- Email: [email protected]
Mesostructured silica based delivery system for a drug with a peptide as a cell-penetrating vector
Microporous and Mesoporous Materials 122 (2009) 201–207
Contents lists available at ScienceDirect
Microporous and Mesoporous Materials journal homepage: www.elsevier.com/locate/micromeso
Mesostructured silica based delivery system for a drug with a peptide as a cell-penetrating vector Chuanbo Gao a,b, Isabel Izquierdo-Barba b, Ikuhiko Nakase c, Shiroh Futaki c,d,*, Juanfang Ruan b, Kazutami Sakamoto e, Yasuhiro Sakamoto b, Kazuyuki Kuroda f, Osamu Terasaki b,*, Shunai Che a,* a
School of Chemistry and Chemical Engineering, State Key Laboratory of Composite Materials, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China Structural Chemistry, Arrhenius Laboratory, Stockholm University, S-10691 Stockholm, Sweden c Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan d SORST, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan e Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, Tokyo, Japan f Department of Applied Chemistry, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan b
a r t i c l e
i n f o
Article history: Received 3 December 2008 Received in revised form 17 February 2009 Accepted 2 March 2009 Available online 11 March 2009 Keywords: Mesostructured silica Drug delivery Cell-penetrating peptide Sigmoidal release Sustained cellular uptake
a b s t r a c t A drug delivery system using mesostructured silica as a reservoir has been developed for the storage and controlled release of a drug with a cell-penetrating peptide (CPP) as a vector. We use fluorescein isothiocyanate (FITC) as the drug model and octaarginine (R8) as a vector to endow the drug with cell-penetrating property. The mesostructured silica reservoir system was prepared by using a one-pot liquid–crystal templating method, which is suitable for the encapsulation of intact FITC-R8 conjugates and sustained release of drugs without hampering their properties. The hydrophobic poly(propyl oxide) (PPO) shell of the pore-filling Pluronic F127 and the electrostatic interaction between R8 and siloxide ions on the pore walls act as the diffusion-limiting factors of the FITC-R8 conjugate. A sigmoidal in vitro release of FITCR8 from mesostructured silica into phosphate buffered saline (PBS, pH 7.4) was observed and the typical release duration was 5 days at 37 °C. Release from the reservoir yielded significant elongation in duration of the FITC signals in DU145 cells by confocal microscopic analysis, compared with a single administration of FITC-R8. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Recently, basic peptides, including human immunodeficiency virus (HIV)-1 Tat (positions 48–60) [1], Antennapedia (positions 43–58) [2] and oligoarginine peptides [3–5], have been reported to have the ability to translocate through cell membranes. These short sequences of peptides are referred to as the ‘‘cell-penetrating peptides (CPPs)” or ‘‘protein transduction domains (PTDs)”, and have been employed for the efficient delivery of exogenous molecules such as proteins [6], DNA [7], liposomes [8] and nanoparticles [9] into living cells. This highlights the applicability of CPPs to drug delivery system in which an enhanced cellular uptake of drugs could be consequently achieved. An example was demonstrated by Rothbard et al. that the conjugate of CPP and cyclosporin A was transported into skin cells of mouse and human, reached dermal T lymphocytes and successfully inhibited inflammation [4]. This drug delivery system using CPPs would become more practical by developing methods of controlled release of the CPP-conjugated * Corresponding authors. Tel.: +86 21 5474 2852; fax: +86 21 5474 1297 (S. Che). E-mail addresses: [email protected] (S. Futaki), [email protected] (O. Terasaki), [email protected] (S. Che). 1387-1811/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.micromeso.2009.03.002
drugs to yield optimized drug concentration to appreciate the maximum therapeutic effects with minimum side effects, as well as to protect the drug-CPP conjugates from possible facile enzymatic or chemical decomposition during storage and administration [10]. Mesostructured silicas, which were initiated by Kuroda and coworkers [11] and the researchers in Mobil Company [12], have opened up new possibilities of applications in the field of drug delivery. The tunable pore sizes in the range of 2–50 nm, high specific surface areas, large pore volumes and non-toxic nature [13] of these materials provide interesting possibilities for the inclusion of molecules of therapeutic value. Therefore, mesostructured silica is a promising candidate as a reservoir ideal for the storage and controlled release of drugs [14–23]. Up to now, the loading of drugs into the channel of mesostructured silica has been attained mainly by post-permeation method, i.e. the mesostructured silica carrier was synthesized and the surfactant was removed first followed by storage of the drug molecules by physical/chemical adsorption [14,17,18]. In the process of post-permeation, the drug incorporation is commonly carried out by soaking of the mesoporous silica in a highly concentrated drug solution and subsequent drying [19]. Therefore, only a portion of the employed drugs can be loaded
202
C. Gao et al. / Microporous and Mesoporous Materials 122 (2009) 201–207
in the silica materials due to the equilibrium and the distribution of the drugs is heterogeneous with possibilities to aggregate outside the particles. Besides, the remaining high porosity after post-permeation may make the drug-CPP compounds less stabilized compared with the case with mesopores filled by surfactant. Researchers also proposed a one-pot method to encapsulate drugs either into the channels of mesostructured silicas or into the polymer-silica assembled capsules [20,21]. Most of the one-pot procedures employed aqueous synthesis of powder forms of mesostructured silicas, and only limited amounts of the given drugs are finally incorporated into silica [22] and the majority of the drugs are segregated out to stay in the aqueous phase, especially in the case using solvent soluble drugs. Moreover, the strong acidic/basic conditions at high synthesis temperatures used in the one-pot method are often rather rigorous and may lead to decomposition of drug-CPP conjugates. Therefore, even though these studies show the applicability of mesostructured silica for the controlled/sustained release of incorporated drugs, new approaches should be employed for the present strategy to charge sufficient amount of intact drug-CPP conjugates into mesostructured channels. To overcome the above shortcomings, we have developed a mesostructured silica system suitable for encapsulating drug-CPP conjugate. Fluorescein isothiocyanate (FITC) was employed as a drug model lacking the membrane permeability, and octaarginine (R8) as a typical cell-penetrating peptide to enhance the cellular uptake. Highly ordered mesostructured surfactant/silica composite has been employed as a reservoir of the FITC-R8 conjugate, and a one-pot liquid–crystal templating [24,25] method was used to incorporate the conjugate into the mesostructured silica. The process was carried out in a nonaqueous system at a low reaction temperature to avoid water/heat mediated reactions of the drugs. Because of the existence of surfactant in the mesopores, it is expected that the FITC-R8 conjugate would be stabilized avoiding enzymatic or chemical decomposition. The copolymer Pluronic F127 (EO106PO70EO106) with low toxicity [26] was employed as the template of the mesopores to avoid the toxicity of the surfactant leached into the body fluid, which ensured the biocompatibility of the drug. The amphiphilic FITC-R8 conjugate is assembled with F127 and hydrolyzed silica source in the formation of highly ordered mesostructured silica in acidic medium, by the means of hydrophobic force, electrostatic interaction of protonated R8 and protonated silanols via counterions (e.g. chloride), and H-bonding of R8 with silanols. This self-assembly mechanism ensures that the FITC-R8 molecules are incorporated in the organic/inorganic interface by using this method. During the release in phosphate buffered saline (PBS, pH 7.4), as shown in Scheme 1, the drug model part (FITC) of the conjugate interacts with F127 micelles, while the positively charged R8 vector interacts electrostatically with the siloxide ions („SiO) covalently tethered on the silica wall; we expect both the hydrophobic poly(propyl oxide) (PPO) shell
and the electrostatic interaction could contribute to the sustained release of FITC-R8 from the carrier matrix [27]. Therefore, it is reasonable that by first conjugating the drugs to a vector (R8) and second incorporating the conjugate into mesostructured silica, the drug is stabilized and has the ability to release in a sustained manner and penetrate the cell membrane more easily. 2. Experimental 2.1. Synthesis of FITC labeled R8 peptide FITC-R8 [FITC-GABA-(Arg)8-amide] was prepared by Fmoc (9fluorenylmethyloxycarbonyl)-solid-phase peptide synthesis on a Rink amide resin as reported previously [28], where c-aminobutyric acid (GABA) was employed as a spacer to connect FITC with the (Arg)8 segment. The peptide was purified and the structure was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOFMS). 2.2. Synthesis of mesostructured silicas containing FITC-R8 In a typical synthesis of the one-pot method, 0.12 g of F127 (BASF) was dissolved in 0.3 g of tetramethyl orthosilicate (TMOS, BASF), and then 0.15 g of H2O/HCl (pH 1.3) was added at 40 °C (F127/TMOS = 0.00454). The hydrolysis of TMOS was achieved in 5 min at 40 °C and H2O molecules were mostly converted into methanol, after which 10.0 mg of FITC-R8 was added into the solution. Volatile chemicals including methanol and HCl were then removed from the synthesis system in vacuum to achieve highly ordered mesostructure [24]. The resultant monolithic gel was then aged at 40 °C for 2 days, ground into powders. To eliminate the particle size effect on the release kinetics, the powders were further sieved using #200 and #300 sieves to get the particles having a diameter of 50–76 lm. The loading amount of FITC-R8 in this mesostructured composite was 38.3 mg/g. A mesostructured silica containing FITC was obtained by using the same procedure and changing FITC-R8 into FITC while keeping the same moles. Nonporous silica material with FITC-R8 incorporated was prepared by the similar means without surfactant as template for mesopore. These were used as control samples in the following in vitro release experiment. 2.3. Release of FITC-R8 from silica FITC-R8 loaded mesostructured silica particles were first washed with PBS (pH 7.4) before in vitro release in order to remove any F127 or FITC-R8 located on the outer surface of the particles and afterwards dried in vacuum. The loading of FITC-R8 in the silica material obtained was recalculated as 38.1 mg/g. Ten milligram of FITC-R8 loaded mesostructured silica particles were added into
Scheme 1. Interactions of drug model FITC-R8 with F127 and silicate as drug diffusion-limiting factors, when mesostructured silica particles were immersed in PBS (pH 7.4).
