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Facile synthesis of the lipid bilayer coated mesoporous silica nanocomposites and their application in drug delivery
Microporous and Mesoporous Materials 219 (2016) 209e218
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Microporous and Mesoporous Materials journal homepage: www.elsevier.com/locate/micromeso
Facile synthesis of the lipid bilayer coated mesoporous silica nanocomposites and their application in drug delivery Ning Han a, 1, Yu Wang b, 1, Junling Bai a, Jiangjun Liu a, Ying Wang a, Yikun Gao c, Tongying Jiang a, Wanjun Kang b, Siling Wang a, * a
Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning Province 110016, PR China Chinese PLA No. 463 Hospital, Shenyang, Liaoning Province 110000, PR China c School of Medical Devices, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning Province 110016, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 May 2015 Received in revised form 5 August 2015 Accepted 6 August 2015 Available online 14 August 2015
The lipid bilayer coated mesoporous silica nanocomposites (LMSNs) were synthesized with aim to obtain better performance in the application of drug delivery. Phospholipids composed of soybean lecithin and DSPE-PEG 2000 were pre-prepared into liposomes, then they were allowed to fuse onto the mesoporous silica nanoparticles (MSNs) forming a surrounding lipid bilayer. The obtained LMSNs had an average particle size of 295 nm, zeta potential of 1.0 mV and a good dispersing stability in saline buffers. Facilitated by the affinity of the lipid bilayer with cell membrane, the internalization of LMSNs by cells was markedly increased. In addition, compared with bare MSNs, the cytotoxicity, hemolysis percentage and nonspecific BSA absorption of LMSNs were significantly reduced, making them become more reliable carriers for drug delivery. When encapsulating a model drug, doxorubicin (DOX) into LMSNs, the loading efficiency can reach as high as 16%. The obtained LMSNs-DOX exhibited a pH-responsive release behavior and the presence of the lipid bilayer did not significantly retard the release of DOX. Furthermore, LMSNs greatly enhanced the cellular accumulation and cytotoxicity of DOX toward the MCF-7 cells. In summary, the lipid bilayer coating was a simple and facile strategy to functionalize MSNs, and the obtained LMSNs exhibiting good biocompatibility were promising nanocarriers in improving the cellular uptake and therapeutic efficacy of anticancer drugs. © 2015 Published by Elsevier Inc.
Keywords: Mesoporous silica Lipid bilayer Drug delivery Cellular uptake Biocompatibility
1. Introduction In recent decades, a variety of nanostructured materials, such as liposomes, micelles and organic/inorganic nanoparticles have attracted great interest as intelligent drug delivery systems and showed promising prospects in a broad range of therapeutic applications [1e4]. Among these, mesoporous silica nanoparticles (MSNs) are particularly attractive due to their unique properties, including a large surface area and pore volume to load drug with high efficiency [5,6]; a uniform and tunable pore size to accommodate molecules with various steric hindrance and an excellent physiochemical stability to protect the encapsulated drugs from degradation by endogenous enzymes [7].
* Corresponding author. Tel./fax: þ86 24 23986348. E-mail address: [email protected] (S. Wang). 1 Authors contributed equally. http://dx.doi.org/10.1016/j.micromeso.2015.08.006 1387-1811/© 2015 Published by Elsevier Inc.
Recently, MSNs have been widely employed as nanocarriers to achieve stimuli-responsive drug release and targeted drug delivery [8e10]. However, the application of MSNs as drug carriers has been limited by some of their properties. Such as easy aggregation in saline buffers, rapid clearance by the reticuloendothelial system (RES) and the risk of inducing hemolysis when given intravenously, a common route of administration [11]. Therefore it is necessary to functionalize MSNs in order to counteract the above disadvantages. Surface modification with polymers, dendrimers [12] or natural materials through chemical reaction or electrostatic attraction (such as layer by layer technique) [13] was reported to be an effective way to change the properties of nanoparticle for various applications. However, previous studies devoting to improve the biocompatibility of MSNs as drug carriers by surface modification were limited and rare, including using polymers such as polyethylene glycol (PEG) or poly(N-vinylcaprolactam-co-methacrylic acid) (P(VCL-s-s-MAA)) [14]. But the functionalization process was complicated and needed to be optimized. In addition, there has
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always been a contradiction in the priority of order between the drug loading and surface functionalization process. If the functionalization comes first, it may cause a reduction in the surface area and pore volume which will adversely affect the drug loading efficiency of MSNs. While if the drug loading is a priority, there will be an inevitable loss of the loaded drugs during the long time chemical reaction and repeated purifying process which are necessary for the successful functionalization. Therefore, it is essential to find a simple and effective approach to modify the MSNs and overcome the above drawbacks of them without adversely affecting the drug loading capacity. Furthermore, as a drug carrier, it is also of great importance to improve the cellular accumulation of the incorporated drugs within the targeted cells to reach effective therapeutic level. Inspired by the supported lipid bilayer which mimics the cell membrane, the lipid bilayer coating can be an innovative functionalization on MSNs by fusion on the surface of them. The preparation method was based on the previous findings by Mornet et al., in which the pre-prepared liposomes can first adhere to MSNs, then undergo gradual deformation, finally rupture and spread all over the nanoparticles to form a continuous coverage [15]. Reasons for using the lipid bilayer coating on mesoporous silica nanoparticles (LMSNs) are as follows: 1) lipids are wellrecognized to be highly biocompatible; 2) the lipid bilayer has a strong affinity with the cell membrane which is advantageous for the subsequent internalization by cells; 3) the PEG modified lipid bilayer can prolong the circulation time and reduce the immunogenicity of MSNs, thereby facilitating the accumulation of drugloaded carriers at the tumor site via the enhanced permeability and retention (EPR) effect. Moreover, compared with other functionalization methods, the lipid bilayer coating is rather simple and facile. Since it can be completed within a short period of time, the loss of the loaded drugs can thereby be markedly reduced. This integrated drug delivery system is expected to inherit the merits from both MSNs and liposomes and exhibit characteristics superior to those of any of its single component. In the nanocomposites, MSNs acting as a drug reservoir can accommodate various drug molecules with a high payload and serve as a supporting skeleton to stabilize the lipid bilayer. While the lipid bilayer coating can in turn serve to improve the dispersing stability and biocompatibility of MSNs and make them more applicable as drug carriers by mitigating their disadvantages. In several other studies, the lipid bilayer present on MSNs was designed as a “gatekeepers” to control drug release or an intermediate for further conjugation of various targeting ligands. There are rarely any literature studied the effect of mere lipid bilayer coating on the properties of MSNs as drug carriers [16e18]. In this study, we innovatively investigated the effect of the lipid bilayer coating on the applicability of MSNs as carriers for intravenous drug delivery. And the superiority of the prepared LMSNs was systematically evaluated by comparing them with bare MSNs in terms of dispersing stability, cellular uptake efficiency and biocompatibility. The prepared LMSNs were then employed to load doxorubicin (DOX), one of the most potent drugs used in clinical chemotherapy. And the in vitro drug release, cellular internalization, and cytotoxicity of LMSNs-DOX were systematically investigated. 2. Materials and methods
Holding Co., Ltd (Tianjin, China). Poloxamer 407 (Lutrol® F-127) was obtained as gift sample from BASF (Ludwigshafen, Germany), Soybean lecithin and cholesterol were purchased from LIPOID GmbH (Ludwigshafen, Germany). 1,2-Distearoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG2000-DSPE) and Rhodamine B PE was obtained from Avanti Polar Lipids (Alabaster, AL). Anticancer drug doxorubicin hydrochloride (DOX) was offered by Zhejiang Hisun Pharmaceutical Co., Ltd (Zhejiang, China). RPMI 1640, fetal bovine serum (FBS) and penicillin/streptomycin solution were attained from GIBCO (BRL, MD, USA). Bovine serum albumin (BSA), fluorescein isothiocyanate (FITC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and trypsin were purchased by SigmaeAldrich (St. Louis, MO). Fluorescent Hoechst 33258 was obtained from Molecular Probes (Eugene, OR, USA). All other chemicals were of analytical grade and used without further purification. 2.2. Synthesis of mesoporous silica nanoparticles (MSNs) As reported previously, mesoporous silica nanoparticles were €ber method [19]. In brief, 0.5 g prepared based on the modified Sto CTAB and 1.0 g triblock copolymer F127 were dissolved in 100 mL distilled water, in which 10 mL 29 wt.% ammonium hydroxide solution and 40 mL ethanol were subsequently added. After complete dissolution, 1.9 mL TEOS was then introduced dropwise to the mixture under intensive stirring, and the reaction was maintained at room temperature for 24 h. The white solid product was collected by centrifugation at 10,000 rpm. For template extraction, the synthesized nanoparticles were re-dispersed in 100 mL absolute ethanol containing 1 g NH4NO3, and allowed to reflux at 80 C overnight with constant stirring to remove the surfactant. The final product was centrifuged, washed with ethanol for 3 times and dried overnight at room temperature in vacuum. 2.3. Preparation of liposomes Liposomes were prepared by the lipid film hydration method, in the experiment 100 mg soybean lecithin, 10 mg DSPE-PEG 2000 and 20 mg cholesterol were dissolved in 5 mL chloroform and evaporated to form a thin lipid film using a rotary evaporator. Then the lipid film was hydrated in PBS of pH 7.4 at concentration of 10 mg/mL and was then extruded for 15 cycles using a mini extruder (Avanti Polar Lipids, Inc., USA) equipped with progressively decreasing pore-sized polycarbonate membrane of 200 nm and 100 nm. The prepared liposomes for further experiment were stored at 4 C for no more than a week. 2.4. Development of the lipid bilayer-mesoporous silica nanocomposites (LMSNs) The LMSNs were prepared by first re-dispersing 20 mg of dried MSNs in 2 mL distilled water under ultrasonic irradiation for 1 min. Then 1 mL of the pre-prepared lipid vesicles was added to the particle suspension, the mixture was well vortexed and left to stand for 1 h at room temperature to allow thorough fusion of liposome on MSNs. Extra liposomes in the supernatant were removed via centrifugation at 10,000 rpm for 10 min. After being washed with PBS for three times, the formed LMSNs were finally dispersed in the PBS solution.
