Recent advances in liposomal nanohybrid cerasomes as promising drug nanocarriers.

CIS-01359; No of Pages 11 Advances in Colloid and Interface Science xxx (2013) xxx–xxx

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Recent advances in liposomal nanohybrid cerasomes as promising drug nanocarriers Xiuli Yue a, Zhifei Dai b,⁎ a b

State Key Laboratory of Urban Water Resources and Environment, School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150001, China Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, 100871, China

a r t i c l e Available online xxxx Keywords: Cerasomes Liposomes Drug delivery Silica nanoparticles Controlled release

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a b s t r a c t Liposomes have been extensively investigated as possible carriers for diagnostic or therapeutic agents due to their unique properties. However, liposomes still have not attained their full potential as drug and gene delivery vehicles because of their insufficient morphological stability. Recently, a super-stable and freestanding hybrid liposomal cerasome (partially ceramic- or silica-coated liposome) has drawn much attention as a novel drug delivery system because its atomic layer of polyorganosiloxane surface imparts higher morphological stability than conventional liposomes and its liposomal bilayer structure reduces the overall rigidity and density greatly compared to silica nanoparticles. Cerasomes are more biocompatible than silica nanoparticles due to the incorporation of the liposomal architecture into cerasomes. Cerasomes combine the advantages of both liposomes and silica nanoparticles but overcome their disadvantages so cerasomes are ideal drug delivery systems. The present review will first highlights some of the key advances of the past decade in the technology of cerasome production and then review current biomedical applications of cerasomes, with a view to stimulating further research in this area of study. © 2013 Elsevier B.V. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . 2. Design and synthesis of cerasome-forming lipids . . . . 3. Fabrication of cerasomes . . . . . . . . . . . . . . . 4. Characterization of cerasomes . . . . . . . . . . . . 5. Stability of cerasomes . . . . . . . . . . . . . . . . 6. Biocompatibility evaluation of cerasomes . . . . . . . 7. Cerasomes for drug and gene carriers . . . . . . . . . 8. Cerasomal porphyrin for photodynamic therapy of cancer 9. Conclusions and perspectives . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction A liposome is one of the most versatile supramolecular assemblies in existence and its applications have been well established in various scientific disciplines, including biophysics, chemistry, colloid science, biochemistry and biology [1]. The industrial applications of liposomes ⁎ Corresponding author at: Peking University, Room 206, Fangzheng Building, No 298 Chengfu Road, Haidian District, Beijing 100871, China. E-mail address: [email protected] (Z. Dai). URL: http://bme.pku.edu.cn/~daizhifei (Z. Dai).

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include drug delivery vehicles in medicine, adjuvants in vaccination, signal enhancers/carriers in medical diagnostics and analytical biochemistry, solubilizers for various ingredients as well as support matrix for various ingredients and penetration enhancer in cosmetics. Especially, liposomes have attracted considerable attention for controlled or targeted release of various drugs and diagnostic agents due to their unique properties [2]. Despite all the work done, liposomes still have not attained their full potential due to their insufficient morphological stability [3]. Depending on their composition, the final liposome formulations may have short shelf-lives partly due to chemical and physical instability. Chemical

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Please cite this article as: Yue X, Dai Z, Recent advances in liposomal nanohybrid cerasomes as promising drug nanocarriers, Adv Colloid Interface Sci (2013), http://dx.doi.org/10.1016/j.cis.2013.11.014

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instability may be caused by hydrolysis of ester bond and/or oxidation of unsaturated acyl chains of lipids. Physical instability may be caused by drug leakage from the vesicles and/or aggregation or fusion of vesicles to form larger particles. When subjected to blood, the morphology of liposomes would be destroyed by interacting with amphiphilic plasma proteins [4]. As drug carriers, drugs such as doxorubicin that possess amphiphilic properties would also work against their stability [5]. The instability of the liposomes could lead to rapid clearance of the vesicles from circulation, often before reaching their target. For these reasons, many researchers are making great efforts to improve the stability of liposomes, such as polymerization of modified lipids that comprised liposomes [6] and coating liposomes with inorganic calcium phosphate [7], silica [8] or polyelectrolytes [9]. Polyethylene glycol (PEG) has been widely used as polymeric steric stabilizer because of many useful properties, such as biocompatibility [10], solubility in aqueous and organic media [10], lack of toxicity, very low immunogenicity and antigenicity [11], and good excretion kinetics [12]. Grafting PEG onto liposomes has demonstrated several biological and technological advantages. The most significant properties of pegylated vesicles are their strongly reduced MPS uptake and their prolonged blood circulation and thus improved distribution in perfused tissues [13]. Yet, the pegylated liposomes appeared to reduce some of the toxic effects caused by the release of their contents, but, unfortunately, new toxic effects appeared because of the presence of the polyethylene glycol [14–17]. For example, the liposomal preparations containing pegylated phospholipids have lead to skin toxicity generally known as “Hand–Foot syndrome,” which results in skin eruptions/ulcers on the palms of the hands and soles of the feet. Another disadvantage with pegylated liposomes is the presence of PEG large molecules on the liposomal surface that may reduce the interactions of liposomes with cells and hinder entry of liposomes into the tumor tissue, thereby possibly reducing the accumulation of liposomal drug in the tumor tissue. Thus, there remains a need for stable, long circulating liposomes that do not cause such deleterious effects such as the “Hand–Foot syndrome” [18]. In recent years, a so-called cerasome, a type of organic–inorganic nanohybrid, was developed through a combination of sol–gel reactions and self-assembly of molecularly designed lipidic organoalkoxysilane in aqueous media to form liposomal bilayer structure covered with an atomic layer of inorganic polyorganosiloxane networks on its surface (Fig. 1) [19]. This silicon strategy provides a simple and widely applicable tool to overcome general problems associated with current liposome technology. A cerasome has a precisely designed organic–inorganic nanohybrid structure. The thickness of both organic and inorganic layers of a cerasome is attributed to the molecular structure of cerasome-