C. Gao et al. / Microporous and Mesoporous Materials 122 (2009) 201–207
203
replaced with an equal volume of fresh PBS. The sample was diluted by 2.7 ml of PBS, and measured by fluorescence spectrophotometer to obtain the concentration of FITC-R8 released. 2.4. Internalization of FITC-R8 by cells DU145 prostate cancer cells were maintained in alpha-minimum essential medium (a-MEM) with 10% heat-inactivated fetal bovine serum as reported [28]. Briefly, 2 105 cells were plated into 35-mm glass-bottomed dishes and cultured for 48 h. The mesostructured silica containing FITC-R8 (3.0 mg) was incubated with the cells at 37 °C with fresh medium (1.0 ml). After 96 h, the DU145 cells were then washed with cold PBS. Distribution of the fluorescently labeled peptides was analyzed without fixing the cells by confocal scanning laser system. The single administration of FITC-R8 was investigated by incubating cells for 3 and 96 h in a-MEM containing 10 lM of FITC-R8. 2.5. Characterizations
Fig. 1. XRD patterns of mesostructured silicas containing FITC-R8 synthesized using F127 as the template. (a) As synthesized, (b) after treatment in PBS at 37 °C for 120 h. The intensity of the as-synthesized mesostructured silica (profile a) was multiplied by five for clarity. The calculated positions of X-ray reflections with indices are also indicated by arrows.
2 ml of PBS (pH 7.4), and afterwards shaken in a water bath at 37 °C. At regular intervals, the release system was centrifuged and 100 ll of the supernate was withdrawn and immediately
X-ray diffraction (XRD) patterns were recorded on a Rigaku X-ray diffractometer D/MAX-2200/PC using Cu Ka radiation (40 kV, 20 mA) at the rate of 0.5° 2h/min over the range of 0.6–5° 2h. High-resolution transmission electron microscopy (HRTEM) was performed with a JEOL JEM-3010 microscope operating at 300 kV (Cs = 0.6 mm, resolution 1.7 Å). The nitrogen adsorption/ desorption isotherms were measured at 77 K with a Quantachrome Nova 4200E porosimeter. Elemental analysis of the mesostructured silica was conducted on a Perkin Elmer PE 2400 II CHNS/O
Fig. 2. HRTEM images of mesostructured silicas containing FITC-R8 synthesized with F127 as the template. (a–c), As synthesized, recorded along [1 0 0], [1 1 0] and [1 1 1] zone axes; (d), After release of FITC-R8 in PBS for 120 h, recorded along [1 0 0] zone axis.
204
C. Gao et al. / Microporous and Mesoporous Materials 122 (2009) 201–207
Analyzer. The concentration of FITC-R8 was determined by using a Varian Fluorescence spectrophotometer Cary Eclipse, excitation: k = 498.05 nm, emission: k = 519.10 nm. Distribution of the FITCR8 in DU145 cells was analyzed by using an Olympus FV300 confocal scanning laser system consisting of an Olympus IX81 microscope.
3. Results and discussion 3.1. Structure and stability of FITC-R8 incorporated mesostructured silica monolithic particles The scanning electron microscopy (SEM) images of the sieved mesostructured silica particles (Supplementary Information Fig. S1) reveals that the particles have irregular morphologies and sizes ranging from 50 to 100 lm randomly. This uniformly distributed mesostructured silica in size simplifies the investigation of the drug release from it because of the elimination of particle size effect on the release kinetics. The XRD patterns of the mesostructured monoliths with FITCR8 incorporated are shown in Fig. 1a. Only one reflection could be observed showing the d-spacing of 10.3 nm. Although no fine peaks showing the detailed structural information have been detected, the sharp X-ray reflection peaks suggest that the material possesses highly ordered mesostructure. As shown in Fig. 1b, after 120 h of the treatment in PBS at 37 °C, the mesostructured silica shows higher X-ray reflection intensity than the as-synthesized one due to the release of both FITC-R8 and the F127 surfactant,
indicating that the mesostructure of the drug reservoir was retained during the drug release. Fig. 2a–c shows the HRTEM images of the thin parts of the mesostructured silica with FITC-R8 incorporated recorded along [1 0 0], [1 1 0] and [1 1 1] zone axes. The mesostructured silica possesses high crystallinity despite the coexistence of disordered mesophase and amorphous impurity. The observed reflection conditions from the Fourier diffractograms of the HRTEM images are hkl: h + k + l = 2n, 0kl: k + l = 2n, hhl: l = 2n, 00l: l = 2n, and thus the space group is determined to be Im3m. Therefore, the X-ray reflections with highest intensity shown in Fig. 1 can be indexed as 1 1 0 structure. The structure of the mesostrucreflection of the Im3m tured monolith after 120 h of drug release in PBS has been con firmed to be cubic Im3m structure and proved intact, as the HRTEM image shown in Fig. 2d reveals. Fig. 3 shows the nitrogen adsorption/desorption isotherm and pore size distributions of the material after treatment in PBS at 37 °C for 168 h and removal of surfactant by calcination. The sorption isotherm clearly shows a H2 type hysteresis loop, indicating a cage type mesopore system, and hence it is SBA-16 type [29,30]. The pore size is 5.8 nm calculated using Barett–Joyner–Halenda (BJH) method according to the adsorption isotherm and 3.8 nm derived from the desorption branch of the isotherm. It is reasonably considered that the mesostructured silica has a mesocage size of 5.8 nm and a size of window between mesocages of 3.8 nm, which are large enough to entrap drug molecules (normally no larger than 5 nm 2 nm) and thus sufficient for the drug storage and delivery system. It has also been suggested in the earlier literatures that the silica matrix may degrade in the drug release which serve as a diffusion-controlling factor and may affect the investigation of drug release kinetics from mesopores [31–33]. In our system, we found that only about 5% of the silica was dissolved into PBS solution (2 ml PBS per 10 mg mesostructured silica, Supplementary Information Fig. S2), which indicates that the silica matrix is stable enough and the effect of the degradation of mesostructured silica on the drug release could be ruled out. 3.2. In vitro release of FITC-R8 from mesostructured silica into PBS
Fig. 3. Nitrogen adsorption/desorption isotherm (A) and BJH pore size distribution (B, from desorption branch (I) and from adsorption branch (II)) of the mesoporous silica. The mesoporous silica containing FITC-R8 was first treated with PBS (pH 7.4, 37 °C) for 168 h and then calcined at 550 °C for 6 h. The H2 hysteresis loop of the isotherm indicates the cage-type mesostructure, and the pore size and window size are 5.8 nm and 3.8 nm, respectively.