2.1. Materials 2.5. Drug loading process and loading efficiency measurement Tetraethyl orthosilicate (TEOS, 98%), hexadecyl-trimethyl ammonium bromine (CTAB, >99%) and ammonium hydroxide (NH4OH, 25e28%) were obtained from Tianjin Bodi Chemical
Drug loading was conducted prior to the lipid bilayer coating, 20 mg of the dried MSNs was ultrasonically re-suspended in 2 mL of
N. Han et al. / Microporous and Mesoporous Materials 219 (2016) 209e218
DOX solution dissolved in distilled water (5 mg/mL) and incubated for 24 h under intermittent stirring to fully absorb the drugs. To obtain LMSNs-DOX, the DOX loaded MSNs were first centrifuged, then the lipid vesicles were added to fuse onto MSNs following the procedure as described in Section 2.3. Samples denoted as MSNsDOX which were prepared in the same way but without lipid bilayer coating was used as control groups in the subsequent experiments. The drug loading efficiency was calculated according to the formula. The weight of encapsulated DOX can be determined by subtracting the mass of DOX in supernatant from the total mass of drug in the initial solution.
LE % ¼
weight of encapsulated DOX 100% weight of DOX loaded LMSNs
2.6. Characterization of the nanoparticles The structure and morphology of MSNs were characterized by the field-emission transmission electron microscopy (TEM) using FEI Tecnai G2 F30 (Netherlands) operated at an acceleration voltage of 200 kV and the scanning electron microscopy (SEM) using ZEISS SUPRA 35 (Germany) after spraying the desiccative nanoparticles with gold on a carbon grid. In order to confirm the lipid bilayer present on MSNs, the cryo-TEM images of LMSNs were taken by depositing 10 mL aliquots of the prepared LMSNs suspension on the cupper grids enveloped with carbon film and quickly frozen in liquid ethane. The grids were placed onto a Gatan 626 cryo-holder and inserted into an FEI Tecnai F20 microscope for observation. Confocal fluorescence images of the FITC-labeled MSNs, Rhodamine B-labeled lipid bilayer, and the merged image were taken by Leica DM-6000 CS microscope (Leica Instruments Inc., Wetzlar, Germany) to further confirm the presence of lipid bilayer on MSNs. The nitrogen adsorption/desorption analysis was conducted to detect the specific surface area and the pore size of the carriers using an adsorption analyzer (V-Sorb 2800P, China). Thermogravimetric analysis (TGA) was also performed on the TGA-50 instrument (Shimadzu, Japan) to investigate the weight loss after the lipid bilayer coating with a heating rate of 5 C/min under a nitrogen flow. The differential scanning calorimeter (DSC) was employed to measure the gel-to-fluid phase transition temperature (Tm) of lipid bilayer on MSNs using DSC 1 STARe (Mettler Toledo, Switzerland). Samples of 40 mL containing approximately 2 mg lipids were placed in the aluminum pan and the experiment was performed from 30 C to 15 C at a heating rate of 2 C/min under a nitrogen flow of 40 ml/min. And the particle size distribution and zeta potential of the MSNs and LMSNs were recorded using the Zetasizer Nano (Malvern Instruments Ltd., United Kingdom).
2.7. Drug release behavior of DOX from MSNs, and LMSNs The release study of DOX from MSNs and LMSNs was performed in pH 7.4 and pH 5.0 PBS. Formulations containing DOX relevant to 3 mg were transferred into a dialysis bag (MWCO 14,000) and sealed with a dialysis bag holder, the dialysis bags were then immersed into 30 mL PBS with constant shaking at 100 rpm/min at 37 C. At predetermined time intervals, 1 mL of the release medium was withdrawn to monitor the release extent of DOX by an UVeVis spectrophotometer (UV-2000, UNICO, USA) at a wavelength of 480 nm using a pre-established calibration curve. Simultaneously the fresh medium with the same volume was replenished. The results were averaged with three measurements.
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2.8. Cell culture The human breast cancer cell line MCF-7 used in this study was purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in RPMI 1640 medium containing 10% (v/v) fetal bovine serum (FBS) and 1% penicillinestreptomycin at 37 C in a humidified atmosphere with 5% CO2. 2.9. In vitro cellular uptake To compare the cellular uptake efficiency of blank MSNs and LMSNs, and investigate the intracellular accumulation of DOX encapsulated MSNs and LMSNs, MCF-7 cells were seeded on round glass coverslips in 24-well plates. After 12 h of attachment, the culture medium was replaced with fresh serum-free RPMI 1640 containing FITC-labeled blank carriers at 20 mg/mL or various DOX formulations containing DOX of 5 mg/mL. After incubation for 2 h, the cells were rinsed with cold PBS for three times, and fixed with 4% formaldehyde. The nuclei were afterward stained by Hoechst 33258 for 20 min and the fixed cells were observed using CLSM. 2.10. Cell viability assessment The in vitro cell viability of MCF-7 cells after incubation with the blank nanocarriers and DOX-encapsulated samples was evaluated by MTT assay. Briefly, cells were seeded into 96-well plates at a density of 1.0 104 cells/well and attached for 24 h. Then the culture medium was removed and cells were subject to DOX-Sol, MSNs-DOX and LMSNs-DOX nanocarrier solutions in serum free medium at equivalent DOX concentrations of 0.01, 0.1, 1, 10, 100 mg/ mL to investigate the cytotoxic effect of different DOX formulations. In addition, the cytotoxicity of the corresponding amount of blank MSNs and LMSNs contained in the DOX formulations at serial tested concentrations were also examined. At the time point of 48 h, 50 mL of MTT solution (2 mg/mL) was introduced to the culture medium and incubated for extra 4 h to quantify cell viability. The supernatant was gently removed and 150 mL of DMSO was added to each well. After being agitated for 10 min to completely dissolve the formed formazan crystals, the 96-well plates were finally placed into an iMark™ microplate reader (Bio-RAD, CA, USA) to monitor the absorbance at wavelength of 570 nm. Cells without treatment were set as control. 2.11. Hemolysis study and BSA absorption measurement In order to assess and compare the safety of MSNs and LMSNs for in vivo applications, hemolysis evaluation was performed by isolating the rabbit red blood cells (RBCs) at 1000 rpm for 10 min and washed three times with sterile saline. The centrifuged RBCs were then diluted with saline to a concentration of 2% (v/v). Afterward, 2 mL of MSNs and LMSNs suspension in saline containing various amount of nanoparticles was added to equal volume of RBCs suspension to make the final nanoparticle concentrations of 20, 50, 100, 200, 500, 1000 and 1500 mg/mL. The mixture was briefly vortexed before left static for 4 h at room temperature. After that, the mixture was centrifuged and the clear supernatant was measured at 541 nm on an UVevis spectrophotometer. The hemolysis of RBCs incubated with distilled water and saline were set as the positive and negative control, separately. Hemolysis percentage was ascertained as follows: hemolysis (%) ¼ (sample absorbance absorbance of negative control)/(absorbance of the positive control absorbance of negative control) 100%. All samples were measured in triplicate. The BSA absorption measurements were performed according to the procedure reported previously to simulate the protein
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Scheme 1. Schematic representation of lipid bilayer coated mesoporous silica nanocomposites (LMSNs).
Fig. 1. SEM (A) and TEM (B) images of MSNs, cryo-TEM image of LMSNs (C).
absorption on silica under physiological condition. Typically, 30 mg BSA was dissolved in 50 mL of deionized water. After dispersing 10 mg of MSNs and LMSNs in saline buffer with concentration of 2 mg/mL, 5 mL of BSA solution was subsequently added. The mixture was put into a shaker with a shaking rate of 135 rpm at 37 C. After a time period of 4 h, 1 mL of Coomassie brilliant blue solution was added to the collected supernatant following the centrifugation of the above mixture. The BSA concentration was quantified by measuring the absorbance at 595 nm on an UVeVis spectrophotometer. And the adsorbed BSA was calculated according to the following equation: q ¼ (Ci Cf)V/m, where Ci and Cf respectively represent the initial and final BSA concentration in the solution. V means the total volume of the mixture and m is the weight of nanoparticles added. 3. Results and discussion 3.1. Preparation and characterization of LMSNs The preparation process of LMSNs was shown in Scheme 1. Monodisperse spherical MSNs were firstly synthesized following
the previously reported method using TEOS as the silica source, CTAB as the pore template and triblock copolymer F127 as a dispersing agent and particle size tailor. MSNs with a range of particle sizes can be prepared by altering the amount of F127 added. The more F127 used, the smaller the size of the MSNs can be obtained [19]. It was reported in many other studies that nanoparticles within the size range of 50e500 nm can guarantee an ideal EPR effect [20]. Hence MSNs with an intermediate particle size of 200 nm were prepared by adding a moderate amount of F127, giving weight ratio of CTAB which was 2:1. The resultant MSNs had a uniform size of ca. 200 nm as can be seen in the SEM image (Fig. 1A). Also, the two-dimensional pore arrangement of MSNs can be clearly observed in the TEM images (Fig. 1B). For LMSNs, cryoTEM images were taken to confirm the presence of lipid bilayer on the periphery of MSNs (Fig. 1C). As can be seen, the LMSNs appeared to be surrounded by an electron-dense ring of about 7 nm, which corresponded to the thickness of lipid bilayer demonstrating the successful coverage of the lipid bilayer on MSNs by the fusion method. Fig. 2 shows the confocal image of LMSNs, where the silica core was labeled with FITC and the phospholipid was labeled with
Fig. 2. CLSM images of LMSNs, FITC labeled MSNs (A), Rhodamine B labeled lipid bilayer (B) and merged image (C).
