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Hybrid liposomes composed of amphiphilic chitosan and phospholipid: Preparation, stability and bioavailability as a carrier for curcumin
Accepted Manuscript Title: Hybrid Liposomes Composed of Amphiphilic Chitosan and Phospholipid: Preparation, Stability and Bioavailability as a Carrier for Curcumin Author: Shengfeng Peng Liqiang Zou Weilin Liu Ziling Li Wei Liu Xiuting Hu Xing Chen Chengmei Liu PII: DOI: Reference:
S0144-8617(16)31114-6 http://dx.doi.org/doi:10.1016/j.carbpol.2016.09.060 CARP 11587
To appear in: Received date: Revised date: Accepted date:
30-6-2016 11-8-2016 16-9-2016
Please cite this article as: Peng, Shengfeng., Zou, Liqiang., Liu, Weilin., Li, Ziling., Liu, Wei., Hu, Xiuting., Chen, Xing., & Liu, Chengmei., Hybrid Liposomes Composed of Amphiphilic Chitosan and Phospholipid: Preparation, Stability and Bioavailability as a Carrier for Curcumin.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.09.060 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Hybrid Liposomes Composed of Amphiphilic Chitosan and Phospholipid: Preparation, Stability and Bioavailability as a Carrier for Curcumin
Author names and affiliations: Shengfeng Peng a, Liqiang Zou a, Weilin Liu b, Ziling Li ac, Wei Liu *a，Xiuting Hu a, Xing Chena, Chengmei Liu a.
a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang
330047, Jiangxi, PR China b
College of Food and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, Zhejiang, PR China
c
School of Life Science, Jiangxi Science and Technology Normal University, Nanchang,
330013, Jiangxi, PR China
*Corresponding author: Tel: +86 791 88305872(8106), Fax: +86 791 88334509, E-mail address: [email protected]
Highlights
Hybrid liposomes composed of amphiphilic chitosan and phospholipid were prepared.
The hybrid liposomes exhibited excellent ionic and thermal stability.
Curcumin hybrid liposomes showed improved stability and sustained release.
Bioavailability of curcumin was improved when loaded in hybrid liposomes. 1
ABSTRACT: Hybrid liposomes, composed of amphiphilic chitosan and phospholipid, were prepared and used to evaluate the effect of amphiphilic polymers on the properties of liposomes. Successful preparation of the hybrid liposomes was confirmed using physicochemical characteristics, including morphology, particle size and zeta potential. Physical stability studies (exposure to solutions of increasing ionic strength and heat treatment) indicated that the hybrid liposomes had better ionic stability than amphiphilic chitosan-based polymeric liposomes and higher thermal stability than traditional phospholipid liposomes. Curcumin was then encapsulated in the hybrid liposomes. Compared with phospholipid liposomes, the hybrid liposomes displayed better storage stability and more sustained curcumin release. Cellular uptake experiments showed that the hybrid liposomes significantly increased the bioavailability of curcumin. The study highlights the potential of well-designed stable hybrid liposomes that increase the stability and bioavailability of lipophilic bioactive, such as curcumin.
Keywords: Hybrid liposomes; Amphiphilic Chitosan; Curcumin; Cellular Uptake; Physical Stability.
1. Introduction Phospholipid liposomes (PLLs), which were spherical vesicles composed of a phospholipid bilayer membrane and an aqueous core, have been extensively investigated as a delivery system for both hydrophilic and hydrophobic components (Shin, Chung, Kim, Joung & Park, 2013). The practical application of PLLs has, however, been limited by their poor physical and chemical stability during preparation and storage. One of the strategy to improve 2
stability is to replace the phospholipid membrane with a membrane composed of amphiphilic polymers, which can self-assemble into polymeric liposomes (PMLs) (Discher & Eisenberg, 2002). Chitosan is widely used in biomedical, pharmaceutical and food technology because of its lack of nontoxicity, high biodegradability and biocompatibility. Several studies have highlighted the potential of chitosan as a stability-enhancing agent material for coating liposomal surfaces (Karewicz et al., 2013; Shin, Chung, Kim, Joung & Park, 2013). Amphiphilic chitosan derivatives are soluble in both organic and aqueous solvents and are able to self-assemble under appropriate conditions. PMLs based on amphiphilic chitosan derivatives have been studied as deliver systems for both genes and natural products, such as salidroside (Liang, Li, Chang, Duan & Li, 2013; Liang et al., 2008; Peng et al., 2014). In a previous study, we prepared dextran sulfate-coated PMLs based on N, N-dimethylhexadecyl carboxymethyl chitosan (DCMC) and investigated their properties (Zou, Peng, Liu, Chen & Liu, 2015). In the present study, we have prepared hybrid liposomes (HBLs), composed of amphiphilic chitosan and phospholipid since we speculated that hydrophobic force between amphiphilic chitosan and phospholipid would stabilize liposomal membrane. Curcumin is a hydrophobic polyphenol that has numerous health benefits, including antioxidant, antibacterial, anti-inflammatory and anti-proliferative properties (Khanna, 1999). Curcumin, however, has poor bioavailability due to its poor solubility, poor absorption, rapid metabolism, and rapid systemic elimination (Anand, Kunnumakkara, Newman & Aggarwal, 2007). A number of attempts have been made to improve the solubility, stability and bioavailability of curcumin, the most promising of which was to encapsulate the curcumin in vectors, such as emulsions (Lin et al., 2014), liposomes (Hasan et al., 2016; Karewicz et al., 3
2013), micelles (Sarika, James, Kumar, Raj & Kumary, 2015), nanoparticles (Sarika & James, 2016), hydrogels (Koop, de Freitas, de Souza, Savi-Jr & Silveira, 2015) and excipient emulsions (Zou, Liu, Liu, Xiao & McClements, 2015a; Zou, Liu, Liu, Xiao & McClements, 2015b). In our earlier study, we showed that, although PLLs improved the solubility and physical stability of curcumin, they resulted in lower cellular uptake than free curcumin (dissolved in DMSO) and had poor storage stability. (Chen et al., 2015). For this reason, we used curcumin to evaluate the properties of HBLs as a carrier system. HBLs composed of amphiphilic chitosan and phospholipid were prepared by a combination of the traditional dry film rehydration method and dynamic high-pressure microfluidization. PLLs and PMLs were also prepared as controls. Physicochemical characteristics of HBLs including morphology, particle size, size distribution and zeta potential, were measured and the stability of HBLs towards solutions of different ionic strength and heat treatment were evaluated by assessing variations in particle size. Storage stability of the curcumin-loaded hybrid liposomes (Cur-HBLs) was evaluated together with in vitro release and cellular uptake of curcumin.
2. Materials and methods 2.1. Chemicals Carboxymethyl chitosan (molecular weight 10 kDa, 85% degree of deacetylation, and 80% degree of carboxylation) purchased from Zhejiang Aoxing Biotechnology (Taizhou, China) were used without further purification. Epoxychloropane was obtained from Aladdin Chemistry Co., Ltd. (Shanghai, China). N, N-Dimethylhexadecylamine was purchased from Aoke 4
Industrial Co., Ltd. (Shanghai, China). Curcumin (98%) was purchased from Aladdin industrial corporation
(Shanghai
China).
Phospholipid
S75
(soybean
lecithin
at
71%
of
phosphatidylcholine and 10% of phosphatidylethanolamine) was provided by Lipoid GmbH (Ludwigshafen, Germany). Cholesterol, Fluorescein 5(6)-isothiocyanate (FITC), 1,6-Diphenyl1,3,5-hexatriene (DPH) and Nile Red were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Fetal bovine serum was obtained from Shanghai ExCell Biology.Inc. Ethanol, Tween 80, Methanol, and other reagent chemicals were all of analytical grade.
2.2. Synthesis and characterization of DCMC DCMC (N, N-dimethylhexadecyl carboxymethyl chitosan) was synthesized and characterized by FT-IR as described in our previous study (Zou, Peng, Liu, Chen & Liu, 2015). To further confirm the structure and substitution degree (SD) of DCMC, 1H NMR spectra of CMC and DCMC were obtained on a spectrometer (Bruker DPX300, Bruker Corporation, Karlsruhe, Germany) with D2O as the solvent. SD of DCMCs was calculated using the following equation (Li, Liu, Huang & Xue, 2011). SD = 3AH/30AC
(1)
AH and AC was corresponded to the area of methylene protons on hexadecyl (δ = 1.22) and the C3–C5 protons (δ= 3.25–3.60) on chitosan backbone.