The time-course analysis of FITC-R8 release from the mesostructured reservoir into PBS (pH 7.4) at 37 °C was then conducted by measuring florescence intensity, and the release of the FITC-R8 conjugate was confirmed by MALDI-TOFMS. After 96 h of FITC-R8 release, calculated m/z for M + H+ was: 1742.1, found: 1351.98(R8) and 1742.05 (FITC-R8). The height ratio of MALDITOFMS two peaks about FITC-cleaved R8 and FITC-R8 was 8:5. For such a long-term release there were still significant amount of intact FITC-R8 in the aqueous release system. It can be reasonably regarded as newly released FITC-R8 conjugate, which was protected before release from the outer water molecules by the hydrophobic mesochannels filled with PPO. Nevertheless, FITC-R8 which was exposed in PBS for a long time decomposed into FITC and R8 because of the instability of FITC-R8 when exposed to water. It confirmed that safety storage of FITC-R8 conjugates could be achieved by using mesostructured organic/inorganic composite as reservoir. Fig. 4 shows the percentile cumulative release profile of FITC-R8 from mesostructured silica into PBS. It follows a sigmoidal release which is characterized by an initial lag in the release reducing or avoiding the ‘‘burst” effect and afterwards an accelerating release [34,35]. This profile could be well fitted to the following equation [36,37]:
ln
X ¼ k t k tmax T X
ð1Þ
C. Gao et al. / Microporous and Mesoporous Materials 122 (2009) 201–207
205
changes of mesostructured silica was investigated in details. Fig. 5 shows the XRD patterns of the mesostructured silicas at different stages of the drug release. An increase in the X-ray diffraction was observed with the time of PBS treatment, which suggests retention of the mesostructure and also a leach of surfactant during release that gives rise to a greater contrast between mesochannel and silica wall. This leach of surfactant produces porosity in the mesostructured F127/silica composite, which acts as an accelerating factor of the FITC-R8 molecules to diffuse out into PBS. Fig. 6 shows the nitrogen adsorption/desorption isotherms of the solids obtained from the release system at different
Fig. 4. Percentile cumulative release of FITC-R8 from mesostructured silica into PBS (pH 7.4, 37 °C) and its sigmoidal curve fitted from Eq. (1).
where X is the fraction of the released FITC-R8 at time t, T is the total released fraction of the FITC-R8 at time infinity, k is the rate constant and tmax is the time to maximum rate of the FITC-R8 release in the sigmoidal release. The model parameters representing the induction and accelerating release were determined and collected in Table 1. According to Fig. 4 and Table 1, it can be concluded that: (1) The release has a induction time of about 36 h, and during this period the release of FITC-R8 from mesostructured silica is very slow; (2) After 36 h, the release was accelerated and reached its maximum rate at 73.5 h; (3) 85% of the FITC-R8 incorporated in the mesostructured silica was released and the release duration was more than 5 days. Interconnected mesochannels have a profound effect on the drug release. No drugs could be released from silica without mesopore (Supplementary Information Fig. S3) indicating its vital role in the drug release. It confirms that the mesochannel is the route for the drug-CPP conjugates to diffuse into the outer PBS solution. The octaarginine is positively charged and the silica is negatively charged at pH 7.4, and therefore during the release, the electrostatic interaction between FITC-R8 and siloxide ions („SiO) on the wall of mesopores will decrease the diffusion rate of FITC-R8. On the other hand, FITC-R8 co-assembles with F127 and silicate during the synthesis, and as a result, the FITC-R8 molecules locate on the organic/inorganic interface. The FITC-R8 molecules would first penetrate the hydrophobic PPO shell formed by the pore-filling surfactant in the diffusion into PBS. This also serves as a diffusion-limiting factor and contributes to the sustained release of FITC-R8. As expected, in the case of FITC storage and release without conjugation with R8 using the same mesostructured F127/silica system as the reservoir, a quick and complete release was observed in less than an hour (Supplementary Information Fig. S4). It emphasizes the effect of both the electrostatic interaction between R8 and siloxide ions and the pore-filling surfactant on the sustained release, because in this system a lack of interaction with silica wall leads to a distribution of FITC in the hydrophobic core of F127 micelles, which makes the FITC molecules easier to diffuse into PBS. In order to discover the cause of the sigmoidal release kinetics of FITC-R8 from mesostructured silica into PBS, the structural
Fig. 5. XRD patterns of the mesostructured silicas at different stages of the drug release. (a–e) The mesostructured silicas were treated with PBS (pH 7.4, 37 °C) for (a) 24 h, (b) 48 h, (c) 72 h, (d) 96 h and (e) 120 h, respectively.
Table 1 Values of kinetics parameters for the release of FTIC-R8 from mesostructured particles fitted from Eq. (1). T
k (h1)
tmax (h)
r2
85.1%
0.05635
73.5
0.9985
Fig. 6. Nitrogen adsorption/desorption isotherms of the mesostructured silicas at different stages of the drug release. (a–e) The mesostructured silicas were treated with PBS (pH 7.4) at 37 °C for (a) 24 h, (b) 48 h, (c) 72 h, (d) 96 h and (e) 120 h, respectively. Isotherm e was offset vertically by 50 cm3 g1 STP for clarity.
206
C. Gao et al. / Microporous and Mesoporous Materials 122 (2009) 201–207
stages of the drug release and the porous properties are summarized in Table 2. It can be inferred that the surface area and the pore volume of the mesostructured solids increase with the treatment time, and the increment of the porosity reaches its maximum value at about 72 h, which is coincident with the rate of FITC-R8 release. It confirms that the porosity resulting from the leaching of pore-filling surfactant dramatically accelerates the release of the drugs. Table 3 shows the carbon content of mesostructured silica solids sampled at different stages of the drug release. The data were derived from the CHN elemental analysis. A decrease of carbon content can be inferred owing to the leach of both the surfactant F127 as well as FITC-R8. Considering that F127 contributes to the most of the carbon decreasing, the leaching amount of the F127 has been estimated and the results are demonstrated in Table 3. It is worth noting that only a part of F127 in the organic/inorganic composite (<25%) has been released during the treatment in PBS, and it is adequate for the production of porosity and acceleration of the drug release. The sigmoidal kinetics has been observed in polymer based homogeneous distributed large particle systems where diffusion
Table 2 Porous properties of mesostructured silicas at different stages of the drug release. Time of the treatment in PBS (h)
Surface area (m2 g1)
Pore volume (cm3 g1)
24 48 72 96 120
12 29 100 197 215
0.02 0.05 0.15 0.29 0.30
Table 3 Carbon content of mesostructured silicas at different stages of the drug release. The data were collected by CHN elemental analysis. A decrease in the carbon content of the mesostructured silicas was observed during the FITC-R8 release. Time of the treatment in PBS (h)
Carbon content (%)
Leaching percentage of F127(%)a
0 24 48 72 96 120
26.43 21.96 21.59 20.82 20.24 20.20
0 16.9 18.3 21.2 23.4 23.6
a Estimated from the carbon content of the silicas given that F127 constitutes the majority of the organic composition and has a large carbon content.
and polymer degradation governed the drug release [36,37]. It can be concluded from the discussion above that in our system, the hydrophobic PPO shell formed by the pore-filling surfactant and the electrostatic interaction between the drug and silica serves as the diffusion-limiting factors, which hindered the drug from releasing in the induction time (ca. 36 h). After this period, an accelerated release of FITC-R8 lasting about 4 days was achieved as a result of the leaching of the surfactant and thus a dramatic increase in mesoporosity of the organic/inorganic composite. And therefore, a sigmoidal release profile of the FITC-R8 was observed. 3.3. Internalization of FITC-R8 by cells In order to examine if the above features in controlled release were actually reflected in the cellular uptake of the drugs, as well as if the CPPs really enhanced cellular uptake of the conjugated drugs, the FITC-R8-containing mesostructured silica was incubated with DU145 cells. The silica particles containing FITC-R8 were soaked in the culture media of DU145 cells and incubated together at 37 °C. The mesostructured silica particles shows little cytotoxity to the DU145 cells. The internalization of the FITC-R8 was assessed by confocal microscopic observation (Fig. 7). When the cells were incubated with silica particle containing FITC-R8, significant fluorescent signals were observed at as long as 96 h (Fig. 7c), whereas no signals were observed from the cells treated with FITC without conjugated with R8 peptide at any of the time points (not shown), exemplifying the effectiveness of conjugation with CPPs for the intracellular delivery of membraneimpermeable drugs. On the other hand, a single administration of 10 lM FITC-R8 without entrapped in the mesostructured reservoir yielded FITC signals at 3 h (Fig. 7a) but very little signals were observed at 96 h after administration (Fig. 7b). This clearly shows that by encapsulation in the mesostructured reservoir, intact storage and sustained release of the drug-CPP conjugate were achieved and eventually led to the efficient intracellular delivery yielding an elongated bioactivity. The above results also suggest that the procedures employed for the reservoir formation were not severe to give damages for the structures and properties of the R8 peptide and FITC, which would be attained by the procedures of conventional mesostructured silica formation for these chemically rather fragile species. This enhanced cellular uptake of the drug-CPP conjugate suggests that, this mesostructured silica system, which provides the intact storage and controlled release of the conjugate, is suitable to be potentially used as a prototype for the transdermal drug delivery. Furthermore, injection is also feasible for this drug delivery system, which could also have the high effectiveness of cellular
Fig. 7. FITC-R8 released from mesostructured silica yielded sustained signals of the model drug (FITC) in DU145 (prostate cancer) cells. (a–b) Confocal micrographs of DU145 cells after incubation at 37 °C in the presence of 10 lM FITC-R8 for (a) 3 h and (b) 96 h, (c) Confocal micrographs of DU145 cells after incubation at 37 °C in the presence of FITC-R8 incorporated mesostructured silica for 96 h. Arrows indicate signals of the model drug (FITC) internalized into cells. Scale bar: 20 lm.