N. Han et al. / Microporous and Mesoporous Materials 219 (2016) 209e218
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Fig. 3. Nitrogen adsorptionedesorption isotherms and pore diameter distribution of MSNs and LMSNs.
Rhodamine B. After merging images of A and B in Fig. 2, the green and red fluorescence completely overlapped, which further confirmed the lipid bilayer on MSNs. According to the nitrogen adsorptionedesorption isotherm curves and pore size distribution of MSNs and LMSNs shown in Fig. 3, the blank MSNs had a total surface area of 800 m2/g, a cumulative pore volume of 1.0 cm3/g and an average pore size of 2.3 nm. After incubating with the pre-prepared liposomes for 1 h, the surface area of MSNs markedly reduced to 50 m2/g, and the pore volume became negligible further supporting the successful coating of the lipid bilayer. Thereafter, drug loading should be performed prior to the lipid bilayer covering on MSNs. The Fourier transform infrared spectroscopy (FT-IR) was further used to confirm the presence of lipid bilayer on MSNs, and the corresponding data was provided in Supplementary data. Thermogravimetric analysis (TGA) was also conducted to investigate the weight percentage of the lipid bilayer on MSNs and the resultant curves are shown in Fig. 4A. Over the temperature range from 50 C to 150 C hardly any weight loss was observed for MSNs, indicating that the surfactant templates have been completely removed. After incubating the MSNs with liposomes for 0.5 h, a weight loss of about 32.4% was observed for LMSNs, which was due to the lipid bilayer existing on MSNs. On extending the incubation time to 1 h, the weight loss only increased by 2.7%, which implied that the lipid bilayer coating on MSNs can almost be
completed within 1 h. Since it can be completed within such a short period of time, the loss of the pre-loaded drugs was greatly eliminated. As seen from the curve of MSNs-DOX, when incorporating DOX into the MSNs, the loading efficiency can reach 23%. And after the lipid bilayer coating, the prepared LMSNs-DOX showed an increased weight loss of about 53%, demonstrating that drug loading would not affect the lipid bilayer coating on MSNs. The DSC results in Fig. 4B showed that the prepared liposome had a Tm of 20.23 C, higher than the Tm of 21.53 C and 21.83 C for the LMSNs and LMSNs-DOX, respectively. It was reported that the reduction in Tm is an indicator of the forming of the lipid bilayer on MSNs, since the fluidity of lipid bilayer will increase due to the interfacial interaction with the porous silica support [16,21]. Therefore, based on above the results, the lipid bilayer can be formed on both the drug free and drug loaded MSNs. And the lipid bilayer was in the fluid phase at the experimental temperature. The change in particle size (Fig. 5A) also confirmed the result above. The MSNs had a hydrodynamic diameter of ca 254 nm, a little larger than the diameter observed in TEM due to the existence of a hydration layer in aqueous solutions. As for LMSNs, the hydrodynamic diameter increased to 295 nm which illustrated the presence of the lipid bilayer on MSNs. And as shown in Fig. 5B, MSNs had a zeta potential of 25 mV due to the ionization of the silanol groups on the surface. While after the lipid bilayer covering, the zeta potential of LMSNs increased to around 0 mV due to the
Fig. 4. The TGA curves of MSNs, LMSNs after 0.5 h and 1 h incubation with liposomes, MSNs-DOX and LMSNs-DOX (A); the DSC curves of liposome, LMSNs and LMSNs-DOX (B).
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Fig. 5. The particle size (A), zeta potential (B), and dispersing stability in PBS (C) of the DOX free and DOX loaded MSNs and LMSNs.
shielding effect of the neutrally charged and PEG grafted lipid bilayer. And the same changing trend in particle size and zeta potential was also observed when preparing the LMSNs-DOX, indicating that the lipid bilayer coating could be well performed on the drug loaded MSNs. It is worth noting that MSNs with a charge stabilized property via the ionization of hydroxyl groups has confronted limitations in their bio-applications. It was found that MSNs can be destabilized easily and aggregated readily in saline buffers with a high ionic strength (Fig. 5C). And the aggregation will expedite following the absorption of the proteins in the serum. Such phenomenon can consequently lead to the rapid clearance of nanocarriers by the reticuloendothelial system (RES) and cause potential safety hazard, which is certainly unwanted for intravenous administration [22]. However, after covering the MSNs with the PEG-modified lipid bilayer, the silanol groups were concealed and the obtained LMSNs can be stably dispersed in PBS (Fig. 5C). And the hydrodynamic diameter of LMSNs in PBS was almost the same with that in water. It indicated that the PEG-modified lipid bilayer can greatly improve the dispersing stability of MSNs, a premise for the safe administration of nanoparticles through the intravenous route. Also, the PEG chains on the lipid bilayer can form mushroom-like clusters on the nanocarriers to prevent them from being recognized and captured by the RES. And a prolonged circulation time and enhanced accumulation of drug loaded carriers at the tumor site can thus be achieved by the EPR effect [23].
were quite stable under the same condition, less than 10% of the incorporated drug was released within 24 h due to the drug diffusion being hindered by the lipid bilayer. When exposing the DOX-MSNs and DOX-LMSNs to pH 5.0 PBS, the release rate of DOX from both carriers markedly accelerated and the release percentage at 24 h was 72% and 68%, respectively. This acid triggered release can be explained as that the electrostatic interactions between the MSNs and DOX were pH-dependent, DOX molecules tended to detach from MSNs more easily under low pH conditions [24,25]. As for LMSNs, a comparable sustained release of DOX can be observed at pH 5.0 demonstrating that the presence of the lipid bilayer can only slightly retard the release of DOX due to the diffusion hindrance it provided. But the lipid bilayer will not totally block the release of DOX at the acidic diseased site, since the drug release driven by the concentration gradient overwhelmed the retardation effect provided by the lipid bilayer. As a consequence, most of the encapsulated DOX was released within 24 h. In summary, compared with MSNs, LMSNs had a stronger retaining effect of DOX in normal physiological conditions and same release responsiveness to the pH change in the environment. Since the pH value in cancer cells and the endosomal compartment is lower than the pH level in healthy cells [2], a preferential release of DOX at the tumor site can be achieved. To study the kinetics mechanism of DOX release from MSNs and LMSNs, equations of zero order, first order, Higuchi and Weibull
3.2. Drug loading efficiency and pH-responsive drug release Drug loading was carried out before the lipid bilayer coating on MSNs. Since the MSNs have a large surface area and pore volume, they can accommodate a loading of as much as 22% DOX molecules by physical absorption, consistent with the result measured by TGA. After coating with the lipid bilayer, the drug loading efficiency decreased to 16%. By tracking the concentration of DOX in the supernatant during the coating process, we found that the loss of drug was negligible, it was the increased weight after the lipid bilayer coverage that led to the decline of LE% in the calculation. Even so, the LE% of DOX-LMSNs was still higher than that of the DOX encapsulated liposomes prepared by the conventional ammonium phosphate gradient method which had a loading efficiency of 9%. The in vitro release behavior of LMSNs-DOX was subsequently investigated. The DOX-loaded LMSNs were placed in pH 5.0 and pH 7.4 PBS and the release profiles are shown in Fig. 6. For comparison, the release of MSNs-DOX was also assessed. As can be seen, DOXMSNs exhibited a gradual release of DOX in pH 7.4 PBS and the release extent was as high as 22% after 24 h. While DOX-LMSNs
Fig. 6. In vitro DOX release from MSNs and LMSNs in pH 7.4 and pH 5.0 PBS.
N. Han et al. / Microporous and Mesoporous Materials 219 (2016) 209e218 Table 1 In vitro release kinetics parameters. Formulations
MSNs-DOX pH 7.4 LMSNs-DOX pH 7.4 MSNs-DOX pH 5.0 LMSNs-DOX pH 5.0
Zero-order
First-order
Higuchi
R2
R2
R2
KH
Weibull R2
0.8116 0.9349 0.8352 0.8791
0.8300 0.9405 0.9305 0.9485
0.9701 0.9963 0.9879 0.9811
6.391 2.555 21.47 20.60
0.9438 0.9775 0.9725 0.9664
were utilized to fit the accumulative release data. The obtained results were listed in Table 1. The highest value of regression coefficient (R2) was observed when the release profiles were described using the Higuchi model (Eq. (1)), in which the drug release from carriers is proportional to the square root of time. Q is the amount of drug released in time t, and KH is the Higuchi rate constant. According to the equation, it is feasible to believe that the release mechanism of DOX from these particles was diffusion controlled [26]. In addition, the KH of MSNs-DOX was larger than that of LMSNs-DOX in both PBS of pH 7.4 and pH 5.0, which again confirmed the slight barrier effect of the lipid bilayer during the drug release process.
Q ¼ KH t 1=2
(1)
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the fluorescence intensity in cells treated with LMSNs was significantly enhanced demonstrating that the presence of the lipid bilayer can greatly promote the cellular uptake of silica nanoparticles. The reason for this was that the neutrally charged lipid bilayer could reach the cell surface more easily and possessed an inherent affinity with the cell membrane since they have the similar structure. So it is favorable for the adhesion and internalization of nanoparticles. The intracellular release of DOX was also examined using the CLSM. As can be seen in Fig. 8, the red fluorescence of DOX in cells treated with free DOX was not as strong as that in those treated with MSNs-DOX. The reason for this phenomenon was that MSNs could facilitate the entry of the encapsulated drugs through the carrier mediated endocytosis pathway. However, the highest fluorescence signal was found to be within the cells treated with LMSNs-DOX, which indicates that LMSNs could further improve the accumulation of DOX in MCF-7 cells by promoting the cellular uptake efficiency of the drug loaded carriers. After entering the cells, DOX was gradually released from the nanoparticles as confirmed by the wide distribution of red fluorescence throughout the entire cytoplasm and the final location at the nucleus. To summarize, the presence of the lipid bilayer can promote the cellular internalization of LMSNs nanoparticles and the encapsulated drugs, and the lipid bilayer will not hinder the intracellular drug release, which are very helpful for improving the therapeutic efficacy.