2.3. Preparation of PLLs, HBLs, PMLs, Cur-PLLs and Cur-HBLs PLLs, HBLs and PMLs were prepared according to our earlier study (Liu, Ye, Liu, Liu & Singh, 2012; Zou, Peng, Liu, Chen & Liu, 2015). HBLs were prepared by dissolving 5
phospholipid and DCMC (5:1 w/w) in absolute ethanol, and subsequently evaporating the solution under vacuum. The resultant thin film was hydrated with super pure water at 45°C (1 mL per 12 mg total lipid). After complete lipid hydration and formation of liposomes, the coarse dispersion treated with a microfluidizer (M-110EH30, Microfluidic Corp., Newton, MA, USA) at 120 MPa for two cycles. PLLs and PMLs were also prepared as described above. In addition, under the similar procedures, curcumin-loaded liposomes were prepared by dissolving curcumin in the absolute ethanol with the other materials (Chen et al., 2015). The hydrate medium of blank liposomes and curcumin-loaded liposomes were different. Blank liposomes (PLLs, HBLs and PMLs) were hydrated with super pure water to avoid extra ion influences because they were prepared to evaluate their ionic stability. While curcumin-loaded liposomes (Cur-PLLs and Cur-HBLs) were hydrated with Phosphate-buffered saline (PBS; 0.05M pH 6.5). PBS could maintain curcumin solutions in acid conditions (pH 6.5) and prevent degradation of curcumin in alkaline pH conditions during preparation and storage. Fig. 1 showed the schematics of samples formation and the photos of Cur-PLLs and Cur-HBLs.
2.4. Characterizations of PLLs, HBLs and PMLs 2.4.1 AFM study Atomic Force Microscope (AFM) was utilized to analyze the morphology of the liposomes according to our previous method (Zou et al., 2014). Briefly, samples were prepared by drying a drop of 500-fold diluted solutions on mica plate and the images were captured using the AFM (Agilent 5500, Agilent Technologies, Santa Clara, CA, USA) with a silicon cantilever of force constant of 0.58 N m1− in tapping mode at room temperature. 6
2.4.2 Size distribution and zeta potential The size distribution of PLLs, PMLs and HBLs were measured at 25°C using a dynamic laser light scattering (DLS) instrument (Nicomp 380 ZLS, Santa Barbara, CA, USA) according to our previous method (Liu, Liu, Liu, Li & Liu, 2013). The intensity was detected at an angle of 90°. The zeta potential of PLLs, PMLs and HBLs were measured by Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK). Each individual zeta potential measurement was calculated from the mean of at least 10 readings from each sample. Samples were diluted 10fold with water in the size distribution and zeta potential measurement. 2.4.3 Membrane fluidity The membrane fluidity of PLLs, HBLs and PMLs were evaluated according to Suga et.al (Suga, Akizaki & Umakoshi, 2016). Briefly, 200 μL of liposomes suspensions (12 mg/mL of lipid) and 5 μL of DPH solution (2 μM in ethanol) were diluted in 4.8 mL of PBS buffer (pH 7.4). The fluorescence polarization of DPH (Ex = 360 nm, Em = 430 nm) was measured by F7000 fluorescence spectrophotometer (Hitachi High-Technologies, Tokyo, Japan) after incubation at 25°C for 30 min. The polarization (P) of DPH was then calculated by using equation (2): 𝑃 = (𝐼0,0 − G𝐼0,90 )/(𝐼0,0 + G𝐼0,90 )
𝐺 = 𝐼90,0 /𝐼90,90
(2)
Where I0,0 and I0,90 are the fluorescence intensities of the emitted light polarized parallel and vertical to the exciting light, respectively, and G is the grating correction coefficient. Since polarization is inversely proportional to fluidity, membrane fluidity is expressed as 1/P. 2.4.4 Confocal Laser Scanning Microscopy Since DCMC and phospholipid both could form liposomes, confocal laser scanning 7
microscopy (CLSM) was employed to confirm whether a uniform HBLs or two kind of liposomes (PLLs and PMLs) were obtained. Nile Red and FITC were used as fluorescent probe of lipid and DCMC, respectively. A 1.4 mg/mL FITC stock solution was made in methanol and diluted 200-fold in the crude HBLs solution. It was then incubated in the dark at ambient temperature for 30 min to enable the fluorescent labeling of the DCMC (Yin, Fei, Cui, Tang & Yin, 2007). Stock solution of Nile Red in ethanol (2.5 mg/mL) was injected to the phospholipid in the darkness, and the finial concentration of Nile Red was the label 50 µg/mL (Banerjee, Sen, Pal & Guha, 2012). These samples were dialyzed against double distilled water overnight to remove free fluorescent label. Finally, these samples were observed by CLSM (LSM710, Zeiss, Germany).
2.5. Physical stability of PLLs, HBLs and PMLs Our previous study found PMLs immediately precipitated after mixed with simulated intestinal fluid. Therefore, the influence of ionic kind (Cl–, SO32– and PO43–) on the stability of PLLs, PMLs and HBLs were determined. Stock solution of sodium chloride (1 M) and sodium sulfate (1 M) were prepared by dissolve sodium chloride or sodium sulfate in double distilled water. The stock solutions were adjusted to pH 6.5 using either 0.1 M hydrochloric acid or 0.1 M sodium hydroxide. PBS stock solution (0.5 M, pH 6.5) was composed of 0.1575 mM Na2HPO4 and 0.3425 mM NaH2PO4. Samples were treated with Cl– (25, 50, 75, 100, 150, 200 mM), SO32– (10, 20, 30, 40, 50, 75, 100 mM) and PO43– (10, 20, 30, 40, 50, 75, 100 mM) by diluted with ion stock solutions and double distilled water. The size of the samples was measured after 0.5h equilibrium at room temperature. 8
A heat treatment (121°C for 20min) was conducted to evaluate the thermal stability of PLLs and HBLs in the presence of different PBS concentrations (10, 25, 50, 75, 100, 125, 150 mM). The thermal stability of PMLs was not determined since it was unstable in the present of PBS.
2.6. Characterizations of Cur-PLLs and Cur-HBLs 2.6.1 Size distribution and zeta potential Size distribution and zeta potential of Cur-PLLs and Cur-HBLs were determined according to the protocol as described in section 2.4.2. 2.6.2 Loading efficiency The loading efficiency (LE) was measured by UV-Vis on an ultraviolet-visible spectroscopy (Pgeneral T6, China) according to Huang et.al (Huang et al., 2014). Curcuminloaded liposomes were centrifuged at 3000 rpm (1248 × g) for 10 min and 20 μL of supernatant were taken and dissolved in 3.8 mL of anhydrous ethanol. Absorbance at 420 nm was measured by UV-Vis and the concentration of loaded curcumin was calculated from the calibration curve. Standard curcumin curve was obtained through curcumin ethanol solutions in a concentration gradient (y = 0.1436x + 0.005, R2 = 0.9999), where y and x correspond to absorbance and curcumin concentration (μg/mL), respectively. The LE was obtained from the equation (3): 𝐿𝐸 = (loaded curcumin⁄total amount of lipids) × 100%
(3)
2.7. Storage stability of Cur-PLLs and Cur-HBLs In order to evaluate the storage stability of Cur-PLLs and Cur-HBLs, samples were stored 9
at 4°C and 25°C, respectively, for 1 month in sealed and dark condition. The average diameter and leakage ratio of curcumin were determined every 5 days. The average diameter was determined by DLS as described in the previous Section 2.4.2. The leakage ratio of curcumin was determined by measuring the loading efficiency (LE) during the storage time compared to the LE before storage (as described in Section 2.6.2), then calculated according to the following Eq. (4). Leakage ratio = (1 − 𝐿𝐸𝑑𝑢𝑟𝑖𝑛𝑔 𝑠𝑡𝑜𝑟𝑎𝑔𝑒 ⁄𝐿𝐸𝑏𝑒𝑓𝑜𝑟𝑒 𝑠𝑡𝑜𝑟𝑎𝑔𝑒 ) × 100%
(4)
2.8. In vitro release of Cur-PLLs and Cur-HBLs In vitro release profile of curcumin from curcumin-loaded liposomes were done by direct dispersion method as explained in literature (Mohanty & Sahoo, 2010). Briefly, samples were diluted 10 folds with PBS (0.05 M, pH 7.4) and solution were divided in Eppendorf tubes. The tubes were incubated in a shaker at 150 rpm at 37°C. At definite time intervals, one set of tubes were taken out and centrifuged at 3000 rpm for 10 min to pelletize the released curcumin. The encapsulated curcumin (supernatant) was diluted 10 fold with ethanol. Finally, amount of curcumin loaded was quantified spectrophotometerically at a wavelength of 420 nm.
2.9. Cellular uptake assays of Cur-PLLs and Cur-HBLs The cellular uptake of liposomal formulation was evaluated in Caco-2 cells by qualitative and quantitative methods according to our previous study (Chen et al., 2015). Caco-2 cells were seeded in 12-well plates at a density of 5 × 104 cells per well and incubated for 24 h. The cells were then treated with Cur-PLLs and Cur-HBLs with a final curcumin concentration of 4 μg/mL. 10
At predetermined time intervals of 0.5, 1, 2 and 4 h, the medium was removed and the cells were washed with PBS twice. Curcumin uptake was visualized under an inverted phase contrast microscope (Nikon Eclipse Ti-U; Nikon Instruments, Kanagawa, Japan) using autofluorescence (excitation wavelength: 480 nm, emission wavelength: 510 nm). To further quantify the difference in cellular uptake, the medium was removed after incubation with Cur-PLLs and Cur-HBLs for 0.5, 1, 2 and 4 h. Cells were then washed twice with PBS, trypsinized, centrifuged and collected in 1 mL PBS. The cell suspension was injected into an Accuri C6 flow cytometer (Accuri Cytometers, Inc., Ann Arbor, MI, USA) in the FL1 channel (488 excitation, blue laser, 530 ± 15 nm, FITC/GFP) to determine the fluorescence intensity (Yallapu, Jaggi & Chauhan, 2010).
