Alizarin Complexone Functionalized Mesoporous Silica

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Alizarin Complexone Functionalized Mesoporous Silica Nanoparticles: A Smart System Integrating Glucose-Responsive Double Drugs Release and Real-Time Monitoring Capabilities Zhen Zou, Dinggeng He, Linli Cai, Xiaoxiao He, Kemin Wang, Xue Yang, Liling Li, Siqi Li, and Xiaoya Su ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b12576 • Publication Date (Web): 21 Mar 2016 Downloaded from http://pubs.acs.org on March 24, 2016

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ACS Applied Materials & Interfaces

Alizarin

Complexone

Functionalized

Mesoporous

Silica

Nanoparticles: A Smart System Integrating Glucose-Responsive Double Drugs Release and Real-Time Monitoring Capabilities

Zhen Zou, Dinggeng He, Linli Cai, Xiaoxiao He*, Kemin Wang*, Xue Yang, Liling Li, Siqi Li, Xiaoya Su

College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082 (China)

* Address correspondence to these authors at: State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P.R. China. Tel: 86-731-88821566; Fax: 86-731-88821566; E-mail: [email protected]; [email protected].

ACS Paragon Plus Environment


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Abstract The outstanding progress of nanoparticles-based delivery systems capable of releasing hypoglycemic drugs in response to glucose has dramatically changed the outlook of diabetes management. However, the developed glucose-responsive systems have not offered real-time monitoring capabilities for accurate quantifying hypoglycemic drugs released. In this study, we present a multifunctional delivery system that integrates both delivery/monitoring issues using glucose-triggered competitive binding scheme on alizarin complexone (ALC) functionalized mesoporous silica nanoparticles (MSN). In this system, ALC is modified on the surface of MSN as the signal reporter. Gluconated

insulin

(G-Ins)

is

then

introduced

onto

MSN-ALC

via

benzene-1,4-diboronic acid (BA) mediated esterification reaction, where G-Ins not only blocks drugs inside the mesopores but also works as a hypoglycemic drug. In the absence of glucose, the sandwich-type boronate ester structure formed by BA binding to the diols of ALC and G-Ins remains intact, resulting in an fluorescence emission peak at 570 nm and blockage of pores. Following a competitive binding, the presence of glucose cause the dissociation of boronate ester between ALC and BA, which lead to the pores opening and disappearance of fluorescence. As proof of concept, rosiglitazone maleate (RSM), an insulin-sensitising agent, was doped into the MSN to form a multifunctional MSN (RSM@MSN-ALC-BA-Ins) integrating with double drugs loading, glucose-responsive performance and real-time monitoring capability. It has been demonstrated that the glucose-responsive release behaviors of insulin and RSM in buffer or in human serum can be quantified in real-time through evaluating


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the changes of fluorescence signal. We believe that this developed multifunctional system shed light on the invention of new generation of smart nanoformulations for optical diagnosis, individualized treatment and non-invasive monitoring of diabetes managment. Keywords: alizarin complexone, monitoring drug release, glucose-responsive, competitive binding, mesoporous silica nanoparticles

Introduction Persistent controlling glycemic within healthy ranges is a determining link for long-term outcomes of diabetes managment.1, 2 Inadequate glycemic control can cause kidney and heart disease, blindness, nerve degeneration, and increased susceptibility to infection.3 On the contrary, overtreatment episodes can result in seizures, coma or death.4 Nanoparticles-based drug delivery holds substantial potential within diabetes treatment by providing a continuous and feedback-controlled delivery system to realize optimal treatments.5-7 Generally, an ideal drug delivery system for diabetes treatment should include two crucial attributes: (i) in order to eliminate the lag between glycemic measurement and drug dosing, the system can track the changes of glycemic levels and self-regulate drug release in response to these changes; (ii) it is very desirable that the glycemic-responsive release behaviors can be quantitativly monitored in real-time so that inadequate or excessive drug dosages can be avoided. To date, a myriad of novel nanoparticles formulations, such as liposomes,8 polymer nanoparticles,9 hydro-gel microspheres,10 and inorganic nanoparticles,11, 12 have been