3.3. Cell internalization and intracellular drug release 3.4. Cell viability The efficient cellular uptake of the drug loaded carriers and the preferably intracellular release of drugs are crucial for the drug delivery and an effective therapy. To investigate the effect of the lipid bilayer on the cellular uptake efficiency of MSNs, the FITC labeled MSNs and LMSNs were prepared and allowed to incubate with MCF-7 cells for 2 h. And the fluorescence signal within cells observed by CLSM was shown in Fig. 7. For MSNs, the green fluorescence signal was rather weak, and the comparably low uptake efficiency was mainly due to the repulsive force between the negatively charged MSNs and the cell membrane which is the same to carry a negative charge. Therefore, there was an energy barrier to overcome for the internalization of MSNs by the cells. In contrast,
To monitor the effect of the lipid bilayer coating on the biocompatibility of the carrier and the therapeutic efficacy of the encapsulated drug, the cell viability of MCF-7 cells after incubation with various DOX formulations and the corresponding amount of blank MSNs and LMSNs was evaluated by MTT essay. And the IC50 (concentration of test samples to inhibit cell growth by 50%) was calculated using SPSS software 13.0 (SPSS Inc., Chicago, IL, USA). The results are listed in Table 2. The profile in Fig. 9A shows that the cytotoxic effect of blank MSNs on MCF-7 was much higher compared with that of LMSNs. The cell viability of the group treated with MSNs at the highest test concentration decreased dramatically
Fig. 7. Confocal microscopy images of MCF-7 cells after treatment with FITC-labeled MSNs and LMSNs for 2 h. Scale bar represents 50 mm.
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Fig. 8. Internalization and accumulation of DOX in MCF-7 cells after treatment with DOX-Sol, DOX-MSNs and DOX-LMSNs for 2 h measured by confocal microscopy. Scale bar represents 50 mm.
to around 62% after an incubation time of 48 h. While for the group cultured with LMSNs at the same concentration, the cell viability remained above 90%. It has been reported that the abundant silanol groups on the outer surface of MSNs may cause structural damage to the membrane lipids and proteins [27]. However, after the coverage of the lipid bilayer, the cytotoxic effect of the nanoparticles was significantly reduced due to the concealing of the silanol groups and the excellent biocompatibility of natural lipids. The cytotoxicity of all DOX formulations was found to be concentration-dependent. The IC50 of DOX-Sol was 0.686 mg/mL, 1.7 times higher than the IC50 of MSNs-DOX calculated to be 0.401 mg/ mL, demonstrating that nanoparticles can assist the entry of the loaded drugs into cells through the carrier mediated endocytosis. In addition, after the coverage of the lipid bilayer, the cytotoxic effect of LMSNs-DOX further increased by 1.3 fold and the IC50 decreased to 0.306 mg/mL. Reasons for this result were as follows. On one Table 2 The IC50 values of different DOX formulations against MCF-7 cells. Formulations
DOX-Sol
MSNs-DOX
LMSNs-DOX
IC50 (mg/mL)
0.686
0.401
0.306
hand, the lipid bilayer could facilitate the cellular uptake of the DOX-loaded nanoparticles due to the high affinity with cell membrane which was in agreement with the phenomenon observed in Fig. 8. On the other hand, the retardant effect of the lipid bilayer could suppress the pre-leakage of DOX during the incubation process and achieve an acid-triggered DOX release after being internalized into the cytoplasm of cancerous cells. 3.5. Hemolysis measurement and BSA absorption The hemolysis percentage of MSNs and LMSNs over the concentration range from 20 to 1500 mg/mL is shown in Fig. 9B. For blank MSNs, the hemolysis ratio of RBCs escalated with the increasing of the tested concentration. When the concentration of MSNs reached 500, 1000 and 1500 mg/mL, the hemolysis ratio was 16.5%, 43.6% and 65.8%, respectively. This severe hemolysis puts great limitations to the safe application of MSNs as intravenous drug delivery systems. The reason for the silica-induced hemolysis was that the silica has high affinity to bind with the tetra-alkyl ammonium groups abundant on RBCs membranes. And this property could subsequently lead to the generation of reactive oxygen species (ROS) and the denaturation of membrane proteins after
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Fig. 9. (A) Cytotoxicity of various DOX formulations and corresponding amount of blank MSNs and LMSNs against MCF-7 cells at serial concentrations. (B) Hemolysis percentage of MSNs and LMSNs at different concentrations (mg/mL).
close contact with silicate [28,29]. However, after being covered by the lipid bilayer, the LMSNs nanoparticles exhibited stable dispersibility in saline solution containing 2% blood cells (V/V). The hemolysis percentage was negligible at low concentrations and remained below 10% even at the highest concentration of 1500 mg/ mL. Since the lipid bilayer can shield the silanol groups and prevent them coming into contact with the RBCs membranes, the biocompatibility of the nanoparticles was dramatically improved making them more reliable for intravenous drug delivery. It is widely accepted that nanoparticles will be rapidly cleared by the RES systems once a mass of proteins in the serum is absorbed on the particle surface which is very undesirable for the accumulation of drug-incorporated carriers at the disease site. In order to evaluate the effectiveness of lipid bilayer coating in preventing the nonspecific protein absorption, the amount of BAS adsorbed on the surface of MSNs and LMSNs was quantified. The degree of BSA absorption was calculated to be 2.06 wt.% for MSNs, and this markedly fell to 0.33 wt.% after the lipid bilayer coating for LMSNs. Such phenomenon would be attributed to the steric hindrance provided by the PEG modified lipid bilayer. Hence, the lipid bilayer coating was a simple and effective way to reduce the nonspecific protein adsorption and improve the dispersing stability and biocompatibility of MSNs. And the PEG modified lipid bilayer could serve to prolong the circulation time and improve the passive targeting efficiency of the MSNs based nanocarriers through the EPR effect. 4. Conclusion Lipid bilayer coating is a facile and effective way to functionalize MSNs. The obtained LMSNs have exhibited many appealing features, such as greatly improved dispersing stability and cellular internalization as well as markedly decreased non-specific protein absorption, hemolytic activity and cytotoxicity. Also, when DOX was used as a model drug, it could be encapsulated within LMSNs with a high LE % and be preferentially released in the cytoplasm of cancerous cells. Furthermore, the LMSNs nanoparticles could increase the cytotoxic effect of DOX toward cancerous cells by elevating the cellular accumulation of the drug loaded carriers. In conclusion, the presence of lipid bilayer could mitigate the drawbacks of MSNs making them more reliable in the application of drug delivery. And the prepared LMSNs have shown to be a
promising drug carrier with improved biocompatibility, a high LE % and the ability to promote the cellular uptake of the encapsulated drugs to achieve better therapeutic efficacy. Conflicts of interest The authors report no conflicts of interest. Acknowledgments We thank Dr. Xiaoju Huang and Dr. Gang Ji for technical support in cryo-TEM sample preparation and data collection. We also gratefully acknowledge the use of TEM facilities at the Center for Biological Imaging (CBI), Institute of Biophysics, Chinese Academy of Science. This work was supported by National Basic Research Program of China (973 Program) (No. 2015CB932100), National Natural Science Foundation of China (No. 81473165), and Basic Research Project of Key Laboratory of Liaoning Provincial Education Department (No. LZ2015068). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.micromeso.2015.08.006. References [1] Z. Deng, F. Yan, Q. Jin, F. Li, J. Wu, X. Liu, H. Zheng, J. Control. Release 174 (2014) 109e116. [2] J. Wang, J. Sun, Q. Chen, Y. Gao, L. Li, H. Li, D. Leng, Y. Wang, Y. Sun, Y. Jing, S. Wang, Z. He, Biomaterials 33 (2012) 6877e6888. [3] Y. Zhang, Z. Zhi, T. Jiang, J. Zhang, Z. Wang, S. Wang, J. Control. Release 145 (2010) 257e263. [4] H. Zhu, H. Chen, X. Zeng, Z. Wang, X. Zhang, Y. Wu, Y. Gao, J. Zhang, K. Liu, R. Liu, Biomaterials 35 (2014) 2391e2400. [5] F. Tang, L. Li, D. Chen, Adv. Mater. 24 (2012) 1504e1534. [6] A. Kierys, M. Rawski, J. Goworek, Microporous Mesoporous Mater. 193 (2014) 40e46. [7] Y. Wang, Q. Zhao, N. Han, L. Bai, J. Li, J. Liu, E. Che, L. Hu, Q. Zhang, T. Jiang, S. Wang, Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 313e327. [8] P. Yang, S. Gai, J. Lin, Chem. Soc. Rev. 41 (2012) 3679e3698. [9] Q. Zhao, C. Wang, Y. Liu, J. Wang, Y. Gao, X. Zhang, T. Jiang, S. Wang, Int. J. Pharm. 477 (2014) 613e622.
