Hydrophobically modified spherical MCM-41 as nanovalve system for controlled drug delivery

Microporous and Mesoporous Materials 200 (2014) 124–131

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Hydrophobically modified spherical MCM-41 as nanovalve system for controlled drug delivery Aneesh Mathew, Surendran Parambadath, Sung Soo Park, Chang-Sik Ha ⇑ Department of Polymer Science and Engineering, Pusan National University, Geumjeong-gu, Busan 609-735, Republic of Korea

a r t i c l e

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Article history: Received 16 June 2014 Accepted 14 August 2014 Available online 23 August 2014 Keywords: MCM-41 Hydrophobic Nanovalve pH responsive Drug delivery

a b s t r a c t Spherical MCM-41 nanovalve having hydrophobically modified pore channels was synthesized via surfactant assisted sol–gel methodology and post modification process. The spherical MCM-41 has been tailored as a smart pH responsive drug carrier system by the insertion of N-3-(trimethoxysilyl)propyl aniline (TMSPA) at the pore opening before extracting the surfactant and further with phenyltrimethoxysilane (PTMS) to impart hydrophobicity on the inner surfaces of the pore channels. The surfactant extracted MCM-41 exhibits excellent textural properties such as very high specific surface area (1307 m2 g 1), pore diameter (24 Å) and pore volume (0.65 cm3 g 1). The transmission electron microscope (TEM) and scanning electron microscope (SEM) images of mesosphere reflect the highly uniform and mono-dispersed spherical morphology having a particle size of 500 nm. 5-Fluorouracil (5-Fu) and famotidine have been loaded into the hydrophobically modified channels followed with b-cyclodextrin (b-CD) as the gatekeeper to make the material as a pH responsive drug delivery system. The drug delivery has been carried out under in vitro condition at pH 4 and the amount of drug released from the nanovalve system was monitored by UV–Vis spectroscopy under regular intervals. The hydrophobically modified nanovalve was found to have delayed drug release of both 5-Fu and famotidine in comparison to the drug delivery from the nanovalve having unmodified pore channels synthesized from spherical MCM-41 under similar experimental conditions. The significance of functionalization as well as capping has been verified by the comparison of drug delivery behaviors among hydrophobically modified, unmodified, b-CD capped and uncapped nanocontainers. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction The exciting discovery of new molecular sieves, generically called M41S [1] in the early 1990s, evoked new windows for the exploration in material research. Cationic surfactant micelles act as templates as well as structure directing agents in mesoporous material synthesis through an electrostatic interaction with the polymerizing silica components. Owing to its convenient synthesis procedure, stupendous mesoporous structure and surface silanol groups, mesoporous silica materials possess exclusive properties such as large surface area, high pore volume, uniform and tunable pore size, low mass density, non-toxic nature, easily modifiable surface properties and good biocompatibility [2–7]. Among the mesoporous materials MCM-41 received much attention due to its tunable nature i.e., its capacity to form different morphologies

⇑ Corresponding author. Tel.: +82 51 510 2407; fax: +82 51 513 7720. E-mail address: [email protected] (C.-S. Ha). http://dx.doi.org/10.1016/j.micromeso.2014.08.033 1387-1811/Ó 2014 Elsevier Inc. All rights reserved.

such as disk, rod, sphere, hexagonal plates, gyroids, crescent-like and worm-shaped particles depending on the various combination of silica precursor/surfactant/water/co-solvent [8]. The spherical morphology has become a passion of modern research in the biomedical field, especially in the drug delivery application. It is possible to tune the size of the sphere and pore diameter by altering synthesis conditions such as amount of silica precursor/water/base or type of surfactant without disturbing the radially aligned mesopores from the center to the surface of the spherical particles for extended applications [9]. Selective functionalization [10–12] of the mesoporous materials play the pivotal role in equipping the modified materials with enhanced hydrothermal stability and cargo loading. Vallet-Regi et al. reported two MCM-41 materials with different pore diameters for the controlled delivery of ibuprofen and found that the release rate of the drug was linearly dependent on the pore size [13]. The study proclaimed that mesoporous silica materials also can fulfill the conditions for homogenous distribution of the drug all over the silica matrix in contrast to the conventionally used polymeric materials. The appropriate

