancer (Schemes 1, 2). The LCST (b37 °C) of DAHBC NPs could be adjusted by changing the grafted density of hydrophobic DOCA groups.
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Scheme 1. Preparation of DAHBC NPs. (A) The composition of amphiphilic DAHBC. The hydrophilic domain contains hydroxybutyl groups. Deoxycholic acid constitutes the hydrophobic domain. (B) The DAHBC self-assembled and loaded hydrophobic CUR to form DAHBC NPs.
The particle shape, size and in vitro drug release were evaluated in the simulated GI tract. The cellular uptake efficiency of DAHBC NPs by Caco-2 cells in different concentration and time was observed via CLSM. Finally, we also studied intestinal absorption pathway of DAHBC NPs using Caco-2 cell monolayers. Thus, DAHBC NPs might show its potential as oral drug delivery system for colorectal cancer therapy.
2. Materials and methods 2.1. Materials Chitosan (molecular weight 1050 kDa, 85% degree of deacetylation) was purchased from Haili Biotechnology Co. Ltd. (Shandong, China). Deoxycholate (DOCA), 1,2-butene oxide, curcumin (CUR), N-(3-
Scheme 2. The principle of thermo and pH dual-responsive morphology transformation of DAHBC NPs by oral delivery. (A) In the stomach, DAHBC NPs were correspondingly stable due to the balance between pH-responsive expansion and thermo-responsive shrinkage. (B) In the intestine, DAHBC NPs exhibited the drug release activity owing to thermo-responsive shrinkage. (C) Oral delivery of DAHBC NPs in the gastrointestinal tract.
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dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), Nhydroxysuccinimide (NHS) and Dulbecco's modified Eagle's medium (DMEM) were obtained from Sigma-Aldrich (St. Louis, USA). Other reagent chemicals were all of analytical grade. 2.2. Synthesis and characterization of deoxycholic acid and hydroxybutyl modified chitosan (DAHBC) 2.2.1. Synthesis of DAHBC Hydroxybutyl modified chitosan (HBC) was prepared by using 1, 2butene oxide as the etherifying agent and connecting hydroxybutyl groups onto chitosan chain under alkaline conditions [25]. Then, DOCA conjugated to the amino groups of HBC based on a carbodiimide reaction [26]. Briefly, HBC dissolved into distilled water followed by dilution with 3-times methanol at 4 °C. After activated the γ-COOH group of DOCA, different amounts of DOCA were added dropwise into the HBC solution (DOCA:HBC = 1:1, 1.2:1, 1.6:1 and 2:1, w/w). Then the resultant mixtures were stirred for 24 h at room temperature, dialyzed to remove methanol and lyophilized. 2.2.2. 1H NMR spectroscopy 1 H NMR spectra (400 MHz) of CS, HBC and DAHBC in D2O were recorded on a Bruker ARX 400 MHz spectrometer. The chemical structures of CS, HBC and DAHBC were proved by NMR measurements. 2.2.3. Elemental analysis Elemental analysis was carried out to characterize the degree of substitution (DS) of DOCA groups and hydroxybutyl groups which defined as the group number per 100 sugar residues of CS. The DS of hydroxybutyl groups (DSHB) and DOCA (DSDOCA) were calculated as follows: DSHB ¼
H CS H%− =H HB =MHB 100 Mcs
DSDOCA ¼
H CS DSHB HD %− −HHB M cs M HB H DOCA 100 MDOCA
ð1Þ
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Table 1 The LCST of DAHBC in PBS (0.02 M, pH 7.4) (n = 3). Samples
DAHBC33
DAHBC31
DAHBC29
DAHBC27
DOCA:HBC (w/w) DS of DOCA (%) LCST (°C)
1:1 7.1 ± 0.4 33.2 ± 0.4
1.2:1 7.5 ± 0.2 31.1 ± 0.2
1.6:1 7.9 ± 0.3 29.4 ± 0.3
2:1 8.2 ± 0.1 27.3 ± 0.1
MDOCA were the molecular weight of CS units, hydroxybutyl groups and DOCA residues, respectively. 2.2.4. Lower critical solution temperature (LCST) The DAHBC was dissolved into PBS (0.02 M, pH 7.4). After equilibrated at the special temperatures for 10 min, the transmittance of DAHBC solutions at 500 nm was measured using a UV–vis spectrophotometer (UV-1200 MAPADA, China). The LCST of polymer solutions was defined as the temperature when the transmittance producing a 50% decrease in optical transmittance [27]. 2.3. The size variation of DAHBC NPs at simulating gastrointestinal environments DAHBC NPs were prepared using a dialysis method [24]. Briefly, DAHBC aqueous dispersions in DMSO were added dropwise into PBS (0.02 M, pH 7.4) under sustaining stir. The resultant mixtures dialyzed against PBS for 24 h to remove DMSO. The size variation of DAHBC NPs at simulated gastrointestinal environments was investigated via transmission electron microscope (TEM, JEM-1200EX JEOL100 Ltd., Japan) and dynamic laser light scattering technique (DLS) (Zetasizer ZEN3600, Malvern 95 Instruments Ltd, UK). The NPs dispersions (1 mg/mL) were prepared in PBS (0.02 M, pH 7.4) solution. After being incubated at specified conditions, the morphology and hydrodynamic size of DAHBC NPs were then measured. The data was analyzed with Zetasizer software (version 7.1). 2.4. Biocompatibility
ð2Þ
where H% and HD% were the percentages of H in HBC and DAHBC, respectively; HCS, HHB, HDOCA were the amounts of H in CS units, hydroxybutyl groups and DOCA residues, respectively; MCS, MHB,
2.4.1. Cytotoxicity assay The in vitro cytotoxicity of DAHBC NPs was evaluated using Caco-2 cell lines. Caco-2 cells were seeded in 96-well culture plates (1 × 104 cells/well) and cultured in DMEM supplemented with 10% fetal bovine serum at 37 °C in 5% CO2 for 12 h. Then cells were treated with DAHBC NPs at different concentrations (0.5–0.0313 mg/mL). After
Fig. 1. The 1H NMR spectra and structure of CS, HBC, DAHBC.
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where ODsample and ODcontrol were the absorbance of the test samples and the control group, respectively. Meanwhile, the cells were seeded in 24-well plates and cultured for 12 h. Then the cells were incubated with DAHBC NPs (0.5 mg/mL) for 24 and 48 h. After stained with calcein-AM, the samples were observed by confocal laser scanning microscope (CLSM, LSM510, Zeiss Ltd., Germany). 2.4.2. Hemolysis test The different concentration of DAHBC NPs suspensions (0.5–0.0313 mg/mL) with 20 μL rabbit blood suspension were incubated for 1 h at 37 °C. Subsequently, the mixtures were centrifugated at 2000 rpm for 10 min, and the absorbance of supernatants at 545 nm was detected using a Microplate Reader (PerkinElmer, Boston, MA, USA) [29]. Negative control was the saline and blood suspension, whereas the positive control was the distilled water and blood suspension. Hemolytic rate (HR) was calculated as follows: HRð%Þ ¼ Fig. 2. The transmittance profiles of DAHBC in PBS (0.02 M, pH 7.4) from 18 °C to 40 °C. The results were calculated as mean ± SD (n = 3).
24 h and 48 h, 10 μL CCK-8 was added to each well and cultured for 4 h [28]. The absorbance of each well was measured using a Microplate reader (PerkinElmer, Boston, MA, USA) at 450 nm. Cell viability (CV) was calculated as follows: CV ð%Þ ¼
ODsample 100 ODcontrol
ð3Þ
ODS −ODNC 100 ODPC −ODNC
ð4Þ
where ODS, ODNC and ODPC were the absorbance of tested sample, negative control and the positive control, respectively. 2.4.3. Protein adsorption test Nanoparticles suspensions (1 mg/mL) were incubated with Bovine Serum Albumin (BSA) solution (500 μg/mL) for 0.5 h at 37 °C and ultracentrifuged at 12,000 rpm for 0.5 h at 4 °C. Then the NPs were washed twice by distilled water. The adsorbed protein was desorbed from the DAHBC NPs surface in PBS solution containing 1% SDS by sonication and measured (λ = 595 nm) with Coomassie Brilliant Blue G-
Fig. 3. Cell viability (A) and fluorescence photomicrographs (B) of DAHBC NPs in Caco-2 cells for 24 h and 48 h. The results were calculated as mean ± SD (n = 5).