forming lipids (CFLs), and the vesicular size of a cerasome is basically controllable by applying conventional methodologies for preparing monodispersed liposomes. As a new drug delivery system, cerasomes take a number of advantages: (1) The siloxane surface adds remarkably high mechanical stability and heat resistance compared with conventional liposome [20]; (2) The siloxane surface can facilitate the stabilization of cerasomes in an environment with a slightly alkaline pH or a significant salt concentration; (3) The presence of a liposomal bilayer structure reduces the overall rigidity and density of cerasomes greatly compared to silica NPs, which is expected to enhance the stability of such particles in aqueous systems against precipitation; (4) Cerasomes may be biodegraded through the biochemical decomposition of the Si\C bond; (5) Cerasomes can be loaded with hydrophilic, hydrophobic as well as amphiphilic drugs without destroying their morphological stability; (6) The silanols located on the exterior surface can be functionalized to allow the easy bioconjugation of biomolecules with silane-coupler chemistry. Such unique advantages give wide applicability to the cerasomes in roles as gene carriers [21], drug delivery systems [22], other biomedical applications and biological energy transfer [20]. The present review will briefly outline the design, preparation and morphological characterization of cerasomes. In particular, we revisit the literature relating to high-stability, biocompatibility, drug and gene delivery, photodynamic therapy of cancer, and future trends concerning novel cerasomes.

2. Design and synthesis of cerasome-forming lipids CFLs were designed based on the concept of the critical packing parameters for lipid assemblies [23,24]. It is important to introduce the connector part to form intermolecular hydrogen bonding to add morphological stability to the lipid assembly between the hydrophobic alkyl chains and the hydrophilic part in the lipid molecule [25]. Murakami and Kikuchi reported the connector part of so-called peptide lipids, which have amino acid or oligopeptide moieties between the hydrophobic and hydrophilic parts [26]. To take into account these points, Kikuchi et al. designed and synthesized CFLs with triethoxysilane headgroups and hydrophobic alkyl chains [19,27]. The general molecular structure of CFLs is shown in Fig. 1. Triethoxysilane headgroups are stable in the air. Upon hydrolysis of the triethoxysilyl groups, the CFLs became amphiphilic to form lipid bilayer in aqueous solution. Followed by condensation among the silanol groups, a silica-like framework developed on the surface of vesicles, resulting in the formation of cerasomes.

Fig. 1. Fabrication procedure and typical structure of a cerasome.



Along this design concept, Kikuchi and Yasuhara synthesized a series of CFLs [28], as shown in Fig. 2. CFL 1 was synthesized by simple condensation reactions between the two molecular units of 3-aminopropyltriethoxylsilane and N,N-dihexadecylsuccinamic acid obtained by coupling dihexadecyamine and succinic anhydride. CFLs 2 and 3 having one urea group and one amide group, respectively, were synthesized with the relating intermediates. Cationic CFLs 4 and 5 used the same framework of the typical peptide lipids [26], in which the dihexadecylamine unit and quaternary ammonium group sandwiched amino acid unit and pentamethylene chain, and one of the methyl groups of the trimethylammonium group was replaced by 3-triethoxypropyl group. Contrary to nonionic lipids, cationic CFLs 4 and 5 can act as an amphiphilic molecule even if its silanol group is capped by ethoxy groups. They show similar physical parameters such as phase-transition temperature as those of the corresponding peptide lipid. CFL 6 with two triethoxysilyl heads was designed to tune the siloxane network on the cerasome surface. CFL 7, a phospholipid with triethoxysilyl headgroup, was also synthesized, providing us capability to fabricate biodegradable cerasomes. After hydrolyzing the triethoxysilyl group, all these CFLs are expected to become suitable structure to form lipid bilayer. Recently, our group designed and synthesized a series of new CFLs (8, 9, 10) with different numbers of triethoxysilane headgroups and hydrophobic alkyl chains covalently attached to each other via glycerol and pentaerythritol [29], as presented in Fig. 2. Two different strategies were applied for structure-guided synthesis of CFLs. For CFLs 8 and 9, the number of alkyl chains was set to two, and the triethoxysilyl headgroups were varied from two to three. On the other hand, CFL 10 has one triethoxysilyl headgroup but three alkyl chains. According to the synthetic routes as illustrated in Fig. 3, N,N-dihexadecylsuccinamic acid as an initiator was first reacted with excess glycerol, and a synthetic precursor with two hydroxyl groups was thus obtained. Next, this precursor was further allowed to react with triethoxy(3-isocyanatopropyl)silane under the catalysis of dibutyltin dilaurate, and consequently gave rise to a new CFL 8 bearing two trialkoxy-silane headgroups and two alkyl chains, which were linked by glycerol. CFL 9 with three trialkoxysilane headgroups and two alky chains was synthesized according to the similar procedure. For the synthesis of CFL 10, pentaerythritol was first coupled with 3 equivalents of bromohexadecane to give an intermediate bearing three alkyl chains and one hydroxyl group, which