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fabricated for glucose-responsive hypoglycemic drug delivery.13 For example, Chou et al. synthesized a class of glucose-responsive insulin derivatives by covalently conjugating an aliphatic domain and a phenylboronic acid (PBA) domain to insulin.14 Shi et al. constructed a core-shell hybrid nanogels by packaged small Ag nanoparticles in poly(4-vinylphenylboronic acid-co-2-(dimethylamino)ethylacrylate) nanogel network chains for integrating optical detection of glucose with self-regulated insulin release.15 Zhao et al. developed a glucose-responsive nanocarrier for deliver insulin and cyclic adenosine monophosphate (cAMP) through encapsulating cAMP molecules inside the mesopores of mesoporous silica particles (MSN) and capping gluconic acid-modified insulin (G-Ins) proteins as novel pore blocker.16 Despite the significant progress of the field, almost all existing hypoglycemic drug delivery systems are powerless of quantitative monitoring drug release in real-time, which may lead to therapy failure for diabetes because the real value of drug concentrations at some focus is not known. The widely strategies for characterizing drug release can be classified as employing fluorescent dyes/drugs as guest molecules,17 radiolabelling of drug’s structure (for example,

11

C, and

15

O),16 or modifing fluorophores to drugs.18

Such strategies have presented disadvantages in terms of dramatic difference between the release of dyes and that of the actual drugs; restriction of the use of fluorescent drugs as guest molecules but most hypoglycemic agents are nonfluorescent; and the potential risks on the curative effect caused by drug modifications.19 So, it is highly preferred in clinic to develop multifunctional delivery systems integration of glucose-responsive hypoglycemic agents delivery within real-time monitoring drug


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release. Unfortunately, as all we know, there have no report of the delivery system with the above two capabilities yet. Notably, in order to ensure the effectiveness and success of cancer therapy, a series of monitoring platforms have emerged for quantitative investigating the release of anticancer drugs.20-25 The common technique reported so far have mainly focused on fluorescence resonance energy transfer (FRET) monitoring.26-31 But this technique is not suitable for hypoglycemic drug delivery systems because too complicated designs are needed to meet such harsh preconditions that the donor-to-acceptor separation distance in the range of 10−80 Å and sensitive to the changes of glycemic levels.32 Recentlly, Balaconis et al. designed a series of fluorescent sensors for monitoring glucose levels using alizarin as a fluorescent reporter on the basis of the principle of competitive binding between a hydrophobic boronic acid, glucose, and the reporter alizarin.33, 34 This competitive binding mechanism not only ensures the response to glucose, but also can cause the change of fluorescence signal simultaneously. Herein, we have sought to develop a novel multifunctional mesoporous silica nanoparticles (MSN) system with integrated glucose-responsive double drugs release and fluorescent real-time monitoring capabilities by taking advantage of this unique feature. As illustrated in Scheme 1, alizarin complexone (ALC) is modified onto the surface of amino-functionalized MSN (MSN-NH2) as the signal reporter. The gluconated insulin (G-Ins), which served as hypoglycemic drug and capping agent, is immobilized on MSN via benzene-1,4-diboronic acid (BA) mediated esterification reaction. In the absence of glucose, the sandwich-type boronate ester structure formed



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by BA binding to the diols of ALC and G-Ins remains intact, leading to the blockage of mesopores and inhibition of the release of drugs. Simultaneously, the boronate ester produced by BA and ALC displays an emission peak at 570 nm under excitation of 460 nm-light at this stage. When glucose is introduced, it will competitively bound with BA and cause the dissociation of boronate ester. As a result, G-Ins is departed from the MSN-ALC, triggering the opening of mesopores and the diffusion of G-Ins and the drugs wrapped in the MSN. Along with the dissociation of boronate ester, the fluorescence intensity appeared to be markedly decreased. Since both the close/open event of mesopores and the change of fluorescence signal are depend on regulating the structure of gating molecular, we can quantitatively monitor the drugs release from MSN. As a proof-of-principle, rosiglitazone maleate (RSM), a well-known hypoglycemic drug working as an insulin sensitizer, was chosen as a model drug molecule.35 It was demonstrated the release ratio of RSM and G-Ins is closely related to the change of glucose concentration, along with a concurrent change in fluorescence signal. It is a good start for developing new generation of nanoformulations for diabetes treatment. Results and discussion Synthesis and characterization of delivery system. Following the design above, MSN was first prepared through a base-catalyzed sol-gel process as reported in the literature.38 Then, the as-made nanoparticles were decorated with 3-aminopropyltriethoxysilane (APTES) by post-synthetic grafting and functionalized with ALC via an amide bond. As shown in the transmission electron