2.10. Statistical analysis Statistical analysis was performed using ORIGIN 8.0 (OriginLab Inc., Northampton, MA, USA). The data are expressed as means ± standard deviations of at least three independent determinations on one sample for each time period, and were analyzed by one-way analysis of variance. Statistical significance was established at P < 0.05.
3. Results and discussion 3.1. Synthesis and structural characterization of DCMC The preparation of DCMC is illustrated in Fig. 2A. The structure of DCMC was confirmed by 1H NMR spectroscopy. The spectrum of chitosan had a single peak at δ 1.82 (H-2), multiple peaks at δ 3.25–3.60 (H-3 to H-5), δ 3.11 (H-6) and a small single peak at δ 4.44 (H-1) (Fig. 11
2B). The signals at δ 0.82, 0.99−1.46, 3.11 and 3.83 were attributed to protons in the long alkyl chains of the quaternary ammonium substituents, indicating successful preparation of DCMC. Using equation (1), the SD value of DCMC was calculated to be 0.815.
3.2. Preparation and characterization of PLLs, HBLs and PMLs AFM images of PLLs, HBLs and PMLs (Fig. 3A, 3B and 3C, respectively) indicated that uniform liposomes had been successfully prepared in each case. The PLLs, HBLs and PMLs were all well-distributed and spherical, with smooth surfaces. The average diameter of PLLs was 93.9 ± 2.7 nm, with a polydispersity index (PDI) of 0.382 ± 0.051 and the average diameter of PMLs was 93.9 ± 2.3 nm, with a PDI of 0.242 ± 0.031 (Fig. 4A). HBLs had a smaller diameter (78.2 ± 1.7 nm) than PLLs and PMLs and a PDI of 0.292 ± 0.041. DCMC acts as a polymeric surfactant in the liposomal structure and thus reduce the particle size of HBLs. The zeta potential of PLLs, PMLs and HBLs are shown in Fig. 4B. HBLs showed a change in zeta potential from -16.3 ± 1.7 mV to +26.3 ± 2.3 mV, which was due to the positive charged quaternary ammonium group of DCMC. The zeta potential of HBLs was also much lower than that of PMLs (+58.3 ± 4.3 mV), because the positively charged DCMC moiety was incorporated into the phospholipid membrane.
Liposomal fluidity refers to the relative motional freedom of the lipid molecules in the membrane bilayer (Arora, Byrem, Nair & Strasburg, 2000). The fluidity of phospholipid membranes reflects the order and dynamics of the phospholipid alkyl chains in the bilayer and 12
regulates liposomes permeation (Hasan et al., 2014). The membrane fluidities (1/P) of PLLs, PMLs and HBLs were 6.10 ± 0.11, 5.41 ± 0.12 and 3.54 ± 0.10, respectively. The membrane fluidities of PLLs are significantly (P < 0.01) higher than those of PMLs because of the higher molecular mobility of the micromolecule (phospholipid) compared with the macromolecule (DCMC). HBLs have significantly (P < 0.01) lower membrane fluidity than PLLs, likely, because hydrophobic interaction between DCMC and phospholipid restrict the motion of the polar phospholipid head-groups. Considering the distribution of DCMC in the phospholipid membrane, the lateral diffusion of phospholipid could also be restricted by steric hindrance of the DCMC backbones. CLSM images are shown in Fig. 4. The green fluorescence of FITC indicates DCMC and the red fluorescence of Nile Red indicates phospholipid. Red and green fluorescence were both observed in HBLs (Fig. 4C and Fig. 4D, respectively). Both red and green fluorescence were observed in every single HBL particle (Fig. 4E). This result, together with the size and zeta potential results, indicates that uniform hybrid liposomes, composed of DCMC and phospholipid, had been successfully prepared.
3.3. Physical stability of PLLs, HBLs and PMLs The diameter of liposomes in the presence of Cl–, SO42– and PO43– ions (Fig. 5A, 5B and 5C, respectively) was used to investigate stability under different ionic conditions. When PLLs and HBLs were treated with different ions, the particle size initially decreased and then remained constant with increasing salt concentration. PMLs showed a slight decrease in size at 13
low ion concentrations and then increased in size along with increasing ion concentrations. The decrease in particle size at low ionic strength might be explained by the osmotic shrinking of the vesicles (Hupfeld, Moen, Ausbacher, Haas & Brandl, 2010). When treated with 200 mM Cl–, the diameter of PMLs increased slightly from 93.9 nm to 119.3 nm. This effect could be attributed to the ability of the counter-ions in NaCl to screen electrostatic repulsive forces acting between the particles, resulting in particles fusion. The diameter of PMLs increased dramatically, however, when treated with 50 mM SO42– or PO43– (from 93.9 nm to 1949.9 nm or to 2564.3 nm, respectively). The dramatic increase in particle size indicated that large aggregates had been formed. The aggregation could be caused by association of the quaternary ammonium group of DCMC, induced by polyvalent anions (SO42– and PO43–). A single PO43– ion could bond simultaneously with three quaternary ammonium groups on DCMC and thus change the well-organized liposomal structure. HBLs were stable in the present of Cl–, SO42– and PO43–, perhaps because of the relatively strong hydrophobic bond interaction between DCMC and the phospholipid membrane. These results demonstrate that incorporation into HBLs improves the ionic stability of DCMC. PMLs were found to be extremely sensitive to ionic strength and precipitated when diluted with PBS or cell culture medium (Fig. 5E).Other properties of PMLs were, therefore, not evaluated. The effect of the ionic strength of the medium on thermal behavior of PLLs and HBLs is shown in Fig. 5D. PLLs and HBLs were both stable after heated at 121°C for 20 min in pure water but a gradual increase in average diameter of both particles was observed in PBS as the concentration increased from 10 to 150 mM. The diameter of PLLs and HBLs increased to 1011.1 nm and 159.3 nm, respectively, after heated at 121°C for 20 min in the presence of 14
50 mM PBS. HBLs thus showed better thermal stability than PLLs in the presence of PBS. PLLs were stable after heated at 121°C for 20 min in pure water, perhaps because electrostatic repulsive forces prevented PLLs from fusing and aggregating (Liu et al., 2016). The diameter of PLLs increased, however, after heated at 121°C for 20 min in the presence of PBS. It has been reported that a rapid decrease in the liposomal negative zeta potential observed on increasing ionic strength (Sabin, Prieto, Ruso, Hidalgo-Alvarez & Sarmiento, 2006). The reduction in zeta potential indicates a reduction in electrostatic repulsive forces between PLLs, resulting in reduced thermal stability. Compared with phospholipid membranes, DCMC increases the hydrophilicity of the hybrid membranes because of the carboxymethyl groups attached to DCMC. Steric forces between HBLs could also improve their thermal stability.
3.4. Characterization of Cur-PLLs and Cur-HBLs The maximum LE of Cur-PLLs and Cur-HBLs were determined by adding extra amount of curcumin to the phospholipid ethanol solutions (Section 2.3). The maximum LE of Cur-PLLs and Cur-HBLs were 6.52% ± 0.44% and 8.08% ± 0.18%, respectively. Cur-PLLs and CurHBLs were, however, extremely unstable when loaded with very high amounts of curcumin. The LE of PLLs and HBLs decreased gradually to 4.21% and 5.35%, respectively, after storage at 4°C for 15 days. This phenomenon was attributed to increased phospholipid bilayer fluidity, caused by molecular interaction between curcumin and liposome (Liu, Liu, Zhu, Gan & Le, 2015). Curcumin (0.5 mg/ml) was, therefore, loaded into both PLLs and HBLs to provide a consistent LE (4.2%). Both PLLs and HBLs were relatively stable when stored in a refrigerator 15
for about one month. The average diameters of Cur-PLLs and Cur-HBLs were 51.5 ± 1.6 nm and 54.1 ± 2.4 nm, respectively. The average diameters of PLLs and HBLs in the presence of 50 mM PBS were 48.7 ± 1.6 nm and 46.5 ± 1.2 nm, respectively (Fig. 5C). The diameter of curcumin-loaded liposomes was slightly larger than blank liposomes, which was attributed to the incorporation of curcumin into the liposome bilayer. The zeta potential of Cur-PLLs and Cur-HBLs were 16.1 ± 1.8 mV and +26.7 ± 2.0 mV, respectively. The incorporation of curcumin in liposomes did not, therefore, change the zeta potential of the liposomes.