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250. The SDS-PAGE was performed and visualized by staining with Coomassie Brilliant Blue R-250 to detect the washed-off protein. 2.5. Drug loading efficiency CUR-loaded nanoparticles were prepared by dissolving CUR together with DAHBC (CUR: DAHBC = 0.25:1, w/w) in DMSO before dialysis. The freshly prepared DAHBC NPs were collected by ultracentrifugation at 12,000 rpm for 30 min. The free CUR in supernatants was detected by a Microplate Reader (PerkinElmer, Boston, MA, USA) at Ex/Em 425/475 nm, whose concentration was determined by a standard calibration curve [30]. The encapsulation efficiency (EE) and drug loading efficiency (LE) were calculated as follows: EEð%Þ ¼
Ct−Cf 100 Ct
ð5Þ
LEð%Þ ¼
Ct−Cf 100 Cn
ð6Þ
where Ct was the total amount of CUR; Cf was the amount of free CUR; Cn was the weight of CUR-loaded nanoparticles. 2.6. Drug release under simulating gastrointestinal environments The drug release studies of CUR from DAHBC NPs were carried out at simulated gastrointestinal environments by a dialysis method. The free
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CUR solution and CUR-loaded DAHBC NPs (1 mL) dispersions were transferred into a dialysis tube, immersed in 49 mL different simulated fluids and then gently stirred at 37 °C for 2 h. A total of 8 h of dialysis was performed in the gastrointestinal mimetic fluid, including simulated gastric fluid (SGF, pH 1.2, 2.0 g NaCl, 7 mL HCl and 3.2 g pepsin), simulated duodenum fluid (pH 6.0, obtained by mixing SGF and SIF), simulated proximal ileum fluid (pH 7.0, obtained by adjusting the pH of SIF) and simulated intestinal fluid (SIF, pH 7.4, 6.8 g KH2PO4, 0.2 N NaOH (190 mL) and 10.0 g trypsin) [31]. At predetermined times, 5 mL of the sample was removed for analysis and 5 mL of fresh medium was then added to the tube to maintain a constant volume. All the measurements were performed in the dark. The CUR concentrations were quantified using a standard CUR calibration curve, which determined by fluorescence microplate reader at Ex/Em 425/475 nm. 2.7. Cellular uptake assays Caco-2 cells were cultured in six-well culture plate for 24 h. The culture media was then replaced with D-Hanks (pH 7.4) and preincubated at 37 °C for 30 min. After equilibration, free CUR or DAHBC NPs solutions were added into each well at an equivalent dose of CUR in D-Hanks (pH 7.4). At predetermined times (i.e. 30, 60, 90, or 120 min), the cells were washed three times with D-Hanks (pH 7.4) and the nuclei was stained with 4, 6-diamidino-2-phenylindolyl hydrochloride (DAPI, Sigma, 1 μg/mL) in D-Hanks. Cover slides were then washed three times with D-Hanks and observed by confocal laser scanning microscope (CLSM, LSM510, Zeiss Ltd., Germany).
Fig. 4. (A) Hemolysis rate and (B) hemolysis test of DAHBC NPs at different concentrations (0.5–0.0313 mg/mL). PC represented positive control. NC represented negative control. The results were calculated as mean ± SD (n = 3). (C) Protein adsorption rate of DAHBC NPs (1 mg/mL). (D) SDS-PAGE electrophoresis gel stained by Coomassie Brilliant Blue. Lane 0: 500 μg/mL BSA; Lane 1–4: BSA adsorbed on DAHBC27, DAHBC29, DAHBC31, DAHBC33 NPs, respectively.
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To quantify the uptake of CUR by Caco-2 cells, Caco-2 cells were cultured in 96-well culture plates and incubated for 24 h. Then the culture medium was replaced by D-Hanks (pH 7.4) and preincubated at 37 °C for 30 min. After equilibration, free CUR or DAHBC NPs solutions were added into each well at an equivalent dose of CUR in D-Hanks (pH 7.4). At predetermined time, the cells were washed with ice-cold PBS (pH 7.4) and lysed with 0.2 mL of 0.5% Triton X-100 in 0.2 M NaOH solution [32]. Then the CUR concentrations were determined by fluorescence microplate reader at Ex/Em 425/475 nm. Cellular uptake efficiency (UE) was calculated as follows:
UEð%Þ ¼
Cu 100 Ci
ð7Þ
where Cu was the uptake concentrations of CUR, and Ci was the initial concentrations of CUR.
2.8. Intestinal absorption by Caco-2 cell monolayer 2.8.1. The transepithelial electrical resistance (TEER) measurement The TEER value was measured with a Millicell-Electrical Resistance System (Millipore Corp., Bedford, MA) in order to evaluate and determine the monolayer integrity of the Caco-2 cell monolayer in the Transwell plates [33]. The grown cell monolayers were incubated with free CUR or DAHBC NPs solutions containing equivalent doses of CUR. Before measuring the values of each well, the cell monolayers were washed twice with pre-warmed D-Hanks (pH 7.4).
Fig. 5. TEM images (A) and the size (B) of DAHBC NPs at RT/pH 7.4, RT/pH 2.0, 37 °C/pH 7.4 and 37 °C/pH 2.0 after incubation 2 h, 1 mg/mL. The results were calculated as mean ± SD (n = 3).
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2.8.2. Transcellular transport Caco-2 cells were grown into a differentiated monolayer for 21 days in the Transwell plate. The monolayer was washed twice with prewarmed D-Hanks (37 °C, pH 7.4, apical 0.5 mL and basolateral 1.5 mL) after the culture medium was withdrawn. Then the D-Hanks in the apical side were replaced with 0.5 mL free CUR or DAHBC NPs solutions containing equivalent doses of CUR. After incubated for 2 h, the polycarbonate membrane was collected, washed three times with ice-cold PBS (pH 7.4) and lysed with 0.2 mL of 0.5% Triton X-100 in 0.2 M NaOH solution. Then the CUR concentrations were determined by fluorescence microplate reader at Ex/Em 425/475 nm. 2.8.3. Paracellular transport Caco-2 cells were seeded in Transwell plate for 21 days. The monolayer was washed twice with pre-warmed D-Hanks (37 °C, pH 7.4, apical 0.5 mL and basolateral 1.5 mL). Then the D-Hanks in the apical side were replaced with 0.5 mL free CUR or DAHBC NPs solutions containing equivalent doses of CUR. The sample solution (0.5 mL) in the basolateral chamber was collected after the chamber was incubated for 2 h, and measured the fluorescence intensity by fluorescence microplate reader at Ex/Em 425/475 nm.
2.9. Statistical analysis Experiments were performed in triplicate and presented as mean ± standard deviation (SD). Student's t-test was used to analyzing the data. The level of significance was set at probabilities of *P b 0.05. 3. Results and discussion 3.1. Characterization of DAHBC 3.1.1. 1H NMR spectra of DAHBC DAHBC was prepared by conjugating hydroxybutyl and DOCA groups to CS. The chemical structure of DAHBC was characterized with 1 H NMR (Fig. 1). HBC was achieved, as evidenced by peaks arising at 0.6–1.7 ppm (peaks 4, 5), compared with the 1H NMR spectrum of CS [34]. The signals of DAHBC at 2.0–3.6 ppm (peaks 1, 2, 3) were appeared due to the protons of –CH3 groups at position 18, 19 and 20 of the steroid skeleton of DOCA, respectively [35]. The results of 1H NMR spectra indicated that the hydrophobic molecule of DOCA and the hydrophilic molecule of hydroxybutyl were successfully grafted onto CS.
Fig. 7. The cumulative release profiles of CUR from DAHBC NPs and free CUR at simulated GI fluid. Each release curve is calculated as mean ± SD (n = 3).
3.1.2. Elemental analysis of the DAHBC The DS of DOCA and hydroxybutyl groups were evaluated via percentages of each element (C, H, O and N) and known structural formula. The DS of hydroxybutyl groups was calculated to be 104.7 ± 2.8 per 100 anhydroglucose units of CS. With the weight ratios of DOCA to HBC increasing from1:1 to 2:1, the DS of DOCA increased from 7.1% to 8.2% (Table 1), indicating that a high proportion of DOCA was favorable to obtain DAHBC with high DS.