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could effectively react with succinic anhydride followed by coupling with 3-aminopropyltriethoxysilane to yield the targeting CFL 10 containing one trialkoxy-silane headgroup and three alkyl chains. A biocompatible and biodegradable CFL 11, cholesteryl-succinyl silane, was also synthesized by simple condensation reactions between the three molecular units, cholesterol, succinic anhydride, and 3-aminopropyltriethoxylsilane. 3. Fabrication of cerasomes Similar to liposomes, cerasomes are spherical vesicles and the thickness of the lipid bilayer membrane is around 4 nm. The fabrication procedure of cerasomes with a multilamellar vesicular structure is basically analogous to that of the liposomes prepared from phospholipids. Conventional or unmodified liposomes with multilamellar or unilamellar vesicular structure are composed of phospholipids (e.g. phosphatidylcholines) and also cholesterol which is often included as a constituent. The latter improves the rigidity of the bilayer membrane and in doing so it reduces the permeability for encapsulated molecules and enhances the stability of the bilayer in the presence of biological fluids [1]. On the contrary, no cholesterol is needed for the fabrication of cerasomes. Two typical methods were adopted by Kikuchi group for the fabrication of cerasomes. In the first method, cerasomes were produced by direct dispersion of CFLs in aqueous solution using vortex-mixing, resulting in the formation of a liposome which self-rigidifies via in situ sol–gel processes (Si-OCH3CH2 + H2O → Si-OH + CH3CH2OH followed by 2Si-OH → Si-O-Si + H2O) on the surface [19]. As for the waterinsoluble CFLs 1–3, 6–11, gradual hydrolysis of the triethoxysilyl head group convertes proamphiphile amphiphilic, resulting in a selfassembly of the liposome-like bilayer membrane in aqueous solution (Fig. 1). In addition, the condensation among the silanol groups on the relatively hydrophobic membrane surface proceeds spontaneously to develop siloxane network. However, if the hydrolysis and subsequent condensation are much faster prior to the self-assembly, the formation of the bilayer structure may be disturbed. Therefore, it is essential for the formation of cerasomes to control the reaction rate in the sol–gel process, especially the hydrolysis, which depends on the pH value of aqueous solution. For the water-insoluble CFLs 1–3, 6–11, hydrolysis of the ethoxysilyl groups is gently proceeded by mild acid catalysis under moderate acidic conditions at pH 3, resulting in a translucent

Fig. 2. Structural formulae of CFLs.


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OH OH OH

O

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N

HO O

OCN

O HO HO

Si(OEt)3

N

O O

O

O (EtO)3Si

N O H O

(EtO)3Si

N H

N

O

O

O CFL 8

Fig. 3. Synthetic route of CFL 8.

solution characteristic of a liposomal dispersion. However, under stronger acidic conditions at pH 1, precipitation is observed immediately and no stable dispersion is obtained since the hydrolysis and subsequent condensation reaction are so fast that the formation of the vesicular structure is prevented. Nevertheless, the hydrolysis of the triethoxysilyl head group is extremely slow under a neutral pH condition. On the other hand, the hydrolysis seems to proceed heterogeneously under basic conditions. Some particular molecules are preferentially hydrolyzed, while the other molecules remain as unreacted species. Thus, basic conditions seem to be unsuitable for the preparation of cerasomes. For the lipids with good water solubility, such as CFLs 4 and 5, cerasomes can be conveniently prepared using the direct dispersion method in water [27]. The second procedure of cerasome preparation is the ethanol sol injection method, where the hydrolysis of CFLs is performed by incubating an acidic ethanol solution of the lipids for an appropriate time. The sol thus obtained is injected into water, and the solution is incubated for an additional 24 h. This method is used for CFLs with poor water solubility. In general, the size of the multilamellar cerasomes is in the range of sub-micrometers. Upon ultrasonication of the aqueous dispersion of cerasome 4 using a probe-type sonicator at 30 W power above the phase transition temperature of the lipids for 3 min, the multilamellar vesicles (MLVs) converted into the corresponding small unilamellar vesicles (SUVs) with a diameter less than 100 nm [27]. Yet, ultrasonication did not alter the structure of cerasome 1 from MLVs to SUVs. Cell-sized cerasomes with a diameter larger than 1 μm can also be prepared by employing the preparation method of giant liposomes.

4. Characterization of cerasomes The morphological structure of cerasomes can be evaluated by means of various microscopic measurements, such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic microscopy (AFM). The SEM image showed that the paclitaxel loaded cerasome 1 prepared from CFL 1 were spherical and the vesicular size ranged from 150 to 300 nm (Fig. 4a), well consistent with the hydrodynamic diameter (214 nm) as evaluated by dynamic light scattering (DLS) measurements [22,30]. The TEM images of cerasome 4 are shown in Fig. 4b. An internal view of the multilamellar vesicles (MLVs) with a bilayer thickness of about 5 nm was clearly confirmed [19]. The formation of siloxane bonds on the cerasome surface was proved by Fourier transform infrared (FT-IR) spectroscopy. Stretching bands assigned to the Si\O\Si and Si\OH groups were observed around 1100 and 920 cm−1, respectively [22,31]. The former peak intensity was much stronger than the latter in cerasomes in the dry state. Thus it is suggested that the cerasomes have an inorganic silicate framework with high degree of polymerization. The detectable species of the lipid oligomers in cerasome 1 were evaluated by matrix assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) spectra [19]. Trimethylsilylation was performed for the aqueous dispersion samples of the cerasome prepared after 15 min and 10 h. After 15 min, oligomers with molecular weight (MW) such as tetramers (MW: 2957.3 for cyclic or branched; 3117.4 for linear) and pentamers (MW: 3695.3 for cyclic or branched)

Fig. 4. (a) SEM micrograph of the paclitaxel-loaded cerasome 1; (b) TEM micrograph of cerasome 4.