3.5. Storage stability of Cur-PLLs and Cur-HBLs Liposomes are thermodynamically unstable, and have a high tendency to degrade, aggregate and fuse, leading to leakage of entrapped compounds during storage (Tan et al., 2014). To investigate the storage stability of curcumin-loaded liposomes, the leakage ratio and particle sizes of Cur-PLLs and Cur-HBLs were monitored during storage at 4°C and 25°C. The initial average diameters of Cur-PLLs and Cur-HBLs were 51.1 nm and 54.1 nm, respectively (Fig. 6). When stored at 4°C, the mean diameter and LE of curcumin did not change noticeably after 30 days. When stored for 30 days at 25°C, however, the mean diameter of Cur-PLLs increased dramatically from 51.1 nm to 109.9 nm, and leakage ratio of curcumin from PLLs was 35.3%. The increase in diameter of Cur-HBLs (from 54.1 nm to 66.2 nm) was much smaller than that of Cur-PLLs, and only 17.1% of curcumin leaked from HBLs. Cur-HBLs exhibit improved storage stability compared with Cur-PLLs. In summary, Cur-PLLs and Cur-HBLs have favorable storage stability with no curcumin 16
leakage and little change in particle size at 4°C since decomposition of the liposomes are inhibited at low temperature (Li et al., 2009). The stability of Cur-PLLs, in terms of aggregation, fusion and leakage, was much poorer than that of Cur-HBLs at 25°C. Crommelin (Crommelin, 1984) reported that incorporation of charged components into the liposome membranes reduced the tendency to aggregate because of increased electrostatic repulsion between the particles. Suspensions with larger absolute value are more stable, perhaps because the charged particles repel each other and overcome the natural tendency to aggregate (Heurtault, Saulnier, Pech, Proust & Benoit, 2003; Zhou et al., 2014). In the present study, the absolute value of zeta potential for PLLs and HBLs were 16.3 mV and 26.3 mV, respectively, suggesting that CurHBLs have greater physical stability than Cur-PLLs. Lower membrane fluidity could also reduce membrane fusion between HBLs, contributing to stability.
3.6. In vitro release of Cur-PLLs and Cur-HBLs The extremely low solubility of curcumin in water and its instability at physiological pH are major obstacles to its delivery to cancerous tissue. It is, therefore, important to confirm the release of curcumin from liposomes under physiological conditions. The in vitro release profiles of Cur-PLLs and Cur-HBLs are shown in Fig. 7. The diameter and zeta potential of the particles did not changed after incubation at 37°C for 24 h. The release of curcumin from both Cur-PLLs and Cur-HBLs displayed a fast initial burst within the first 12 h, followed by a slow and sustained release. Approximately 36.8% and 41.6% of curcumin was released from PLLs at 12 and 24 h, respectively. HBLs more effectively entrap the curcumin and only 13.1% and 15.5% curcumin was released from HBLs at 12 and 24 h, respectively. Drug release from liposomes 17
is a complex process which depends on drug physicochemical properties, carrier integrity and diffusion and dissolution between the phospholipid bilayer and the drug. Calvagno found that the release of cargo from liposomes is determined by two factors. The first of these is the strength of the interaction between liposomal membrane and cargo, i.e. stronger interaction lead to less desorption and hence the burst effect. The second factor is the fluidity of the bilayer, i.e. more fluid bilayers allow greater and more rapid cargo leakage from liposomes (Grazia Calvagno et al., 2007). As described above, DCMC distributes in the membrane of HBLs, with lipophilic moieties inserting into the phospholipid bilayer and thus decreasing their permeability. The reduced bilayer fluidity would reduce diffusion of curcumin from the bilayers and ultimately reduce leakage of curcumin from Cur-HBLs.
3.7. Cellular uptake of Cur-PLLs and Cur-HBLs Caco-2 cells, which are epithelial cells derived from human colon adenocarcinoma, were used to evaluate the cellular uptake of Cur-PLLs and Cur-HBLs (Huang & Kuo, 2016). Fluorescence microscopy images showed that the fluorescence intensity of the Caco-2 cells increased and then remained unchanged after incubation for 2 h (Fig. 8A). The fluorescence intensity of Caco-2 cells treated with Cur-HBLs was noticeably higher than that of those treated with Cur-PLLs, indicating that DCMC significantly improved the cellular uptake of liposomes. Cellular uptake was next quantified by flow cytometry. Cellular uptake rates of Cur-PLLs were 25.2%, 30.6%, 37.7% and 40.6% and uptake rate of Cur-HBLs were 31.8%, 49.5%, 64.1% and 66.2% after incubation for 0.5, 1, 2 and 4 h, respectively (Fig. 8B, C). Both fluorescence microscopy and flow cytometry thus demonstrate a higher cellular uptake rate for Cur-HBLs 18
compared with Cur-PLLs. The transfer of drugs encapsulated in liposomes into cells relies mainly on endocytosis (Un, Sakai-Kato, Oshima, Kawanishi & Okuda, 2012) and absorption of liposomes onto the cell membrane is the first and critical step. Compared with PLLs, HBLs have increased positive charge, and decreased hydrophobicity. This should lead to higher absorption activity and higher cellular uptake. The surface carboxyl groups originating from DCMC, which decrease the surface the surface hydrophobicity, could also facilitate internalization of HBLs and improve cellular uptake (Xiao, Nian & Huang, 2015). The curcumin released from PLLs and HBLs would also influence drug uptake by cells. The burst release of curcumin from PLLs (Section 3.4) resulted in decreased curcumin bioavailability since free curcumin is insoluble in aqueous solutions and thus not available for uptake by cells.
4. Conclusion Novel HBLs containing both DCMC and phospholipid have been successfully prepared. The particles were spherical and positively charged, and had a narrow size distribution, and low membrane fluidity. HBLs were stable in the presence of different ions and when heated at 121°C for 20 min. When loaded with curcumin, Cur-HBLs had good storage stability, and provided sustained release with good cellular uptake. The present study indicates that HBLs are a promising delivery system for curcumin. To further evaluate the bioavailability of HBLs, more model drugs will be encapsulated in this novel carrier.
19
Acknowledgments We appreciate the financial support by the National Science Foundation of China (No. 21266021), Technological expertise and academic leaders training plan of Jiangxi Province and the Youth Fund of Department of Education of Jiangxi Province (N0.150089).
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Figure captions
Fig. 1. Schematics representation of phospholipid liposomes (PLLs), hybrid liposomes (HBLs), polymeric liposomes (PMLs), curcumin-loaded phospholipid liposomes (Cur-PLLs) and curcumin-loaded hybrid liposomes (Cur-HBLs) and photos of Cur-PLLs and Cur-HBLs.
Fig. 2. The synthesis process of DCMC (A) and 1H NMR spectra of DCMC (B).
Fig. 3. AFM images of (A) PLLs, (B) HBLs and (C) PMLs.
Fig. 4. (A) Size distribution and (B) zeta potential of PLLs, HBLs and PMLs. CLSM images of HBLs: (C) FITC channel, (D) Nile Red channel, (E) Merged image of FITC and Nile Red.
Fig. 5. Effect of (A) Cl-, (B) SO42– and (C) PO43– on the average diameter of PLLs, HBLs and PMLs. (D) Effect of heat treatment on the average diameter of PLLs and HBLs in the presence of different concentration of PBS. (E) The photography of PLLs, HBLs and PMLs after treated with 50 mM NaCl, 50 mM Na2SO4 and 50 mM PBS, and heated in the presence of 100 mM PBS.
Fig. 6. The changes of average diameter (A) and leakage ratio of (B) Cur-PLLs and Cur-HBLs during storage at 4 and 25°C.
27
Fig. 7. In vitro release profile of curcumin from Cur-PLLs and Cur-HBLs.
Fig. 8. Fluorescence microscope (A), Flow cytometric (B) and Uptaking ratio (C) analysis of Caco-2 cells treated with 4 μg/mL Cur-PLLs and Cur-HBLs after a defined period of time.
28
Fig. 1. Schematics representation of phospholipid liposomes (PLLs), hybrid liposomes (HBLs), polymeric liposomes (PMLs), curcumin-loaded phospholipid liposomes (Cur-PLLs) and curcumin-loaded hybrid liposomes (Cur-HBLs) and photos of Cur-PLLs and Cur-HBLs.
29
Fig. 2. The synthesis process of DCMC (A) and 1H NMR spectra of DCMC (B).
30
Fig. 3. AFM images of (A) PLLs, (B) HBLs and (C) PMLs.
31
Fig. 4. (A) Size distribution and (B) zeta potential of PLLs, HBLs and PMLs. CLSM images of HBLs: (C) FITC channel, (D) Nile Red channel, (E) Merged image of FITC and Nile Red.
32
Fig. 5. Effect of (A) Cl-, (B) SO42– and (C) PO43– on the average diameter of PLLs, HBLs and PMLs. (D) Effect of heat treatment on the average diameter of PLLs and HBLs in the presence of different concentration of PBS. (E) The photography of PLLs, HBLs and PMLs after treated with 50 mM NaCl, 50 mM Na2SO4 and 50 mM PBS, and heated in the presence of 100 mM PBS.
33
Fig. 6. The changes of average diameter (A) and leakage ratio of (B) Cur-PLLs and CurHBLs during storage at 4 and 25 °C.
34
Fig. 7. In vitro release profile of curcumin from Cur-PLLs and Cur-HBLs.