3.1.3. The LCST of the DAHBC Variations in the transmittance of DAHBC with temperature were evaluated using UV–vis spectroscopy. As the temperature increased from 18 °C to 40 °C, the transmittance of DAHBC was decreased (Fig. 2). The LCST was defined as the temperature when the transmittance produced a 50% decrease in optical transmittance. The reduced extent in DAHBC transmittance was increased with the weight ratio of hydrophobic DOCA to hydrophilic HBC decreased, and in consequence, LCST showed an ascending trend, which may be due to the increased number of H-bonds between the DAHBC polymers [23]. According to the LCST (summarized in Table 1), four types of DAHBC were named as DAHBC27, DAHBC29, DAHBC31 and DAHBC33, respectively. Therefore, the control of the LCST of the polymer could be manipulated by tuning of the hydrophobicity and grafted moieties.
Fig. 6. Drug encapsulation efficiency (A) and loading capacity (B) of DAHBC NPs. The results were calculated as mean ± SD (n = 3).
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3.2. Biocompatibility 3.2.1. Cytotoxicity To evaluate the cytotoxicity of the DAHBC NPs, a range of concentrations of DAHBC NPs from 0.5 to 0.0313 mg/mL towards Caco-2 cells were evaluated via CCK-8 assay. The cell viability of Caco-2 cells treaded with four DAHBC NPs was N80% at all concentrations during 24 h and 48 h (Fig. 3A). The morphology micrographs of cells further illustrated cytocompatibility of DAHBC NPs (Fig. 3B). The most cells of all DAHBC NPs (0.5 mg/mL) treatment groups showed uniformly spread state as same as control group at 24 and 48 h. These results indicated DAHBC NPs did not influence on cell viability and had satisfactory cytocompatibility. 3.2.2. Blood compatibility The hemolysis test and protein adsorption capacity with DAHBC NPs were detected as important index of the blood compatibility. Less than 5% hemolysis was regarded as a nontoxic effect level in this blood compatibility experiment [36]. The hemolysis rate (HR%) of four DAHBC NPs at different concentrations (0.5–0.0313 mg/mL) was all below 5% after incubating for 1 h (Fig. 4A). Meanwhile, unconspicuous erythrocyte hemolysis was observed in the presence of DAHBC NPs (Fig. 4B), which was considered as non-hemolysis. The adsorption of plasma proteins
occurred when blood contacted with a foreign material surface [37]. Protein adsorption rate of DAHBC NPs (1 mg/mL) was all below 10% (Fig. 4C). In addition, DAHBC NPs did not perform apparent bands (lane 1–4) compared with the BSA group (lane 0) (Fig. 4D), which indicated that the DAHBC NPs caused no adverse effects on the blood components. These results exhibited the DAHBC NPs had good blood compatibility. 3.3. The size variation of DAHBC NPs at simulated gastrointestinal environments The size variation of DAHBC NPs at simulated gastrointestinal environments was investigated to examine the stability (Fig. 5). DAHBC NPs had an approximately spherical shape at room temperature (pH 7.4). The diameters of DAHBC27 NPs (~61 nm) were smaller than that of the DAHBC29 (~75 nm), DAHBC31 (~109 nm) and DAHBC33 NPs (~151 nm), due to increased ratio of hydrophobic DOCA groups. When the pH transformed into pH 2.0, a considerable swelling of DAHBC27 NPs (with hydrodynamic size of ~185 nm) was clearly observed. Meanwhile, the spherical structure of DAHBC29 NPs was gradually loose, DAHBC31, DAHBC33 NPs almost disintegrated. The pHresponsive particle morphology changes were clearly observed, which could be caused by the amino groups on CS (pKa: ~6.5) were protonized
Fig. 8. (A) CLSM images of Caco-2 cells that were incubated with DAHBC27 NPs and free CUR for 2 h. Cell nuclei were stained with DAPI (blue). (B) Cellular uptake efficiency of CUR from DAHBC27 NPs and free CUR for 2 h with different concentration: 15, 30, 60 μg/mL. The results were calculated as mean ± SD (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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in the strongly acidic condition, reduced the chain–chain interaction and increased the hydrophilic performance with polymer chain [38]. At pH 2.0/37 °C (simulating gastric conditions), DAHBC27 NPs still showed a spherical shape and the hydrodynamic sizes increased to ~105 nm. But DAHBC29, DAHBC31 and DAHBC33 NPs transformed into more loose structure with the diameter of ~174 nm, ~202 nm and ~357 nm, respectively. The lowest LCST of DAHBC27 NPs led to the furthest offset of size change between the shrinkage caused by temperature (NLCST) and the expansion in acid environment, thus DAHBC27 NPs were stable at simulating gastric conditions. At pH 7.4/37 °C (simulating intestinal conditions), DAHBC27 NPs began to shrink and the particle sizes was ~40 nm. The decrease of DAHBC NPs size was due to the reinforced hydrogen bond within the polymer chain at 37 °C (NLCST), resulting in the enhanced hydrophobic effect of hydroxybutyl moieties [39]. The size change rules of DAHBC29, DAHBC31 and DAHBC33 NPs showed the same descending trend. With the increase of LCST, the degree of shrinkage decreased, owing to reduced hydrophobic DOCA groups. Therefore, DAHBC27 NPs were suitable for oral drug delivery through the gastric environments to the intestine.
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simulated gastric conditions and to release the loaded CUR (~73%) at simulated intestinal tract. Based on these results, the DAHBC27 NPs were selected for further studies. 3.6. Cellular uptake of DAHBC NPs The cellular uptake capacity of DAHBC27 NPs was studied by Caco-2 cells after incubation for 2 h. CUR with a green fluorescence can be used as probe to monitor the cellular uptake effect. The green fluorescence signal was detected after incubated with DAHBC27 NPs and free CUR (Fig. 8A), indicated that the CUR was effectively uptake by Caco-2 cells, this also implied the uptake activity of Caco-2 cells treated with DAHBC27 NPs was as high as free CUR. The cell uptake efficiency of CUR was ~10.2% at 15 μg/mL and then increased to ~23.7% at 60 μg/mL at DAHBC27 NPs treatment group, demonstrated that the amounts of
3.4. Drug loading efficiency To investigate drug-loading efficiency of DAHBC NPs, CUR was chosen as a model drug encapsulated into nanoparticles. The encapsulation efficiency of DAHBC NPs decreased from ~59% to ~52% as the LCST of DAHBC changed from 27 °C to 33 °C (Fig. 6A). A similar pattern was observed for CUR-loading efficiency of DAHBC NPs, which decreased from ~14% to ~12% (Fig. 6B), may attributed to the reduced amount of hydrophobic DOCA groups. DAHBC27 NPs showed the maximum encapsulation efficiency (~59%) and loading capacity (~14%). These results indicated that this variation tendency of encapsulation efficiency and loading efficiency was opposite to the LCST. 3.5. CUR release in vitro The release behavior of CUR from DAHBC NPs and free CUR was evaluated at simulated GI fluid. A sustained release of CUR from DAHBC NPs was observed over a period of 8 h (Fig. 7). In the first 2 h of incubation at SIF (pH 1.2), the cumulative release amount of CUR from DAHBC27, DAHBC29, DAHBC31 and DAHBC33 NPs was ~11%, ~15%, ~25% and ~38%, respectively. However, the free CUR group indicated the abundant release (N95%) in 2 h (Fig. 7). The phenomenon illustrated that the prepared DAHBC NPs was capable of protecting the CUR and alleviating the rapid metabolism of CUR under simulated gastric conditions [40]. DAHBC29, DAHBC31 and DAHBC33 NPs exhibited a faster release behavior compared to DAHBC27 NPs at SIF (pH 1.2), suggesting that higher LCST resulted in more drug release. Within 2–8 h of incubation at simulated duodenum fluid (pH 6.0), simulated proximal ileum fluid (pH 7.0) and SIF (pH 7.4), four DAHBC NPs displayed similar release tendency and the sustained CUR release curve was observed. The ultimate CUR release profiles were ~73%, ~74%, ~81% and ~82% for DAHBC27, DAHBC29, DAHBC31 and DAHBC33 NPs at SIF (pH 7.4), respectively. However, the cumulative CUR release from DAHBC27 NPs was ~62% during 2–8 h while DAHBC29, DAHBC31 and DAHBC33 NPs released ~60%, ~56% and ~44%, respectively. The phenomenon could be explained by the pH-responsive and thermo-responsive characteristics in nanoparticles. After incubation for 2 h in strong acidic environment, the nanoparticles were positively charged and hydrophilicity increased, leading to pH-responsive swell. Meanwhile, CUR could be caught by the strongly interaction between pH-responsive swell and thermoresponsive shrinkage. When the pH transformed into near neutral condition, the particles became compact in body temperature (NLCST) and hydrophobicity increased. CUR escaped from nanoparticles by thermoresponsive shrinkage, which was confirmed by the morphology and size in Section 3.2. These results suggested that DAHBC27 NPs with the lowest LCST had the ability to reduce drug leakage (~11%) under