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physicochemical modifications in particle morphology and channel functionalization (hydrophilic/hydrophobic) are essential because the mesoporous silica surface covered with silanol groups is not selective enough to adsorb drug molecules with different functionalities. Even in the presence of such modifications, an immediate or uncontrolled release has been experienced by several researchers, which strengthened the requirement of more advanced and synergic manipulation to attain a perfect drug delivery system. The premature release of drug molecules from the pore channels should result an untargeted drug release that could cause undesirable side effects to normal cells and organs [14]. An overview of the drug delivery studies showed that most of the drug carriers are developed based on the hydrophilically modified inner surfaces since hydrophilic surface favors the high loading and delayed cargo release. But, the drugs utilizing for practical uses generally fall either in hydrophilic or hydrophobic categories. So, it is necessary to investigate the activity of hydrophobically modified surface toward drug delivery from a scientific viewpoint. Unfortunately, only few reports are available under this category. Mesoporous materials with different particle morphology, pore geometry and surface organic composition have been widely investigated as drug delivery system in order to achieve high drug loading capacity and well-defined drug delivery profile. Previous studies revealed that the mesoporous silica carrier modified with special functional groups have great benefits for controlled drug release [15–21]. Several studies pointed out that the release rate of ibuprofen, which has been widely investigated as model drug, could be modulated by varying the density of the surface organic amino groups, changing the chain length of amino groups, or using different species of amino groups, e.g. aminopropyl, aminoethylaminopropyl and so on [15– 17]. The drug delivery systems were much professionalized only after the discovery of stimuli-responsive methods for controlling the drug access to and from the nanopores such as pH, temperature, light, magnetism, enzyme, redox agents, etc. These gatekeepers include inorganic nanoparticles, organic molecules, biological macromolecules and supra molecular assemblies, which can keep guest molecules in the pores until they are removed by external stimuli. Grafting suitable gatekeepers onto the surface of mesoporous silica can prevent the premature release of the cargo before reaching the target. These methods range from coating the nanoparticles with polymers to controlling individual nanopores with molecules that undergo largeamplitude motions to the immobilization of small molecules on the pore opening by chemical method. The latter offers the highest degree of control because the ‘‘gatekeeper’’ molecules are bonded covalently inside or at the entrances of the nanopores. When modulating the properties such as morphology, particle size, pore channel functionalization and gatekeeper modifications obviously promise a better candidate for drug delivery application. Herein, we have synthesized hydrophobically modified spherical MCM-41 nanovalve system in a multi-step synthesis strategy. The conventional sol–gel synthesis method was adopted for the synthesis of mesoporous spherical MCM-41 and the pore openings have been modified using TMSPA in dry toluene. Further the pore channels were hydrophobically tailored using PTMS after the surfactant removal. 5-Fu and famotidine were loaded into the modified channels and the drug delivery was carried out under in vitro condition at pH 4. We have compared the results obtained from hydrophobically modified drug delivery system with the nanovalve having unmodified pore channels synthesized from spherical MCM-41 under similar experimental conditions. To the best of our knowledge there are no studies regarding hydrophobically modified MCM-41 nanovalve systems for controlled drug delivery applications.