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microscopy (TEM) image (Figure 1A), the as-synthesized MSN-ALC had a typical hexagonal channel-like pore. From scanning electron microscope (SEM) image (Figure 1B), the particle size of MSN-ALC was 60~100 nm, which is small enough to allow long blood circulation.36 The small-angle X-ray diffraction (XRD) analysis (Fig. 1D) confirmed the hexagonal ordering of the mesopores, which can be characterized as (100), (110), and (200) Bragg peaks. Fourier Transform Infrared (FTIR) spectra and Zeta-Sizer Nano were subsequently used to confirm the successful modification of ALC. As shown in Figure S1, the MSN-ALC exhibited a number of characteristic spectral bands. The absorption band around 3300 cm−1 in the sample was assigned to the N–H stretching. The absorption at 3064 cm−1 is associated with for C–H stretching amide B. The emerging absorption band of MSN-ALC at around 1544 cm−1 were attributed to C–N stretching and N–H deformation for amide III groups. Moreover, in contrast to MSN-NH2 with the zeta potential of 40.7 ± 7.7 mV, the zeta potential of MSN-ALC was changed into 10.3 ± 4.9 mV (Figure S2). All of these results demonstrated the successful modification of ALC. Then G-Ins was grafted on the surface of MSN-ALC as the pore blocker. The resulting G-Ins capped MSN-ALC (MSN-ALC-BA-G-Ins) nanoparticles can still keep their morphology after the functionalization. But the mesopore arrays cannot be observed after the introduction of G-Ins in aqueous solution because of the masking of G-Ins (Figure 1C). The zeta potential was changed into -6.65 ± 1.7 mV. Quantification of all modification processes were accomplished by thermogravimetric analysis (TGA). As depicted in Figure S3, the weight loss values of unloaded MSN-NH2, MSN-ALC, and



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MSN-ALC-BA-G-Ins were 7.0%, 20.5%, 34.6%, respectively, when the temperature increased to 900 °C. 7.0 %, weight loss for MSN-NH2 was from the loss of water from the silica and a few residual CTAB. 12.5% weight loss for MSN-ALC indicate the decorating amount of ALC on MSN was 324 µmol g-1 SiO2. Over 14.1% weight loss for MSN-ALC-BA-G-Ins was also reflected the successful modification of G-Ins. Fluorescenct characterization of the glucose-responsivenesses of the system. Ideally, the fluorescence properties of the nanoparticles would changed upon the modification of BA. After BA binding with nonfluorescent ALC containing diol moieties, the produced boronate ester has an emission peak at 570 nm under 460 nm excitation (Figure 2A). The results of the experiment are consistent with our expectations. As depicted in Figure 2B, an overall emission of light around 500~700 nm with a emission peak at 570 nm were observed. The capping of G-Ins has no impact on the fluorescence signal. The according fluorescence emission images under UV-light excitation are also in line with the above results (inset in Figure 2A ). The reaction between the boric acid and diol moieties is reversible.37 When glucose is introduced, a competitive binding between glucose and BA was occurred. As shown in Figure 2B, when 500 mM glucose was added, the fluorescence sigal at 570 nm showed a significant decrease. The color of the MSN-ALC-BA-G-Ins was changed from bright orange to weak fluorescence under UV-light irradiation. These results demonstrated that the glucose-responsive property of the MSN-ALC-BA-G-Ins can be evaluated by examining the changes in fluorescence signal in the presence of glucose.