C. Gao et al. / Microporous and Mesoporous Materials 122 (2009) 201–207
uptake, if the mesostructured silica is prepared into the form of nanoparticles. 4. Conclusions In conclusion, we have succeeded in developing a drug delivery system by using mesostructured silica as a reservoir and CPP as a vector. The mesostructured silica reservoir system prepared by a one-pot liquid-crystal templating method is suitable for the encapsulation of drug-CPP conjugates and enables stable storage and sustained release of drugs conjugated with R8. The encapsulated surfactant and the siloxide ions on mesochannels contributed to the sustained release of drugs conjugated with cationic R8. The typical in vitro release duration was 5 days in PBS (pH 7.4) at 37 °C. Release from the mesostructured silica reservoir yielded significant elongation in duration of the FITC signals in DU145 cells by confocal microscopic analysis in comparison with a single administration of FITC-R8, which suggests a more effective and sustainable cellular uptake of the drug. Acknowledgments This work was supported by Strategic International Cooperative Project with Sweden (Novel Transdermal Drug Delivery System: Designing Meso-Structured Materials for Controlled and Triggered Release) from Japan Science and Technology Agency (JST) and Swedish Governmental Agency for Innovation Systems (VINNOVA)/Swedish Foundation for Strategic Research (SSF), the National Natural Science Foundation of China (Grant No. 20425102, 20890121 and 20821140537), 973 project (2009CB930403) of China and A3 Foresight Program and NSF. O.T. and Y.S. thank Swedish Research Council (VR) and Japan Science and Technology Agency (JST) for financial support. The authors are grateful to the lab center for basic chemistry, Shanghai Jiao Tong University, for taking the fluorescence spectroscopy. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.micromeso.2009.03.002. References [1] S. Fawell, J. Seery, Y. Daikh, C. Moore, L.L. Chen, B. Pepinsky, J. Barsoum, Proc. Natl. Acad. Sci. USA 91 (1994) 664. [2] D. Derossi, A.H. Joliot, G. Chassaing, A. Prochiantz, J. Biol. Chem. 269 (1994) 10444.
207
[3] S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda, Y. Sugiura, J. Biol. Chem. 276 (2001) 5836. [4] J.B. Rothbard, S. Garlington, Q. Lin, T. Kirschberg, E. Kreider, P.L. McGrane, P.A. Wender, P.A. Khavari, Nature Med. 6 (2000) 1253. [5] S. Futaki, Biopolymers 84 (2006) 241. [6] S.R. Schwarze, A. Ho, A. Vocero-Akbani, S.F. Dowdy, Science 285 (1999) 1569. [7] E.L. Snyder, S.F. Dowdy, Curr. Opin. Mol. Ther. 3 (2001) 147. [8] V.P. Torchilin, R. Rammohan, V. Weissig, T.S. Levchenko, Proc. Natl. Acad. Sci. USA 98 (2001) 8786. [9] L. Josephson, C.H. Tung, A. Moore, R. Weissleder, Bioconjug. Chem. 10 (1999) 186. [10] C. Palm, M. Jayamanne, M. Kjellander, M. Hällbrink, Biochim. Biophys. Acta 1768 (2007) 1769. [11] T. Yanagisawa, T. Shimizu, K. Kuroda, C. Kato, Bull. Chem. Soc. Jpn. 63 (1990) 988. [12] C.T. Kresge, M.E. Leonowicz, W.J. Roth, J.C. Vartuli, J.S. Beck, Nature 359 (1992) 710. [13] C. Tourné-Péteilh, D.A. Lerner, C. Charnay, L. Nicole, S. Begu, J.M. Devoisselle, ChemPhysChem 4 (2003) 281. [14] M. Vallet-Regi, A. Rámila, R.P. del Real, J. Pérez-Pariente, Chem. Mater. 13 (2001) 308. [15] B.G. Trewyn, S. Giri, I.I. Slowing, V.S.-Y. Lin, Chem. Commun. (2007) 3236. [16] K. Ariga, A. Vinu, J.P. Hill, T. Mori, Coord. Chem. Rev. 251 (2007) 2562. [17] C.-Y. Lai, B.G. Trewyn, D.M. Jeftinija, K. Jeftinija, S. Xu, S. Jeftinija, V.S.-Y. Lin, J. Am. Chem. Soc. 125 (2003) 4451. [18] A.L. Doadrio, E.M.B. Sousa, J.C. Doadrio, J. Pérez Pariente, I. Izquierdo-Barba, M. Vallet-Regí, J. Control. Release 97 (2004) 125. [19] M. Vallet-Regí, F. Balas, D. Arcos, Angew. Chem. Int. Ed. 47 (2007) 7548. [20] F. Qu, G. Zhu, H. Lin, J. Sun, D. Zhang, S. Li, S. Qiu, Eur. J. Inorg. Chem. 19 (2006) 3943. [21] Y.-H. Son, M. Park, Y.B. Choy, F.R. Choi, D.S. Kim, K.C. Park, J.-H. Choy, Chem. Commun. (2007) 2799. [22] C. Charnay, S. Begu, C. Tourne-Peteilh, L. Nicole, D.A. Lerner, J.M. Devoisselle, Eur. J. Pharm. Biopharm. 57 (2004) 533. [23] Q. Ji, M. Miyahara, J.P. Hill, S. Acharya, A. Vinu, S.B. Yoon, J.-S. Yu, K. Sakamoto, K. Ariga, J. Am. Chem. Soc. 130 (2008) 2376. [24] G.S. Attard, J.C. Glyde, C.G. Göltner, Nature 378 (1995) 366. [25] S.A. El-Safty, T. Hanaoka, Chem. Mater. 15 (2003) 2892. [26] S.F. Khattak, S.R. Bhatia, S.C. Roberts, Tissue Eng. 11 (2005) 974. [27] J. Kirstein, B. Platschek, C. Jung, R. Brown, T. Bein, C. Bräuchle, Narure Mater. 6 (2007) 303. [28] I. Nakase, M. Niwa, T. Takeuchi, K. Sonomura, N. Kawabata, Y. Koike, M. Takehashi, S. Tanaka, K. Ueda, J.C. Simpson, A.T. Jones, Y. Sugiura, S. Futaki, Mol. Ther. 10 (2004) 1011. [29] D. Zhao, Q. Huo, J. Feng, B.F. Chmelka, G.D. Stucky, J. Am. Chem. Soc. 120 (1998) 6024. [30] Y. Sakamoto, M. Kaneda, O. Terasaki, D.Y. Zhao, J.M. Kim, G. Stucky, H.J. Shin, R. Ryoo, Nature 408 (2000) 449. [31] M. Ahola, P. Kortesuo, I. Kangasniemi, J. Kiesvaara, A. Yli-Urpo, Int. J. Pharm. 195 (2000) 219. [32] T. Czuryszkiewicz, J. Ahvenlammi, P. Kortesuo, M. Ahola, F. Kleitz, M. Jokinen, M. Lind, J.B. Rosenholm, J. Non-Cryst. Solids 306 (2002) 1. [33] J.D. Bass, D. Grosso, C. Boissiere, E. Belamie, T. Coradin, C. Sanchez, Chem. Mater. 19 (2007) 4349. [34] C. Berkland, K. Kim, D.W. Pack, Pharm. Res. 20 (2003) 1055. [35] J.F. Fitzgerald, O.I. Corrigan, J. Control. Release 42 (1996) 125. [36] J.F. Fitzgerald, O.I. Corrigan, in: M.A. El-Nokaly, D.M. Piatt, B.A. Charpentier (Eds.), Polymeric Delivery Systems, Properties and Applications, ACS Symposium Series, vol. 520, American Chemical Society, Washington, DC, 1993, p. 311. [37] K.M. Gallagher, O.I. Corrigan, J. Control. Release 69 (2000) 261.