35
Fig. 8. Fluorescence microscope (A), Flow cytometric (B) and uptaking ratio (C) analysis of Caco-2 cells treated with 4 μg/mL Cur-PLLs and Cur-HBLs after a defined period of time.
36
S0144-8617(16)31114-6 http://dx.doi.org/doi:10.1016/j.carbpol.2016.09.060 CARP 11587
To appear in: Received date: Revised date: Accepted date:
30-6-2016 11-8-2016 16-9-2016
Please cite this article as: Peng, Shengfeng., Zou, Liqiang., Liu, Weilin., Li, Ziling., Liu, Wei., Hu, Xiuting., Chen, Xing., & Liu, Chengmei., Hybrid Liposomes Composed of Amphiphilic Chitosan and Phospholipid: Preparation, Stability and Bioavailability as a Carrier for Curcumin.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.09.060 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Hybrid Liposomes Composed of Amphiphilic Chitosan and Phospholipid: Preparation, Stability and Bioavailability as a Carrier for Curcumin
Author names and affiliations: Shengfeng Peng a, Liqiang Zou a, Weilin Liu b, Ziling Li ac, Wei Liu *a，Xiuting Hu a, Xing Chena, Chengmei Liu a.
a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang
330047, Jiangxi, PR China b
College of Food and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, Zhejiang, PR China
c
School of Life Science, Jiangxi Science and Technology Normal University, Nanchang,
330013, Jiangxi, PR China
*Corresponding author: Tel: +86 791 88305872(8106), Fax: +86 791 88334509, E-mail address: [email protected]
Highlights
Hybrid liposomes composed of amphiphilic chitosan and phospholipid were prepared.
The hybrid liposomes exhibited excellent ionic and thermal stability.
Curcumin hybrid liposomes showed improved stability and sustained release.
Bioavailability of curcumin was improved when loaded in hybrid liposomes. 1
ABSTRACT: Hybrid liposomes, composed of amphiphilic chitosan and phospholipid, were prepared and used to evaluate the effect of amphiphilic polymers on the properties of liposomes. Successful preparation of the hybrid liposomes was confirmed using physicochemical characteristics, including morphology, particle size and zeta potential. Physical stability studies (exposure to solutions of increasing ionic strength and heat treatment) indicated that the hybrid liposomes had better ionic stability than amphiphilic chitosan-based polymeric liposomes and higher thermal stability than traditional phospholipid liposomes. Curcumin was then encapsulated in the hybrid liposomes. Compared with phospholipid liposomes, the hybrid liposomes displayed better storage stability and more sustained curcumin release. Cellular uptake experiments showed that the hybrid liposomes significantly increased the bioavailability of curcumin. The study highlights the potential of well-designed stable hybrid liposomes that increase the stability and bioavailability of lipophilic bioactive, such as curcumin.
Keywords: Hybrid liposomes; Amphiphilic Chitosan; Curcumin; Cellular Uptake; Physical Stability.
1. Introduction Phospholipid liposomes (PLLs), which were spherical vesicles composed of a phospholipid bilayer membrane and an aqueous core, have been extensively investigated as a delivery system for both hydrophilic and hydrophobic components (Shin, Chung, Kim, Joung & Park, 2013). The practical application of PLLs has, however, been limited by their poor physical and chemical stability during preparation and storage. One of the strategy to improve 2
stability is to replace the phospholipid membrane with a membrane composed of amphiphilic polymers, which can self-assemble into polymeric liposomes (PMLs) (Discher & Eisenberg, 2002). Chitosan is widely used in biomedical, pharmaceutical and food technology because of its lack of nontoxicity, high biodegradability and biocompatibility. Several studies have highlighted the potential of chitosan as a stability-enhancing agent material for coating liposomal surfaces (Karewicz et al., 2013; Shin, Chung, Kim, Joung & Park, 2013). Amphiphilic chitosan derivatives are soluble in both organic and aqueous solvents and are able to self-assemble under appropriate conditions. PMLs based on amphiphilic chitosan derivatives have been studied as deliver systems for both genes and natural products, such as salidroside (Liang, Li, Chang, Duan & Li, 2013; Liang et al., 2008; Peng et al., 2014). In a previous study, we prepared dextran sulfate-coated PMLs based on N, N-dimethylhexadecyl carboxymethyl chitosan (DCMC) and investigated their properties (Zou, Peng, Liu, Chen & Liu, 2015). In the present study, we have prepared hybrid liposomes (HBLs), composed of amphiphilic chitosan and phospholipid since we speculated that hydrophobic force between amphiphilic chitosan and phospholipid would stabilize liposomal membrane. Curcumin is a hydrophobic polyphenol that has numerous health benefits, including antioxidant, antibacterial, anti-inflammatory and anti-proliferative properties (Khanna, 1999). Curcumin, however, has poor bioavailability due to its poor solubility, poor absorption, rapid metabolism, and rapid systemic elimination (Anand, Kunnumakkara, Newman & Aggarwal, 2007). A number of attempts have been made to improve the solubility, stability and bioavailability of curcumin, the most promising of which was to encapsulate the curcumin in vectors, such as emulsions (Lin et al., 2014), liposomes (Hasan et al., 2016; Karewicz et al., 3
2013), micelles (Sarika, James, Kumar, Raj & Kumary, 2015), nanoparticles (Sarika & James, 2016), hydrogels (Koop, de Freitas, de Souza, Savi-Jr & Silveira, 2015) and excipient emulsions (Zou, Liu, Liu, Xiao & McClements, 2015a; Zou, Liu, Liu, Xiao & McClements, 2015b). In our earlier study, we showed that, although PLLs improved the solubility and physical stability of curcumin, they resulted in lower cellular uptake than free curcumin (dissolved in DMSO) and had poor storage stability. (Chen et al., 2015). For this reason, we used curcumin to evaluate the properties of HBLs as a carrier system. HBLs composed of amphiphilic chitosan and phospholipid were prepared by a combination of the traditional dry film rehydration method and dynamic high-pressure microfluidization. PLLs and PMLs were also prepared as controls. Physicochemical characteristics of HBLs including morphology, particle size, size distribution and zeta potential, were measured and the stability of HBLs towards solutions of different ionic strength and heat treatment were evaluated by assessing variations in particle size. Storage stability of the curcumin-loaded hybrid liposomes (Cur-HBLs) was evaluated together with in vitro release and cellular uptake of curcumin.
2. Materials and methods 2.1. Chemicals Carboxymethyl chitosan (molecular weight 10 kDa, 85% degree of deacetylation, and 80% degree of carboxylation) purchased from Zhejiang Aoxing Biotechnology (Taizhou, China) were used without further purification. Epoxychloropane was obtained from Aladdin Chemistry Co., Ltd. (Shanghai, China). N, N-Dimethylhexadecylamine was purchased from Aoke 4
Industrial Co., Ltd. (Shanghai, China). Curcumin (98%) was purchased from Aladdin industrial corporation
(Shanghai
China).
Phospholipid
S75
(soybean
lecithin
at
71%
of
phosphatidylcholine and 10% of phosphatidylethanolamine) was provided by Lipoid GmbH (Ludwigshafen, Germany). Cholesterol, Fluorescein 5(6)-isothiocyanate (FITC), 1,6-Diphenyl1,3,5-hexatriene (DPH) and Nile Red were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Fetal bovine serum was obtained from Shanghai ExCell Biology.Inc. Ethanol, Tween 80, Methanol, and other reagent chemicals were all of analytical grade.
2.2. Synthesis and characterization of DCMC DCMC (N, N-dimethylhexadecyl carboxymethyl chitosan) was synthesized and characterized by FT-IR as described in our previous study (Zou, Peng, Liu, Chen & Liu, 2015). To further confirm the structure and substitution degree (SD) of DCMC, 1H NMR spectra of CMC and DCMC were obtained on a spectrometer (Bruker DPX300, Bruker Corporation, Karlsruhe, Germany) with D2O as the solvent. SD of DCMCs was calculated using the following equation (Li, Liu, Huang & Xue, 2011). SD = 3AH/30AC
(1)
AH and AC was corresponded to the area of methylene protons on hexadecyl (δ = 1.22) and the C3–C5 protons (δ= 3.25–3.60) on chitosan backbone.
2.3. Preparation of PLLs, HBLs, PMLs, Cur-PLLs and Cur-HBLs PLLs, HBLs and PMLs were prepared according to our earlier study (Liu, Ye, Liu, Liu & Singh, 2012; Zou, Peng, Liu, Chen & Liu, 2015). HBLs were prepared by dissolving 5
phospholipid and DCMC (5:1 w/w) in absolute ethanol, and subsequently evaporating the solution under vacuum. The resultant thin film was hydrated with super pure water at 45°C (1 mL per 12 mg total lipid). After complete lipid hydration and formation of liposomes, the coarse dispersion treated with a microfluidizer (M-110EH30, Microfluidic Corp., Newton, MA, USA) at 120 MPa for two cycles. PLLs and PMLs were also prepared as described above. In addition, under the similar procedures, curcumin-loaded liposomes were prepared by dissolving curcumin in the absolute ethanol with the other materials (Chen et al., 2015). The hydrate medium of blank liposomes and curcumin-loaded liposomes were different. Blank liposomes (PLLs, HBLs and PMLs) were hydrated with super pure water to avoid extra ion influences because they were prepared to evaluate their ionic stability. While curcumin-loaded liposomes (Cur-PLLs and Cur-HBLs) were hydrated with Phosphate-buffered saline (PBS; 0.05M pH 6.5). PBS could maintain curcumin solutions in acid conditions (pH 6.5) and prevent degradation of curcumin in alkaline pH conditions during preparation and storage. Fig. 1 showed the schematics of samples formation and the photos of Cur-PLLs and Cur-HBLs.