Fig. 9. (A) CLSM images and (B) cellular uptake efficiency of CUR from DAHBC27 NPs and free CUR for 30, 60, 90, 120 min. Cell nuclei were stained with DAPI (blue). The results were calculated as mean ± SD (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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CUR in Caco-2 cells basically raised with the increase of concentration (Fig. 8B). The cellular uptake of CUR increased with an increase in times was observed at DAHBC27 NPs treatment group (Fig. 9A), which were well accordant with the qualitative study (Fig. 9B). The results indicated that the cellular uptake of DAHBC27 NPs by Caco-2 cells was a concentration- and time-dependent process. 3.7. The intestinal absorption by Caco-2 cell monolayer In order to evaluate the intestinal absorption capacity of the DAHBC27 NPs, the present study was undertaken using Caco-2 cell monolayers. The monolayers integrity is an essential condition of the study of drugs transport via the intestinal membrane [41]. TEER is the standard for quantifying the integrity of cell-tight monolayers. As the culture time increased to 10 days, the TEER value of the Caco-2 cell monolayers reached 300 Ω cm2 (Fig. 10A), implying the integrity of the cell monolayer during the intestinal absorption study. The TEER values decreased when the cell monolayers were incubated with DAHBC27 NPs for 2 h, due to the disrupting of tight junctions (TJ). The phenomenon probably mediated by an ionic interaction between positively charged amino groups of chitosan and the negatively charged components on the surface of the epithelial cells [42], which induced a structural readjustment of TJ-associated proteins (such as JAM-1, ZO1), thus promoted the opening of TJ [43,44]. The extents in TEER reduction produced by DAHBC27 NPs were 4-fold higher than that produced by free CUR, indicated the high destruction efficiency of dense monolayer integrity of Caco-2 cells. After removing of the DAHBC27 NPs, the transient decreased TEER values can gradually return to normal levels within a short time (Fig. 10B), indicating that DAHBC27 NPs can reversibly open TJ. The result could be explained by the fact that the activity of reversible tight junctions opening is established to involve the translocation of trans-membrane protein JAM-1 [44].
The Caco-2 cell monolayers in the upper wells were treated with DAHBC27 NPs and free CUR for 2 h. The uptake of DAHBC27 NPs and free CUR by transcellular transport across Caco-2 cell monolayers was visualized using fluorescence images of Transwell chamber polycarbonate membrane (Fig. 10C). The green fluorescence signals of polycarbonate membrane were observed at DAHBC27 NPs and free CUR treatment groups. The intensity of green fluorescence incubated with DAHBC27 NPs for 2 h was weaker than with free CUR. DAHBC27 NPs loaded with CUR could be uptaken by Caco-2 cells and the uptake amount of CUR was 20.9%. While, the amount of CUR absorbed was 25.8% in free CUR treatment group, which did not show significant difference compared with DAHBC27 NPs treatment group (Fig. 10D). Moreover, DAHBC27 NPs opened TJs of Caco-2 cell monolayer to allow the transport of CUR along the paracellular pathway. Thus, the quantification of CUR at the basolateral side was conducted. The amount of CUR in the basolateral chamber was used as a criterion for evaluating the effect of paracellular transport. The amount of CUR loaded into DAHBC27 NPs transported across cell monolayers was 24.6% (Fig. 8D), which was about 10-fold higher (P b 0.05) than that of free CUR treatment group. This could be explained by the decrease in the TEER values of DAHBC27 NPs treated Caco-2 cell monolayers and consequently improved cell monolayer permeability through the opened TJ, thus boosting paracellular transport amount of CUR. The results indicated that DAHBC27 NPs was efficient for enhancing the intestinal absorption of CUR mainly via paracellular pathway and the NPs could be an effective drug delivery system. 4. Conclusions In summary, we have designed a series of thermo and pH dualresponsive DAHBC NPs with different LCST (b37 °C) as an oral anticancer drugs delivery system. DAHBC27 NPs exhibited an excellent stability
Fig. 10. (A) Time course of transepithelial electrical resistance (TEER) of Caco-2 cell monolayers. (B) Time course of TEER of Caco-2 cell monolayers that were incubated with DAHBC27 NPs and free CUR. (C) Fluorescence intensity of the cells thought transcellular transport CUR and (D) the transport amount of CUR thought transcellular and paracellular pathway after 2 h incubation in the presence of DAHBC27 NPs and free CUR. The results were calculated as mean ± SD (n = 3, P b 0.05).
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under simulated gastric environments owing to offset of size change between pH-responsive expansion and thermo-responsive shrinkage. Moreover, the thermo-sensitive shrinkage characteristics of the DAHBC27 NPs facilitated the sustaining release of CUR in the intestinal tract. The cellular uptake of DAHBC27 NPs by Caco-2 cells was a concentration- and time-dependent process. DAHBC27 NPs could be absorbed by Caco-2 cell monolayer via transcellular and paracellular transport and enhanced intestinal absorption efficiency of CUR mainly by paracellular transport pathway. Thus, the study suggested DAHBC27 NPs was a promising drug delivery system for colorectal cancer therapy.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Gastric environment-stable oral nanocarriers for in situ colorectal cancer therapy Ting Wang a, Fangqin Wang a, Mengjie Sun a, Meiping Tian a, Yuzhi Mu a, Xiguang Chen a,b, Ya Liu a,⁎ a b
College of Marine Life Science, Ocean University of China, Qingdao 266003, PR China Qingdao National Laboratory for Marine Science and Technology, Qingdao 266000, PR China
a r t i c l e
i n f o
Article history: Received 11 April 2019 Received in revised form 3 August 2019 Accepted 10 August 2019 Available online 11 August 2019 Keywords: Gastric environment Stable Nanocarriers Oral delivery Colorectal cancer
a b s t r a c t Colorectal cancer (CRC) is a prevalent and fatal cancer. Oral administration provided the potential for in situ treatment of the colorectal cancer. However, drugs couldn't be well-absorbed mainly due to its degradation in the gastric area and poor intestinal permeability. In this study, we synthesized deoxycholic acid and hydroxybutyl decorated chitosan nanoparticles (DAHBC NPs) as oral curcumin (CUR) delivery system for colorectal cancer treatment. DAHBC with lower critical solution temperature (LCST) below 37 °C (27–33 °C) was obtained. DAHBC NPs were correspondingly stable in simulated gastric conditions (pH 1.2, 37 °C), due to the offset of size change between pH-responsive expansion and thermo-responsive shrinkage. In simulated intestinal tract (pH 7.0–7.4, 37 °C), DAHBC NPs exhibited burst release of CUR owing to the onefold effect of thermoresponsive shrinkage. DAHBC27 NPs showed the minimum CUR leakage (~10%) in simulated gastric conditions, because a furthest temperature-sensitive shrinkage caused by the lowest LCST offset the expansion in acid environment. DAHBC27 NPs induced ~10-fold increased (P b 0.05) CUR absorption by paracellular transport pathway, compared to the free CUR. Thus, DAHBC NPs stabilized in the gastric environment may be a promising oral drugs delivery system for effective in situ colorectal cancer therapy. © 2019 Published by Elsevier B.V.