Contents lists available at ScienceDirect
Microporous and Mesoporous Materials journal homepage: www.elsevier.com/locate/micromeso
Mesostructured silica based delivery system for a drug with a peptide as a cell-penetrating vector Chuanbo Gao a,b, Isabel Izquierdo-Barba b, Ikuhiko Nakase c, Shiroh Futaki c,d,*, Juanfang Ruan b, Kazutami Sakamoto e, Yasuhiro Sakamoto b, Kazuyuki Kuroda f, Osamu Terasaki b,*, Shunai Che a,* a
School of Chemistry and Chemical Engineering, State Key Laboratory of Composite Materials, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China Structural Chemistry, Arrhenius Laboratory, Stockholm University, S-10691 Stockholm, Sweden c Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan d SORST, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan e Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, Tokyo, Japan f Department of Applied Chemistry, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan b
a r t i c l e
i n f o
Article history: Received 3 December 2008 Received in revised form 17 February 2009 Accepted 2 March 2009 Available online 11 March 2009 Keywords: Mesostructured silica Drug delivery Cell-penetrating peptide Sigmoidal release Sustained cellular uptake
a b s t r a c t A drug delivery system using mesostructured silica as a reservoir has been developed for the storage and controlled release of a drug with a cell-penetrating peptide (CPP) as a vector. We use fluorescein isothiocyanate (FITC) as the drug model and octaarginine (R8) as a vector to endow the drug with cell-penetrating property. The mesostructured silica reservoir system was prepared by using a one-pot liquid–crystal templating method, which is suitable for the encapsulation of intact FITC-R8 conjugates and sustained release of drugs without hampering their properties. The hydrophobic poly(propyl oxide) (PPO) shell of the pore-filling Pluronic F127 and the electrostatic interaction between R8 and siloxide ions on the pore walls act as the diffusion-limiting factors of the FITC-R8 conjugate. A sigmoidal in vitro release of FITCR8 from mesostructured silica into phosphate buffered saline (PBS, pH 7.4) was observed and the typical release duration was 5 days at 37 °C. Release from the reservoir yielded significant elongation in duration of the FITC signals in DU145 cells by confocal microscopic analysis, compared with a single administration of FITC-R8. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Recently, basic peptides, including human immunodeficiency virus (HIV)-1 Tat (positions 48–60) [1], Antennapedia (positions 43–58) [2] and oligoarginine peptides [3–5], have been reported to have the ability to translocate through cell membranes. These short sequences of peptides are referred to as the ‘‘cell-penetrating peptides (CPPs)” or ‘‘protein transduction domains (PTDs)”, and have been employed for the efficient delivery of exogenous molecules such as proteins [6], DNA [7], liposomes [8] and nanoparticles [9] into living cells. This highlights the applicability of CPPs to drug delivery system in which an enhanced cellular uptake of drugs could be consequently achieved. An example was demonstrated by Rothbard et al. that the conjugate of CPP and cyclosporin A was transported into skin cells of mouse and human, reached dermal T lymphocytes and successfully inhibited inflammation [4]. This drug delivery system using CPPs would become more practical by developing methods of controlled release of the CPP-conjugated * Corresponding authors. Tel.: +86 21 5474 2852; fax: +86 21 5474 1297 (S. Che). E-mail addresses: [email protected] (S. Futaki), [email protected] (O. Terasaki), [email protected] (S. Che). 1387-1811/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.micromeso.2009.03.002
drugs to yield optimized drug concentration to appreciate the maximum therapeutic effects with minimum side effects, as well as to protect the drug-CPP conjugates from possible facile enzymatic or chemical decomposition during storage and administration [10]. Mesostructured silicas, which were initiated by Kuroda and coworkers [11] and the researchers in Mobil Company [12], have opened up new possibilities of applications in the field of drug delivery. The tunable pore sizes in the range of 2–50 nm, high specific surface areas, large pore volumes and non-toxic nature [13] of these materials provide interesting possibilities for the inclusion of molecules of therapeutic value. Therefore, mesostructured silica is a promising candidate as a reservoir ideal for the storage and controlled release of drugs [14–23]. Up to now, the loading of drugs into the channel of mesostructured silica has been attained mainly by post-permeation method, i.e. the mesostructured silica carrier was synthesized and the surfactant was removed first followed by storage of the drug molecules by physical/chemical adsorption [14,17,18]. In the process of post-permeation, the drug incorporation is commonly carried out by soaking of the mesoporous silica in a highly concentrated drug solution and subsequent drying [19]. Therefore, only a portion of the employed drugs can be loaded
202
C. Gao et al. / Microporous and Mesoporous Materials 122 (2009) 201–207
in the silica materials due to the equilibrium and the distribution of the drugs is heterogeneous with possibilities to aggregate outside the particles. Besides, the remaining high porosity after post-permeation may make the drug-CPP compounds less stabilized compared with the case with mesopores filled by surfactant. Researchers also proposed a one-pot method to encapsulate drugs either into the channels of mesostructured silicas or into the polymer-silica assembled capsules [20,21]. Most of the one-pot procedures employed aqueous synthesis of powder forms of mesostructured silicas, and only limited amounts of the given drugs are finally incorporated into silica [22] and the majority of the drugs are segregated out to stay in the aqueous phase, especially in the case using solvent soluble drugs. Moreover, the strong acidic/basic conditions at high synthesis temperatures used in the one-pot method are often rather rigorous and may lead to decomposition of drug-CPP conjugates. Therefore, even though these studies show the applicability of mesostructured silica for the controlled/sustained release of incorporated drugs, new approaches should be employed for the present strategy to charge sufficient amount of intact drug-CPP conjugates into mesostructured channels. To overcome the above shortcomings, we have developed a mesostructured silica system suitable for encapsulating drug-CPP conjugate. Fluorescein isothiocyanate (FITC) was employed as a drug model lacking the membrane permeability, and octaarginine (R8) as a typical cell-penetrating peptide to enhance the cellular uptake. Highly ordered mesostructured surfactant/silica composite has been employed as a reservoir of the FITC-R8 conjugate, and a one-pot liquid–crystal templating [24,25] method was used to incorporate the conjugate into the mesostructured silica. The process was carried out in a nonaqueous system at a low reaction temperature to avoid water/heat mediated reactions of the drugs. Because of the existence of surfactant in the mesopores, it is expected that the FITC-R8 conjugate would be stabilized avoiding enzymatic or chemical decomposition. The copolymer Pluronic F127 (EO106PO70EO106) with low toxicity [26] was employed as the template of the mesopores to avoid the toxicity of the surfactant leached into the body fluid, which ensured the biocompatibility of the drug. The amphiphilic FITC-R8 conjugate is assembled with F127 and hydrolyzed silica source in the formation of highly ordered mesostructured silica in acidic medium, by the means of hydrophobic force, electrostatic interaction of protonated R8 and protonated silanols via counterions (e.g. chloride), and H-bonding of R8 with silanols. This self-assembly mechanism ensures that the FITC-R8 molecules are incorporated in the organic/inorganic interface by using this method. During the release in phosphate buffered saline (PBS, pH 7.4), as shown in Scheme 1, the drug model part (FITC) of the conjugate interacts with F127 micelles, while the positively charged R8 vector interacts electrostatically with the siloxide ions („SiO) covalently tethered on the silica wall; we expect both the hydrophobic poly(propyl oxide) (PPO) shell
and the electrostatic interaction could contribute to the sustained release of FITC-R8 from the carrier matrix [27]. Therefore, it is reasonable that by first conjugating the drugs to a vector (R8) and second incorporating the conjugate into mesostructured silica, the drug is stabilized and has the ability to release in a sustained manner and penetrate the cell membrane more easily. 2. Experimental 2.1. Synthesis of FITC labeled R8 peptide FITC-R8 [FITC-GABA-(Arg)8-amide] was prepared by Fmoc (9fluorenylmethyloxycarbonyl)-solid-phase peptide synthesis on a Rink amide resin as reported previously [28], where c-aminobutyric acid (GABA) was employed as a spacer to connect FITC with the (Arg)8 segment. The peptide was purified and the structure was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOFMS). 2.2. Synthesis of mesostructured silicas containing FITC-R8 In a typical synthesis of the one-pot method, 0.12 g of F127 (BASF) was dissolved in 0.3 g of tetramethyl orthosilicate (TMOS, BASF), and then 0.15 g of H2O/HCl (pH 1.3) was added at 40 °C (F127/TMOS = 0.00454). The hydrolysis of TMOS was achieved in 5 min at 40 °C and H2O molecules were mostly converted into methanol, after which 10.0 mg of FITC-R8 was added into the solution. Volatile chemicals including methanol and HCl were then removed from the synthesis system in vacuum to achieve highly ordered mesostructure [24]. The resultant monolithic gel was then aged at 40 °C for 2 days, ground into powders. To eliminate the particle size effect on the release kinetics, the powders were further sieved using #200 and #300 sieves to get the particles having a diameter of 50–76 lm. The loading amount of FITC-R8 in this mesostructured composite was 38.3 mg/g. A mesostructured silica containing FITC was obtained by using the same procedure and changing FITC-R8 into FITC while keeping the same moles. Nonporous silica material with FITC-R8 incorporated was prepared by the similar means without surfactant as template for mesopore. These were used as control samples in the following in vitro release experiment. 2.3. Release of FITC-R8 from silica FITC-R8 loaded mesostructured silica particles were first washed with PBS (pH 7.4) before in vitro release in order to remove any F127 or FITC-R8 located on the outer surface of the particles and afterwards dried in vacuum. The loading of FITC-R8 in the silica material obtained was recalculated as 38.1 mg/g. Ten milligram of FITC-R8 loaded mesostructured silica particles were added into
Scheme 1. Interactions of drug model FITC-R8 with F127 and silicate as drug diffusion-limiting factors, when mesostructured silica particles were immersed in PBS (pH 7.4).
