Supramolecular Core–Shell Nanosilica@ Liposome Nanocapsules for

Article pubs.acs.org/Langmuir

Supramolecular Core−Shell Nanosilica@Liposome Nanocapsules for Drug Delivery Mingxian Liu, Lihua Gan,* Liuhua Chen, Zijie Xu, Dazhang Zhu, Zhixian Hao, and Longwu Chen Department of Chemistry, Tongji University, 1239 Siping Road, Shanghai 200092, P. R. China ABSTRACT: The fabrication of core−shell structural nanosilica@liposome nanocapsules as a drug delivery vehicle is reported. SiO2 nanoparticles are encapsulated within liposomes by a W/O/W emulsion approach to form supramolecular assemblies with a core of colloidal particles enveloped by a lipid bilayer shell. A nanosilica core provides charge compensation and architectural support for the lipid bilayer, which significantly improves their physical stability. A preliminary application of these core−shell nanocapsules for hemoglobin (Hb) delivery is described. Through the H-bonding interaction between the hydroxyl groups on nanosilicas and the amino nitrogens of Hb, Hb−SiO2 nanocomplexes in which the saturated adsorption amount of Hb on SiO2 is 0.47 g g−1 are coated with lipids to generate core−shell Hb−SiO2@liposome nanocapsules with mean diameters of 60−500 nm and Hb encapsulation efficiency of 48.4−87.9%. Hb−SiO2@liposome supramolecular nanovehicles create a mode of delivery that stabilizes the encapsulated Hb and achieves long-lasting release, thereby improving the efficacy of the drug. Compared with liposome-encapsulated Hb and Hb-loaded SiO2 particles, such core−shell nanovehicles show substantially enhanced release performance of Hb in vitro. This finding opens up a new window of liposome-based formulations as drug delivery nanovehicles for widespread pharmaceutical applications. rupture, which makes their elaboration hardly reproducible.18 Besides, this poor physical stability also makes liposomes have the shortages like low encapsulation efficiency and fast release of the cargo. This means drug leakage before reaching to the target tissues, and thus it faces the difficulty of achieving long circulation times required to fulfill the clinical efficacy of the drug.19,20 In a word, the inherent instability of common liposomes causes much difficulty in the development of liposome-based formulations as pharmaceutically acceptable delivery nanovehicles. For the successful therapeutic application of liposomes, their mechanical stability is a critical factor that has to be considered and optimized. Recently, some liposome-based formulations such as polymer-coated liposomes,21 hydrogel/liposome composites,22 nanoparticle-stabilized liposomes,23 etc. have been developed to improve the stability or release properties of liposomes. For example, MacKinnon et al. found that immobilization of liposomes onto polymeric hydrogel microbeads enabled the controlled release of liposomal cargoes via coupling of triggered swelling/deswelling of the hydrogel. Besides, because the dimension of hydrogel microbeads is similar with the pore size in tissue engineering scaffolds, it was beneficial to embed hydrogel microbeads with their attached liposomes, which effectively localize liposome-sequestered species at the site of action and provide a reservoir for prolonged release.22 Liu et al. described silica nanoparticle

1. INTRODUCTION Besides providing the desired therapeutic effects, many drugs have an unacceptable risk of adverse side effects.1 Consequently, substantial efforts were made to minimize the side effects and to optimize the dosage regimen without compromising the therapeutic efficacy.2 The foundation was laid in 1952 by Smith et al., who introduced the first timerelease capsule of Dexedrine for narcolepsy.3 In the 1990s, the appearance of new drugs with large molecular dimensions, high dose sensitivities, and often poor stabilities led to a strong push toward the development of efficient encapsulation and controlled-release techonologies.4 Subsequently, many novel drug delivery systems were originated including nanoscaled capsules, spheres, or particles made of biodegradable natural and synthetic materials, hydrogels, micelles, cells, lipoproteins, liposomes, etc., which aim to achieve sustained and essential controlled release of drugs.5−11 Liposomes have been extensively studied as potential drug delivery vehicles.10,12 They are artificial lipid vesicles consisting of one or multiple concentric phospholipid bilayers entrapping aqueous compartments. Liposomes are capable of encapsulating hydrophilic drugs in the inner aqueous phase or lipophilic drugs in the bilayer walls.13,14 A cell membrane-like environment of liposome is available for protecting drugs and controlling their release. Therefore, liposomes are considered as an attractive drug delivery system for the treatment of many diseases including cancer.15,16 The successful use of liposomes as drug delivery nanovehicles is highly dependent on their physical and/or chemical stability.17 However, conventional liposomes often suffer from vesicle aggregation, fusion, or © 2012 American Chemical Society