1. Introduction Colorectal cancer (CRC) is among the most commonly cancer and a major source of cancer-related deaths worldwide [1]. Most chemotherapy regimens administered through intravenous injection (i.v.), leading to the side effects and limited therapeutic efficacy [2]. Oral administration is a convenient and safe route that make drug acted directly on the intestinal tract, avoiding the cytotoxicity on normal tissues to consequently minimize the side effects [3,4]. Curcumin (CUR), a natural plant-derived therapeutic agent, demonstrated its antitumor activity in various cancer cell models [5–8]. However, the bioavailability of CUR is restricted by the instability and poor intestinal permeability in the gastrointestinal (GI) tract [9–11]. Nano-engineered drug delivery systems had gained sustaining attention for improving oral bioavailability of anticancer drugs because they provided the possibility of loading hydrophobic compounds [12–14]. To enhance the performance of the oral drug nanocarriers, the protection of drug in gastric environment and the drug release in intestinal conditions were essential [15,16]. Therefore, nanoparticles
⁎ Corresponding author at: College of Marine Life Science, Ocean University of China, 5# Yushan Road, Qingdao 266003, PR China. E-mail address: [email protected] (Y. Liu).
https://doi.org/10.1016/j.ijbiomac.2019.08.088 0141-8130/© 2019 Published by Elsevier B.V.
(NPs) stabilized in gastric environment were desired as oral drug delivery platforms for colorectal cancer therapy. Chitosan (CS) had been commonly used as a material for oral drugs delivery systems [17–19]. Its amino could be protonated under acidic conditions to carry a positive charge in response to pH stimulation, led to morphology expansion of NPs [20,21]. In addition, CS could open the tight junctions between intestinal epithelium and enhance the membrane permeability of drug rely on a translocation of tight junction proteins from the membrane to the cytoskeleton [22]. In our previous research, we prepared thermo-responsive CS nanoparticles, which could shrink at a temperature higher than the lower critical solution temperature (LCST), due to heightened intermolecular hydrogen bonding interactions and enhanced hydrophobic effect, finally resulting in the temperature-sensitive drug release [23,24]. Based on the opposite effects of pH-responsive expansion and thermo-responsive shrinkage in particle size, we hypothesized that thermo and pH dual-responsive nanoparticles with appropriate LCST would be efficient for the protection and the stable existence of drugs in gastric environment and on-demand release in the intestinal tract. Herein, hydrophobic deoxycholic acid (DOCA) and hydrophilic hydroxybutyl decorated chitosan nanoparticles (DAHBC NPs) were prepared as oral CUR delivery system for the in situ treatment of colorectal cancer (Schemes 1, 2). The LCST (b37 °C) of DAHBC NPs could be adjusted by changing the grafted density of hydrophobic DOCA groups.
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Scheme 1. Preparation of DAHBC NPs. (A) The composition of amphiphilic DAHBC. The hydrophilic domain contains hydroxybutyl groups. Deoxycholic acid constitutes the hydrophobic domain. (B) The DAHBC self-assembled and loaded hydrophobic CUR to form DAHBC NPs.
The particle shape, size and in vitro drug release were evaluated in the simulated GI tract. The cellular uptake efficiency of DAHBC NPs by Caco-2 cells in different concentration and time was observed via CLSM. Finally, we also studied intestinal absorption pathway of DAHBC NPs using Caco-2 cell monolayers. Thus, DAHBC NPs might show its potential as oral drug delivery system for colorectal cancer therapy.
2. Materials and methods 2.1. Materials Chitosan (molecular weight 1050 kDa, 85% degree of deacetylation) was purchased from Haili Biotechnology Co. Ltd. (Shandong, China). Deoxycholate (DOCA), 1,2-butene oxide, curcumin (CUR), N-(3-
Scheme 2. The principle of thermo and pH dual-responsive morphology transformation of DAHBC NPs by oral delivery. (A) In the stomach, DAHBC NPs were correspondingly stable due to the balance between pH-responsive expansion and thermo-responsive shrinkage. (B) In the intestine, DAHBC NPs exhibited the drug release activity owing to thermo-responsive shrinkage. (C) Oral delivery of DAHBC NPs in the gastrointestinal tract.
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dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), Nhydroxysuccinimide (NHS) and Dulbecco's modified Eagle's medium (DMEM) were obtained from Sigma-Aldrich (St. Louis, USA). Other reagent chemicals were all of analytical grade. 2.2. Synthesis and characterization of deoxycholic acid and hydroxybutyl modified chitosan (DAHBC) 2.2.1. Synthesis of DAHBC Hydroxybutyl modified chitosan (HBC) was prepared by using 1, 2butene oxide as the etherifying agent and connecting hydroxybutyl groups onto chitosan chain under alkaline conditions [25]. Then, DOCA conjugated to the amino groups of HBC based on a carbodiimide reaction [26]. Briefly, HBC dissolved into distilled water followed by dilution with 3-times methanol at 4 °C. After activated the γ-COOH group of DOCA, different amounts of DOCA were added dropwise into the HBC solution (DOCA:HBC = 1:1, 1.2:1, 1.6:1 and 2:1, w/w). Then the resultant mixtures were stirred for 24 h at room temperature, dialyzed to remove methanol and lyophilized. 2.2.2. 1H NMR spectroscopy 1 H NMR spectra (400 MHz) of CS, HBC and DAHBC in D2O were recorded on a Bruker ARX 400 MHz spectrometer. The chemical structures of CS, HBC and DAHBC were proved by NMR measurements. 2.2.3. Elemental analysis Elemental analysis was carried out to characterize the degree of substitution (DS) of DOCA groups and hydroxybutyl groups which defined as the group number per 100 sugar residues of CS. The DS of hydroxybutyl groups (DSHB) and DOCA (DSDOCA) were calculated as follows: DSHB ¼
H CS H%− =H HB =MHB 100 Mcs
DSDOCA ¼
H CS DSHB HD %− −HHB M cs M HB H DOCA 100 MDOCA
ð1Þ
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Table 1 The LCST of DAHBC in PBS (0.02 M, pH 7.4) (n = 3). Samples
DAHBC33
DAHBC31
DAHBC29
DAHBC27
DOCA:HBC (w/w) DS of DOCA (%) LCST (°C)
1:1 7.1 ± 0.4 33.2 ± 0.4
1.2:1 7.5 ± 0.2 31.1 ± 0.2
1.6:1 7.9 ± 0.3 29.4 ± 0.3
2:1 8.2 ± 0.1 27.3 ± 0.1
MDOCA were the molecular weight of CS units, hydroxybutyl groups and DOCA residues, respectively. 2.2.4. Lower critical solution temperature (LCST) The DAHBC was dissolved into PBS (0.02 M, pH 7.4). After equilibrated at the special temperatures for 10 min, the transmittance of DAHBC solutions at 500 nm was measured using a UV–vis spectrophotometer (UV-1200 MAPADA, China). The LCST of polymer solutions was defined as the temperature when the transmittance producing a 50% decrease in optical transmittance [27]. 2.3. The size variation of DAHBC NPs at simulating gastrointestinal environments DAHBC NPs were prepared using a dialysis method [24]. Briefly, DAHBC aqueous dispersions in DMSO were added dropwise into PBS (0.02 M, pH 7.4) under sustaining stir. The resultant mixtures dialyzed against PBS for 24 h to remove DMSO. The size variation of DAHBC NPs at simulated gastrointestinal environments was investigated via transmission electron microscope (TEM, JEM-1200EX JEOL100 Ltd., Japan) and dynamic laser light scattering technique (DLS) (Zetasizer ZEN3600, Malvern 95 Instruments Ltd, UK). The NPs dispersions (1 mg/mL) were prepared in PBS (0.02 M, pH 7.4) solution. After being incubated at specified conditions, the morphology and hydrodynamic size of DAHBC NPs were then measured. The data was analyzed with Zetasizer software (version 7.1). 2.4. Biocompatibility
ð2Þ
where H% and HD% were the percentages of H in HBC and DAHBC, respectively; HCS, HHB, HDOCA were the amounts of H in CS units, hydroxybutyl groups and DOCA residues, respectively; MCS, MHB,
2.4.1. Cytotoxicity assay The in vitro cytotoxicity of DAHBC NPs was evaluated using Caco-2 cell lines. Caco-2 cells were seeded in 96-well culture plates (1 × 104 cells/well) and cultured in DMEM supplemented with 10% fetal bovine serum at 37 °C in 5% CO2 for 12 h. Then cells were treated with DAHBC NPs at different concentrations (0.5–0.0313 mg/mL). After
Fig. 1. The 1H NMR spectra and structure of CS, HBC, DAHBC.