were detected. As incubation time increases the degree of polymerization of the siloxane network becomes higher. The zeta potentials of cerasome 1 and silica nanoparticles (NPs) in water were −24.2 ± 1.0 mV and −26.3 ± 2.1 mV, respectively, indicating that cerasomes possessed similar surface properties with silica NPs. There was a slight agglomeration shown by increase in particle size when nanoparticles were dispersed in 1640 culture medium with or without serum [32]. Also, zeta potential for both cerasomes and silica NPs decreased in both culture mediums. These results may arise from the high ionic strength or the presence of serum in the culture medium which causes the decrease of surface charge of nanoparticles [33]. The zeta-potential of cerasome 1 changed from +10 to −70 mV depending on the medium pH. The isoelectric point (IEP) of the cerasomes was found to be 4.3. Cerasome 1 had large negative charges under neutral and basic conditions, reflecting deprotonation of the silanol groups on the cerasome surface. It is well known that the IEP values for the typical silica particles derived from the sol–gel method lie in the region of 2–3 and have a zeta-potential ranging from +20 to −80 mV in the analogous pH region [34,35]. The IEP value for the cerasome was somewhat larger than those for the silica particles, presumably due to the electron-donating character of the alkyl group bound to the silicon atom in the former. Therefore, cerasome 1 is weakly negatively charged in the physiological condition, ensuring the potential applicability of this delivery system in vivo. Nevertheless, the IEP value shifted to 12.0 for cerasome 4. In a pH range lower than 12.0, the zeta-potential of the cerasome increased with a decrease in pH to reach + 70 mV at pH 6. This value was considerably higher than the maximal zetapotential of cerasome 1. Such difference is attributed to the existence of a quaternary ammonium group in CFL 4. At neutral pH, cerasome 1 acts as a polyanionic vesicular particle, whereas cerasome 4 is polycationic. Thus we can control the IEP value of the cerasome to a desired value between 4 and 12 by mixing CFLs 1 and 4 in an appropriate ratio. The hydrodynamic diameter did not change in the regions below pH 4 and above pH 6. In a pH region around the IEP value, however, the hydrodynamic diameter was increased drastically to form the low dispersive vesicular aggregates due to the loss of charges on the cerasome surface.

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change at all even in the presence of 40 equivalents of Triton X-100. The cerasome vesicles retained their vesicular structure upon the addition of 40 equiv of Triton X-100. In addition, it was found that cerasomes freshly prepared were stable in alkaline condition, but sensitive to acid. The inorganic siloxane network was not well developed on the surface of the freshly prepared cerasomes so the resistance of cerasomes toward acidic solution was not sufficient immediately after preparation. Interestingly, the cerasomes prepared after 24 h exhibited remarkable morphological resistance toward both acidic and alkaline conditions. The particle size of the cerasome had almost no change even after 24 h incubation in acidic solution. Nevertheless, the same acidic or alkaline treatment could destroy conventional liposomes. The cerasome is composed of a spherical lipid-bilayer membrane having an internal aqueous compartment, like a so-called liposome, and additionally covered with a siloxane network on its surface. The siloxane network is viewed as a protection toward surfactant solubilization, long-term storage, acidic media, and all factors susceptible to destabilize bare liposomes. The silica layer of cerasomes greatly reduces liposome fusion and results in a substantial increase in their long-term stability [19,42]. The three-dimensional integration of lipid vesicular nanoparticles on a substrate was achieved by layer-by-layer (LbL) assembly of a pair of cationic and anionic cerasomes prepared from CFLs 1 and 4 [43–46]. These cerasomes perform as polyanionic and polycationic nanoparticles in neutral pH region, since the isoelectric points determined by pH dependence of the zeta-potential were 4.3 and 12.0 [46–49]. The cerasome particles closely packed like a stone pavement were clearly observed in both layers. In addition, difference in the particle size for each layer indicates the cationic and anionic cerasomes undoubtedly formed the LbL assembly. The layered paving of the vesicular nanoparticles was seen in every layer at least up to tenth adsorption steps. It is noteworthy that the layered paving superstructure was never observed by replacement of the cerasome to other bilayer vesicles formed with phospholipids or synthetic lipids. Therefore, the morphological stability of cerasomes seems to be extremely higher than those of other bilayer vesicles, since the membrane surface of the cerasome is covered with the siloxane network to prevent collapse and fusion of the vesicle. 6. Biocompatibility evaluation of cerasomes

5. Stability of cerasomes A typical phenomenon of instability in the conventional liposome formulation is the increase in particle size due to the aggregation or the fusion of unstable liposomes during the formulation processing storage [36–38]. An increase in particle size of liposomes generally results in rapid uptake by the reticuloendothelial system (RES) with subsequent rapid clearance and a short half-life. Thus, controlling and maintaining liposomes at small and uniform sizes are critical in developing a viable pharmaceutical product [39–41]. The shelf-life stability of pharmaceutical and cosmetic products is determined by the physical and chemical stability of the liposomes (uniformity of size distribution and encapsulation efficiency, and minimal degradation of all compounds, respectively). By optimizing the size distribution and pH and ionic strength, the stability of liquid liposome formulations can be improved greatly. Nevertheless, the solution of cerasomes could be stored at 4 °C for several months and showed almost no change in particle size after long-term storage, indicating no aggregation or fusion of cerasomes [20]. Surfactant solubilization is a useful method to evaluate morphological stability of liposomes in aqueous media. The resistance of cerasomes against a cationic surfactant Triton X-100 was determined by the light scattering intensity of the vesicles with conventional liposomes from dimyristoylphosphatidylcholine (DMPC) as a reference. When three equivalents of Triton X-100 were added to the liposomes, the Dhy decreased drastically, indicating a collapse of the vesicles. In contrast, the cerasomes exhibited remarkable morphological resistance toward Triton X-100. The Dhy of the cerasomes prepared from CFL 1 did not