Received: May 28, 2012 Revised: June 28, 2012 Published: June 29, 2012 10725

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was added into a round-bottom flask. The water phase containing emulsifier was added into the oil phase containing lecithin to get a water-in-oil (W/O) emulsion under stirring. Then an additional 100 mL of water which is used as the outer water phase was added into the W/O emulsion to form a W/O/W double emulsion. Finally, chloroform in the system was vacuum evaporated to prepare core− shell nanosilica@liposome nanocapsules. The formation of nanosilica@liposome nanocapsules was mainly based on the directional adsorption of amphiphiles at the interface and hydrophobic interaction among lecithin molecules, as shown in Scheme 2. First, when the water phase (nanosilica particles and water) was mixed with the oil phase (chloroform and lecithin) in the presence of CTAB, a primary W/O emulsion was obtained. Generally, common emulsion is a thermodynamically unstable system, and the mass ratio among the components affects its stability.30 The system of CHCl3(lecithin)/ CTAB/H2O(nanosilica) was found to form a stable W/O emulsion at a mass ratio of about 55:5−15:30−45. Subsequently, the primary W/O emulsion was encapsulated within the outer water phase to form a W/ O/W double emulsion. After the removal of the organic agent by vacuum evaporation, the tail chains of amphiphiles moved closer through hydrophobic interaction. Finally, core−shell nanosilica@ liposome nanocapsules were fabricated with SiO2 particles as cores enveloped by lipids organized in layers. 2.3. Characterization. N2 adsorption and desorption analysis was operated on a Micromeritics TriStar 3000. The adsorption data was taken at −196 °C to calculate the specific surface area by the Brunauer−Emmett−Teller (BET) model. Surface wetability measurement was performed on a JC2000A contact angle meter (Shanghai Zhongcheng Digital Technology Co., Ltd.). FT-IR analysis was performed on a Nexus Fourier transform infrared spectrometer. UV− vis analysis was conducted on an Agilent 8453 UV−vis spectrometer at room temperature. TEM images were obtained with a JEOL JEM1230 microscope. The liposome sample was absorbed on copper mesh grids coated with Formvar film for 15 min and then was negatively stained for 2 min with phosphor tungstenic acid solution for TEM observation. A NanoZS900 laser particle size distribution analyzer was used for determining the size of liposomes. 2.4. Nanosilica@Liposome Nanocapsules for Hb Delivery. The nanosilica@liposome nanocapsules were used as a drug delivery vehicle by selecting hydrophilic Hb as a simulated drug. Nanosilica particles were added into bovine Hb aqueous solution under stirring (the concentrations of nanosilica and Hb were 2 mg mL−1 and 5−50 μmol L−1, respectively). The mixture was placed in a refrigerator at 4 °C for 24 h, followed by centrifugal filtration and freeze-drying to obtain Hb−SiO2 nanocomplexes. The concentrations of the Hb−SiO2 nanocomplexes are varied from 0.10 to 0.30 mg mL−1. The Hb−SiO2 nanocomplexes were used instead of nanosilica to prepare core−shell Hb−SiO2@liposome nanocapsules according to the same procedure for nanosilica@liposome nanocapsules. 2.5. Encapsulation Efficiency of Hb in Hb−SiO2@Liposome Nanocapsules. A certain amount of Hb−SiO2@liposome nanocapsules (the initial mass of Hb−SiO2 used for preparing these nanocapsules is M1) was added into methanol under ultrasonic condition until the mixture solution was clear. The solution then was centrifugated, freeze-dried and weighed to calculate the mass of Hb− SiO2 encapsulated by liposome (M2). The value that divides the loaded amount of Hb by the added amount of Hb could be applied to define the Hb encapsulation efficiency (EE, %) in the Hb−SiO2@ liposome nanocapsules. Because Hb was first adsorbed on the surfaces of SiO2 nanoparticles to obtain Hb−SiO2 nanocomplexes (the unabsorbed Hb molecules were centrifugal filtrated), and then the nanocomplexes were encapsulated by liposomes. The loaded amount of Hb−SiO2 divided by the added amount of Hb−SiO2 is equal to that of the loaded amount of Hb divided by the added amount of Hb. Therefore, the EE value can be calculated as follows:

supported lipid bilayers for gene delivery.24 SiO2 particles distributed on the surface of the lipid bilayers stabilize the liposome based on the electrostatic attraction between cationic lipids and negatively charged SiO2 particles. Besides, they also reported that fusion of liposomes with mesosilicas synergistically seals a negatively charged cargo.25 Adjustment of the liposome composition/charge allows control of the cargo content; however, relatively small pore sizes of mesosilicas (1.95 nm) make these liposomes unsuitable for loading and releasing of drugs with large molecular dimensions. Hemoglobin (Hb) is an essential component of red blood cells, the main physiological function of which is oxygencarrying.26−28 Liposome-encapsulated Hb (LEH) is being examined as an oxygen carrier that mimics membrane enclosed cellular structure of red blood cells29 but suffers from the aforementioned instability of liposomes. Herein, we report the construction of supramolecular core−shell nanosilica@liposome nanocapsules by a water-in-oil-in-water (W/O/W) emulsion method and an initial application of these materials for Hb delivery. SiO2 nanoparticles provide charge compensation for liposome due to their negative surface charges which could electrostaticly interact with cationic lipids. Besides, SiO2 served as a rigid core offer an architectural support for the lipid bilayer shell. Such nanocapsules show higher physical stability compared with empty liposomes. Compared with common liposomes, these core−shell nanovehicles are more stable and take advantage of the colloidal core to control payload and release of drugs, whereas compared with other nanoparticle vehicles, the nanocapsules are simple and take advantage of biocompatibility and mimic the cell membrane environment of liposomes. The nanosilica@liposome nanocapsules are expected to control drug release spatially and temporally. Hbloaded nanosilica@liposome nanovehicles significantly stabilize the entrapped Hb for sustained and controlled release in vitro. Therefore, we believe the introduction of nanosilica into liposomes shows good potential for the application of liposome-based drug delivery systems.

2. EXPERIMENTAL SECTION 2.1. Materials. L-Alpha-phosphatidyl choline (soybean lecithin, C44H86NO8P, 98%, schematic structure was shown in Scheme 1),

Scheme 1. Molecular Structure of L-Alpha-Phosphatidyl Choline

methanol (AR), bovine Hb (BR, M = 64 500 Da), chloroform (AR), and cetyltrimethylammonium bromide (CTAB, AR) were purchased from Sinopharm Chemical Reagent Co. Ltd. SiO2 nanoparticles (∼10 nm) were purchased from Zhejiang Yuda Chemical Co., Ltd. A dialysis bag (MWCO 100, 000) was purchased from Shanghai Yibaiju Biology Co., Ltd. Phosphate buffer (PBS) used in the experiment has a pH value of 7.4. The water used was ultrapure (18.2 MΩ cm−1). 2.2. Fabrication of Core−Shell Nanosilica@Liposome Nanocapsules. A total of 0.021 g of SiO2 nanoparticles was mixed with 2.5 mL of water in which 0.3 g of CTAB was dissolved, followed by an ultrasonic dispersion for about 5 min. The mixture composed of water and silica particles serves as the water phase for the following emulsion. Chloroform (2.3 mL) which acts as the oil phase was mixed with L-alpha-phosphatidyl choline (lecithin, 0.021 g), and the mixture

EE(%) =

M2 × 100 M1

(1)

2.6. In Vitro Release of Hb from Hb−SiO2@Liposome Nanocapsules. The release of Hb from Hb−SiO2@liposome 10726

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Scheme 2. Schematic Formation of Nanosilica@Liposome Hybrid Nanocapsules with a Core−Shell Supramolecular Structure

nanocapsules in vitro was measured by a dynamic dialysis method. A total of 2 mL of Hb−SiO2@liposome nanocapsules was put into a dialysis bag and then they were immerged in 100 mL of phosphate buffer (PBS, pH 7.4). The test tube was placed in an incubator shaker maintained at a temperature of 37 ± 1 °C and a rotational speed of 100 r min−1. A 5 mL solution was collected at the fixed time and meanwhile equivalent PBS was added into the test tube. The Hb concentration was determined by Agilent 8453 UV−vis spectrometer at room temperature. The cumulative release percentage of Hb from Hb−SiO2@liposome nanocapsules (CR, %) in vitro can be calculated by the following equation:

CR(%) =

Wrelease × 100 Wtotal

average diameter in the range of 60−500 nm dependent on the specific experiment conditions, as we will discuss later. When chloroform was vacuum evaporated, the emulsion droplets become smaller because the amphiphile tail chains moved much closer through hydrophobic interaction. These SiO2 particle supported liposomes show no breaking or fusion with each other and exhibit good dispersion stability. Zeta-potential measurements show that the empty liposomes (mainly composed of L-alpha-phosphatidyl choline, Scheme 1) and SiO2 nanoparticles take positively charges and negatively charges, respectively (pH 7.0). The zeta-potential is about 22 mV for empty liposomes and −18 mV for SiO2 nanoparticles. Their electrostatic interaction increases the stability of liposome. More importantly, the nanosilica core offers a rigid framework to support liposome and effectively to prevent their fusion or rupture. Therefore, the nanosilica@liposome nanocapsules with a supramolecular core−shell structure exhibit much higher stability than conventional liposomes. For a comparison, a TEM image of empty liposomes was shown in Figure 1B. It can be seen that the empty liposomes are easy to be fused or broken in the absence of a solid core. Such liposomes suffer from intrinsically poor stability that limits both their route of administration and shelf life. Much of the literature also reported that similar phenomena existed in empty liposome or LEH.31 To avoid aggregation and fusion of liposomes (or LEH) and improve their stability, Rameez and Palmer used poly(ethylene glycol) (PEG) to modify the surface of LEH. The resultant PEG-LEH dispersion was colloidally stable for 4−5 months.21 On the other hand, the core−shell hybrid nanocapsules prepared with various concentrations of Hb−SiO2 nanocomplexes and lecithin are stable for several months. As a comparison, blank liposomes without a SiO2 particle core appear precipitation in about one month in our study. These results indicate that silica nanoparticles inside the liposomes provide charge compensation and a rigid framework for the lipid bilayer and thus improve the physical stability of liposomes. 3.2. Adsorption of Hb on the Surface of Nanosilica Particles. For the use of these supramolecular assemblies as

(2)

where Wrelease is the time-depending accumulatively released Hb from Hb−SiO2@liposome nanocapsules and Wtotal is the total weight of Hb encapsulated by liposomes.

3. RESULTS AND DISCUSSION 3.1. Morphology and Stability of Core−Shell Nanosilica@Liposome Nanocapsules. Figure 1A shows a typical TEM image of the core−shell structural nanosilica@liposome nanocapsules. The hybrid nanocapsules have a mean diameter of about 60 nm with cores of SiO2 nanoparticles and shells of lipid bilayer membrane. Actually, the nanocapsules show an

Figure 1. TEM images of typical core−shell structural nanosilica@ liposome nanocapsules (A) and empty liposomes (B). 10727

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Hb delivery nanovehicles, Hb molecules were first mixed with nanosilica particles to obtain Hb−SiO2 nanocomplexes. Figure 2 shows FT-IR spectra of Hb and Hb−SiO2 complexes. In the

Figure 3. N2 adsorption and desorption isotherms of nanosilica particles (black square) and Hb-loaded nanosilica particles with an Hb concentration of 5.0 (red circle), 10.0 (pink triangle), and 20.0 μmol L−1(blue triangle). Figure 2. FT-IR spectra of Hb and Hb−SiO2 nanocomplexes.