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2. Experimental 2.1. Reagents and materials Tetramethylorthosilicate (TMOS), cetyltrimethylammonium bromide (CTABr), N-[3-(trimethoxysilyl)propyl]aniline (TMSPA), phenyltrimethoxysilane (PTMS), 5-fluorouracil (5-Fu), famotidine, b-cyclodextrin (b-CD) and anhydrous toluene were purchased from Sigma–Aldrich. All chemicals were used as received without further purification. 2.2. Synthesis of spherical MCM-41 The spherical MCM-41 has been synthesized using an earlier procedure with suitable adaptation [9]. In a typical synthesis, CTABr (3.52 g, 9.65 mmol) was dissolved in 800 g of methanol/ water (50/50w/w) mixture containing 4 g of 1 M sodium hydroxide solution. After stirring for 1 h, TMOS (1.32 g, 8.68 mmol) was added to the homogenous solution with constant stirring. Immediately a white precipitate was formed and was allowed to stir for 8 h at room temperature, subsequently aged for overnight at static condition. The white precipitate was filtered off, washed several time with water, and ethanol then dried at 50 °C for 24 h. 2.3. Surface functionalization of spherical MCM-41 Functionalization process was carried out using previously reported procedure [12,14]. As-synthesized spherical MCM-41 (1 g) was functionalized with TMSPA (0.135 g, 0.53 mmol) in dry toluene at reflux temperature for 24 h under inert atmosphere. The functionalized material was filtered and washed with toluene and dichloromethane, then dried under vacuum for overnight at 60 °C. The surfactant extraction was carried out [22] using conc. HCl (3 g, 36%) in 100 ml ethanol per gram of spherical MCM-41. The mixture was stirred at 60 °C for 24 h. The surfactant removed spherical MCM-41 was filtered, washed with water and ethanol, further dried under vacuum at 60 °C for overnight. The TMSPA functionalized and surfactant removed spherical MCM-41 was named as P-MCM-41. PTMS functionalization on the inner surface of P-MCM-41 was carried out in a similar way as discussed previously. 1 g of dried P-MCM-41 was taken in 100 ml of toluene and 1 ml of PTMS was injected and further refluxed for 24 h. After the surface treatment, the solid was filtered, washed and dried overnight in vacuum at 60 °C and named as PP-MCM-41. 2.4. Drug loading and release 5-Fu loading was carried out as per our previous reports [12,14]. A stock solution was made by dissolving a known amount of 5-Fu in water (10 mg/ml). 100 mg of P-MCM-41/PP-MCM-41 nanocontainer was mixed with 12 ml of drug solution. The suspension was stirred for 24 h at room temperature and the pH of the solution was adjusted to 7 before the addition of b-CD (220 mg). The mixture was allowed to stir for another 72 h after that the b-CD capped nanocontainer was filtered off, washed with water and absolute ethanol, dried overnight at 60 °C under vacuum. The b-CD capped systems were named as PA-MCM-41 and PPA-MCM-41, respectively for P-MCM-41 and PP-MCM-41. The above procedure was followed for loading of famotidine except a methanolic stock solution was used instead of water (methanol/water, 50/50w/w). The amount of drug loading was determined by UV–Visible spectroscopic analysis. In vitro drug release experiments were carried out by placing 100 mg of drug loaded material in a dialysis membrane bag (molecular weight cut-off 5000 KDa) and immersed it into 20 ml of water (pH adjusted to 4 with aqueous solution of

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0.2 M HCl) [22]. The released concentration as a function of time was analyzed by UV–Visible spectroscopy at 269/282 nm in the releasing medium. Also, we have conducted the drug release studies to evaluate the effect of b-CD capping efficiency for trapping molecules inside the pore channels. To examine the described effect, it is necessary to have a release experiment for both drugs from the P-MCM-41 and PP-MCM-41. 2.5. Characterization X-ray powder diffraction (XRD, Bruker AXN) was performed using Cu-Ka radiation. The XRD patterns were collected in the low-angle range from 1.2° to 10° 2h. Transmission electron microscopy (TEM, JEOL 2010) was performed at an accelerating voltage of 200 kV. Scanning electron microscopy (SEM, JEOL 6400) images were collected at an operating voltage of 20 kV. The N2 adsorption–desorption isotherms were measured using a Nova 4000e surface area and pore size analyzer. The samples were degassed at 120 °C for 12 h before the measurements. The Brunauer–Emmet–Teller (BET) method was used to calculate the specific surface area. The pore size distribution curve was obtained from an analysis of the adsorption branch using the Barrett–Joyner–Halenda (BJH) method. Fourier transform infrared (FT-IR, JASCO FTIR 4100) spectra were measured from KBr pellets in the frequency range 4000–600 cm 1. Thermogravimetric analysis (TGA, Perkin Elmer Pyris Diamond) was carried out at a heating rate of 10 °C min 1 in air. 13C CP (Cross-polarization) and 29Si MAS (Magic Angle Spinning) NMR (Bruker DSX 400) spectra were obtained with a 4 mm zirconia rotor spinning at 6 kHz (resonance frequencies of 79.5 and 100.6 MHz for 29Si and 13C CP MAS NMR, respectively; 90o pulse width of 5 ms, contact time of 2 ms, recycle delay of 3 s for both 29Si MAS and 13C CP MAS NMR, Korea Basic Science Institute Daejeon Center). The absorption spectra of the samples were obtained from UV–Visible spectrophotometer (U-2010, HITACHI Co.). 3. Results and discussion 3.1. Powder X-ray diffraction (XRD) pattern The X-ray diffraction patterns obtained for (a) P-MCM-41 and (b) PP-MCM-41 are shown in Fig. 1 [23,25]. Inset showed the same for as-synthesized MCM-41, which shows the presence of a single