C. Gao et al. / Microporous and Mesoporous Materials 122 (2009) 201–207
203
replaced with an equal volume of fresh PBS. The sample was diluted by 2.7 ml of PBS, and measured by fluorescence spectrophotometer to obtain the concentration of FITC-R8 released. 2.4. Internalization of FITC-R8 by cells DU145 prostate cancer cells were maintained in alpha-minimum essential medium (a-MEM) with 10% heat-inactivated fetal bovine serum as reported [28]. Briefly, 2 105 cells were plated into 35-mm glass-bottomed dishes and cultured for 48 h. The mesostructured silica containing FITC-R8 (3.0 mg) was incubated with the cells at 37 °C with fresh medium (1.0 ml). After 96 h, the DU145 cells were then washed with cold PBS. Distribution of the fluorescently labeled peptides was analyzed without fixing the cells by confocal scanning laser system. The single administration of FITC-R8 was investigated by incubating cells for 3 and 96 h in a-MEM containing 10 lM of FITC-R8. 2.5. Characterizations
Fig. 1. XRD patterns of mesostructured silicas containing FITC-R8 synthesized using F127 as the template. (a) As synthesized, (b) after treatment in PBS at 37 °C for 120 h. The intensity of the as-synthesized mesostructured silica (profile a) was multiplied by five for clarity. The calculated positions of X-ray reflections with indices are also indicated by arrows.
2 ml of PBS (pH 7.4), and afterwards shaken in a water bath at 37 °C. At regular intervals, the release system was centrifuged and 100 ll of the supernate was withdrawn and immediately
X-ray diffraction (XRD) patterns were recorded on a Rigaku X-ray diffractometer D/MAX-2200/PC using Cu Ka radiation (40 kV, 20 mA) at the rate of 0.5° 2h/min over the range of 0.6–5° 2h. High-resolution transmission electron microscopy (HRTEM) was performed with a JEOL JEM-3010 microscope operating at 300 kV (Cs = 0.6 mm, resolution 1.7 Å). The nitrogen adsorption/ desorption isotherms were measured at 77 K with a Quantachrome Nova 4200E porosimeter. Elemental analysis of the mesostructured silica was conducted on a Perkin Elmer PE 2400 II CHNS/O
Fig. 2. HRTEM images of mesostructured silicas containing FITC-R8 synthesized with F127 as the template. (a–c), As synthesized, recorded along [1 0 0], [1 1 0] and [1 1 1] zone axes; (d), After release of FITC-R8 in PBS for 120 h, recorded along [1 0 0] zone axis.
204
C. Gao et al. / Microporous and Mesoporous Materials 122 (2009) 201–207
Analyzer. The concentration of FITC-R8 was determined by using a Varian Fluorescence spectrophotometer Cary Eclipse, excitation: k = 498.05 nm, emission: k = 519.10 nm. Distribution of the FITCR8 in DU145 cells was analyzed by using an Olympus FV300 confocal scanning laser system consisting of an Olympus IX81 microscope.
3. Results and discussion 3.1. Structure and stability of FITC-R8 incorporated mesostructured silica monolithic particles The scanning electron microscopy (SEM) images of the sieved mesostructured silica particles (Supplementary Information Fig. S1) reveals that the particles have irregular morphologies and sizes ranging from 50 to 100 lm randomly. This uniformly distributed mesostructured silica in size simplifies the investigation of the drug release from it because of the elimination of particle size effect on the release kinetics. The XRD patterns of the mesostructured monoliths with FITCR8 incorporated are shown in Fig. 1a. Only one reflection could be observed showing the d-spacing of 10.3 nm. Although no fine peaks showing the detailed structural information have been detected, the sharp X-ray reflection peaks suggest that the material possesses highly ordered mesostructure. As shown in Fig. 1b, after 120 h of the treatment in PBS at 37 °C, the mesostructured silica shows higher X-ray reflection intensity than the as-synthesized one due to the release of both FITC-R8 and the F127 surfactant,
indicating that the mesostructure of the drug reservoir was retained during the drug release. Fig. 2a–c shows the HRTEM images of the thin parts of the mesostructured silica with FITC-R8 incorporated recorded along [1 0 0], [1 1 0] and [1 1 1] zone axes. The mesostructured silica possesses high crystallinity despite the coexistence of disordered mesophase and amorphous impurity. The observed reflection conditions from the Fourier diffractograms of the HRTEM images are hkl: h + k + l = 2n, 0kl: k + l = 2n, hhl: l = 2n, 00l: l = 2n, and thus the space group is determined to be Im3m. Therefore, the X-ray reflections with highest intensity shown in Fig. 1 can be indexed as 1 1 0 structure. The structure of the mesostrucreflection of the Im3m tured monolith after 120 h of drug release in PBS has been con firmed to be cubic Im3m structure and proved intact, as the HRTEM image shown in Fig. 2d reveals. Fig. 3 shows the nitrogen adsorption/desorption isotherm and pore size distributions of the material after treatment in PBS at 37 °C for 168 h and removal of surfactant by calcination. The sorption isotherm clearly shows a H2 type hysteresis loop, indicating a cage type mesopore system, and hence it is SBA-16 type [29,30]. The pore size is 5.8 nm calculated using Barett–Joyner–Halenda (BJH) method according to the adsorption isotherm and 3.8 nm derived from the desorption branch of the isotherm. It is reasonably considered that the mesostructured silica has a mesocage size of 5.8 nm and a size of window between mesocages of 3.8 nm, which are large enough to entrap drug molecules (normally no larger than 5 nm 2 nm) and thus sufficient for the drug storage and delivery system. It has also been suggested in the earlier literatures that the silica matrix may degrade in the drug release which serve as a diffusion-controlling factor and may affect the investigation of drug release kinetics from mesopores [31–33]. In our system, we found that only about 5% of the silica was dissolved into PBS solution (2 ml PBS per 10 mg mesostructured silica, Supplementary Information Fig. S2), which indicates that the silica matrix is stable enough and the effect of the degradation of mesostructured silica on the drug release could be ruled out. 3.2. In vitro release of FITC-R8 from mesostructured silica into PBS
Fig. 3. Nitrogen adsorption/desorption isotherm (A) and BJH pore size distribution (B, from desorption branch (I) and from adsorption branch (II)) of the mesoporous silica. The mesoporous silica containing FITC-R8 was first treated with PBS (pH 7.4, 37 °C) for 168 h and then calcined at 550 °C for 6 h. The H2 hysteresis loop of the isotherm indicates the cage-type mesostructure, and the pore size and window size are 5.8 nm and 3.8 nm, respectively.