absorption spectrum of Hb, the absorption peak of 3458 cm−1 reflects the stretching vibration of N−H bond, and the absorption peaks of 1655, 1542, and 1372 cm−1 indicate amide I (CO stretching vibration), amide II (N−H bending vibration) and amide IV absorption band of Hb main chain, respectively.32 The absorption spectrum of Hb−SiO2 complexes also shows the characteristic absorption peaks of Hb. Besides, the peaks of 1099, 795, and 480 cm−1 could be ascribed to the asymmetric stretching vibration, symmetric stretching vibration and bending vibration of Si−O−Si. The absorption peak of 962 cm−1 is assigned to the stretching vibration of Si−OH. Therefore, combined absorption peaks of Hb and nanosilica in Hb−SiO2 complexes suggest that Hb was successfully adsorbed on the surfaces of nanosilica particles. There are both hydrophilic and hydrophobic amino acids in Hb molecules. Generally, the hydrophilic amino acids locate in the outer layer to ensure the solubility of Hb in water, and the hydrophobic ones distribute in the inner to prevent the movement of water molecules into the heme cavity. The hydroxyl groups of nanosilica particles could form H-bonding with the amino nitrogens of hydrophilic amino acids, which facilitate the adsorption of Hb molecules on SiO2 surfaces. Figure 3 shows the N2 adsorption and desorption isotherms of SiO2 nanoparticles and Hb−SiO2 nanocomplexes. The isotherms are type IV curves according to the International Union of Pure and Applied Chemistry classification.33 They show an obvious hysteresis loop at high relative pressure region (P/P0 = 0.8−1.0), which is a characteristic associated with capillary condensation. The specific surface area of SiO2 particles calculated by using BET model is 110.2 m2 g−1. The BET surface areas of Hb−SiO2 ocomplexes decrease due to the adsorption of Hb molecules on SiO2 surfaces. When mixing of 2 mg L−1 SiO2 particles with Hb which respectively has a concentration of 5, 10, and 20 μmol L−1, the BET areas of the resultant Hb−SiO2 complexes correspond to 73.0, 71.4, and 70.7 m2 g−1. This result also indicates successful adsorption of Hb molecules on nanosilica particles. The UV−vis adsorption spectra of Hb with different concentrations were shown in Figure 4. There are two adsorption peaks, 278 and 406 nm, in the spectra. The absorption peak at 278 nm is characteristic of the dynamic motion of Hb. Each subunit of Hb was composed of a

Figure 4. UV−vis adsorption spectra of Hb with a concentration of 5.0 (a), 10.0 (b), 25.0 (c), 30.0 (d), and 50.0 μmol L−1 (e). The inset shows a relationship between the absorbance and the concentration of Hb at 406 nm.

hemachrome molecule and a peptide chain. The hemachrome molecule has a porphyrin structure. The absorption peak located at 406 nm reflects the characteristic absorption peaks of the porphyrin ring.34 With increasing concentration of Hb, the intensity of the absorption peak at 406 nm also increases. The inset of Figure 4 shows a good linear relationship between the absorbance and the concentration of Hb. According to above linear relationship, we can calculate the adsorption efficiency of Hb molecules on SiO2 particles. Hb adsorption rate (AR, %) and adsorption amount (AA, g g−1) could be expressed as follows: W − Wfree AR(%) = Hb × 100 WHb (3) AA(g g −1) =

WHb − Wfree Wnanosilica

(4)

where WHb and Wfree denote the total weight of Hb and the weight of Hb unabsorbed by SiO2 nanoparticles, respectively. Wnanosilica is the mass of nanosilica particles. Table 1 reflects the effects of Hb concentration on the adsorption ratio and adsorption amount of Hb on the SiO2 surface. A total of 1 g of nanosilica particles could adsorb 0.12 g of Hb when the concentration of Hb is 5.0 μmol L−1. With Hb concentration increasing, the Hb adsorption amount also increases with a decreasing adsorption rate. The saturated adsorption amount of 10728

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Table 1. Relationship between Hb Concentration (CHb) and the Adsorption Ratio (AR) and Adsorption Amount (AA) of Hb on the Surfaces of Nanosilica Particles CHb (μmol L−1)

AR (%)

AAa (g g−1)

AAb (g g−1)

5.0 10.0 20.0 50.0

75.0 74.5 73.1 28.8

0.12 0.24 0.47 0.47

0.113c/0.145d 0.118/0.151 0.120/0.154

Scheme 3. Monolayer (left) and Multilayer (right) Adsorption of Hb on SiO2 Surfaces

a

The values calculated from UV−vis spectra. bThe values calculated from BET analysis dates. cThe values calculated by using the biggest molecular areas of Hb. dThe values calculated by using the smallest molecular areas of Hb.

Hb is 0.47 g g−1 on SiO2 particles with an adsorption rate of 73.1% in case of 20 μmol L−1 Hb concentration. According to the Langmuir and BET adsorption theory, gas molecules will adsorb and desorb at the position of the solid surface where it is not saturated because of a residual force.35,36 In the case of collision of N2 molecules to the blank solid surfaces of SiO2 unabsorbed by Hb, some of them will be absorbed and release adsorption heat. The bovine Hb molecule has a dimension size of 6.4 nm ×5.5 nm ×5.0 nm,26 and its biggest and smallest molecular areas in natural conformation are 35.20 nm2 (6.4 nm ×5.5 nm) and 27.5 nm2 (5.0 nm ×5.5 nm), respectively. Provided that Hb molecules absorbed on the SiO2 surface are monolayer, through the change of the specific surface areas of SiO2 particles and Hb−SiO2 complexes, the Hb adsorption amount on the SiO2 surface could be estimated by the following equation: AA(g g −1) =