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where ODsample and ODcontrol were the absorbance of the test samples and the control group, respectively. Meanwhile, the cells were seeded in 24-well plates and cultured for 12 h. Then the cells were incubated with DAHBC NPs (0.5 mg/mL) for 24 and 48 h. After stained with calcein-AM, the samples were observed by confocal laser scanning microscope (CLSM, LSM510, Zeiss Ltd., Germany). 2.4.2. Hemolysis test The different concentration of DAHBC NPs suspensions (0.5–0.0313 mg/mL) with 20 μL rabbit blood suspension were incubated for 1 h at 37 °C. Subsequently, the mixtures were centrifugated at 2000 rpm for 10 min, and the absorbance of supernatants at 545 nm was detected using a Microplate Reader (PerkinElmer, Boston, MA, USA) [29]. Negative control was the saline and blood suspension, whereas the positive control was the distilled water and blood suspension. Hemolytic rate (HR) was calculated as follows: HRð%Þ ¼ Fig. 2. The transmittance profiles of DAHBC in PBS (0.02 M, pH 7.4) from 18 °C to 40 °C. The results were calculated as mean ± SD (n = 3).
24 h and 48 h, 10 μL CCK-8 was added to each well and cultured for 4 h [28]. The absorbance of each well was measured using a Microplate reader (PerkinElmer, Boston, MA, USA) at 450 nm. Cell viability (CV) was calculated as follows: CV ð%Þ ¼
ODsample 100 ODcontrol
ð3Þ
ODS −ODNC 100 ODPC −ODNC
ð4Þ
where ODS, ODNC and ODPC were the absorbance of tested sample, negative control and the positive control, respectively. 2.4.3. Protein adsorption test Nanoparticles suspensions (1 mg/mL) were incubated with Bovine Serum Albumin (BSA) solution (500 μg/mL) for 0.5 h at 37 °C and ultracentrifuged at 12,000 rpm for 0.5 h at 4 °C. Then the NPs were washed twice by distilled water. The adsorbed protein was desorbed from the DAHBC NPs surface in PBS solution containing 1% SDS by sonication and measured (λ = 595 nm) with Coomassie Brilliant Blue G-
Fig. 3. Cell viability (A) and fluorescence photomicrographs (B) of DAHBC NPs in Caco-2 cells for 24 h and 48 h. The results were calculated as mean ± SD (n = 5).
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250. The SDS-PAGE was performed and visualized by staining with Coomassie Brilliant Blue R-250 to detect the washed-off protein. 2.5. Drug loading efficiency CUR-loaded nanoparticles were prepared by dissolving CUR together with DAHBC (CUR: DAHBC = 0.25:1, w/w) in DMSO before dialysis. The freshly prepared DAHBC NPs were collected by ultracentrifugation at 12,000 rpm for 30 min. The free CUR in supernatants was detected by a Microplate Reader (PerkinElmer, Boston, MA, USA) at Ex/Em 425/475 nm, whose concentration was determined by a standard calibration curve [30]. The encapsulation efficiency (EE) and drug loading efficiency (LE) were calculated as follows: EEð%Þ ¼
Ct−Cf 100 Ct
ð5Þ
LEð%Þ ¼
Ct−Cf 100 Cn
ð6Þ
where Ct was the total amount of CUR; Cf was the amount of free CUR; Cn was the weight of CUR-loaded nanoparticles. 2.6. Drug release under simulating gastrointestinal environments The drug release studies of CUR from DAHBC NPs were carried out at simulated gastrointestinal environments by a dialysis method. The free
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CUR solution and CUR-loaded DAHBC NPs (1 mL) dispersions were transferred into a dialysis tube, immersed in 49 mL different simulated fluids and then gently stirred at 37 °C for 2 h. A total of 8 h of dialysis was performed in the gastrointestinal mimetic fluid, including simulated gastric fluid (SGF, pH 1.2, 2.0 g NaCl, 7 mL HCl and 3.2 g pepsin), simulated duodenum fluid (pH 6.0, obtained by mixing SGF and SIF), simulated proximal ileum fluid (pH 7.0, obtained by adjusting the pH of SIF) and simulated intestinal fluid (SIF, pH 7.4, 6.8 g KH2PO4, 0.2 N NaOH (190 mL) and 10.0 g trypsin) [31]. At predetermined times, 5 mL of the sample was removed for analysis and 5 mL of fresh medium was then added to the tube to maintain a constant volume. All the measurements were performed in the dark. The CUR concentrations were quantified using a standard CUR calibration curve, which determined by fluorescence microplate reader at Ex/Em 425/475 nm. 2.7. Cellular uptake assays Caco-2 cells were cultured in six-well culture plate for 24 h. The culture media was then replaced with D-Hanks (pH 7.4) and preincubated at 37 °C for 30 min. After equilibration, free CUR or DAHBC NPs solutions were added into each well at an equivalent dose of CUR in D-Hanks (pH 7.4). At predetermined times (i.e. 30, 60, 90, or 120 min), the cells were washed three times with D-Hanks (pH 7.4) and the nuclei was stained with 4, 6-diamidino-2-phenylindolyl hydrochloride (DAPI, Sigma, 1 μg/mL) in D-Hanks. Cover slides were then washed three times with D-Hanks and observed by confocal laser scanning microscope (CLSM, LSM510, Zeiss Ltd., Germany).
Fig. 4. (A) Hemolysis rate and (B) hemolysis test of DAHBC NPs at different concentrations (0.5–0.0313 mg/mL). PC represented positive control. NC represented negative control. The results were calculated as mean ± SD (n = 3). (C) Protein adsorption rate of DAHBC NPs (1 mg/mL). (D) SDS-PAGE electrophoresis gel stained by Coomassie Brilliant Blue. Lane 0: 500 μg/mL BSA; Lane 1–4: BSA adsorbed on DAHBC27, DAHBC29, DAHBC31, DAHBC33 NPs, respectively.
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To quantify the uptake of CUR by Caco-2 cells, Caco-2 cells were cultured in 96-well culture plates and incubated for 24 h. Then the culture medium was replaced by D-Hanks (pH 7.4) and preincubated at 37 °C for 30 min. After equilibration, free CUR or DAHBC NPs solutions were added into each well at an equivalent dose of CUR in D-Hanks (pH 7.4). At predetermined time, the cells were washed with ice-cold PBS (pH 7.4) and lysed with 0.2 mL of 0.5% Triton X-100 in 0.2 M NaOH solution [32]. Then the CUR concentrations were determined by fluorescence microplate reader at Ex/Em 425/475 nm. Cellular uptake efficiency (UE) was calculated as follows:
UEð%Þ ¼
Cu 100 Ci
ð7Þ
where Cu was the uptake concentrations of CUR, and Ci was the initial concentrations of CUR.
2.8. Intestinal absorption by Caco-2 cell monolayer 2.8.1. The transepithelial electrical resistance (TEER) measurement The TEER value was measured with a Millicell-Electrical Resistance System (Millipore Corp., Bedford, MA) in order to evaluate and determine the monolayer integrity of the Caco-2 cell monolayer in the Transwell plates [33]. The grown cell monolayers were incubated with free CUR or DAHBC NPs solutions containing equivalent doses of CUR. Before measuring the values of each well, the cell monolayers were washed twice with pre-warmed D-Hanks (pH 7.4).
Fig. 5. TEM images (A) and the size (B) of DAHBC NPs at RT/pH 7.4, RT/pH 2.0, 37 °C/pH 7.4 and 37 °C/pH 2.0 after incubation 2 h, 1 mg/mL. The results were calculated as mean ± SD (n = 3).