2.4. Characterizations of PLLs, HBLs and PMLs 2.4.1 AFM study Atomic Force Microscope (AFM) was utilized to analyze the morphology of the liposomes according to our previous method (Zou et al., 2014). Briefly, samples were prepared by drying a drop of 500-fold diluted solutions on mica plate and the images were captured using the AFM (Agilent 5500, Agilent Technologies, Santa Clara, CA, USA) with a silicon cantilever of force constant of 0.58 N m1− in tapping mode at room temperature. 6
2.4.2 Size distribution and zeta potential The size distribution of PLLs, PMLs and HBLs were measured at 25°C using a dynamic laser light scattering (DLS) instrument (Nicomp 380 ZLS, Santa Barbara, CA, USA) according to our previous method (Liu, Liu, Liu, Li & Liu, 2013). The intensity was detected at an angle of 90°. The zeta potential of PLLs, PMLs and HBLs were measured by Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK). Each individual zeta potential measurement was calculated from the mean of at least 10 readings from each sample. Samples were diluted 10fold with water in the size distribution and zeta potential measurement. 2.4.3 Membrane fluidity The membrane fluidity of PLLs, HBLs and PMLs were evaluated according to Suga et.al (Suga, Akizaki & Umakoshi, 2016). Briefly, 200 μL of liposomes suspensions (12 mg/mL of lipid) and 5 μL of DPH solution (2 μM in ethanol) were diluted in 4.8 mL of PBS buffer (pH 7.4). The fluorescence polarization of DPH (Ex = 360 nm, Em = 430 nm) was measured by F7000 fluorescence spectrophotometer (Hitachi High-Technologies, Tokyo, Japan) after incubation at 25°C for 30 min. The polarization (P) of DPH was then calculated by using equation (2): 𝑃 = (𝐼0,0 − G𝐼0,90 )/(𝐼0,0 + G𝐼0,90 )
𝐺 = 𝐼90,0 /𝐼90,90
(2)
Where I0,0 and I0,90 are the fluorescence intensities of the emitted light polarized parallel and vertical to the exciting light, respectively, and G is the grating correction coefficient. Since polarization is inversely proportional to fluidity, membrane fluidity is expressed as 1/P. 2.4.4 Confocal Laser Scanning Microscopy Since DCMC and phospholipid both could form liposomes, confocal laser scanning 7
microscopy (CLSM) was employed to confirm whether a uniform HBLs or two kind of liposomes (PLLs and PMLs) were obtained. Nile Red and FITC were used as fluorescent probe of lipid and DCMC, respectively. A 1.4 mg/mL FITC stock solution was made in methanol and diluted 200-fold in the crude HBLs solution. It was then incubated in the dark at ambient temperature for 30 min to enable the fluorescent labeling of the DCMC (Yin, Fei, Cui, Tang & Yin, 2007). Stock solution of Nile Red in ethanol (2.5 mg/mL) was injected to the phospholipid in the darkness, and the finial concentration of Nile Red was the label 50 µg/mL (Banerjee, Sen, Pal & Guha, 2012). These samples were dialyzed against double distilled water overnight to remove free fluorescent label. Finally, these samples were observed by CLSM (LSM710, Zeiss, Germany).
2.5. Physical stability of PLLs, HBLs and PMLs Our previous study found PMLs immediately precipitated after mixed with simulated intestinal fluid. Therefore, the influence of ionic kind (Cl–, SO32– and PO43–) on the stability of PLLs, PMLs and HBLs were determined. Stock solution of sodium chloride (1 M) and sodium sulfate (1 M) were prepared by dissolve sodium chloride or sodium sulfate in double distilled water. The stock solutions were adjusted to pH 6.5 using either 0.1 M hydrochloric acid or 0.1 M sodium hydroxide. PBS stock solution (0.5 M, pH 6.5) was composed of 0.1575 mM Na2HPO4 and 0.3425 mM NaH2PO4. Samples were treated with Cl– (25, 50, 75, 100, 150, 200 mM), SO32– (10, 20, 30, 40, 50, 75, 100 mM) and PO43– (10, 20, 30, 40, 50, 75, 100 mM) by diluted with ion stock solutions and double distilled water. The size of the samples was measured after 0.5h equilibrium at room temperature. 8
A heat treatment (121°C for 20min) was conducted to evaluate the thermal stability of PLLs and HBLs in the presence of different PBS concentrations (10, 25, 50, 75, 100, 125, 150 mM). The thermal stability of PMLs was not determined since it was unstable in the present of PBS.
2.6. Characterizations of Cur-PLLs and Cur-HBLs 2.6.1 Size distribution and zeta potential Size distribution and zeta potential of Cur-PLLs and Cur-HBLs were determined according to the protocol as described in section 2.4.2. 2.6.2 Loading efficiency The loading efficiency (LE) was measured by UV-Vis on an ultraviolet-visible spectroscopy (Pgeneral T6, China) according to Huang et.al (Huang et al., 2014). Curcuminloaded liposomes were centrifuged at 3000 rpm (1248 × g) for 10 min and 20 μL of supernatant were taken and dissolved in 3.8 mL of anhydrous ethanol. Absorbance at 420 nm was measured by UV-Vis and the concentration of loaded curcumin was calculated from the calibration curve. Standard curcumin curve was obtained through curcumin ethanol solutions in a concentration gradient (y = 0.1436x + 0.005, R2 = 0.9999), where y and x correspond to absorbance and curcumin concentration (μg/mL), respectively. The LE was obtained from the equation (3): 𝐿𝐸 = (loaded curcumin⁄total amount of lipids) × 100%
(3)
2.7. Storage stability of Cur-PLLs and Cur-HBLs In order to evaluate the storage stability of Cur-PLLs and Cur-HBLs, samples were stored 9
at 4°C and 25°C, respectively, for 1 month in sealed and dark condition. The average diameter and leakage ratio of curcumin were determined every 5 days. The average diameter was determined by DLS as described in the previous Section 2.4.2. The leakage ratio of curcumin was determined by measuring the loading efficiency (LE) during the storage time compared to the LE before storage (as described in Section 2.6.2), then calculated according to the following Eq. (4). Leakage ratio = (1 − 𝐿𝐸𝑑𝑢𝑟𝑖𝑛𝑔 𝑠𝑡𝑜𝑟𝑎𝑔𝑒 ⁄𝐿𝐸𝑏𝑒𝑓𝑜𝑟𝑒 𝑠𝑡𝑜𝑟𝑎𝑔𝑒 ) × 100%
(4)
2.8. In vitro release of Cur-PLLs and Cur-HBLs In vitro release profile of curcumin from curcumin-loaded liposomes were done by direct dispersion method as explained in literature (Mohanty & Sahoo, 2010). Briefly, samples were diluted 10 folds with PBS (0.05 M, pH 7.4) and solution were divided in Eppendorf tubes. The tubes were incubated in a shaker at 150 rpm at 37°C. At definite time intervals, one set of tubes were taken out and centrifuged at 3000 rpm for 10 min to pelletize the released curcumin. The encapsulated curcumin (supernatant) was diluted 10 fold with ethanol. Finally, amount of curcumin loaded was quantified spectrophotometerically at a wavelength of 420 nm.
2.9. Cellular uptake assays of Cur-PLLs and Cur-HBLs The cellular uptake of liposomal formulation was evaluated in Caco-2 cells by qualitative and quantitative methods according to our previous study (Chen et al., 2015). Caco-2 cells were seeded in 12-well plates at a density of 5 × 104 cells per well and incubated for 24 h. The cells were then treated with Cur-PLLs and Cur-HBLs with a final curcumin concentration of 4 μg/mL. 10
At predetermined time intervals of 0.5, 1, 2 and 4 h, the medium was removed and the cells were washed with PBS twice. Curcumin uptake was visualized under an inverted phase contrast microscope (Nikon Eclipse Ti-U; Nikon Instruments, Kanagawa, Japan) using autofluorescence (excitation wavelength: 480 nm, emission wavelength: 510 nm). To further quantify the difference in cellular uptake, the medium was removed after incubation with Cur-PLLs and Cur-HBLs for 0.5, 1, 2 and 4 h. Cells were then washed twice with PBS, trypsinized, centrifuged and collected in 1 mL PBS. The cell suspension was injected into an Accuri C6 flow cytometer (Accuri Cytometers, Inc., Ann Arbor, MI, USA) in the FL1 channel (488 excitation, blue laser, 530 ± 15 nm, FITC/GFP) to determine the fluorescence intensity (Yallapu, Jaggi & Chauhan, 2010).