Snanosilica − SHb − nanosilica MHb SHbNa

(5)

Figure 5. Photographs of water droplet on SiO2 particles (A) and Hb−SiO2 complexes (B).

in which Snanosilica and SHb‑nanosilica respectively represent the BET surface areas of SiO2 particles and Hb−SiO2 complexes (m2 g−1), SHb denotes the single molecular area of Hb (35.20 or 27.5 × 10−18 m2), Na is the Avogadro constant (6.02 × 1023), and MHb is the molar mass of Hb (64500 g mol−1). The calculated adsorption amount of Hb molecules on nanosilicas according to eq 5 was also shown in Table 1. Hb adsorption amount calculated respectively from UV−vis spectra and N2 analysis dates is quite similar at low Hb concentration (5 μmol L−1). However, these values estimated from the latter are much smaller than those obtained from the former when the concentration of Hb is more than 5.0 μmol L−1. This result indicates that the adsorption of Hb molecules on SiO2 surfaces is monolayer at low Hb concentration and multilayer at higher concentration, as shown in Scheme 3. As a result, the BET areas of Hb−SiO2 complexes do not change obviously when the Hb concentration is increased from 5 to 20.0 μmol L−1, although the Hb adsorption amount on SiO2 increases from 0.12 to a maximum of 0.47 g g−1. Silica nanoparticles inside lipsomes have intrinsic hydrophilicity and biocompatibility as well as good adsorption ability to bind hydrophilic molecules, which makes the supramolecular core−shell nanosilica@liposome nanocapsules available for controlled drug delivery application. The water contact angle measurement reflects the surface wettability of materials. Figure 5 shows the photographs of water droplet on the surface of SiO2 nanoparticles and Hb− SiO2 nanocomplexes. The contact angle of SiO2 particles for water is very small due to their strongly hydrophilic hydroxyl groups on their surface, while that of Hb−SiO2 complexes is about 82°. The formation of H-bonds between hydroxyl groups

of nanosilicas and amino nitrogens of Hb decreases exposed hydroxyl groups on Hb−SiO2 complexes. Besides, the molecular dimension of Hb is similar with the particle size of SiO2. Thus, the surface wetability of Hb−SiO2 complexes mainly depends on that of Hb molecules. Their hydrophilic property seems less than hydroxyl groups, resulting in increasing contact angle of Hb−SiO2 complexes. However, Hb−SiO2 complexes are still hydrophilic, and they could be dispersed in water to be encapsulated by lipsomes during the formation of the W/O/W emulsion. Table 2 shows the effects of the concentration of Hb−SiO2 nanocomplexes and lecithin on the mean diameters of Hb− SiO2@liposome nanocapsules and Hb encapsulation efficiency (EE%) in the nanocapsules. The hybrid nanocapsules have an average diameter in the range of about 60−500 nm. Figure 6 provides a TEM image of sample 1 shown in Table 2 which has a similar preparation condition with the sample shown in Figure 1A (the only difference is that one has Hb and the other one has not). The additional encapsulation of Hb as well as the concentrations of Hb−SiO2 show no obvious influence on the diameters of liposomes. The increasing of lecithin concentration results in the formation of liposomes with larger diameters. When the lecithin concentrations increase from 0.20 to 0.79 mg mL−1, the mean diameters of Hb−SiO2@liposome nanocapsules increase from 60 to about 500 nm. Liposomes exhibit a good encapsulated ability to Hb−SiO2 complexes; most of the EE% reaches beyond 70% or even higher. As a comparison, Li et al. prepared liposome-encapsulated actin− 10729

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Table 2. Effects of the Concentration of Hb−SiO2 Nanocomplexes (CHb‑nanosilica) and Lecithin (Clecthin) on the Mean Diameters (D) of Hb−SiO2@Liposome Nanocapsules and Hb Encapsulation Efficiency (EE%) in the Nanocapsules sample

CHb‑nanosilica (mg mL−1)

Clecthin (mg mL−1)

D (nm)

EE (%)

1 2 3 4 5 6 7 8 9

0.20 0.20 0.20 0.20 0.20 0.10 0.15 0.25 0.30

0.20 0.27 0.40 0.54 0.79 0.79 0.79 0.79 0.79

60 120 205 329 512 498 502 520 518

48.4 52.6 63.7 70.2 79.3 87.9 83.2 75.4 68.5

Figure 7. In vitro release profiles (A) and schematic release process (B) of Hb from LEH, Hb−SiO2 nanocomplexes, and Hb−SiO2@ liposome nanocapsules (sample 5 shown in Table 2) in phosphate buffer at 37 ± 1 °C.