To effectively develop cerasomes for biomedical applications, the biocompatibility of cerasomes with an average diameter of 153.7 ± 8.0 nm was evaluated [32]. In order to eliminate the effect of particle size on cell behavior, the silica NPs were prepared with an average diameter of 153.2 ± 7.3 nm, similar to the size range of cerasomes. It was observed that the uptake of cerasomes by human umbilical vein endothelial cells (HUVECs) increased notably with increasing concentrations of cerasomes and got saturated at 400 μg/mL after 4 h incubation. The kinetics of cell association with NPs revealed that the uptake of cerasomes by HUVECs increased as the incubation time during the first 6 h. But the uptake amount reached maximum at 6 h and no more uptake was observed even after 19 h incubation, which appeared to derive from saturation of the uptake system. Confocal laser scanning microscopy (CLSM) was used to verify the cellular localization of the cerasomes. Fluorescent cerasomes were clearly observed inside the cells as green spots and showed a cytosolic localization, indicating that the cerasomes were indeed endocytosed. It was found that the uptake of cerasomes was obviously inhibited after treatment at 4 °C with respect to the same experiment carried out at 37 °C, indicating that the cellular internalization of cerasomes follows an energy dependent or independent pathway. After HUVECs were treated by sucrose and K+-free buffer, the uptake of cerasomes by cells decreased from 73% to 75%, respectively, suggesting that the uptake of cerasomes occurred possibly through a clathrin-dependent endocytosis pathway. Nanoparticles are transported into the cell by specific or nonspecific cellular uptake mechanisms depending on the surface properties of NPs. Cerasomes were negatively charged and did not possess


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any specific ligands on their surfaces. As a result, a nonspecific cellular uptake would participate in the process of cerasome uptake [43]. Both cerasomes and silica NPs exhibited growth inhibition in a concentration dependent manner in both HUVECs and Hela cells. Cell viability decreased with increasing NPs concentrations. Nevertheless, cerasome-treated cells showed higher viability than cells incubated with silica NPs of the same concentration. In another word, cells treated with cerasomes had lower cytotoxicity than silica NPs. It is reported that large amount of silicic acid released by the silica NPs would probably increase the cytotoxicity by reducing local extracellular or intracellular pH value. There was a small increase in cell viability for Hela cells treated with silica NPs of low concentrations for 24 h, which might arise from the stimulation of cells in the presence of silica NPs [50]. After treatment with DPPC liposomes of various concentrations for 24 h, HUVECs exhibited higher cell viability than those treated with cerasomes. However, no apparent difference in cell viability was seen between cerasomes and liposomes after 72 h incubation. This was probably due to the insufficient morphological stability of liposomes. Effects of cerasomes on cell viability were also time dependent. After 24 h incubation, both the cell lines exhibited good viability when incubated with cerasomes. After 72 h, cerasomes treated cells showed lower viability than 24 h treatment with a similar trend for concentration dependent cytotoxic effects against these two cell lines. Moreover, when treated with cerasomes, the cell viability for Hela cells was higher than that for HUVECs. After 24 h incubation with Hela cells, high cell viability was observed. Even at a concentration of 1 mg/mL, more than 80% Hela cells were viable. These results indicated that in addition to concentration and time, effect on cell viability of cerasomes as well as silica NPs is cell-type dependent. The differences suggested that cancerous cells presented several features different from healthy cells to promote their survival and may also attribute to the differences in uptake levels in cancer cells [51]. In cell cycle study, the different effects on the percent of G0/G1 phase between silica NPs and cerasomes indicated that silica NPs were significantly more toxic than the corresponding cerasomes by inducing

G0/G1 arrest in HUVECs. It has been reported that some NPs inhibit cell growth due to its ability to enhance intracellular reactive oxygen species (ROS) and the changes of ROS generation strongly influenced cell proliferation and gave rise to necrosis in cells incubated with NPs [52–54]. Our study showed that more endogenous ROS of cells was stimulated in response to the treatment with silica NPs than cerasomes. Together with the results from the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and cell apoptosis studies, it could be concluded that the lower cell viability and higher percent of necrotic cells may be attributed to the higher endogenous ROS level caused by silica NPs than cerasomes. Compared with cerasomes, silica NPs caused increased adhesiveness shown by induction of intercellular adhesion molecule (ICAM-1). Moreover, silica NPs induced ICAM-1 expression in a concentration- and time-dependent manner. The ICAM-1 expression increased with silica NP concentrations and incubation time (Fig. 5). Thus, exposure to silica NPs may be a significant risk for the development of inflammatory disease [55–57]. In contrast, cerasome is a less potent activator of inflammatory reaction than silica NPs for ICAM-1 expression. In a word, cerasomes were internalized via non-specific or clathrinrelated endocytosis and affected different aspects of cell functions to a smaller extent than silica NPs of the similar size, including cell proliferation, cell cycle, cell apoptosis, endogenous ROS level and ICAM-1 expression. The introduction of liposomal architecture into cerasomes boosts the biocompatibility. Cerasome's biocompatibility is as good as traditional liposome but better than silica NPs, indicating great potential for drug and gene delivery applications [58]. 7. Cerasomes for drug and gene carriers Nowadays, controlled drug delivery technology represents one of the frontier areas of modern medicine and pharmaceutics due to their great capability to regulate drug release to improve therapeutic efficacy and reduce side effects [59–61]. A big challenge of engineering drug

Fig. 5. ICAM-1 expression of HUVECs exposed to cerasome and silica NPs for (a) 6 h, (b) 12 h and (c) 24 h. The data are represented as mean ± SD. Three samples were measured for each treatment: *p b 0.05 and **p b 0.01.