2.10. Statistical analysis Statistical analysis was performed using ORIGIN 8.0 (OriginLab Inc., Northampton, MA, USA). The data are expressed as means ± standard deviations of at least three independent determinations on one sample for each time period, and were analyzed by one-way analysis of variance. Statistical significance was established at P < 0.05.
3. Results and discussion 3.1. Synthesis and structural characterization of DCMC The preparation of DCMC is illustrated in Fig. 2A. The structure of DCMC was confirmed by 1H NMR spectroscopy. The spectrum of chitosan had a single peak at δ 1.82 (H-2), multiple peaks at δ 3.25–3.60 (H-3 to H-5), δ 3.11 (H-6) and a small single peak at δ 4.44 (H-1) (Fig. 11
2B). The signals at δ 0.82, 0.99−1.46, 3.11 and 3.83 were attributed to protons in the long alkyl chains of the quaternary ammonium substituents, indicating successful preparation of DCMC. Using equation (1), the SD value of DCMC was calculated to be 0.815.
3.2. Preparation and characterization of PLLs, HBLs and PMLs AFM images of PLLs, HBLs and PMLs (Fig. 3A, 3B and 3C, respectively) indicated that uniform liposomes had been successfully prepared in each case. The PLLs, HBLs and PMLs were all well-distributed and spherical, with smooth surfaces. The average diameter of PLLs was 93.9 ± 2.7 nm, with a polydispersity index (PDI) of 0.382 ± 0.051 and the average diameter of PMLs was 93.9 ± 2.3 nm, with a PDI of 0.242 ± 0.031 (Fig. 4A). HBLs had a smaller diameter (78.2 ± 1.7 nm) than PLLs and PMLs and a PDI of 0.292 ± 0.041. DCMC acts as a polymeric surfactant in the liposomal structure and thus reduce the particle size of HBLs. The zeta potential of PLLs, PMLs and HBLs are shown in Fig. 4B. HBLs showed a change in zeta potential from -16.3 ± 1.7 mV to +26.3 ± 2.3 mV, which was due to the positive charged quaternary ammonium group of DCMC. The zeta potential of HBLs was also much lower than that of PMLs (+58.3 ± 4.3 mV), because the positively charged DCMC moiety was incorporated into the phospholipid membrane.
Liposomal fluidity refers to the relative motional freedom of the lipid molecules in the membrane bilayer (Arora, Byrem, Nair & Strasburg, 2000). The fluidity of phospholipid membranes reflects the order and dynamics of the phospholipid alkyl chains in the bilayer and 12
regulates liposomes permeation (Hasan et al., 2014). The membrane fluidities (1/P) of PLLs, PMLs and HBLs were 6.10 ± 0.11, 5.41 ± 0.12 and 3.54 ± 0.10, respectively. The membrane fluidities of PLLs are significantly (P < 0.01) higher than those of PMLs because of the higher molecular mobility of the micromolecule (phospholipid) compared with the macromolecule (DCMC). HBLs have significantly (P < 0.01) lower membrane fluidity than PLLs, likely, because hydrophobic interaction between DCMC and phospholipid restrict the motion of the polar phospholipid head-groups. Considering the distribution of DCMC in the phospholipid membrane, the lateral diffusion of phospholipid could also be restricted by steric hindrance of the DCMC backbones. CLSM images are shown in Fig. 4. The green fluorescence of FITC indicates DCMC and the red fluorescence of Nile Red indicates phospholipid. Red and green fluorescence were both observed in HBLs (Fig. 4C and Fig. 4D, respectively). Both red and green fluorescence were observed in every single HBL particle (Fig. 4E). This result, together with the size and zeta potential results, indicates that uniform hybrid liposomes, composed of DCMC and phospholipid, had been successfully prepared.
3.3. Physical stability of PLLs, HBLs and PMLs The diameter of liposomes in the presence of Cl–, SO42– and PO43– ions (Fig. 5A, 5B and 5C, respectively) was used to investigate stability under different ionic conditions. When PLLs and HBLs were treated with different ions, the particle size initially decreased and then remained constant with increasing salt concentration. PMLs showed a slight decrease in size at 13
low ion concentrations and then increased in size along with increasing ion concentrations. The decrease in particle size at low ionic strength might be explained by the osmotic shrinking of the vesicles (Hupfeld, Moen, Ausbacher, Haas & Brandl, 2010). When treated with 200 mM Cl–, the diameter of PMLs increased slightly from 93.9 nm to 119.3 nm. This effect could be attributed to the ability of the counter-ions in NaCl to screen electrostatic repulsive forces acting between the particles, resulting in particles fusion. The diameter of PMLs increased dramatically, however, when treated with 50 mM SO42– or PO43– (from 93.9 nm to 1949.9 nm or to 2564.3 nm, respectively). The dramatic increase in particle size indicated that large aggregates had been formed. The aggregation could be caused by association of the quaternary ammonium group of DCMC, induced by polyvalent anions (SO42– and PO43–). A single PO43– ion could bond simultaneously with three quaternary ammonium groups on DCMC and thus change the well-organized liposomal structure. HBLs were stable in the present of Cl–, SO42– and PO43–, perhaps because of the relatively strong hydrophobic bond interaction between DCMC and the phospholipid membrane. These results demonstrate that incorporation into HBLs improves the ionic stability of DCMC. PMLs were found to be extremely sensitive to ionic strength and precipitated when diluted with PBS or cell culture medium (Fig. 5E).Other properties of PMLs were, therefore, not evaluated. The effect of the ionic strength of the medium on thermal behavior of PLLs and HBLs is shown in Fig. 5D. PLLs and HBLs were both stable after heated at 121°C for 20 min in pure water but a gradual increase in average diameter of both particles was observed in PBS as the concentration increased from 10 to 150 mM. The diameter of PLLs and HBLs increased to 1011.1 nm and 159.3 nm, respectively, after heated at 121°C for 20 min in the presence of 14
50 mM PBS. HBLs thus showed better thermal stability than PLLs in the presence of PBS. PLLs were stable after heated at 121°C for 20 min in pure water, perhaps because electrostatic repulsive forces prevented PLLs from fusing and aggregating (Liu et al., 2016). The diameter of PLLs increased, however, after heated at 121°C for 20 min in the presence of PBS. It has been reported that a rapid decrease in the liposomal negative zeta potential observed on increasing ionic strength (Sabin, Prieto, Ruso, Hidalgo-Alvarez & Sarmiento, 2006). The reduction in zeta potential indicates a reduction in electrostatic repulsive forces between PLLs, resulting in reduced thermal stability. Compared with phospholipid membranes, DCMC increases the hydrophilicity of the hybrid membranes because of the carboxymethyl groups attached to DCMC. Steric forces between HBLs could also improve their thermal stability.
3.4. Characterization of Cur-PLLs and Cur-HBLs The maximum LE of Cur-PLLs and Cur-HBLs were determined by adding extra amount of curcumin to the phospholipid ethanol solutions (Section 2.3). The maximum LE of Cur-PLLs and Cur-HBLs were 6.52% ± 0.44% and 8.08% ± 0.18%, respectively. Cur-PLLs and CurHBLs were, however, extremely unstable when loaded with very high amounts of curcumin. The LE of PLLs and HBLs decreased gradually to 4.21% and 5.35%, respectively, after storage at 4°C for 15 days. This phenomenon was attributed to increased phospholipid bilayer fluidity, caused by molecular interaction between curcumin and liposome (Liu, Liu, Zhu, Gan & Le, 2015). Curcumin (0.5 mg/ml) was, therefore, loaded into both PLLs and HBLs to provide a consistent LE (4.2%). Both PLLs and HBLs were relatively stable when stored in a refrigerator 15
for about one month. The average diameters of Cur-PLLs and Cur-HBLs were 51.5 ± 1.6 nm and 54.1 ± 2.4 nm, respectively. The average diameters of PLLs and HBLs in the presence of 50 mM PBS were 48.7 ± 1.6 nm and 46.5 ± 1.2 nm, respectively (Fig. 5C). The diameter of curcumin-loaded liposomes was slightly larger than blank liposomes, which was attributed to the incorporation of curcumin into the liposome bilayer. The zeta potential of Cur-PLLs and Cur-HBLs were 16.1 ± 1.8 mV and +26.7 ± 2.0 mV, respectively. The incorporation of curcumin in liposomes did not, therefore, change the zeta potential of the liposomes.