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[20] P. Decuzzi, B. Godin, T. Tanaka, S.-Y. Lee, C. Chiappini, X. Liu, M. Ferrari, J. Control. Release 141 (2010) 320e327. [21] S. Savarala, S. Ahmed, M.A. Ilies, S.L. Wunder, Langmuir 26 (2010) 12081e12088. [22] M.M. van Schooneveld, E. Vucic, R. Koole, Y. Zhou, J. Stocks, D.P. Cormode, C.Y. Tang, R.E. Gordon, K. Nicolay, A. Meijerink, Nano Lett. 8 (2008) 2517e2525. [23] S. Hak, E. Helgesen, H.H. Hektoen, E.M. Huuse, P.A. Jarzyna, W.J. Mulder, O. Haraldseth, ACS Nano 6 (2012) 5648e5658. [24] Y. Zhu, T. Ikoma, N. Hanagata, S. Kaskel, Small 6 (2010) 471e478. [25] M. Xie, H. Shi, Z. Li, H. Shen, K. Ma, B. Li, S. Shen, Y. Jin, Colloids Surf., B: Biointerfaces 110 (2013) 138e147. [26] W.M. Li, S.Y. Chen, D.M. Liu, Acta Biomater. 9 (2013) 5360e5368. [27] Z. Tao, B.B. Toms, J. Goodisman, T. Asefa, Chem. Res. Toxicol. 22 (2009) 1869e1880. [28] I.I. Slowing, C.W. Wu, J.L. Vivero-Escoto, V.S.Y. Lin, Small 5 (2009) 57e62. [29] T. Yu, A. Malugin, H. Ghandehari, ACS Nano 5 (2011) 5717e5728.
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Microporous and Mesoporous Materials journal homepage: www.elsevier.com/locate/micromeso
Facile synthesis of the lipid bilayer coated mesoporous silica nanocomposites and their application in drug delivery Ning Han a, 1, Yu Wang b, 1, Junling Bai a, Jiangjun Liu a, Ying Wang a, Yikun Gao c, Tongying Jiang a, Wanjun Kang b, Siling Wang a, * a
Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning Province 110016, PR China Chinese PLA No. 463 Hospital, Shenyang, Liaoning Province 110000, PR China c School of Medical Devices, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning Province 110016, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 May 2015 Received in revised form 5 August 2015 Accepted 6 August 2015 Available online 14 August 2015
The lipid bilayer coated mesoporous silica nanocomposites (LMSNs) were synthesized with aim to obtain better performance in the application of drug delivery. Phospholipids composed of soybean lecithin and DSPE-PEG 2000 were pre-prepared into liposomes, then they were allowed to fuse onto the mesoporous silica nanoparticles (MSNs) forming a surrounding lipid bilayer. The obtained LMSNs had an average particle size of 295 nm, zeta potential of 1.0 mV and a good dispersing stability in saline buffers. Facilitated by the affinity of the lipid bilayer with cell membrane, the internalization of LMSNs by cells was markedly increased. In addition, compared with bare MSNs, the cytotoxicity, hemolysis percentage and nonspecific BSA absorption of LMSNs were significantly reduced, making them become more reliable carriers for drug delivery. When encapsulating a model drug, doxorubicin (DOX) into LMSNs, the loading efficiency can reach as high as 16%. The obtained LMSNs-DOX exhibited a pH-responsive release behavior and the presence of the lipid bilayer did not significantly retard the release of DOX. Furthermore, LMSNs greatly enhanced the cellular accumulation and cytotoxicity of DOX toward the MCF-7 cells. In summary, the lipid bilayer coating was a simple and facile strategy to functionalize MSNs, and the obtained LMSNs exhibiting good biocompatibility were promising nanocarriers in improving the cellular uptake and therapeutic efficacy of anticancer drugs. © 2015 Published by Elsevier Inc.
Keywords: Mesoporous silica Lipid bilayer Drug delivery Cellular uptake Biocompatibility
1. Introduction In recent decades, a variety of nanostructured materials, such as liposomes, micelles and organic/inorganic nanoparticles have attracted great interest as intelligent drug delivery systems and showed promising prospects in a broad range of therapeutic applications [1e4]. Among these, mesoporous silica nanoparticles (MSNs) are particularly attractive due to their unique properties, including a large surface area and pore volume to load drug with high efficiency [5,6]; a uniform and tunable pore size to accommodate molecules with various steric hindrance and an excellent physiochemical stability to protect the encapsulated drugs from degradation by endogenous enzymes [7].
* Corresponding author. Tel./fax: þ86 24 23986348. E-mail address: [email protected] (S. Wang). 1 Authors contributed equally. http://dx.doi.org/10.1016/j.micromeso.2015.08.006 1387-1811/© 2015 Published by Elsevier Inc.
Recently, MSNs have been widely employed as nanocarriers to achieve stimuli-responsive drug release and targeted drug delivery [8e10]. However, the application of MSNs as drug carriers has been limited by some of their properties. Such as easy aggregation in saline buffers, rapid clearance by the reticuloendothelial system (RES) and the risk of inducing hemolysis when given intravenously, a common route of administration [11]. Therefore it is necessary to functionalize MSNs in order to counteract the above disadvantages. Surface modification with polymers, dendrimers [12] or natural materials through chemical reaction or electrostatic attraction (such as layer by layer technique) [13] was reported to be an effective way to change the properties of nanoparticle for various applications. However, previous studies devoting to improve the biocompatibility of MSNs as drug carriers by surface modification were limited and rare, including using polymers such as polyethylene glycol (PEG) or poly(N-vinylcaprolactam-co-methacrylic acid) (P(VCL-s-s-MAA)) [14]. But the functionalization process was complicated and needed to be optimized. In addition, there has
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always been a contradiction in the priority of order between the drug loading and surface functionalization process. If the functionalization comes first, it may cause a reduction in the surface area and pore volume which will adversely affect the drug loading efficiency of MSNs. While if the drug loading is a priority, there will be an inevitable loss of the loaded drugs during the long time chemical reaction and repeated purifying process which are necessary for the successful functionalization. Therefore, it is essential to find a simple and effective approach to modify the MSNs and overcome the above drawbacks of them without adversely affecting the drug loading capacity. Furthermore, as a drug carrier, it is also of great importance to improve the cellular accumulation of the incorporated drugs within the targeted cells to reach effective therapeutic level. Inspired by the supported lipid bilayer which mimics the cell membrane, the lipid bilayer coating can be an innovative functionalization on MSNs by fusion on the surface of them. The preparation method was based on the previous findings by Mornet et al., in which the pre-prepared liposomes can first adhere to MSNs, then undergo gradual deformation, finally rupture and spread all over the nanoparticles to form a continuous coverage [15]. Reasons for using the lipid bilayer coating on mesoporous silica nanoparticles (LMSNs) are as follows: 1) lipids are wellrecognized to be highly biocompatible; 2) the lipid bilayer has a strong affinity with the cell membrane which is advantageous for the subsequent internalization by cells; 3) the PEG modified lipid bilayer can prolong the circulation time and reduce the immunogenicity of MSNs, thereby facilitating the accumulation of drugloaded carriers at the tumor site via the enhanced permeability and retention (EPR) effect. Moreover, compared with other functionalization methods, the lipid bilayer coating is rather simple and facile. Since it can be completed within a short period of time, the loss of the loaded drugs can thereby be markedly reduced. This integrated drug delivery system is expected to inherit the merits from both MSNs and liposomes and exhibit characteristics superior to those of any of its single component. In the nanocomposites, MSNs acting as a drug reservoir can accommodate various drug molecules with a high payload and serve as a supporting skeleton to stabilize the lipid bilayer. While the lipid bilayer coating can in turn serve to improve the dispersing stability and biocompatibility of MSNs and make them more applicable as drug carriers by mitigating their disadvantages. In several other studies, the lipid bilayer present on MSNs was designed as a “gatekeepers” to control drug release or an intermediate for further conjugation of various targeting ligands. There are rarely any literature studied the effect of mere lipid bilayer coating on the properties of MSNs as drug carriers [16e18]. In this study, we innovatively investigated the effect of the lipid bilayer coating on the applicability of MSNs as carriers for intravenous drug delivery. And the superiority of the prepared LMSNs was systematically evaluated by comparing them with bare MSNs in terms of dispersing stability, cellular uptake efficiency and biocompatibility. The prepared LMSNs were then employed to load doxorubicin (DOX), one of the most potent drugs used in clinical chemotherapy. And the in vitro drug release, cellular internalization, and cytotoxicity of LMSNs-DOX were systematically investigated. 2. Materials and methods
Holding Co., Ltd (Tianjin, China). Poloxamer 407 (Lutrol® F-127) was obtained as gift sample from BASF (Ludwigshafen, Germany), Soybean lecithin and cholesterol were purchased from LIPOID GmbH (Ludwigshafen, Germany). 1,2-Distearoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG2000-DSPE) and Rhodamine B PE was obtained from Avanti Polar Lipids (Alabaster, AL). Anticancer drug doxorubicin hydrochloride (DOX) was offered by Zhejiang Hisun Pharmaceutical Co., Ltd (Zhejiang, China). RPMI 1640, fetal bovine serum (FBS) and penicillin/streptomycin solution were attained from GIBCO (BRL, MD, USA). Bovine serum albumin (BSA), fluorescein isothiocyanate (FITC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and trypsin were purchased by SigmaeAldrich (St. Louis, MO). Fluorescent Hoechst 33258 was obtained from Molecular Probes (Eugene, OR, USA). All other chemicals were of analytical grade and used without further purification. 2.2. Synthesis of mesoporous silica nanoparticles (MSNs) As reported previously, mesoporous silica nanoparticles were €ber method [19]. In brief, 0.5 g prepared based on the modified Sto CTAB and 1.0 g triblock copolymer F127 were dissolved in 100 mL distilled water, in which 10 mL 29 wt.% ammonium hydroxide solution and 40 mL ethanol were subsequently added. After complete dissolution, 1.9 mL TEOS was then introduced dropwise to the mixture under intensive stirring, and the reaction was maintained at room temperature for 24 h. The white solid product was collected by centrifugation at 10,000 rpm. For template extraction, the synthesized nanoparticles were re-dispersed in 100 mL absolute ethanol containing 1 g NH4NO3, and allowed to reflux at 80 C overnight with constant stirring to remove the surfactant. The final product was centrifuged, washed with ethanol for 3 times and dried overnight at room temperature in vacuum. 2.3. Preparation of liposomes Liposomes were prepared by the lipid film hydration method, in the experiment 100 mg soybean lecithin, 10 mg DSPE-PEG 2000 and 20 mg cholesterol were dissolved in 5 mL chloroform and evaporated to form a thin lipid film using a rotary evaporator. Then the lipid film was hydrated in PBS of pH 7.4 at concentration of 10 mg/mL and was then extruded for 15 cycles using a mini extruder (Avanti Polar Lipids, Inc., USA) equipped with progressively decreasing pore-sized polycarbonate membrane of 200 nm and 100 nm. The prepared liposomes for further experiment were stored at 4 C for no more than a week. 2.4. Development of the lipid bilayer-mesoporous silica nanocomposites (LMSNs) The LMSNs were prepared by first re-dispersing 20 mg of dried MSNs in 2 mL distilled water under ultrasonic irradiation for 1 min. Then 1 mL of the pre-prepared lipid vesicles was added to the particle suspension, the mixture was well vortexed and left to stand for 1 h at room temperature to allow thorough fusion of liposome on MSNs. Extra liposomes in the supernatant were removed via centrifugation at 10,000 rpm for 10 min. After being washed with PBS for three times, the formed LMSNs were finally dispersed in the PBS solution.