Figure 6. TEM image of Hb−SiO2@liposome nanocapsules.

hemoglobin (LEAcHb) artificial blood substitutes, and the EE% was 30.1−46.0%.37 Therefore, the improved stability of asprepared core−shell nanocapsules stabilizes the encapsulated drug and enhances the EE%. Lecithin concentrations greatly affect the EE%. When the concentration of the Hb−SiO2 complexes was fixed to 2.0 mg mL−1, the EE% increases with the increasing of lecithin concentration because more lecithin molecules facilitates the formation of more or bigger liposomes to encapsulate Hb−SiO2. The EE% increases from 48.4 to 79.3% when lecithin the concentration increases from 0.20 to 0.79 mg mL−1. Besides, the decrease of the concentration of Hb−SiO2 complexes to 0.10 mg mL−1 would enhance the EE% (87.9%). However, the practical encapsulated amount of Hb decreases in this case. 3.3. In Vitro Release of Hb from Hb−SiO2@Liposome Nanocapsules. In vitro release profiles of Hb molecules from LEH, Hb−SiO2 nanocomplexes, and Hb−SiO2@liposome hybrid nanocapsules (sample 5 shown in Table 2) in phosphate buffer at 37 ± 1 °C are presented in Figure 7A. After Hb loading, the cumulative release curves of Hb could be roughly divided into the first (I), the second (II), and the third (III) stages. The three nanovehicles show distinct release performances. From the first step of the release curves, LEH show a burst release at the beginning of 3 h with a cumulative release amount of 39.8%, much higher than that of 21.2% for Hb−SiO2 nanocomplexes and 15.0% for Hb−SiO2@liposome nanocapsules. LEH is featured with spatial isolation of Hb by lipid bilayer which enables to some degree the controlled release of therapeutic agents, but it often suffers from inherent physical instability. Vehicle breaking or fusion facilitates the drug fast diffusion, and thereby the initial rapid release is unavoidable. Regarding Hb−SiO2 nanocomplexes, the release of Hb is much

lower than that of LEH. Silica particles themselves are biocompatible, stable, and “stealthy” drug delivery vehicles due to a high available BET area,4 the formation of hydrogen bonding between Hb molecules and SiO2 nanoparticles effectively impedes the drug release. Compared with LEH and Hb−SiO2 nanocomplexes, the core−shell Hb−SiO2@ liposome nanocapsules display the lowest release percentage at the first stage. This could be ascribed to the following two aspects. First, SiO2 nanoparticles served as a supported core for liposomes significantly improves the stability of the lipid bilayers by preventing their rapture and fusion. As a result, these hybrid liposomes are stable and not immediately eliminated. Second, H-bonding interaction existing in Hb− SiO2 complexes enables the controlled release of Hb. Thus, by combination of the advantages of liposomes with a cell membrane-like environment and SiO2 particles with multifunctions of charge compensation, architectural supporting as well as good adsorption ability, such core−shell Hb−SiO2@ liposome supramolecular assemblies hinder the release process to a greater extent and show a long-circulating property, as shown in Figure7B. At the second release stage, Hb molecules are released from LEH with relative slower rate because the concentration gradient of Hb has been reduced after the initial burst. For Hb−SiO2 complexes and Hb−SiO2@liposome nanocapsules, Hb release is in a sustained state since the adsorption of Hb molecules on SiO2 surface is multilayer. After the relatively fast release of Hb located at the out layer which is not tightly adsorbed by the SiO2 core, the release of Hb becomes slow due to the H-bond interaction between Hb and SiO2. At the third stage, the release rate of Hb from the three nanovehicles gets 10730

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delivery systems. Consequently, the nanosilica@liposome hybrid nanocapsules which show the ability to control drug release both spatially and temporally provide an ideal candidate for drug delivery application. In-depth studies of these core− shell hybrid nanocapsules are in progress in our group.