3.5. Storage stability of Cur-PLLs and Cur-HBLs Liposomes are thermodynamically unstable, and have a high tendency to degrade, aggregate and fuse, leading to leakage of entrapped compounds during storage (Tan et al., 2014). To investigate the storage stability of curcumin-loaded liposomes, the leakage ratio and particle sizes of Cur-PLLs and Cur-HBLs were monitored during storage at 4°C and 25°C. The initial average diameters of Cur-PLLs and Cur-HBLs were 51.1 nm and 54.1 nm, respectively (Fig. 6). When stored at 4°C, the mean diameter and LE of curcumin did not change noticeably after 30 days. When stored for 30 days at 25°C, however, the mean diameter of Cur-PLLs increased dramatically from 51.1 nm to 109.9 nm, and leakage ratio of curcumin from PLLs was 35.3%. The increase in diameter of Cur-HBLs (from 54.1 nm to 66.2 nm) was much smaller than that of Cur-PLLs, and only 17.1% of curcumin leaked from HBLs. Cur-HBLs exhibit improved storage stability compared with Cur-PLLs. In summary, Cur-PLLs and Cur-HBLs have favorable storage stability with no curcumin 16
leakage and little change in particle size at 4°C since decomposition of the liposomes are inhibited at low temperature (Li et al., 2009). The stability of Cur-PLLs, in terms of aggregation, fusion and leakage, was much poorer than that of Cur-HBLs at 25°C. Crommelin (Crommelin, 1984) reported that incorporation of charged components into the liposome membranes reduced the tendency to aggregate because of increased electrostatic repulsion between the particles. Suspensions with larger absolute value are more stable, perhaps because the charged particles repel each other and overcome the natural tendency to aggregate (Heurtault, Saulnier, Pech, Proust & Benoit, 2003; Zhou et al., 2014). In the present study, the absolute value of zeta potential for PLLs and HBLs were 16.3 mV and 26.3 mV, respectively, suggesting that CurHBLs have greater physical stability than Cur-PLLs. Lower membrane fluidity could also reduce membrane fusion between HBLs, contributing to stability.
3.6. In vitro release of Cur-PLLs and Cur-HBLs The extremely low solubility of curcumin in water and its instability at physiological pH are major obstacles to its delivery to cancerous tissue. It is, therefore, important to confirm the release of curcumin from liposomes under physiological conditions. The in vitro release profiles of Cur-PLLs and Cur-HBLs are shown in Fig. 7. The diameter and zeta potential of the particles did not changed after incubation at 37°C for 24 h. The release of curcumin from both Cur-PLLs and Cur-HBLs displayed a fast initial burst within the first 12 h, followed by a slow and sustained release. Approximately 36.8% and 41.6% of curcumin was released from PLLs at 12 and 24 h, respectively. HBLs more effectively entrap the curcumin and only 13.1% and 15.5% curcumin was released from HBLs at 12 and 24 h, respectively. Drug release from liposomes 17
is a complex process which depends on drug physicochemical properties, carrier integrity and diffusion and dissolution between the phospholipid bilayer and the drug. Calvagno found that the release of cargo from liposomes is determined by two factors. The first of these is the strength of the interaction between liposomal membrane and cargo, i.e. stronger interaction lead to less desorption and hence the burst effect. The second factor is the fluidity of the bilayer, i.e. more fluid bilayers allow greater and more rapid cargo leakage from liposomes (Grazia Calvagno et al., 2007). As described above, DCMC distributes in the membrane of HBLs, with lipophilic moieties inserting into the phospholipid bilayer and thus decreasing their permeability. The reduced bilayer fluidity would reduce diffusion of curcumin from the bilayers and ultimately reduce leakage of curcumin from Cur-HBLs.
3.7. Cellular uptake of Cur-PLLs and Cur-HBLs Caco-2 cells, which are epithelial cells derived from human colon adenocarcinoma, were used to evaluate the cellular uptake of Cur-PLLs and Cur-HBLs (Huang & Kuo, 2016). Fluorescence microscopy images showed that the fluorescence intensity of the Caco-2 cells increased and then remained unchanged after incubation for 2 h (Fig. 8A). The fluorescence intensity of Caco-2 cells treated with Cur-HBLs was noticeably higher than that of those treated with Cur-PLLs, indicating that DCMC significantly improved the cellular uptake of liposomes. Cellular uptake was next quantified by flow cytometry. Cellular uptake rates of Cur-PLLs were 25.2%, 30.6%, 37.7% and 40.6% and uptake rate of Cur-HBLs were 31.8%, 49.5%, 64.1% and 66.2% after incubation for 0.5, 1, 2 and 4 h, respectively (Fig. 8B, C). Both fluorescence microscopy and flow cytometry thus demonstrate a higher cellular uptake rate for Cur-HBLs 18
compared with Cur-PLLs. The transfer of drugs encapsulated in liposomes into cells relies mainly on endocytosis (Un, Sakai-Kato, Oshima, Kawanishi & Okuda, 2012) and absorption of liposomes onto the cell membrane is the first and critical step. Compared with PLLs, HBLs have increased positive charge, and decreased hydrophobicity. This should lead to higher absorption activity and higher cellular uptake. The surface carboxyl groups originating from DCMC, which decrease the surface the surface hydrophobicity, could also facilitate internalization of HBLs and improve cellular uptake (Xiao, Nian & Huang, 2015). The curcumin released from PLLs and HBLs would also influence drug uptake by cells. The burst release of curcumin from PLLs (Section 3.4) resulted in decreased curcumin bioavailability since free curcumin is insoluble in aqueous solutions and thus not available for uptake by cells.
4. Conclusion Novel HBLs containing both DCMC and phospholipid have been successfully prepared. The particles were spherical and positively charged, and had a narrow size distribution, and low membrane fluidity. HBLs were stable in the presence of different ions and when heated at 121°C for 20 min. When loaded with curcumin, Cur-HBLs had good storage stability, and provided sustained release with good cellular uptake. The present study indicates that HBLs are a promising delivery system for curcumin. To further evaluate the bioavailability of HBLs, more model drugs will be encapsulated in this novel carrier.
19
Acknowledgments We appreciate the financial support by the National Science Foundation of China (No. 21266021), Technological expertise and academic leaders training plan of Jiangxi Province and the Youth Fund of Department of Education of Jiangxi Province (N0.150089).
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Figure captions
Fig. 1. Schematics representation of phospholipid liposomes (PLLs), hybrid liposomes (HBLs), polymeric liposomes (PMLs), curcumin-loaded phospholipid liposomes (Cur-PLLs) and curcumin-loaded hybrid liposomes (Cur-HBLs) and photos of Cur-PLLs and Cur-HBLs.
Fig. 2. The synthesis process of DCMC (A) and 1H NMR spectra of DCMC (B).
Fig. 3. AFM images of (A) PLLs, (B) HBLs and (C) PMLs.
Fig. 4. (A) Size distribution and (B) zeta potential of PLLs, HBLs and PMLs. CLSM images of HBLs: (C) FITC channel, (D) Nile Red channel, (E) Merged image of FITC and Nile Red.
Fig. 5. Effect of (A) Cl-, (B) SO42– and (C) PO43– on the average diameter of PLLs, HBLs and PMLs. (D) Effect of heat treatment on the average diameter of PLLs and HBLs in the presence of different concentration of PBS. (E) The photography of PLLs, HBLs and PMLs after treated with 50 mM NaCl, 50 mM Na2SO4 and 50 mM PBS, and heated in the presence of 100 mM PBS.
Fig. 6. The changes of average diameter (A) and leakage ratio of (B) Cur-PLLs and Cur-HBLs during storage at 4 and 25°C.
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Fig. 7. In vitro release profile of curcumin from Cur-PLLs and Cur-HBLs.
Fig. 8. Fluorescence microscope (A), Flow cytometric (B) and Uptaking ratio (C) analysis of Caco-2 cells treated with 4 μg/mL Cur-PLLs and Cur-HBLs after a defined period of time.
28
Fig. 1. Schematics representation of phospholipid liposomes (PLLs), hybrid liposomes (HBLs), polymeric liposomes (PMLs), curcumin-loaded phospholipid liposomes (Cur-PLLs) and curcumin-loaded hybrid liposomes (Cur-HBLs) and photos of Cur-PLLs and Cur-HBLs.
29
Fig. 2. The synthesis process of DCMC (A) and 1H NMR spectra of DCMC (B).
30
Fig. 3. AFM images of (A) PLLs, (B) HBLs and (C) PMLs.
31
Fig. 4. (A) Size distribution and (B) zeta potential of PLLs, HBLs and PMLs. CLSM images of HBLs: (C) FITC channel, (D) Nile Red channel, (E) Merged image of FITC and Nile Red.
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Fig. 5. Effect of (A) Cl-, (B) SO42– and (C) PO43– on the average diameter of PLLs, HBLs and PMLs. (D) Effect of heat treatment on the average diameter of PLLs and HBLs in the presence of different concentration of PBS. (E) The photography of PLLs, HBLs and PMLs after treated with 50 mM NaCl, 50 mM Na2SO4 and 50 mM PBS, and heated in the presence of 100 mM PBS.
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Fig. 6. The changes of average diameter (A) and leakage ratio of (B) Cur-PLLs and CurHBLs during storage at 4 and 25 °C.
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Fig. 7. In vitro release profile of curcumin from Cur-PLLs and Cur-HBLs.
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Fig. 8. Fluorescence microscope (A), Flow cytometric (B) and uptaking ratio (C) analysis of Caco-2 cells treated with 4 μg/mL Cur-PLLs and Cur-HBLs after a defined period of time.
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