2.1. Materials 2.5. Drug loading process and loading efficiency measurement Tetraethyl orthosilicate (TEOS, 98%), hexadecyl-trimethyl ammonium bromine (CTAB, >99%) and ammonium hydroxide (NH4OH, 25e28%) were obtained from Tianjin Bodi Chemical
Drug loading was conducted prior to the lipid bilayer coating, 20 mg of the dried MSNs was ultrasonically re-suspended in 2 mL of
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DOX solution dissolved in distilled water (5 mg/mL) and incubated for 24 h under intermittent stirring to fully absorb the drugs. To obtain LMSNs-DOX, the DOX loaded MSNs were first centrifuged, then the lipid vesicles were added to fuse onto MSNs following the procedure as described in Section 2.3. Samples denoted as MSNsDOX which were prepared in the same way but without lipid bilayer coating was used as control groups in the subsequent experiments. The drug loading efficiency was calculated according to the formula. The weight of encapsulated DOX can be determined by subtracting the mass of DOX in supernatant from the total mass of drug in the initial solution.
LE % ¼
weight of encapsulated DOX 100% weight of DOX loaded LMSNs
2.6. Characterization of the nanoparticles The structure and morphology of MSNs were characterized by the field-emission transmission electron microscopy (TEM) using FEI Tecnai G2 F30 (Netherlands) operated at an acceleration voltage of 200 kV and the scanning electron microscopy (SEM) using ZEISS SUPRA 35 (Germany) after spraying the desiccative nanoparticles with gold on a carbon grid. In order to confirm the lipid bilayer present on MSNs, the cryo-TEM images of LMSNs were taken by depositing 10 mL aliquots of the prepared LMSNs suspension on the cupper grids enveloped with carbon film and quickly frozen in liquid ethane. The grids were placed onto a Gatan 626 cryo-holder and inserted into an FEI Tecnai F20 microscope for observation. Confocal fluorescence images of the FITC-labeled MSNs, Rhodamine B-labeled lipid bilayer, and the merged image were taken by Leica DM-6000 CS microscope (Leica Instruments Inc., Wetzlar, Germany) to further confirm the presence of lipid bilayer on MSNs. The nitrogen adsorption/desorption analysis was conducted to detect the specific surface area and the pore size of the carriers using an adsorption analyzer (V-Sorb 2800P, China). Thermogravimetric analysis (TGA) was also performed on the TGA-50 instrument (Shimadzu, Japan) to investigate the weight loss after the lipid bilayer coating with a heating rate of 5 C/min under a nitrogen flow. The differential scanning calorimeter (DSC) was employed to measure the gel-to-fluid phase transition temperature (Tm) of lipid bilayer on MSNs using DSC 1 STARe (Mettler Toledo, Switzerland). Samples of 40 mL containing approximately 2 mg lipids were placed in the aluminum pan and the experiment was performed from 30 C to 15 C at a heating rate of 2 C/min under a nitrogen flow of 40 ml/min. And the particle size distribution and zeta potential of the MSNs and LMSNs were recorded using the Zetasizer Nano (Malvern Instruments Ltd., United Kingdom).
2.7. Drug release behavior of DOX from MSNs, and LMSNs The release study of DOX from MSNs and LMSNs was performed in pH 7.4 and pH 5.0 PBS. Formulations containing DOX relevant to 3 mg were transferred into a dialysis bag (MWCO 14,000) and sealed with a dialysis bag holder, the dialysis bags were then immersed into 30 mL PBS with constant shaking at 100 rpm/min at 37 C. At predetermined time intervals, 1 mL of the release medium was withdrawn to monitor the release extent of DOX by an UVeVis spectrophotometer (UV-2000, UNICO, USA) at a wavelength of 480 nm using a pre-established calibration curve. Simultaneously the fresh medium with the same volume was replenished. The results were averaged with three measurements.
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2.8. Cell culture The human breast cancer cell line MCF-7 used in this study was purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in RPMI 1640 medium containing 10% (v/v) fetal bovine serum (FBS) and 1% penicillinestreptomycin at 37 C in a humidified atmosphere with 5% CO2. 2.9. In vitro cellular uptake To compare the cellular uptake efficiency of blank MSNs and LMSNs, and investigate the intracellular accumulation of DOX encapsulated MSNs and LMSNs, MCF-7 cells were seeded on round glass coverslips in 24-well plates. After 12 h of attachment, the culture medium was replaced with fresh serum-free RPMI 1640 containing FITC-labeled blank carriers at 20 mg/mL or various DOX formulations containing DOX of 5 mg/mL. After incubation for 2 h, the cells were rinsed with cold PBS for three times, and fixed with 4% formaldehyde. The nuclei were afterward stained by Hoechst 33258 for 20 min and the fixed cells were observed using CLSM. 2.10. Cell viability assessment The in vitro cell viability of MCF-7 cells after incubation with the blank nanocarriers and DOX-encapsulated samples was evaluated by MTT assay. Briefly, cells were seeded into 96-well plates at a density of 1.0 104 cells/well and attached for 24 h. Then the culture medium was removed and cells were subject to DOX-Sol, MSNs-DOX and LMSNs-DOX nanocarrier solutions in serum free medium at equivalent DOX concentrations of 0.01, 0.1, 1, 10, 100 mg/ mL to investigate the cytotoxic effect of different DOX formulations. In addition, the cytotoxicity of the corresponding amount of blank MSNs and LMSNs contained in the DOX formulations at serial tested concentrations were also examined. At the time point of 48 h, 50 mL of MTT solution (2 mg/mL) was introduced to the culture medium and incubated for extra 4 h to quantify cell viability. The supernatant was gently removed and 150 mL of DMSO was added to each well. After being agitated for 10 min to completely dissolve the formed formazan crystals, the 96-well plates were finally placed into an iMark™ microplate reader (Bio-RAD, CA, USA) to monitor the absorbance at wavelength of 570 nm. Cells without treatment were set as control. 2.11. Hemolysis study and BSA absorption measurement In order to assess and compare the safety of MSNs and LMSNs for in vivo applications, hemolysis evaluation was performed by isolating the rabbit red blood cells (RBCs) at 1000 rpm for 10 min and washed three times with sterile saline. The centrifuged RBCs were then diluted with saline to a concentration of 2% (v/v). Afterward, 2 mL of MSNs and LMSNs suspension in saline containing various amount of nanoparticles was added to equal volume of RBCs suspension to make the final nanoparticle concentrations of 20, 50, 100, 200, 500, 1000 and 1500 mg/mL. The mixture was briefly vortexed before left static for 4 h at room temperature. After that, the mixture was centrifuged and the clear supernatant was measured at 541 nm on an UVevis spectrophotometer. The hemolysis of RBCs incubated with distilled water and saline were set as the positive and negative control, separately. Hemolysis percentage was ascertained as follows: hemolysis (%) ¼ (sample absorbance absorbance of negative control)/(absorbance of the positive control absorbance of negative control) 100%. All samples were measured in triplicate. The BSA absorption measurements were performed according to the procedure reported previously to simulate the protein
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Scheme 1. Schematic representation of lipid bilayer coated mesoporous silica nanocomposites (LMSNs).
Fig. 1. SEM (A) and TEM (B) images of MSNs, cryo-TEM image of LMSNs (C).
absorption on silica under physiological condition. Typically, 30 mg BSA was dissolved in 50 mL of deionized water. After dispersing 10 mg of MSNs and LMSNs in saline buffer with concentration of 2 mg/mL, 5 mL of BSA solution was subsequently added. The mixture was put into a shaker with a shaking rate of 135 rpm at 37 C. After a time period of 4 h, 1 mL of Coomassie brilliant blue solution was added to the collected supernatant following the centrifugation of the above mixture. The BSA concentration was quantified by measuring the absorbance at 595 nm on an UVeVis spectrophotometer. And the adsorbed BSA was calculated according to the following equation: q ¼ (Ci Cf)V/m, where Ci and Cf respectively represent the initial and final BSA concentration in the solution. V means the total volume of the mixture and m is the weight of nanoparticles added. 3. Results and discussion 3.1. Preparation and characterization of LMSNs The preparation process of LMSNs was shown in Scheme 1. Monodisperse spherical MSNs were firstly synthesized following
the previously reported method using TEOS as the silica source, CTAB as the pore template and triblock copolymer F127 as a dispersing agent and particle size tailor. MSNs with a range of particle sizes can be prepared by altering the amount of F127 added. The more F127 used, the smaller the size of the MSNs can be obtained [19]. It was reported in many other studies that nanoparticles within the size range of 50e500 nm can guarantee an ideal EPR effect [20]. Hence MSNs with an intermediate particle size of 200 nm were prepared by adding a moderate amount of F127, giving weight ratio of CTAB which was 2:1. The resultant MSNs had a uniform size of ca. 200 nm as can be seen in the SEM image (Fig. 1A). Also, the two-dimensional pore arrangement of MSNs can be clearly observed in the TEM images (Fig. 1B). For LMSNs, cryoTEM images were taken to confirm the presence of lipid bilayer on the periphery of MSNs (Fig. 1C). As can be seen, the LMSNs appeared to be surrounded by an electron-dense ring of about 7 nm, which corresponded to the thickness of lipid bilayer demonstrating the successful coverage of the lipid bilayer on MSNs by the fusion method. Fig. 2 shows the confocal image of LMSNs, where the silica core was labeled with FITC and the phospholipid was labeled with
Fig. 2. CLSM images of LMSNs, FITC labeled MSNs (A), Rhodamine B labeled lipid bilayer (B) and merged image (C).