Paclitaxel-loaded cerasome 1 Paclitaxel-loaded cerasome 8 Paclitaxel-loaded cerasome 9 Paclitaxel-loaded cerasome 10

50

Cumulative release (%)

delivery systems is the controlled assembly of purposefully designed molecules, or ensembles of molecules, into nanoscaled structures to provide increasingly precise control at molecular levels, over the structure, properties and function. Despite numerous drug carriers have been developed and tested over the past decades, clinical success is still relatively rare. Liposomes can minimize the uptake of drug by normal cells and enhance the accumulation of the drug in tumor cells [62–64]. To deliver the molecules to sites of action, the lipid bilayer can fuse with other bilayers such as the cell membrane, thus delivering the liposome contents [65,66]. Some of liposomal drugs have been applied in clinical [67,68]. Various functional molecules showing temperature [69–71], light [72–74], pH [75–77], and even multi-stimuli [78–80] sensitive properties can be incorporated into liposomes to modulate the drug release behavior on demand by chemical conjugation to lipid molecules beforehand. Nevertheless, the primary drawback hampering the application of liposome is the general lack of stability in physiological condition, which often leads to a burst release of the encapsulated drugs, causing undesirable side reactions [81,82]. Therefore, we demonstrated for the first time that the highly stable liposomal cerasomes can be used as a new promising drug delivery system. Different from traditional liposomes, cerasomes can encapsulate not only both hydrophobic and hydrophilic molecules, but also amphiphilic molecules. The entrapment efficiencies of hydrophilic doxorubicin (DOX) and hydrophobic paclitaxel (PTX) were evaluated to define the potential carrier capacity of the various cerasomes with different numbers of the triethoxysilyl headgroups and the alkyl chains. DOX was entrapped into the aqueous compartment of cerasomes with encapsulation efficiencies of 32–45% in the increasing order of cerasome 1 b cerasome 10 b cerasome 8 b cerasome 9, in agreement with the triethoxysilyl headgroup number of the corresponding lipids, due to the presence of the hydrophilic part of silanol moieties on the outer surface and in the inner aqueous compartment of the cerasomes [29]. This may cause an electrostatic adsorption between positively charged DOX and negatively charged cerasomes, hence resulting in an increase in the encapsulation efficacy of water-soluble DOX drugs. However, the change in drug loading contents (1.84–2.43%) of the four cerasomes were not consistent with their encapsulation efficiencies, showing the order of cerasome 9 b cerasome 10 b cerasome 8 b cerasome 1. Similar results can be found for the PTX drug loading contents with the increasing order of cerasome 9 b cerasome 8 b cerasome 10 b cerasome 1, also different from their entrapment efficiency (cerasome 1 b cerasome 8 b cerasome 9 b cerasome 10). The variation in entrapment efficiency was also dependent on lipid compositions, CFLs 1, 8 and 9 were all bearing two alkyl chains, and the presence of more triethoxy-silane headgroups may retained more PTX drug in the cerasome bilayers during the preparation process. In addition, encapsulation efficiency of PTX in cerasomes depended on both the triethoxysilyl headgroups and the alkyl chains. Cerasome 10 has the highest encapsulation efficiency for PTX probably due to the structure of CFL 10, which contained three alkyl chains, leading to the resultant vesicle with more compacted bilayer as compared to the other cerasomes, providing an increase in PTX entrapment efficiency of about 80%. PTX is hydrophobic so it is believed that paclitaxel molecules present in the hydrophobic domain of the lipid bilayer [83]. The incorporation of PTX seemed to cause almost no change in size and morphology of cerasomes. Vesicle composition plays an important role in modulating the drug release behavior. To reveal how the cerasome composition affects the release profiles of PTX, the in vitro PTX release from cerasomes 1, 8, 9 to 10 was examined over an experimental time period of 96 h [29]. Four types of cerasomes had similar release profiles and exhibited no initial burst release due to their high stability (Fig. 6). But their release rates were different. The release rate of PTX from the cerasomes was markedly influenced by the molecular structure of CFLs. From cerasome 1 to cerasome 9, the release rate decreased as the number of triethoxysilane headgroups in CFLs increased. Within 100 h, the total release amount of PTX was up to 36%, 24% and 18% for cerasomes 1, 8

7

40

30

20

10

0 0

20

40

60

80

100

Time (h) Fig. 6. Cumulative release of PTX from cerasomes 1, 8, 9, and 10 as a function of time.

and 9, respectively. The formation of siloxane bonds resulted in a silica-like surface of cerasomes with high degree of polymerization. The density of siloxane networks increased with the number of triethoxysilane headgroups of CFLs. The slower release rate was attributed to the higher density of siloxane networks, which block the drug release channels. Nevertheless, the siloxane network was not so highly developed on the cerasome surface since the length of the Si\O\Si bond was much shorter than the diameter of the cross-section of the alkyl chain segment of the hybrid amphiphiles. This effect perturbed the membrane structure and may induce the formation of pores that were sufficiently large to allow for the leakage of PTX, allowing for the permeation of small molecules across the membrane without an introduction of a pore-forming protein [84]. Therefore, we can modulate the drug release rates from cerasomes by adjusting the number of triethoxysilane headgroup and alkyl chains in CFLs. The magnitude of the initial burst and the rate of the sustained release of DOX from cerasomes can be modulated by incorporating dipalmitoylphosphatidylglycerol (DPPG) in cerasome formation and altering the ratios of CFLs and phospholipids [85–87]. As presented in Fig. 7, cerasomes containing 0%, 10%, 30%, 50%, and 80% DPPG released 32.2 ± 1.8%, 41.4 ± 1.9%, 55.7 ± 2.7%, 66.7 ± 3.0%, and 75.8 ± 2.0% DOX in the first 10 h, respectively. Clearly, the initial burst release of DOX from cerasomes increased as the DPPG content increased. This suggests that DOX interacts more strongly with hydrolyzed CFLs than DPPG. As a result, an increase in the DPPG content weakened DOXcarrier interactions in cerasomes. This resulted in more encapsulated

Fig. 7. Influences of vesicle composition on the sustained release of DOX from DPPG-cerasomes. ■ Liposomes, ♦ 80% DPPG, ▼ 50% DPPG, ▲ 30% DPPG, ● 10% DPPG, Y cerasomes. Data are shown as mean ± SD, n = 5.