intense peak at 2.1° and three weak peaks at 3.7°, 4.2° and 5.5° 2h values and can be assigned as (1 0 0), (1 1 0), (2 0 0) and (2 1 0), respectively. The mesoporosity has been clearly revealed from the (1 0 0) peak, which is characteristic of MCM-41 materials and peaks at (1 1 0), (2 0 0) and (2 1 0) are characteristic of long range ordered hexagonal MCM-41 mesoporous phase. The d1 0 0 diffraction peak shifted to 2.2° 2h values in P-MCM-41 and PP-MCM-41. This is due to the inherent shrinkage of the mesopore after modification processes and not due to the collapse in the pore structure of mesoporous material. The persistence of d1 0 0, d1 1 0, d2 0 0, d2 1 0 for P-MCM-41 and d1 0 0, d1 1 0, d2 0 0 for PP-MCM-41 suggest the framework stability and long range order are still maintained even after several modification steps. 3.2. Transmission electron microscope (TEM) and scanning electron microscope (SEM) Fig. 2(a) and (b) reveals the TEM images of spherical MCM-41. Uniform spheres with mono dispersed particles were obtained as shown in the low-magnification TEM images [9,24]. It is clear that the obtained samples have spherical structure with a particle size of 500 nm. Mesopore channels can be clearly observed on the surface of the sphere. Fig. 2(c) and (d) represents the SEM images of as-synthesized MCM-41. The images tell the spherical appearance of the silica particles, which supports the TEM results. 3.3. Nitrogen sorption analysis Table 1 shows the physicochemical properties of MCM-41 materials obtained from N2 adsorption–desorption measurement. Figs. 3 and 4 represent the low temperature nitrogen adsorption– desorption isotherms and pore size distributions of (a) P-MCM41 and (b) PP-MCM-41, respectively. Both samples exhibited type-IV isotherms with sharp capillary condensation steps at relative pressure (P/Po) range from 0.1 to 0.3 and an H1 type narrow hysteresis loop, which is typical of mesoporous solids [26]. The hysteresis loops are very narrow for both materials, which clearly points out the narrow channels of MCM-41 materials. The surface areas, average pore diameters and total pore volumes of the PMCM-41 and PP-MCM-41 were found to be 1307 m2 g 1, 24.5 Å, 0.65 cm3 g 1 and 1133 m2 g 1, 23.4 Å, 0.44 cm3 g 1, correspondingly. The surface activation of PTMS on the inner pore wall of P-MCM-41 leads to the lowering of surface area from 1307 to 1133 m2 g 1. It is found that when functionalizing phenyl group into P-MCM-41, approximately 14% of the surface area has been decreased. At the same time the pore diameter and pore volume were decreased from 24.5 to 23.4 Å and 0.65 to 0.44 cm3 g 1, respectively. These results demonstrated that the surface area, pore diameter and pore volume of the modified material decreased due to the functionalization of phenyl group inside the channels of P-MCM-41. 3.4. FT-IR spectra analysis

Fig. 1. XRD patterns of (a) P-MCM-41 and (b) PP-MCM-41. Inset: as-synthesized MCM-41.

Both gatekeeper immobilization and subsequent phenyl functionalization are equally important on the drug delivery application. Especially the anchoring moieties have specific bands in the FTIR spectra. The appearance of these bands are the direct evidence for the presence of organic functionalities, and the corresponding intensities can serve as a guide of their relative abundance. Therefore, FTIR is useful to identify the presence of functional groups integrated over the silica framework. Fig. 5 shows the FT-IR spectra of (a) P-MCM-41 and (b) PP-MCM-41. The major peaks of both materials are clearly seen in the FT-IR spectra over the region of 1200–1000 cm 1, which provided a good insight on the silica framework of MCM-41. In both spectra, strong and intense bands

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Fig. 2. As-synthesized MCM-41 samples: (a, b) TEM images and (c, d) SEM images.

Table 1 Physicochemical properties from N2-sorption analysis of P-MCM-41 and PP-MCM-41. Material

Specific surface area (m2 g 1)

Pore diameter (Å)

Pore volume (cm3 g 1)

P-MCM-41 PP-MCM-41

1307 1133

24.5 23.4

0.65 0.44

Fig. 5. FT-IR spectra of (a) P-MCM-41 and (b) PP-MCM-41.

Fig. 3. N2 adsorption–desorption isotherms of (a) P-MCM-41 and (b) PP-MCM-41.