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2.8.2. Transcellular transport Caco-2 cells were grown into a differentiated monolayer for 21 days in the Transwell plate. The monolayer was washed twice with prewarmed D-Hanks (37 °C, pH 7.4, apical 0.5 mL and basolateral 1.5 mL) after the culture medium was withdrawn. Then the D-Hanks in the apical side were replaced with 0.5 mL free CUR or DAHBC NPs solutions containing equivalent doses of CUR. After incubated for 2 h, the polycarbonate membrane was collected, washed three times with ice-cold PBS (pH 7.4) and lysed with 0.2 mL of 0.5% Triton X-100 in 0.2 M NaOH solution. Then the CUR concentrations were determined by fluorescence microplate reader at Ex/Em 425/475 nm. 2.8.3. Paracellular transport Caco-2 cells were seeded in Transwell plate for 21 days. The monolayer was washed twice with pre-warmed D-Hanks (37 °C, pH 7.4, apical 0.5 mL and basolateral 1.5 mL). Then the D-Hanks in the apical side were replaced with 0.5 mL free CUR or DAHBC NPs solutions containing equivalent doses of CUR. The sample solution (0.5 mL) in the basolateral chamber was collected after the chamber was incubated for 2 h, and measured the fluorescence intensity by fluorescence microplate reader at Ex/Em 425/475 nm.
2.9. Statistical analysis Experiments were performed in triplicate and presented as mean ± standard deviation (SD). Student's t-test was used to analyzing the data. The level of significance was set at probabilities of *P b 0.05. 3. Results and discussion 3.1. Characterization of DAHBC 3.1.1. 1H NMR spectra of DAHBC DAHBC was prepared by conjugating hydroxybutyl and DOCA groups to CS. The chemical structure of DAHBC was characterized with 1 H NMR (Fig. 1). HBC was achieved, as evidenced by peaks arising at 0.6–1.7 ppm (peaks 4, 5), compared with the 1H NMR spectrum of CS [34]. The signals of DAHBC at 2.0–3.6 ppm (peaks 1, 2, 3) were appeared due to the protons of –CH3 groups at position 18, 19 and 20 of the steroid skeleton of DOCA, respectively [35]. The results of 1H NMR spectra indicated that the hydrophobic molecule of DOCA and the hydrophilic molecule of hydroxybutyl were successfully grafted onto CS.
Fig. 7. The cumulative release profiles of CUR from DAHBC NPs and free CUR at simulated GI fluid. Each release curve is calculated as mean ± SD (n = 3).
3.1.2. Elemental analysis of the DAHBC The DS of DOCA and hydroxybutyl groups were evaluated via percentages of each element (C, H, O and N) and known structural formula. The DS of hydroxybutyl groups was calculated to be 104.7 ± 2.8 per 100 anhydroglucose units of CS. With the weight ratios of DOCA to HBC increasing from1:1 to 2:1, the DS of DOCA increased from 7.1% to 8.2% (Table 1), indicating that a high proportion of DOCA was favorable to obtain DAHBC with high DS.
3.1.3. The LCST of the DAHBC Variations in the transmittance of DAHBC with temperature were evaluated using UV–vis spectroscopy. As the temperature increased from 18 °C to 40 °C, the transmittance of DAHBC was decreased (Fig. 2). The LCST was defined as the temperature when the transmittance produced a 50% decrease in optical transmittance. The reduced extent in DAHBC transmittance was increased with the weight ratio of hydrophobic DOCA to hydrophilic HBC decreased, and in consequence, LCST showed an ascending trend, which may be due to the increased number of H-bonds between the DAHBC polymers [23]. According to the LCST (summarized in Table 1), four types of DAHBC were named as DAHBC27, DAHBC29, DAHBC31 and DAHBC33, respectively. Therefore, the control of the LCST of the polymer could be manipulated by tuning of the hydrophobicity and grafted moieties.
Fig. 6. Drug encapsulation efficiency (A) and loading capacity (B) of DAHBC NPs. The results were calculated as mean ± SD (n = 3).
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3.2. Biocompatibility 3.2.1. Cytotoxicity To evaluate the cytotoxicity of the DAHBC NPs, a range of concentrations of DAHBC NPs from 0.5 to 0.0313 mg/mL towards Caco-2 cells were evaluated via CCK-8 assay. The cell viability of Caco-2 cells treaded with four DAHBC NPs was N80% at all concentrations during 24 h and 48 h (Fig. 3A). The morphology micrographs of cells further illustrated cytocompatibility of DAHBC NPs (Fig. 3B). The most cells of all DAHBC NPs (0.5 mg/mL) treatment groups showed uniformly spread state as same as control group at 24 and 48 h. These results indicated DAHBC NPs did not influence on cell viability and had satisfactory cytocompatibility. 3.2.2. Blood compatibility The hemolysis test and protein adsorption capacity with DAHBC NPs were detected as important index of the blood compatibility. Less than 5% hemolysis was regarded as a nontoxic effect level in this blood compatibility experiment [36]. The hemolysis rate (HR%) of four DAHBC NPs at different concentrations (0.5–0.0313 mg/mL) was all below 5% after incubating for 1 h (Fig. 4A). Meanwhile, unconspicuous erythrocyte hemolysis was observed in the presence of DAHBC NPs (Fig. 4B), which was considered as non-hemolysis. The adsorption of plasma proteins
occurred when blood contacted with a foreign material surface [37]. Protein adsorption rate of DAHBC NPs (1 mg/mL) was all below 10% (Fig. 4C). In addition, DAHBC NPs did not perform apparent bands (lane 1–4) compared with the BSA group (lane 0) (Fig. 4D), which indicated that the DAHBC NPs caused no adverse effects on the blood components. These results exhibited the DAHBC NPs had good blood compatibility. 3.3. The size variation of DAHBC NPs at simulated gastrointestinal environments The size variation of DAHBC NPs at simulated gastrointestinal environments was investigated to examine the stability (Fig. 5). DAHBC NPs had an approximately spherical shape at room temperature (pH 7.4). The diameters of DAHBC27 NPs (~61 nm) were smaller than that of the DAHBC29 (~75 nm), DAHBC31 (~109 nm) and DAHBC33 NPs (~151 nm), due to increased ratio of hydrophobic DOCA groups. When the pH transformed into pH 2.0, a considerable swelling of DAHBC27 NPs (with hydrodynamic size of ~185 nm) was clearly observed. Meanwhile, the spherical structure of DAHBC29 NPs was gradually loose, DAHBC31, DAHBC33 NPs almost disintegrated. The pHresponsive particle morphology changes were clearly observed, which could be caused by the amino groups on CS (pKa: ~6.5) were protonized
Fig. 8. (A) CLSM images of Caco-2 cells that were incubated with DAHBC27 NPs and free CUR for 2 h. Cell nuclei were stained with DAPI (blue). (B) Cellular uptake efficiency of CUR from DAHBC27 NPs and free CUR for 2 h with different concentration: 15, 30, 60 μg/mL. The results were calculated as mean ± SD (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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in the strongly acidic condition, reduced the chain–chain interaction and increased the hydrophilic performance with polymer chain [38]. At pH 2.0/37 °C (simulating gastric conditions), DAHBC27 NPs still showed a spherical shape and the hydrodynamic sizes increased to ~105 nm. But DAHBC29, DAHBC31 and DAHBC33 NPs transformed into more loose structure with the diameter of ~174 nm, ~202 nm and ~357 nm, respectively. The lowest LCST of DAHBC27 NPs led to the furthest offset of size change between the shrinkage caused by temperature (NLCST) and the expansion in acid environment, thus DAHBC27 NPs were stable at simulating gastric conditions. At pH 7.4/37 °C (simulating intestinal conditions), DAHBC27 NPs began to shrink and the particle sizes was ~40 nm. The decrease of DAHBC NPs size was due to the reinforced hydrogen bond within the polymer chain at 37 °C (NLCST), resulting in the enhanced hydrophobic effect of hydroxybutyl moieties [39]. The size change rules of DAHBC29, DAHBC31 and DAHBC33 NPs showed the same descending trend. With the increase of LCST, the degree of shrinkage decreased, owing to reduced hydrophobic DOCA groups. Therefore, DAHBC27 NPs were suitable for oral drug delivery through the gastric environments to the intestine.