8


DOX molecules existing in the molecularly dispersed state, available for the initial burst release. Interestingly, the sustained release of DOX from cerasomes followed the initial burst also slightly increased as the DPPG content increased. As a result, the total release of DOX from cerasomes containing 0%, 10%, 30%, 50%, and 80% DPPG was 55.2 ± 1.5%, 65.7 ± 5.0%, 85.0 ± 2.9%, 93.2 ± 2.1%, and 97.6 ± 1.3% DOX in 57 h, respectively. This can be ascribed to the difference in the development of siloxane networks in the mixed cerasomes. As the proportion of DPPG increases, the degree of polymerization of siloxane networks decreases, hence the release rate increases. We thus demonstrated that both the initial burst and subsequent sustained release can be modulated by alteration in vesicle composition. Consequently, a wide range of release profiles were achieved. Therefore, the inclusion of DPPG in cerasome formation offers a new mechanism that modulates the release profiles of DOX from cerasomes. We also compared the antitumoral activity of PTX-loaded cerasomes (PLCs) with that of free drug using human ovarian cancer cell line 2774. It was found that cancer cell viability was dependent on the incubation time. PLCs exhibited a much higher IC50 value than free PTX (151.9 nM vs. 69.8 nM) after 24 h incubation. The cytotoxicity of free PTX was higher than that of PLCs and this was most probably attributed to the faster drug release rate of free PTX. After 72 h incubation, both PTX and PLCs showed improved cytotoxicity relative to 24 h incubation. PLCs were found to be as active as the free drug and show a similar IC50 value as free PTX (8.7 nM vs. 8.6 nM). Cancer cell viability was also dependent on the drug concentration. The higher the drug concentration, the more significant effects would be resulted. It is consistent with the result of the release study. The siloxane framework on cerasome surface improved the stability of vesicular lipid bilayer and reduced the release rate of paclitaxel from the cerasomes. As a result, only a fraction of the incorporated drug is released over the experimental period of 24 h when vesicles come into contract with medium and cells. Since the discovery of lipofection [88], cationic lipids, which can form liposomal bilayers in water, have been widely used as transfection agents in gene delivery [89,90]. Polyanionic DNAs are readily bound to the resulting cationic liposomes electrostatically to give lipoplexes which are taken in the cells via endocytosis to ultimately result in expression of the encoded gene. Nevertheless, the bilayer-keeping forces are not very strong, and liposomes are not so rigid or robust. Liposomes (vesicles) easily undergo fusion (aggregation) as induced not only by hydrophobic species but also by simple ions (divalent metal cations and organic dianions for anionic and cationic liposomes, respectively). There is no doubt that DNAs (and RNAs as well) are potential fusioninducers to lead to transfection-irrelevant (low efficiency and high toxicity) big particles [91,92]. Therefore, the surface rigidified cerasomes were applied as a gene carrier [21,27]. It is proved that cerasomes are neither fused nor cross-linked when bound to siRNA (short duplex RNA) but not to plasmid DNA (long duplex DNA) which induces cross-linking [93]. On the contrary, non-ceramic reference liposomes are easily fused by the siRNA. The cerasome can thus be used as a viral-size siRNA-carrier in a wide range of concentration for RNAi silencing of exogenous and endogenous genes. In a word, the silicon strategy thus provides a simple and widely applicable tool to overcome general problems associated with current technology of drug and gene delivery. We therefore anticipate that cerasomes will be a promising candidate as drug carrier with more sophisticated and smarter controlled release behavior. 8. Cerasomal porphyrin for photodynamic therapy of cancer With the aim of improving the efficacy and safety of photodynamic therapy (PDT), liposomes with their flexibility to accommodate photosensitizers (PSs) with variable physicochemical properties came into focus as a valuable carrier and delivery system [94,95]. However, a lipid exchange between the liposomes and lipoproteins leads to an irreversible disintegration of the liposome, resulting in the release of PSs in