Fig. 4. Pore size distributions of (a) P-MCM-41 and (b) PP-MCM-41.

at 1078 and 800 cm 1 are due to the asymmetric and symmetric stretching vibrations of Si–O–Si bridges, in the order. Additionally a broad and weak band at 960 cm 1 appeared in both materials is indexed to the symmetric stretching vibration of Si–OH moieties present in the pore channels. Interestingly the intensity of the above peak is decreased after the functionalization of PTMS in the pore channels of P-MCM-41. A broad band between 3400 and 3650 cm 1 is because of surface silanol groups and a sharp band between 1650 and 1600 cm 1 is due to the bending vibrations of surface O–H groups and water molecules occluded in the pores [27]. In addition to the above bands, both the materials show additional weak bands at around 3200–2800 and 1500–1300 cm 1 which are due to the C–H stretching and bending vibrations of methylene group [7]. C–N stretching vibration absorbance is normally observed around 1200–1000 cm 1 but this band cannot be resolved due to its overlapping with the absorbance of Si–O–Si stretch in the range of 1000–1300 cm 1. But a shoulder peak at 3243 cm 1 has been observed due to asymmetric stretching vibration of N–H group present in TMSPA. Moreover, a strong but overlapped band at 1240 cm 1 in all the samples for the vibration

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of Si–C indicates that the Si–C bond has not been broken during the modification process. Obviously the intensity of the Si–C peak is slightly increasing when coming from P-MCM-41 to PP-MCM-41, which clearly indicates the introduction of hydrophobic phenylsilane inside the pore channels [14,30]. The peaks in the range of 3100–3000 and 690–740 cm 1 could be attributed to stretching and bending vibrations of C–H group present in benzene ring. Also the peaks in between 1450 and 1460 cm 1 are assigned to the benzene ring framework vibrations. The intensity of the above mentioned spectral vibration is considerably increased in PP-MCM-41 due to the presence of additional phenyl moiety from PTMS. These results demonstrate the successful incorporation of the TMSPA and PTMS on the walls of MCM-41 [15].

3.5. Solid state

29

Si MAS and

13

C CP MAS NMR analyses

Fig. 6 shows the 29Si MAS NMR spectra of (a) as-synthesized MCM-41, (b) P-MCM-41 and (c) PP-MCM-41. In Fig. 6(a) the signals due to Q2 site at 90 ppm, Q3 sites at 98 ppm and Q4 sites at 109 ppm were observed [28]. The less intense Q3 peak is the indication of minimum amount of surface silanol groups present in the as-synthesized MCM-41. Q4 has given the evidence of Si–O–Si framework, which confirms the formation of highly cross-linked silica framework. Fig. 6(b) discloses the structural evidence of TMSPA functionalization and then surfactant removed MCM-41. After surfactant removal, the formation of enormous amount of surface silanol group on the inner wall surface will increase the intensity of Q3 peak ( 100 ppm) as well as Q4 and Q2, respectively at 109 and 92 ppm. The surface functionalization leads to the formation of T signal and it is visible in the Si-NMR spectra at 66 and 58 ppm. After PTMS functionalization the intensity of Q3 peak were reduced considerably and appeared at 101 ppm. Furthermore, the disappearance of Q2 signal can be seen from the spectrum. Fig. 7(a) exhibits the 13C CP-MAS NMR spectrum of the P-MCM41. In the spectrum, four peaks (0–60 ppm) are presented, which can be due to the Si–CH2CH2CH2- and –OCH3 signals present in the TMSPA. The spectrum exhibits one of the most prominent peaks at 8.7 ppm, which is responsible for the carbon (C1) atom directly attached to the silicon and demonstrates the presence of the Si–C bond. The resonance at 16.1 ppm represents the second –CH2 from silicon atom (C2 carbon) and resonance at 19.7 ppm (C3) can be assigned to the carbon atom directly attached to the –NH group. The presence of trapped ethanol in the materials during solvent extraction also can be observed in both the spectrum near 50 ppm (⁄C). A set of three peaks at 122, 127 and 137 ppm

Fig. 6. Solid-state MCM-41.

Fig. 7.

13

C CP-MAS NMR spectra of (a) P-MCM-41 and (b) PP-MCM-41.

are responsible for the phenyl group of propyl aniline, which has been numbered as C4, C5 and C6 in P-MCM-41. The absence of intense peak at 63–70 ppm supports the removal of most of the surfactant by solvent extraction and the presence of very negligible amount of unreacted methoxy group is indicated by a small but sharp peak at 58 ppm (C7) that appear as the part of broad peak. In the spectrum of PP-MCM-41 Fig. 7(b) there are no additional peaks were observed after the functionalization of PTMS, further due to the addition of phenyl ring on the surface of the mesoporous channels, which is already present in the gate keeper molecule. As a reason all the same peaks of P-MCM-41 were persisted, indicating the formation of hydrophobically modified silica material [29]. 3.6. Thermogravimetric analysis Fig. 8 shows the TGA profiles of (a) P-MCM-41 and (b) PP-MCM41, which shows weight loss under 100 °C about 2% due to loss of physisorbed water molecules. For P-MCM-41, gradual degradation rate observed at temperatures over the range between 100 and 450 °C corresponds to the removal of the TMPSA groups attached to MCM-41 [30]. Meanwhile, further a sharp decomposition rate was observed at temperatures over the range between 450 and 800 °C due to the thermal dehydroxylation of internal surface

29

Si MAS NMR spectra of (a) MCM-41 and (b) P-MCM-41 (c) PPFig. 8. TGA curves of (a) P-MCM-41 and (b) PP-MCM-41.