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simulated gastric conditions and to release the loaded CUR (~73%) at simulated intestinal tract. Based on these results, the DAHBC27 NPs were selected for further studies. 3.6. Cellular uptake of DAHBC NPs The cellular uptake capacity of DAHBC27 NPs was studied by Caco-2 cells after incubation for 2 h. CUR with a green fluorescence can be used as probe to monitor the cellular uptake effect. The green fluorescence signal was detected after incubated with DAHBC27 NPs and free CUR (Fig. 8A), indicated that the CUR was effectively uptake by Caco-2 cells, this also implied the uptake activity of Caco-2 cells treated with DAHBC27 NPs was as high as free CUR. The cell uptake efficiency of CUR was ~10.2% at 15 μg/mL and then increased to ~23.7% at 60 μg/mL at DAHBC27 NPs treatment group, demonstrated that the amounts of
3.4. Drug loading efficiency To investigate drug-loading efficiency of DAHBC NPs, CUR was chosen as a model drug encapsulated into nanoparticles. The encapsulation efficiency of DAHBC NPs decreased from ~59% to ~52% as the LCST of DAHBC changed from 27 °C to 33 °C (Fig. 6A). A similar pattern was observed for CUR-loading efficiency of DAHBC NPs, which decreased from ~14% to ~12% (Fig. 6B), may attributed to the reduced amount of hydrophobic DOCA groups. DAHBC27 NPs showed the maximum encapsulation efficiency (~59%) and loading capacity (~14%). These results indicated that this variation tendency of encapsulation efficiency and loading efficiency was opposite to the LCST. 3.5. CUR release in vitro The release behavior of CUR from DAHBC NPs and free CUR was evaluated at simulated GI fluid. A sustained release of CUR from DAHBC NPs was observed over a period of 8 h (Fig. 7). In the first 2 h of incubation at SIF (pH 1.2), the cumulative release amount of CUR from DAHBC27, DAHBC29, DAHBC31 and DAHBC33 NPs was ~11%, ~15%, ~25% and ~38%, respectively. However, the free CUR group indicated the abundant release (N95%) in 2 h (Fig. 7). The phenomenon illustrated that the prepared DAHBC NPs was capable of protecting the CUR and alleviating the rapid metabolism of CUR under simulated gastric conditions [40]. DAHBC29, DAHBC31 and DAHBC33 NPs exhibited a faster release behavior compared to DAHBC27 NPs at SIF (pH 1.2), suggesting that higher LCST resulted in more drug release. Within 2–8 h of incubation at simulated duodenum fluid (pH 6.0), simulated proximal ileum fluid (pH 7.0) and SIF (pH 7.4), four DAHBC NPs displayed similar release tendency and the sustained CUR release curve was observed. The ultimate CUR release profiles were ~73%, ~74%, ~81% and ~82% for DAHBC27, DAHBC29, DAHBC31 and DAHBC33 NPs at SIF (pH 7.4), respectively. However, the cumulative CUR release from DAHBC27 NPs was ~62% during 2–8 h while DAHBC29, DAHBC31 and DAHBC33 NPs released ~60%, ~56% and ~44%, respectively. The phenomenon could be explained by the pH-responsive and thermo-responsive characteristics in nanoparticles. After incubation for 2 h in strong acidic environment, the nanoparticles were positively charged and hydrophilicity increased, leading to pH-responsive swell. Meanwhile, CUR could be caught by the strongly interaction between pH-responsive swell and thermoresponsive shrinkage. When the pH transformed into near neutral condition, the particles became compact in body temperature (NLCST) and hydrophobicity increased. CUR escaped from nanoparticles by thermoresponsive shrinkage, which was confirmed by the morphology and size in Section 3.2. These results suggested that DAHBC27 NPs with the lowest LCST had the ability to reduce drug leakage (~11%) under
Fig. 9. (A) CLSM images and (B) cellular uptake efficiency of CUR from DAHBC27 NPs and free CUR for 30, 60, 90, 120 min. Cell nuclei were stained with DAPI (blue). The results were calculated as mean ± SD (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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CUR in Caco-2 cells basically raised with the increase of concentration (Fig. 8B). The cellular uptake of CUR increased with an increase in times was observed at DAHBC27 NPs treatment group (Fig. 9A), which were well accordant with the qualitative study (Fig. 9B). The results indicated that the cellular uptake of DAHBC27 NPs by Caco-2 cells was a concentration- and time-dependent process. 3.7. The intestinal absorption by Caco-2 cell monolayer In order to evaluate the intestinal absorption capacity of the DAHBC27 NPs, the present study was undertaken using Caco-2 cell monolayers. The monolayers integrity is an essential condition of the study of drugs transport via the intestinal membrane [41]. TEER is the standard for quantifying the integrity of cell-tight monolayers. As the culture time increased to 10 days, the TEER value of the Caco-2 cell monolayers reached 300 Ω cm2 (Fig. 10A), implying the integrity of the cell monolayer during the intestinal absorption study. The TEER values decreased when the cell monolayers were incubated with DAHBC27 NPs for 2 h, due to the disrupting of tight junctions (TJ). The phenomenon probably mediated by an ionic interaction between positively charged amino groups of chitosan and the negatively charged components on the surface of the epithelial cells [42], which induced a structural readjustment of TJ-associated proteins (such as JAM-1, ZO1), thus promoted the opening of TJ [43,44]. The extents in TEER reduction produced by DAHBC27 NPs were 4-fold higher than that produced by free CUR, indicated the high destruction efficiency of dense monolayer integrity of Caco-2 cells. After removing of the DAHBC27 NPs, the transient decreased TEER values can gradually return to normal levels within a short time (Fig. 10B), indicating that DAHBC27 NPs can reversibly open TJ. The result could be explained by the fact that the activity of reversible tight junctions opening is established to involve the translocation of trans-membrane protein JAM-1 [44].
The Caco-2 cell monolayers in the upper wells were treated with DAHBC27 NPs and free CUR for 2 h. The uptake of DAHBC27 NPs and free CUR by transcellular transport across Caco-2 cell monolayers was visualized using fluorescence images of Transwell chamber polycarbonate membrane (Fig. 10C). The green fluorescence signals of polycarbonate membrane were observed at DAHBC27 NPs and free CUR treatment groups. The intensity of green fluorescence incubated with DAHBC27 NPs for 2 h was weaker than with free CUR. DAHBC27 NPs loaded with CUR could be uptaken by Caco-2 cells and the uptake amount of CUR was 20.9%. While, the amount of CUR absorbed was 25.8% in free CUR treatment group, which did not show significant difference compared with DAHBC27 NPs treatment group (Fig. 10D). Moreover, DAHBC27 NPs opened TJs of Caco-2 cell monolayer to allow the transport of CUR along the paracellular pathway. Thus, the quantification of CUR at the basolateral side was conducted. The amount of CUR in the basolateral chamber was used as a criterion for evaluating the effect of paracellular transport. The amount of CUR loaded into DAHBC27 NPs transported across cell monolayers was 24.6% (Fig. 8D), which was about 10-fold higher (P b 0.05) than that of free CUR treatment group. This could be explained by the decrease in the TEER values of DAHBC27 NPs treated Caco-2 cell monolayers and consequently improved cell monolayer permeability through the opened TJ, thus boosting paracellular transport amount of CUR. The results indicated that DAHBC27 NPs was efficient for enhancing the intestinal absorption of CUR mainly via paracellular pathway and the NPs could be an effective drug delivery system. 4. Conclusions In summary, we have designed a series of thermo and pH dualresponsive DAHBC NPs with different LCST (b37 °C) as an oral anticancer drugs delivery system. DAHBC27 NPs exhibited an excellent stability
Fig. 10. (A) Time course of transepithelial electrical resistance (TEER) of Caco-2 cell monolayers. (B) Time course of TEER of Caco-2 cell monolayers that were incubated with DAHBC27 NPs and free CUR. (C) Fluorescence intensity of the cells thought transcellular transport CUR and (D) the transport amount of CUR thought transcellular and paracellular pathway after 2 h incubation in the presence of DAHBC27 NPs and free CUR. The results were calculated as mean ± SD (n = 3, P b 0.05).
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under simulated gastric environments owing to offset of size change between pH-responsive expansion and thermo-responsive shrinkage. Moreover, the thermo-sensitive shrinkage characteristics of the DAHBC27 NPs facilitated the sustaining release of CUR in the intestinal tract. The cellular uptake of DAHBC27 NPs by Caco-2 cells was a concentration- and time-dependent process. DAHBC27 NPs could be absorbed by Caco-2 cell monolayer via transcellular and paracellular transport and enhanced intestinal absorption efficiency of CUR mainly by paracellular transport pathway. Thus, the study suggested DAHBC27 NPs was a promising drug delivery system for colorectal cancer therapy.
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