slower until the end compared with the former two stages. For LEH, the release of Hb was basically finished in 30 h with a total release percent of 60.7%. By contrast, the Hb amount released from Hb−SiO2 complexes is 45.3% in 30 h, and thereafter Hb release becomes more and slower with a maximum release percentage of 60.2% in 110 h, whereas core−shell Hb−SiO2@liposome nanocapsules have the slowest release profile with a release content of 40.0% in 30 h. It needs to be pointed out that it basically shows a near line release curve until 110 h with a cumulative percentage of 59.2%, which shows excellent performance for controlled release requirement. The core−shell Hb−SiO2@liposome hybrid nanocapsules have a final release percentage of 61.7% finished in 150 h which is five times as long as that of LEH without nanosilica cores. Therefore, Hb−SiO2@liposome nanovehicles show a better release property for Hb in vitro compared with LEH and Hb−SiO2 complexes. In addition, the release profiles for other samples (samples 1−4) in Table 2 are very similar with that of sample 5 (data not shown). Generally, liposomes have diameters ranging from several dozens to several hundreds of nanometers, and particle size influences the ability of liposomes to encapsulate drug compounds. Large liposomes are more likely to be broken or fractured than small ones,38 which results in drug leakage before reaching to the target tissues, corresponding to a burst release in their release profile, just as shown in Figure 7A. Therefore, liposomes with larger size distribution show relatively faster release speed. As-prepared core−shell Hb−SiO2@liposome hybrid nanovehicles are very stable and show similar release performance independent of the particle diameter, which has the advantage of achieving long circulation times required to fulfill the clinical efficacy of the drug. The controlled release of Hb molecules is important for clinical applications because it is a tetramer composed of two α and two β chains which fast breaks down into two dimers composed of α and β subunits without a red blood cell environment. Relatively fast release of Hb would lead to rapid acute tubular necrosis and renal failure. The results obtained from Figure 7 suggest that the introduction of SiO 2 nanoparticles as a rigid supporting core into liposomes effectively enhances the stability of liposomes and substantially improves the release performance of Hb. Therefore, the core− shell Hb−SiO2@liposome nanocapsules overcome the drawbacks of conventional LEH and thus provide a new example for pharmaceutical applications. It should be pointed out here that this work focuses on the stability of the core−shell nanosilica@ liposome nanocapsules and their release performance in vitro by using Hb as a stimulated drug and thus overleaps the protection process of Hb to avoid oxidation. However, protection of Hb, e.g., under CO gas,21 is needed when practical application of this liposome-based formulation as a LEH. Furthermore, the amphiphilic character of lipids facilitates the envelopment of hydrophilic drugs in the inner aqueous compartment via hydrophilic interactions, and the embedment of hydrophobic cargos in the bilayer walls via hydrophobic effects. Therefore, our method could also be extended to other hydrophilic drugs and lipophilic agents. As-prepared suparmolecular core−shell nanovehicles, on the one hand, are more stable and take advantage of a colloidal core to control loading and release compared with common liposomes, and on the other hand, are pretty simple and take advantage of the low toxicity and biocompatibility as well as cell membrane-like environment of liposomes compared with other nanoparticle

4. CONCLUSIONS In summary, we demonstrate the building of core−shell nanosilica@liposome nanocapsules by a W/O/W emulsion approach and an initial application of these supramolecular assemblies for the controlled release of Hb molecules. The introduction of SiO2 nanoparticles as charge compensation and architectural cores for liposomes significantly improves their physical stability. Besides, silica nanoparticles have intrinsic hydrophilicity, biocompatibility as well as excellent adsorption ability to bind hydrophilic drug compounds. Therefore, these stable core−shell hybrid nanovehicles show substantially enhanced release performance of Hb in vitro compared with conventional LEH and Hb−SiO2 nanocomplexes. Although only Hb was researched as a simulated drug for this paper, we believe that this method could be broadly applied to other hydrophilic drugs as well as lipophilic drugs where controlled or prolonged release is needed. Our methodology would create a mode of delivery that is time release, stabilizes the encapsulated drugs, and reduces the side effects, thereby improving the efficacy of the drugs. This finding highlights the potential of the well-organized core−shell nanosilica@liposome nanocapsules as ideal drug delivery nanovehicles for widespread pharmaceutical applications.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The present work was financially supported by the National Natural Science Foundation of China (Nos. 20673076, 20973127), Shanghai Nanotechnology Promotion Center (No. 11 nm0501000), and the Fundamental Research Funds for the Central Universities (2011KJ023).

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REFERENCES

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