The time-course analysis of FITC-R8 release from the mesostructured reservoir into PBS (pH 7.4) at 37 °C was then conducted by measuring florescence intensity, and the release of the FITC-R8 conjugate was confirmed by MALDI-TOFMS. After 96 h of FITC-R8 release, calculated m/z for M + H+ was: 1742.1, found: 1351.98(R8) and 1742.05 (FITC-R8). The height ratio of MALDITOFMS two peaks about FITC-cleaved R8 and FITC-R8 was 8:5. For such a long-term release there were still significant amount of intact FITC-R8 in the aqueous release system. It can be reasonably regarded as newly released FITC-R8 conjugate, which was protected before release from the outer water molecules by the hydrophobic mesochannels filled with PPO. Nevertheless, FITC-R8 which was exposed in PBS for a long time decomposed into FITC and R8 because of the instability of FITC-R8 when exposed to water. It confirmed that safety storage of FITC-R8 conjugates could be achieved by using mesostructured organic/inorganic composite as reservoir. Fig. 4 shows the percentile cumulative release profile of FITC-R8 from mesostructured silica into PBS. It follows a sigmoidal release which is characterized by an initial lag in the release reducing or avoiding the ‘‘burst” effect and afterwards an accelerating release [34,35]. This profile could be well fitted to the following equation [36,37]:
ln
X ¼ k t k tmax T X
ð1Þ
C. Gao et al. / Microporous and Mesoporous Materials 122 (2009) 201–207
205
changes of mesostructured silica was investigated in details. Fig. 5 shows the XRD patterns of the mesostructured silicas at different stages of the drug release. An increase in the X-ray diffraction was observed with the time of PBS treatment, which suggests retention of the mesostructure and also a leach of surfactant during release that gives rise to a greater contrast between mesochannel and silica wall. This leach of surfactant produces porosity in the mesostructured F127/silica composite, which acts as an accelerating factor of the FITC-R8 molecules to diffuse out into PBS. Fig. 6 shows the nitrogen adsorption/desorption isotherms of the solids obtained from the release system at different
Fig. 4. Percentile cumulative release of FITC-R8 from mesostructured silica into PBS (pH 7.4, 37 °C) and its sigmoidal curve fitted from Eq. (1).
where X is the fraction of the released FITC-R8 at time t, T is the total released fraction of the FITC-R8 at time infinity, k is the rate constant and tmax is the time to maximum rate of the FITC-R8 release in the sigmoidal release. The model parameters representing the induction and accelerating release were determined and collected in Table 1. According to Fig. 4 and Table 1, it can be concluded that: (1) The release has a induction time of about 36 h, and during this period the release of FITC-R8 from mesostructured silica is very slow; (2) After 36 h, the release was accelerated and reached its maximum rate at 73.5 h; (3) 85% of the FITC-R8 incorporated in the mesostructured silica was released and the release duration was more than 5 days. Interconnected mesochannels have a profound effect on the drug release. No drugs could be released from silica without mesopore (Supplementary Information Fig. S3) indicating its vital role in the drug release. It confirms that the mesochannel is the route for the drug-CPP conjugates to diffuse into the outer PBS solution. The octaarginine is positively charged and the silica is negatively charged at pH 7.4, and therefore during the release, the electrostatic interaction between FITC-R8 and siloxide ions („SiO) on the wall of mesopores will decrease the diffusion rate of FITC-R8. On the other hand, FITC-R8 co-assembles with F127 and silicate during the synthesis, and as a result, the FITC-R8 molecules locate on the organic/inorganic interface. The FITC-R8 molecules would first penetrate the hydrophobic PPO shell formed by the pore-filling surfactant in the diffusion into PBS. This also serves as a diffusion-limiting factor and contributes to the sustained release of FITC-R8. As expected, in the case of FITC storage and release without conjugation with R8 using the same mesostructured F127/silica system as the reservoir, a quick and complete release was observed in less than an hour (Supplementary Information Fig. S4). It emphasizes the effect of both the electrostatic interaction between R8 and siloxide ions and the pore-filling surfactant on the sustained release, because in this system a lack of interaction with silica wall leads to a distribution of FITC in the hydrophobic core of F127 micelles, which makes the FITC molecules easier to diffuse into PBS. In order to discover the cause of the sigmoidal release kinetics of FITC-R8 from mesostructured silica into PBS, the structural
Fig. 5. XRD patterns of the mesostructured silicas at different stages of the drug release. (a–e) The mesostructured silicas were treated with PBS (pH 7.4, 37 °C) for (a) 24 h, (b) 48 h, (c) 72 h, (d) 96 h and (e) 120 h, respectively.
Table 1 Values of kinetics parameters for the release of FTIC-R8 from mesostructured particles fitted from Eq. (1). T
k (h1)
tmax (h)
r2
85.1%
0.05635
73.5
0.9985
Fig. 6. Nitrogen adsorption/desorption isotherms of the mesostructured silicas at different stages of the drug release. (a–e) The mesostructured silicas were treated with PBS (pH 7.4) at 37 °C for (a) 24 h, (b) 48 h, (c) 72 h, (d) 96 h and (e) 120 h, respectively. Isotherm e was offset vertically by 50 cm3 g1 STP for clarity.
206
C. Gao et al. / Microporous and Mesoporous Materials 122 (2009) 201–207
stages of the drug release and the porous properties are summarized in Table 2. It can be inferred that the surface area and the pore volume of the mesostructured solids increase with the treatment time, and the increment of the porosity reaches its maximum value at about 72 h, which is coincident with the rate of FITC-R8 release. It confirms that the porosity resulting from the leaching of pore-filling surfactant dramatically accelerates the release of the drugs. Table 3 shows the carbon content of mesostructured silica solids sampled at different stages of the drug release. The data were derived from the CHN elemental analysis. A decrease of carbon content can be inferred owing to the leach of both the surfactant F127 as well as FITC-R8. Considering that F127 contributes to the most of the carbon decreasing, the leaching amount of the F127 has been estimated and the results are demonstrated in Table 3. It is worth noting that only a part of F127 in the organic/inorganic composite (<25%) has been released during the treatment in PBS, and it is adequate for the production of porosity and acceleration of the drug release. The sigmoidal kinetics has been observed in polymer based homogeneous distributed large particle systems where diffusion
Table 2 Porous properties of mesostructured silicas at different stages of the drug release. Time of the treatment in PBS (h)
Surface area (m2 g1)
Pore volume (cm3 g1)
24 48 72 96 120
12 29 100 197 215
0.02 0.05 0.15 0.29 0.30
Table 3 Carbon content of mesostructured silicas at different stages of the drug release. The data were collected by CHN elemental analysis. A decrease in the carbon content of the mesostructured silicas was observed during the FITC-R8 release. Time of the treatment in PBS (h)
Carbon content (%)
Leaching percentage of F127(%)a
0 24 48 72 96 120
26.43 21.96 21.59 20.82 20.24 20.20
0 16.9 18.3 21.2 23.4 23.6
a Estimated from the carbon content of the silicas given that F127 constitutes the majority of the organic composition and has a large carbon content.
and polymer degradation governed the drug release [36,37]. It can be concluded from the discussion above that in our system, the hydrophobic PPO shell formed by the pore-filling surfactant and the electrostatic interaction between the drug and silica serves as the diffusion-limiting factors, which hindered the drug from releasing in the induction time (ca. 36 h). After this period, an accelerated release of FITC-R8 lasting about 4 days was achieved as a result of the leaching of the surfactant and thus a dramatic increase in mesoporosity of the organic/inorganic composite. And therefore, a sigmoidal release profile of the FITC-R8 was observed. 3.3. Internalization of FITC-R8 by cells In order to examine if the above features in controlled release were actually reflected in the cellular uptake of the drugs, as well as if the CPPs really enhanced cellular uptake of the conjugated drugs, the FITC-R8-containing mesostructured silica was incubated with DU145 cells. The silica particles containing FITC-R8 were soaked in the culture media of DU145 cells and incubated together at 37 °C. The mesostructured silica particles shows little cytotoxity to the DU145 cells. The internalization of the FITC-R8 was assessed by confocal microscopic observation (Fig. 7). When the cells were incubated with silica particle containing FITC-R8, significant fluorescent signals were observed at as long as 96 h (Fig. 7c), whereas no signals were observed from the cells treated with FITC without conjugated with R8 peptide at any of the time points (not shown), exemplifying the effectiveness of conjugation with CPPs for the intracellular delivery of membraneimpermeable drugs. On the other hand, a single administration of 10 lM FITC-R8 without entrapped in the mesostructured reservoir yielded FITC signals at 3 h (Fig. 7a) but very little signals were observed at 96 h after administration (Fig. 7b). This clearly shows that by encapsulation in the mesostructured reservoir, intact storage and sustained release of the drug-CPP conjugate were achieved and eventually led to the efficient intracellular delivery yielding an elongated bioactivity. The above results also suggest that the procedures employed for the reservoir formation were not severe to give damages for the structures and properties of the R8 peptide and FITC, which would be attained by the procedures of conventional mesostructured silica formation for these chemically rather fragile species. This enhanced cellular uptake of the drug-CPP conjugate suggests that, this mesostructured silica system, which provides the intact storage and controlled release of the conjugate, is suitable to be potentially used as a prototype for the transdermal drug delivery. Furthermore, injection is also feasible for this drug delivery system, which could also have the high effectiveness of cellular
Fig. 7. FITC-R8 released from mesostructured silica yielded sustained signals of the model drug (FITC) in DU145 (prostate cancer) cells. (a–b) Confocal micrographs of DU145 cells after incubation at 37 °C in the presence of 10 lM FITC-R8 for (a) 3 h and (b) 96 h, (c) Confocal micrographs of DU145 cells after incubation at 37 °C in the presence of FITC-R8 incorporated mesostructured silica for 96 h. Arrows indicate signals of the model drug (FITC) internalized into cells. Scale bar: 20 lm.