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Fig. 3. Nitrogen adsorptionedesorption isotherms and pore diameter distribution of MSNs and LMSNs.
Rhodamine B. After merging images of A and B in Fig. 2, the green and red fluorescence completely overlapped, which further confirmed the lipid bilayer on MSNs. According to the nitrogen adsorptionedesorption isotherm curves and pore size distribution of MSNs and LMSNs shown in Fig. 3, the blank MSNs had a total surface area of 800 m2/g, a cumulative pore volume of 1.0 cm3/g and an average pore size of 2.3 nm. After incubating with the pre-prepared liposomes for 1 h, the surface area of MSNs markedly reduced to 50 m2/g, and the pore volume became negligible further supporting the successful coating of the lipid bilayer. Thereafter, drug loading should be performed prior to the lipid bilayer covering on MSNs. The Fourier transform infrared spectroscopy (FT-IR) was further used to confirm the presence of lipid bilayer on MSNs, and the corresponding data was provided in Supplementary data. Thermogravimetric analysis (TGA) was also conducted to investigate the weight percentage of the lipid bilayer on MSNs and the resultant curves are shown in Fig. 4A. Over the temperature range from 50 C to 150 C hardly any weight loss was observed for MSNs, indicating that the surfactant templates have been completely removed. After incubating the MSNs with liposomes for 0.5 h, a weight loss of about 32.4% was observed for LMSNs, which was due to the lipid bilayer existing on MSNs. On extending the incubation time to 1 h, the weight loss only increased by 2.7%, which implied that the lipid bilayer coating on MSNs can almost be
completed within 1 h. Since it can be completed within such a short period of time, the loss of the pre-loaded drugs was greatly eliminated. As seen from the curve of MSNs-DOX, when incorporating DOX into the MSNs, the loading efficiency can reach 23%. And after the lipid bilayer coating, the prepared LMSNs-DOX showed an increased weight loss of about 53%, demonstrating that drug loading would not affect the lipid bilayer coating on MSNs. The DSC results in Fig. 4B showed that the prepared liposome had a Tm of 20.23 C, higher than the Tm of 21.53 C and 21.83 C for the LMSNs and LMSNs-DOX, respectively. It was reported that the reduction in Tm is an indicator of the forming of the lipid bilayer on MSNs, since the fluidity of lipid bilayer will increase due to the interfacial interaction with the porous silica support [16,21]. Therefore, based on above the results, the lipid bilayer can be formed on both the drug free and drug loaded MSNs. And the lipid bilayer was in the fluid phase at the experimental temperature. The change in particle size (Fig. 5A) also confirmed the result above. The MSNs had a hydrodynamic diameter of ca 254 nm, a little larger than the diameter observed in TEM due to the existence of a hydration layer in aqueous solutions. As for LMSNs, the hydrodynamic diameter increased to 295 nm which illustrated the presence of the lipid bilayer on MSNs. And as shown in Fig. 5B, MSNs had a zeta potential of 25 mV due to the ionization of the silanol groups on the surface. While after the lipid bilayer covering, the zeta potential of LMSNs increased to around 0 mV due to the
Fig. 4. The TGA curves of MSNs, LMSNs after 0.5 h and 1 h incubation with liposomes, MSNs-DOX and LMSNs-DOX (A); the DSC curves of liposome, LMSNs and LMSNs-DOX (B).
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Fig. 5. The particle size (A), zeta potential (B), and dispersing stability in PBS (C) of the DOX free and DOX loaded MSNs and LMSNs.
shielding effect of the neutrally charged and PEG grafted lipid bilayer. And the same changing trend in particle size and zeta potential was also observed when preparing the LMSNs-DOX, indicating that the lipid bilayer coating could be well performed on the drug loaded MSNs. It is worth noting that MSNs with a charge stabilized property via the ionization of hydroxyl groups has confronted limitations in their bio-applications. It was found that MSNs can be destabilized easily and aggregated readily in saline buffers with a high ionic strength (Fig. 5C). And the aggregation will expedite following the absorption of the proteins in the serum. Such phenomenon can consequently lead to the rapid clearance of nanocarriers by the reticuloendothelial system (RES) and cause potential safety hazard, which is certainly unwanted for intravenous administration [22]. However, after covering the MSNs with the PEG-modified lipid bilayer, the silanol groups were concealed and the obtained LMSNs can be stably dispersed in PBS (Fig. 5C). And the hydrodynamic diameter of LMSNs in PBS was almost the same with that in water. It indicated that the PEG-modified lipid bilayer can greatly improve the dispersing stability of MSNs, a premise for the safe administration of nanoparticles through the intravenous route. Also, the PEG chains on the lipid bilayer can form mushroom-like clusters on the nanocarriers to prevent them from being recognized and captured by the RES. And a prolonged circulation time and enhanced accumulation of drug loaded carriers at the tumor site can thus be achieved by the EPR effect [23].
were quite stable under the same condition, less than 10% of the incorporated drug was released within 24 h due to the drug diffusion being hindered by the lipid bilayer. When exposing the DOX-MSNs and DOX-LMSNs to pH 5.0 PBS, the release rate of DOX from both carriers markedly accelerated and the release percentage at 24 h was 72% and 68%, respectively. This acid triggered release can be explained as that the electrostatic interactions between the MSNs and DOX were pH-dependent, DOX molecules tended to detach from MSNs more easily under low pH conditions [24,25]. As for LMSNs, a comparable sustained release of DOX can be observed at pH 5.0 demonstrating that the presence of the lipid bilayer can only slightly retard the release of DOX due to the diffusion hindrance it provided. But the lipid bilayer will not totally block the release of DOX at the acidic diseased site, since the drug release driven by the concentration gradient overwhelmed the retardation effect provided by the lipid bilayer. As a consequence, most of the encapsulated DOX was released within 24 h. In summary, compared with MSNs, LMSNs had a stronger retaining effect of DOX in normal physiological conditions and same release responsiveness to the pH change in the environment. Since the pH value in cancer cells and the endosomal compartment is lower than the pH level in healthy cells [2], a preferential release of DOX at the tumor site can be achieved. To study the kinetics mechanism of DOX release from MSNs and LMSNs, equations of zero order, first order, Higuchi and Weibull
3.2. Drug loading efficiency and pH-responsive drug release Drug loading was carried out before the lipid bilayer coating on MSNs. Since the MSNs have a large surface area and pore volume, they can accommodate a loading of as much as 22% DOX molecules by physical absorption, consistent with the result measured by TGA. After coating with the lipid bilayer, the drug loading efficiency decreased to 16%. By tracking the concentration of DOX in the supernatant during the coating process, we found that the loss of drug was negligible, it was the increased weight after the lipid bilayer coverage that led to the decline of LE% in the calculation. Even so, the LE% of DOX-LMSNs was still higher than that of the DOX encapsulated liposomes prepared by the conventional ammonium phosphate gradient method which had a loading efficiency of 9%. The in vitro release behavior of LMSNs-DOX was subsequently investigated. The DOX-loaded LMSNs were placed in pH 5.0 and pH 7.4 PBS and the release profiles are shown in Fig. 6. For comparison, the release of MSNs-DOX was also assessed. As can be seen, DOXMSNs exhibited a gradual release of DOX in pH 7.4 PBS and the release extent was as high as 22% after 24 h. While DOX-LMSNs
Fig. 6. In vitro DOX release from MSNs and LMSNs in pH 7.4 and pH 5.0 PBS.
N. Han et al. / Microporous and Mesoporous Materials 219 (2016) 209e218 Table 1 In vitro release kinetics parameters. Formulations
MSNs-DOX pH 7.4 LMSNs-DOX pH 7.4 MSNs-DOX pH 5.0 LMSNs-DOX pH 5.0
Zero-order
First-order
Higuchi
R2
R2
R2
KH
Weibull R2
0.8116 0.9349 0.8352 0.8791
0.8300 0.9405 0.9305 0.9485
0.9701 0.9963 0.9879 0.9811
6.391 2.555 21.47 20.60
0.9438 0.9775 0.9725 0.9664
were utilized to fit the accumulative release data. The obtained results were listed in Table 1. The highest value of regression coefficient (R2) was observed when the release profiles were described using the Higuchi model (Eq. (1)), in which the drug release from carriers is proportional to the square root of time. Q is the amount of drug released in time t, and KH is the Higuchi rate constant. According to the equation, it is feasible to believe that the release mechanism of DOX from these particles was diffusion controlled [26]. In addition, the KH of MSNs-DOX was larger than that of LMSNs-DOX in both PBS of pH 7.4 and pH 5.0, which again confirmed the slight barrier effect of the lipid bilayer during the drug release process.