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silanol groups to form siloxane bridges and channel metamorphosis. The initial weight loss of PP-MCM-41 (up to 250 °C) exactly similar to P-MCM-41 is due to the removal of adsorbed water and the initiation of TMPSA degradation. Further a sharp and increased weight loss has been observed as a function of temperature due to the decomposition of internally functionalized hydrophobic phenyl moieties [31]. The total weight loss in P-MCM-41 and PP-MCM-41 are 18% and 25%, respectively, indicating not only the decomposed organic moieties but also the weight loss due to water from silanol condensation. It is difficult to say the amount of phenyl group inside the channel of PP-MCM-41 because the material contains two types of functionality; one is on the pore opening and second is inside the pore channels. With respect to this observation it was concluded that there is enough phenyl moieties inside the pore channels of PP-MCM-41 to make it hydrophobic in nature. Fig. 10. Famotidine release behaviors from (a) PA-MCM-41 and (b) PPA-MCM-41 at pH 4, (c) PA-MCM-41 and (d) PPA-MCM-41 at pH 7.

3.7. Drug delivery To study the performance of hydrophobically modified spherical MCM-41 in a drug delivery application, an acidic drug (5-Fu) and a basic drug (famotidine) were chosen. Figs. 9 and 10 show the in vitro release profile of 5-Fu and famotidine, respectively at 37 °C from PP-MCM-41 into the intestinal fluid at pH 4. For a comparison the release profile from unmodified MCM-41 nanovalve (P-MCM-41) is also given along with the hydrophobically modified material. The capping efficiency was verified by the cargo release at pH 7 for both modified and unmodified nanovalve systems were given in the figures (Figs. 9 and 10). No release of cargo was found even after 60 h. The quantities of 5-Fu/famotidine adsorbed were measured by UV–Vis spectroscopy, which exposed the amounts of 11 and 15 wt% in the case of 5-Fu and 12 and 16 wt% in the case of famotidine for PA-MCM-41 and PPA-MCM-41, respectively. It is significant that when comparing between PA-MCM-41 and PPAMCM-41, the apparent weight of 5-Fu/famotidine molecule packed inside the nanovalve is higher in PPA-MCM-41 than the PA-MCM41 even though the materials have less surface area. These results suggest a solid relation between the functionalization and nature of the drug molecule, besides the particle size, pore diameter and surface area of the host material. It has been shown elsewhere [32] that the driving force for the inclusion of 5-Fu/famotidine inside the MCM-41 nanovalve materials seems to be the hydrogen bonding interaction between the functional groups of 5-Fu/famotidine molecule and the surface hydroxyl groups or the hydrophobic phenyl stacking in the pore channel. It is also expected that a prominent interaction by the amine protons pointing directly at

Fig. 9. 5-Flurouracil release behaviors from (a) PA-MCM-41 and (b) PPA-MCM-41 at pH 4, (c) PA-MCM-41 and (d) PPA-MCM-41 at pH 7.