the bloodstream [95]. This premature release may reduce efficacy of treatment because the release of the PS drugs is not a prerequisite for PDT action unlike conventional chemotherapy [96,97]. Porphyrins are the most popular PSs for PDT. Herein, we fabricated porphyrin bilayer cerasomes (PBCs) for the first time by sol–gel reaction and self-assembly process from a conjugate of porphyrinorganoalkoxysilylated lipid (PORSIL) with dual triethoxysilyl heads, a hydrophobic double-chain segment, a porphyrin moiety and a connector unit among them (Fig. 8) [98]. Compared with the reported cerasomes [99–101], the covalent linkage of porphyrin to cerasomes resulted in the drug loading content of 33.46% in the PBCs, significantly higher than the physically entrapping cerasomes or liposomes (generally less than 10%). In addition, the premature release of PSs can be avoided during systemic circulation. TEM image clearly demonstrated the formation of PBCs with a diameter range from 50 to 100 nm, in agreement with DLS measurements that gave a sharp peak with a narrow distribution (70 ± 13 nm). The vesicular structure of PBCs was further confirmed by encapsulation of a water-soluble fluorescent dye of calcein. The calcein-loaded vesicles had brightly green fluorescent cores and red fluorescent shells, which were clearly seen using CLSM. The red emission resulted from the conjugated porphyrin in the lipid bilayer of PBCs and the green emission from calcein in the aqueous core of PBCs. Thus, the capability to load water-soluble compound into the core of PBCs proved the existence of the vesicular structure. Compared with PORSIL lipid in chloroform, the UV–Vis absorption spectra of PBCs broadened and the absorbance of PBCs increased but the formation of cerasomes did not have major effect on the position of Soret and Q bands of porphyrin. The shape of the absorption spectrum of PBCs is quite similar to that of the solution-phase PORSIL lipid. These indicate that aggregation does not occur in the nanoparticles. Both Soret and Q bands exhibit bathochromic shift of 3 nm because of an alternative arranging mode of porphyrin group and alkyl chains, which can greatly reduce the aggregation of the porphyrin moiety. This is due to the fact that the existence of double-alkyl chains sterically hinders porphyrin moieties approaching each other. Therefore, we believe this method is highly effective for neither preparing photofunctional cerasomes without aggregation nor deteriorating the quality of photoluminescence. It was found that the presence of the double-alkyl chains avoided significant fluorescence self-quenching in PBCs. In addition, the conjugation of porphyrin to cerasomes improved significantly the 1O2 generation. CLSM was used to verify the cellular localization of PBCs (Fig. 9). Fluorescent PBCs were clearly observed inside the cells as red spots distributing in the cytoplasm and mainly localized in the lysosomes. An almost complete co-localization could be observed between red and green fluorescence as it was evident from the pale yellow fluorescence arising from the overlap of the two fluorescence images. After the nuclei of the tumor cells were stained with 4′, 6-diamidino-2-phenylindole (DAPI) (blue in Fig. 9c), PBCs were found to be distributed throughout the entire cytoplasm (Fig. 9d), indicating that PBCs were indeed endocytosed. The cell phototoxicity study shows the percentage of cell survival after treatment of Hela cells with various concentrations of PBCs followed by subsequent irradiation with the light (400–700 nm). Significant phototoxic effect on the cultured cells can be observed. In addition, the uptake amount of PBCs increased with the elevated concentration, further leading to a higher phototoxicity against Hela cells. In contrast, after 24 h incubation, no significant decrease in cell viability in the dark could be observed with the concentration of PBCs below 1.0 μM, indicating low dark toxicity of PBCs. Nevertheless, free porphyrin showed higher dark toxicity and remarkably lower phototoxicity as compared to PBCs. No apparent phototoxicity was observed at the concentrations below 1.0 μM. When the concentration was above 5.0 μM, cell viability decreased, mainly arising from the dark toxicity of the free drug.



9

Fig. 8. Schematic illustration for the formation of PBCs from a PORSIL lipid.

Fig. 9. Subcellular localization of PBCs observed by fluorescent CLSM. Hela cells were incubated with PBCs at the concentration of 5 μM for 4 h at 37 °C. Observations of a) the PBCs channel, b) LysoTracker green (DND-26, to label lysosomes) channel, c) the nuclear dye DAPI channel and d) the overlap of the above images.


10


The blood circulation dynamics of PBCs were investigated in comparison with the porphyrin-loaded DSPC liposomes (PLLs). The blood fluorescence intensity of PLLs decreased to 30% at 6 h and almost no fluorescence was observed at 24 h, exhibiting rapid clearance kinetics. On the contrary, the decrease in the blood fluorescence intensity of PBCs was only 33.5% at 24 h, showing dramatically prolonged and slow clearance kinetics. Thus, it provided evidence that PBCs can maintain a long circulation without PEG chains. The covalent linkage of porphyrins can prevent cerasomes releasing PSs during systemic circulation and thus enhance the outcome of PDT. The orderly arranging mode of porphyrins in the lipid bilayer ensures the efficacy of singlet oxygen production even at an extremely high number of porphyrins and effectively prevents fluorescence loss of porphyrins, permitting a powerful tool for in vivo imaging and photodynamic diagnostics. The capability to load chemotherapy drugs into the internal aqueous core of PBCs makes it possible to develop a drugcarrier system for the synergistic combination of chemotherapy and PDT for the treatment of cancer. The PBCs have shown an avid uptake by malignant cells and cellular phototoxicity under irradiation. There is no doubt that the dual function nature of the PBCs will play an important role in future clinical photodynamics. 9. Conclusions and perspectives Cerasomes own higher stability than conventional liposomes and lower rigidity and density than competing silica nanoparticles. In addition, cerasomes are more biocompatible than silica NPs. They generally offer a large surface area that allows covalent and non-covalent surface functionalization with hydrophilic polymers, therapeutic moieties, and targeting ligands. Cerasomal drugs exhibit reduced toxicities and retain or gain enhanced efficacy compared with their free counterparts. Cerasomes allow enhanced drug delivery to disease sites by virtue of long circulation residence times. Also cerasomes promote targeting to particular diseased cells within the disease site. Particularly, cerasomes show promise as intracellular delivery systems for proteins/peptides, antisense molecules, ribozymes and DNA. Taking into account the great advantages of cerasomes, numerous potential applications of cerasomes may be envisaged. These include the use of cerasomes as diagnostic and therapeutic tools, sensors, information storage and processing systems, “smart” materials of controlled hydrophilicity/ hydrophobicity, nanoscale robots, valves or pistons, and more. Also, the integration of chemically synthesized nanostructures with biomolecules to yield hybrid nanostructures exhibiting multifunctions represents an encouraging path to follow and provides a new facet of nanobiotechnology. Albeit the progress in the field, exciting future developments are ahead of us. We anticipate that cerasomes will soon begin to reach their full potential as an important class of therapeutic agents and will contribute to significant advances in the treatment of many classes of diseases. The development of cerasomes that can be administered systemically and exhibit targeted and fusogenic properties appears to be increasingly within our grasp. All these efforts will certainly require interdisciplinary collaborations of chemists, physicists, biologists, and materials scientists. Acknowledgments This research was financially supported by the State Key Program of National Natural Science of China (No. 81230036), the National Natural Science Foundation of China (No. 21273014, 81371580) and the National Natural Science Foundation for Distinguished Young Scholars (No. 81225011). References [1] Lasic DD. Liposomes: from physics to applications. Amsterdam: Elsevier; 1993. [2] Wang XH, Li F, Liu SX, Pope MT. J Inorg Biochem 2005;99:452–7.

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