Q ¼ KH t 1=2
(1)
215
the fluorescence intensity in cells treated with LMSNs was significantly enhanced demonstrating that the presence of the lipid bilayer can greatly promote the cellular uptake of silica nanoparticles. The reason for this was that the neutrally charged lipid bilayer could reach the cell surface more easily and possessed an inherent affinity with the cell membrane since they have the similar structure. So it is favorable for the adhesion and internalization of nanoparticles. The intracellular release of DOX was also examined using the CLSM. As can be seen in Fig. 8, the red fluorescence of DOX in cells treated with free DOX was not as strong as that in those treated with MSNs-DOX. The reason for this phenomenon was that MSNs could facilitate the entry of the encapsulated drugs through the carrier mediated endocytosis pathway. However, the highest fluorescence signal was found to be within the cells treated with LMSNs-DOX, which indicates that LMSNs could further improve the accumulation of DOX in MCF-7 cells by promoting the cellular uptake efficiency of the drug loaded carriers. After entering the cells, DOX was gradually released from the nanoparticles as confirmed by the wide distribution of red fluorescence throughout the entire cytoplasm and the final location at the nucleus. To summarize, the presence of the lipid bilayer can promote the cellular internalization of LMSNs nanoparticles and the encapsulated drugs, and the lipid bilayer will not hinder the intracellular drug release, which are very helpful for improving the therapeutic efficacy.
3.3. Cell internalization and intracellular drug release 3.4. Cell viability The efficient cellular uptake of the drug loaded carriers and the preferably intracellular release of drugs are crucial for the drug delivery and an effective therapy. To investigate the effect of the lipid bilayer on the cellular uptake efficiency of MSNs, the FITC labeled MSNs and LMSNs were prepared and allowed to incubate with MCF-7 cells for 2 h. And the fluorescence signal within cells observed by CLSM was shown in Fig. 7. For MSNs, the green fluorescence signal was rather weak, and the comparably low uptake efficiency was mainly due to the repulsive force between the negatively charged MSNs and the cell membrane which is the same to carry a negative charge. Therefore, there was an energy barrier to overcome for the internalization of MSNs by the cells. In contrast,
To monitor the effect of the lipid bilayer coating on the biocompatibility of the carrier and the therapeutic efficacy of the encapsulated drug, the cell viability of MCF-7 cells after incubation with various DOX formulations and the corresponding amount of blank MSNs and LMSNs was evaluated by MTT essay. And the IC50 (concentration of test samples to inhibit cell growth by 50%) was calculated using SPSS software 13.0 (SPSS Inc., Chicago, IL, USA). The results are listed in Table 2. The profile in Fig. 9A shows that the cytotoxic effect of blank MSNs on MCF-7 was much higher compared with that of LMSNs. The cell viability of the group treated with MSNs at the highest test concentration decreased dramatically
Fig. 7. Confocal microscopy images of MCF-7 cells after treatment with FITC-labeled MSNs and LMSNs for 2 h. Scale bar represents 50 mm.
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Fig. 8. Internalization and accumulation of DOX in MCF-7 cells after treatment with DOX-Sol, DOX-MSNs and DOX-LMSNs for 2 h measured by confocal microscopy. Scale bar represents 50 mm.
to around 62% after an incubation time of 48 h. While for the group cultured with LMSNs at the same concentration, the cell viability remained above 90%. It has been reported that the abundant silanol groups on the outer surface of MSNs may cause structural damage to the membrane lipids and proteins [27]. However, after the coverage of the lipid bilayer, the cytotoxic effect of the nanoparticles was significantly reduced due to the concealing of the silanol groups and the excellent biocompatibility of natural lipids. The cytotoxicity of all DOX formulations was found to be concentration-dependent. The IC50 of DOX-Sol was 0.686 mg/mL, 1.7 times higher than the IC50 of MSNs-DOX calculated to be 0.401 mg/ mL, demonstrating that nanoparticles can assist the entry of the loaded drugs into cells through the carrier mediated endocytosis. In addition, after the coverage of the lipid bilayer, the cytotoxic effect of LMSNs-DOX further increased by 1.3 fold and the IC50 decreased to 0.306 mg/mL. Reasons for this result were as follows. On one Table 2 The IC50 values of different DOX formulations against MCF-7 cells. Formulations
DOX-Sol
MSNs-DOX
LMSNs-DOX
IC50 (mg/mL)
0.686
0.401
0.306
hand, the lipid bilayer could facilitate the cellular uptake of the DOX-loaded nanoparticles due to the high affinity with cell membrane which was in agreement with the phenomenon observed in Fig. 8. On the other hand, the retardant effect of the lipid bilayer could suppress the pre-leakage of DOX during the incubation process and achieve an acid-triggered DOX release after being internalized into the cytoplasm of cancerous cells. 3.5. Hemolysis measurement and BSA absorption The hemolysis percentage of MSNs and LMSNs over the concentration range from 20 to 1500 mg/mL is shown in Fig. 9B. For blank MSNs, the hemolysis ratio of RBCs escalated with the increasing of the tested concentration. When the concentration of MSNs reached 500, 1000 and 1500 mg/mL, the hemolysis ratio was 16.5%, 43.6% and 65.8%, respectively. This severe hemolysis puts great limitations to the safe application of MSNs as intravenous drug delivery systems. The reason for the silica-induced hemolysis was that the silica has high affinity to bind with the tetra-alkyl ammonium groups abundant on RBCs membranes. And this property could subsequently lead to the generation of reactive oxygen species (ROS) and the denaturation of membrane proteins after
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Fig. 9. (A) Cytotoxicity of various DOX formulations and corresponding amount of blank MSNs and LMSNs against MCF-7 cells at serial concentrations. (B) Hemolysis percentage of MSNs and LMSNs at different concentrations (mg/mL).
close contact with silicate [28,29]. However, after being covered by the lipid bilayer, the LMSNs nanoparticles exhibited stable dispersibility in saline solution containing 2% blood cells (V/V). The hemolysis percentage was negligible at low concentrations and remained below 10% even at the highest concentration of 1500 mg/ mL. Since the lipid bilayer can shield the silanol groups and prevent them coming into contact with the RBCs membranes, the biocompatibility of the nanoparticles was dramatically improved making them more reliable for intravenous drug delivery. It is widely accepted that nanoparticles will be rapidly cleared by the RES systems once a mass of proteins in the serum is absorbed on the particle surface which is very undesirable for the accumulation of drug-incorporated carriers at the disease site. In order to evaluate the effectiveness of lipid bilayer coating in preventing the nonspecific protein absorption, the amount of BAS adsorbed on the surface of MSNs and LMSNs was quantified. The degree of BSA absorption was calculated to be 2.06 wt.% for MSNs, and this markedly fell to 0.33 wt.% after the lipid bilayer coating for LMSNs. Such phenomenon would be attributed to the steric hindrance provided by the PEG modified lipid bilayer. Hence, the lipid bilayer coating was a simple and effective way to reduce the nonspecific protein adsorption and improve the dispersing stability and biocompatibility of MSNs. And the PEG modified lipid bilayer could serve to prolong the circulation time and improve the passive targeting efficiency of the MSNs based nanocarriers through the EPR effect. 4. Conclusion Lipid bilayer coating is a facile and effective way to functionalize MSNs. The obtained LMSNs have exhibited many appealing features, such as greatly improved dispersing stability and cellular internalization as well as markedly decreased non-specific protein absorption, hemolytic activity and cytotoxicity. Also, when DOX was used as a model drug, it could be encapsulated within LMSNs with a high LE % and be preferentially released in the cytoplasm of cancerous cells. Furthermore, the LMSNs nanoparticles could increase the cytotoxic effect of DOX toward cancerous cells by elevating the cellular accumulation of the drug loaded carriers. In conclusion, the presence of lipid bilayer could mitigate the drawbacks of MSNs making them more reliable in the application of drug delivery. And the prepared LMSNs have shown to be a
promising drug carrier with improved biocompatibility, a high LE % and the ability to promote the cellular uptake of the encapsulated drugs to achieve better therapeutic efficacy. Conflicts of interest The authors report no conflicts of interest. Acknowledgments We thank Dr. Xiaoju Huang and Dr. Gang Ji for technical support in cryo-TEM sample preparation and data collection. We also gratefully acknowledge the use of TEM facilities at the Center for Biological Imaging (CBI), Institute of Biophysics, Chinese Academy of Science. This work was supported by National Basic Research Program of China (973 Program) (No. 2015CB932100), National Natural Science Foundation of China (No. 81473165), and Basic Research Project of Key Laboratory of Liaoning Provincial Education Department (No. LZ2015068). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.micromeso.2015.08.006. References [1] Z. Deng, F. Yan, Q. Jin, F. Li, J. Wu, X. Liu, H. Zheng, J. Control. Release 174 (2014) 109e116. [2] J. Wang, J. Sun, Q. Chen, Y. Gao, L. Li, H. Li, D. Leng, Y. Wang, Y. Sun, Y. Jing, S. Wang, Z. He, Biomaterials 33 (2012) 6877e6888. [3] Y. Zhang, Z. Zhi, T. Jiang, J. Zhang, Z. Wang, S. Wang, J. Control. Release 145 (2010) 257e263. [4] H. Zhu, H. Chen, X. Zeng, Z. Wang, X. Zhang, Y. Wu, Y. Gao, J. Zhang, K. Liu, R. Liu, Biomaterials 35 (2014) 2391e2400. [5] F. Tang, L. Li, D. Chen, Adv. Mater. 24 (2012) 1504e1534. [6] A. Kierys, M. Rawski, J. Goworek, Microporous Mesoporous Mater. 193 (2014) 40e46. [7] Y. Wang, Q. Zhao, N. Han, L. Bai, J. Li, J. Liu, E. Che, L. Hu, Q. Zhang, T. Jiang, S. Wang, Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 313e327. [8] P. Yang, S. Gai, J. Lin, Chem. Soc. Rev. 41 (2012) 3679e3698. [9] Q. Zhao, C. Wang, Y. Liu, J. Wang, Y. Gao, X. Zhang, T. Jiang, S. Wang, Int. J. Pharm. 477 (2014) 613e622.
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