the surface phenyl functionality through a hydrogen bond offers high stability to the drug molecules inside the pore channels [33]. From the structural analysis it is found that the percentage of amine functionality is more in the case of famotidine than 5Fu and as a reason the former can provide stronger interaction with the surface phenyl functionality. Moreover, the presence of fluorine atom in 5-Fu makes it weakly acid and the higher amount of amine functionality makes famotidine a weak base [34]. This is the reason why the amount of famotidine loading is higher than 5-Fu for both nanovalve (PA-MCM-41 and PPA-MCM-41) systems under our experimental conditions. It can be observed that all the release profiles are similar and exhibit sustained release behaviors at pH 4. When considering the release profile of 5-Fu from PAMCM-41 and PPA-MCM-41 (Fig. 9), the release from PPA-MCM-41 is more controlled than the PA-MCM-41. It is found that around 14% and 7% of 5-Fu have been released at the initial stage of drug delivery (with in 3 h) from PA-MCM-41 and PPA-MCM-41, respectively after that a sudden increase in the release profile has been observed when the b-CD cap will be removed. The same tendency was observed for famotidine release from PA-MCM-41 and PPAMCM-41, which are 8% and 3%, respectively. The initial delay was due to the diffusion control over the release medium by the limited pore diameter for PA-MCM-41 and hydrophobic surface along with the pore diameter for PPA-MCM-41. After 3 h much faster release has been observed due to the removal of attached drug molecule inside the pore channels. The ionizations to the drug molecules occurred much faster on the pore mouth and the rate become slow when penetrating to the deep. Because of the presence of imino and carbonyl functionality in 5-Fu, a weak interaction (hydrogen bond) in between the drug molecule and the surface hydroxyl group is generated [35]. This interaction will play a key role for making this stage of 5-Fu release as controlled one from PAMCM-41 along with the b-CD. The 5-Fu release is further more controlled in PPA-MCM-41 than PA-MCM-41 under the same experimental conditions. This is due to the hydrophobic stacking interaction between the heterocyclic ring structure/amine functionality of 5-Fu and the aromatic moiety immobilized on the surface of spherical MCM-41. The protonation to the drug will be much slower because of the diffusion control of the releasing medium into the pores due to the hydrophobically rich inner channels even though the b-CD cap will be removed. As a result a highly controlled and slower release has been observed. The maximum 5-Fu released from PA-MCM-41 and PPA-MCM-41after 60 h was found to be 74% and 61%, respectively. Famotidine is much ionic and bulkier molecule with highly basic character due to the presence of four amine and three imine functionalities. From the drug

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release profile (Fig. 10) it is observed that famotidine were released much faster from PA-MCM-41 than from PPA-MCM-41. Fortunately the interaction of famotidine with surface hydroxyl in PA-MCM-41 or aromatic functionality of PPA-MCM-41 has much similarity with the interactions of 5-Fu with the same surfaces. Even though famotidine molecules are attached to the silanol groups of P-MCM-41 through hydrogen bonding, this interaction is very weak in the presence of an acidic medium. The abundance of amino functionality in famotidine favors fast ionization and the proceeding dissolution lead to an immediate diffusion from the channels to the outer solvent medium [36]. As a result a faster and uncontrolled release has been observed from PA-MCM-41. When considering that the release of famotidine from PPA-MCM41 under the same experimental conditions is much slower and kinetically controlled, this behavior is not only due to the hydrophobic ring interaction between the high amount of amine/imine functionality in famotidine with the phenyl functionality of PPMCM-41 but also the sterically hindered diffusion from the highly strained pore channels of PP-MCM-41 due to the size bulkiness of famotidine when comparing with 5-Fu. As a reason the maximum famotidine released from PA-MCM-41 and PPA-MCM-41 after 60 h was found to be 70% and 55%, respectively, which is less than the maximum released amount observed for 5-Fu. It is worthy to consider the release profiles of 5-Fu and famotidine from an uncapped but hydrophobically modified system at pH 4 and 7. Under such circumstance one can get a good insight about the exact interaction behavior of the hydrophobically modified inner channels with the drug molecules and also the necessity to modify the mesoporous material as nanovalve in drug delivery applications. The 5-Fu (Fig. 11(a) and (b)) and famotidine (Fig. 12(a) and (b)) release profiles from PP-MCM-41 at pH 7 and 4 support the loading and release behavior of an uncapped hydrophobic drug delivery system. The drug loading was found to be 11 and 14 wt%, respectively for 5-Fu and famotidine in PP-MCM-41. The 5-Fu and famotidine loading in PP-MCM-41 were found to be 4 and 2 wt% less than that of the loading in PPA-MCM-41 nanovalve system. This may be due to the removal of loaded drug from the immediate vicinity of pore mouth due to the washing with water and it is more in the case of 5-Fu than famotidine due to the smaller size and higher solubility in water. Moreover such a loss is omitted from a nanovalve system due to the b-CD protection. When coming to the general release behavior of 5-Fu and famotidine at pH 4 and 7, no delivery has been observed at the initial hour (wetting time) in the profiles and this is due to the diffusion control over the release medium by the hydrophobically rich pore openings. It is well established that phenyl stacking has less hydrophobicity than an alkyl chain due to its polar nature and it