C. Gao et al. / Microporous and Mesoporous Materials 122 (2009) 201–207
uptake, if the mesostructured silica is prepared into the form of nanoparticles. 4. Conclusions In conclusion, we have succeeded in developing a drug delivery system by using mesostructured silica as a reservoir and CPP as a vector. The mesostructured silica reservoir system prepared by a one-pot liquid-crystal templating method is suitable for the encapsulation of drug-CPP conjugates and enables stable storage and sustained release of drugs conjugated with R8. The encapsulated surfactant and the siloxide ions on mesochannels contributed to the sustained release of drugs conjugated with cationic R8. The typical in vitro release duration was 5 days in PBS (pH 7.4) at 37 °C. Release from the mesostructured silica reservoir yielded significant elongation in duration of the FITC signals in DU145 cells by confocal microscopic analysis in comparison with a single administration of FITC-R8, which suggests a more effective and sustainable cellular uptake of the drug. Acknowledgments This work was supported by Strategic International Cooperative Project with Sweden (Novel Transdermal Drug Delivery System: Designing Meso-Structured Materials for Controlled and Triggered Release) from Japan Science and Technology Agency (JST) and Swedish Governmental Agency for Innovation Systems (VINNOVA)/Swedish Foundation for Strategic Research (SSF), the National Natural Science Foundation of China (Grant No. 20425102, 20890121 and 20821140537), 973 project (2009CB930403) of China and A3 Foresight Program and NSF. O.T. and Y.S. thank Swedish Research Council (VR) and Japan Science and Technology Agency (JST) for financial support. The authors are grateful to the lab center for basic chemistry, Shanghai Jiao Tong University, for taking the fluorescence spectroscopy. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.micromeso.2009.03.002. References [1] S. Fawell, J. Seery, Y. Daikh, C. Moore, L.L. Chen, B. Pepinsky, J. Barsoum, Proc. Natl. Acad. Sci. USA 91 (1994) 664. [2] D. Derossi, A.H. Joliot, G. Chassaing, A. Prochiantz, J. Biol. Chem. 269 (1994) 10444.
207
[3] S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda, Y. Sugiura, J. Biol. Chem. 276 (2001) 5836. [4] J.B. Rothbard, S. Garlington, Q. Lin, T. Kirschberg, E. Kreider, P.L. McGrane, P.A. Wender, P.A. Khavari, Nature Med. 6 (2000) 1253. [5] S. Futaki, Biopolymers 84 (2006) 241. [6] S.R. Schwarze, A. Ho, A. Vocero-Akbani, S.F. Dowdy, Science 285 (1999) 1569. [7] E.L. Snyder, S.F. Dowdy, Curr. Opin. Mol. Ther. 3 (2001) 147. [8] V.P. Torchilin, R. Rammohan, V. Weissig, T.S. Levchenko, Proc. Natl. Acad. Sci. USA 98 (2001) 8786. [9] L. Josephson, C.H. Tung, A. Moore, R. Weissleder, Bioconjug. Chem. 10 (1999) 186. [10] C. Palm, M. Jayamanne, M. Kjellander, M. Hällbrink, Biochim. Biophys. Acta 1768 (2007) 1769. [11] T. Yanagisawa, T. Shimizu, K. Kuroda, C. Kato, Bull. Chem. Soc. Jpn. 63 (1990) 988. [12] C.T. Kresge, M.E. Leonowicz, W.J. Roth, J.C. Vartuli, J.S. Beck, Nature 359 (1992) 710. [13] C. Tourné-Péteilh, D.A. Lerner, C. Charnay, L. Nicole, S. Begu, J.M. Devoisselle, ChemPhysChem 4 (2003) 281. [14] M. Vallet-Regi, A. Rámila, R.P. del Real, J. Pérez-Pariente, Chem. Mater. 13 (2001) 308. [15] B.G. Trewyn, S. Giri, I.I. Slowing, V.S.-Y. Lin, Chem. Commun. (2007) 3236. [16] K. Ariga, A. Vinu, J.P. Hill, T. Mori, Coord. Chem. Rev. 251 (2007) 2562. [17] C.-Y. Lai, B.G. Trewyn, D.M. Jeftinija, K. Jeftinija, S. Xu, S. Jeftinija, V.S.-Y. Lin, J. Am. Chem. Soc. 125 (2003) 4451. [18] A.L. Doadrio, E.M.B. Sousa, J.C. Doadrio, J. Pérez Pariente, I. Izquierdo-Barba, M. Vallet-Regí, J. Control. Release 97 (2004) 125. [19] M. Vallet-Regí, F. Balas, D. Arcos, Angew. Chem. Int. Ed. 47 (2007) 7548. [20] F. Qu, G. Zhu, H. Lin, J. Sun, D. Zhang, S. Li, S. Qiu, Eur. J. Inorg. Chem. 19 (2006) 3943. [21] Y.-H. Son, M. Park, Y.B. Choy, F.R. Choi, D.S. Kim, K.C. Park, J.-H. Choy, Chem. Commun. (2007) 2799. [22] C. Charnay, S. Begu, C. Tourne-Peteilh, L. Nicole, D.A. Lerner, J.M. Devoisselle, Eur. J. Pharm. Biopharm. 57 (2004) 533. [23] Q. Ji, M. Miyahara, J.P. Hill, S. Acharya, A. Vinu, S.B. Yoon, J.-S. Yu, K. Sakamoto, K. Ariga, J. Am. Chem. Soc. 130 (2008) 2376. [24] G.S. Attard, J.C. Glyde, C.G. Göltner, Nature 378 (1995) 366. [25] S.A. El-Safty, T. Hanaoka, Chem. Mater. 15 (2003) 2892. [26] S.F. Khattak, S.R. Bhatia, S.C. Roberts, Tissue Eng. 11 (2005) 974. [27] J. Kirstein, B. Platschek, C. Jung, R. Brown, T. Bein, C. Bräuchle, Narure Mater. 6 (2007) 303. [28] I. Nakase, M. Niwa, T. Takeuchi, K. Sonomura, N. Kawabata, Y. Koike, M. Takehashi, S. Tanaka, K. Ueda, J.C. Simpson, A.T. Jones, Y. Sugiura, S. Futaki, Mol. Ther. 10 (2004) 1011. [29] D. Zhao, Q. Huo, J. Feng, B.F. Chmelka, G.D. Stucky, J. Am. Chem. Soc. 120 (1998) 6024. [30] Y. Sakamoto, M. Kaneda, O. Terasaki, D.Y. Zhao, J.M. Kim, G. Stucky, H.J. Shin, R. Ryoo, Nature 408 (2000) 449. [31] M. Ahola, P. Kortesuo, I. Kangasniemi, J. Kiesvaara, A. Yli-Urpo, Int. J. Pharm. 195 (2000) 219. [32] T. Czuryszkiewicz, J. Ahvenlammi, P. Kortesuo, M. Ahola, F. Kleitz, M. Jokinen, M. Lind, J.B. Rosenholm, J. Non-Cryst. Solids 306 (2002) 1. [33] J.D. Bass, D. Grosso, C. Boissiere, E. Belamie, T. Coradin, C. Sanchez, Chem. Mater. 19 (2007) 4349. [34] C. Berkland, K. Kim, D.W. Pack, Pharm. Res. 20 (2003) 1055. [35] J.F. Fitzgerald, O.I. Corrigan, J. Control. Release 42 (1996) 125. [36] J.F. Fitzgerald, O.I. Corrigan, in: M.A. El-Nokaly, D.M. Piatt, B.A. Charpentier (Eds.), Polymeric Delivery Systems, Properties and Applications, ACS Symposium Series, vol. 520, American Chemical Society, Washington, DC, 1993, p. 311. [37] K.M. Gallagher, O.I. Corrigan, J. Control. Release 69 (2000) 261.