can offer the best control over the diffusion of the release medium into the pore channels along with the delivery of the target molecule than the second. After that the drug release was increasing slowly with progress to the solvent diffusion into the depth of the pore channels and finally most of the drugs were removed at the final hours in the release profile. It is also observed that the rate of drug release (5-Fu/famotidine) at pH 4 is higher than the rate observed at pH 7 due to the fast ionization and subsequent dissolution in the releasing medium. Even though retardation to the drug releases were observed at the initial hours of the delivery, maximum release was attained before 60 h due to the affordable pore size and well defined pore structure of MCM-41. When comparing the drug delivery from nanovalve system (PPA-MCM-41) and non-nanovalve system (PP-MCM-41), the former one acts perfectly like a pH responsive system and the latter one works as diffusion controlled system. The necessity of pore channel modification and b-CD capping were further confirmed by the 5-Fu/famotidine delivery from PMCM-41 (hydrophobically unmodified and uncapped by b-CD but pore mouth modified by TMSPA) under the similar conditions adopted for the previous experiments. The drug loadings were found to be 10 and 13 wt%, respectively for 5-Fu and famotidine. Fig. 13(a) and (b) are the 5-Fu release profiles from P-MCM-41 at pH 7 and 4, respectively. More amounts of 5-Fu release (95%) were observed at pH 4 than pH 7 (79%), as expected, due to the fast ionization and diffusion to the drug molecules in a highly acidic medium. Also a fast release rate was observed at the initial hours of the delivery at pH 4 than pH 7 due to the minimal resistance of the

Fig. 11. 5-Flurouracil release behaviors from PP-MCM-41 at (a) pH 4 and (b) pH 7.

Fig. 13. 5-Flurouracil release behaviors from P-MCM-41 at (a) pH 4 and (b) pH 7.

Fig. 12. Famotidine release behaviors from PP-MCM-41 at (a) pH 4 and (b) pH 7.

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is a combined effect of both narrow pore diameter and hydrophobic nature. Therefore, phenyl groups in the modified nanovalve system could effectively delay the release of both drugs in comparison with the unmodified nanovalve system. The importance of functionalization and the need of capping have been confirmed by the comparison of drug delivery behaviors from synthesized nanocontainers. Acknowledgments The work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Ministry of Science, ICT, and Future Planning, Korea {Pioneer Research Center Program (2010-0019308/2010-0019482); NRF-RFBR Joint Research Program (2013K2A1A7076267); Brain Korea 21 Plus program (21A2013800002)}. Fig. 14. Famotidine release behaviors from P-MCM-41 at (a) pH 4 and (b) pH 7.

capillary diffusion over a highly ionized delivery medium. Further the saturation in delivery was attained hereafter in both pH. Similar trend was observed for famotidine delivery from P-MCM-41 at the same pH conditions (Fig. 14(a) and (b)). Here the maximum deliveries are 71% and 82% at pH 7 and 4, respectively. The maximum delivery amount and the rate of delivery from P-MCM-41 suggest the need of a surface functionalization, and the uncontrolled initial delivery suggests the need of a capped gate system for keeping the drug dosage invariably over the entire period of delivery even in a highly acidic medium. 4. Conclusions In this study we have synthesized highly ordered spherical MCM-41 nanovalve systems with (PP-MCM-41) and without (P-MCM-41) internally functionalized phenyl moieties. The MCM-41 structure of the starting material was confirmed by X-ray diffraction and TEM analyses. The N2 adsorption–desorption isotherms of the prepared P-MCM-41 and PP-MCM-41 phases show typical type IV-curves with specific surface areas of 1307 m2 g 1 and 1133 m2 g 1 and a narrow pore size distribution with an average pore diameters of 24.5 Å and 23.4 Å, respectively. The appearance of Q and T sites in the 29Si MAS NMR spectra before and after functionalization clearly evidenced the formation of different silane environment, which is a clear confirmation for the presence of various silane environments in the materials. 13C CP MAS NMR analysis confirmed the fine distribution of functional groups inside as well as pore opening. TG analysis further supported the sufficient amount of functionality in the nanovalve systems to entrap structurally simple drugs like 5-Fu and famotidine with high loading. The synthesized nanovalve systems have been utilized for the controlled drug delivery of 5-Fu and famotidine drugs at pH 4. When comparing with the unmodified nanovalve system, the phenyl modified system showed delayed release profile for both 5-Fu and famotidine. Famotidine release is more delayed than 5-Fu from unmodified and modified nanovalve system owing to its bigger size and higher content of functionality in the drug molecule. The release profile from the modified nanovalve system at pH 4 showed that these hydrophobic phenyl groups can delay the penetration of releasing medium into the mesopore channels and slow down the process of drug release out of the mesoporous channels. On the other hand, the delayed delivery of drugs from the materials

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