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To assess potential interference by other saccharide triggers, control experiments were implemented by imersing MSN-ALC-BA-G-Ins into the solutions of mannose, maltose, lactose, galactose, and fructose. The fluorescence intensity was measured compared to the buffer control. As demonstrated in Fig 2C, MSN-ALC-BA-G-Ins displayed a good responsiveness toward fructose and glucose. In contrast, other saccharides have little effect on the fluorescence of the system. Although the system showed stronger preference toward fructose than glucose, it is still applicable for glucose-responsive drug delivery because the content of fructose in the body is very low. RSM loading into the pores of MSN-ALC-BA-G-Ins. Once we confirmed glucose-responsive behavior of MSN-ALC-BA-G-Ins, then the loading and release capacity of this system to the drugs was investigated. In this system, G-Ins is not only working as a blocking agent, but also a hypoglycemic drug. As demonstrated in the literature, the bioactivity of G-Ins was similar to unmodified insulin.39 The loaded G-Ins amount were determined to be 49 µmol g-1 SiO2 by the Bradford method. RSM was chosen as another hypoglycemic drug loading into MSN because it could improve the sensitivity of end organs to insulin but may cause a series of side effects, such as hemodilution, anemia, weight gain, and edema as well as increased risk for heart failure and myocardial ischemic events.40, 41 Loading RSM in MSN to form a glucose-responsive control release system are favorable to eliminating these undesired side effects and increasing therapeutic effect. The loading RSM

were

first

confirmed

by

Brunauer−Emmett−Teller


(BET)

and


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Barrett−Joyner−Halenda (BJH) analyses. As depicted in Figure S4 and Table S1, the surface area (11.5 m2 g−1) and pore volume (0.007 cm3 g−1) of RSM loaded MSN-ALC-BA-G-Ins (RSM@MSN-ALC-G-Ins) had reduced significantly compared to the high surface area of 1009.9 m2 g−1 and large pore volume of 1.05 cm3 g−1 for as-synthesized MSN-ALC. This result demonstrated that the mesoporous channels of MSN-ALC-G-Ins were filled with RSM. The loading amount of RSM in MSN-ALC-G-Ins determined by UV-vis absorption measurement was as high as 84.1 µmol g-1 SiO2. Real-time fluorescence monitoring glucose-responsive release of G-Ins and RSM in buffer. Since the drugs release occurs only when the G-Ins detachment away from the RSM@MSN-ALC-BA-G-Ins in response to glucose, which is integrated within the modulation of fluorescence signal. We hypothesize that the changes of fluorescence signal of MSN-ALC-BA-G-Ins can indicate the amount of RSM and G-Ins released. The more RSM and G-Ins that released from RSM@MSN-ALC-BA-G-Ins, the lower the fluorescence intensity is present (Figure 3A). In order to confirm this hypothesis, the correlation between the glucose concentration and the fluorescence intensity of MSN-ALC-BA-G-Ins was evaluated. As depicted in Figure 3B, the increase of glucose concentration led to the gradual decrease of fluorescence intensity. When the glucose concentration raised from 0 mM to 640 mM, the fluorescence intensity was decreased from 3153 a.u. to 1877 a.u., indicating that the release behavior of G-Ins is glucose-concentration-dependent, along with the changes of fluorescence intensity.


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To demonstrate the correlation between the released G-Ins and the fluorescence decreased, a certain amount of RSM@MSN-ALC-BA-G-Ins was dispersed in PBS at various glucose concentrations stirring gently at 37 oC. The released G-Ins amount was determined via centrifuging the suspension at defined time intervals and measuring through the Bradford method. As shown in Figure 3C, in the absence of glucose, about 8% G-Ins was released over a period of 40 min. In contrast, with the increase of glucose concentration, more and more G-Ins molecules are released from RSM@MSN-ALC-BA-G-Ins as time progressed. Simultaneously, the fluorescence signal of RSM@MSN-ALC-BA-G-Ins was tested by the percentage of fluorescence intensity change ((F0 − Fx)/F0×100%), where F0 is the initial fluorescence intensity of the RSM@MSN-ALC-BA-G-Ins at 570 nm in 0 mM glucose and Fx is the final fluorescence intensity in glucose solution with given concentration (Figure 3D). Similar to the trends of G-Ins release, the changes in the fluorescence signal was glucose- and time-dependent. The release of G-Ins triggered by glucose was found to be complete at 40 min, at which the fluorescence signal reached a plateau. So, the correlation between the released G-Ins and percentage of the fluorescence decreased was obtained (Figure 3E), thus strongly demonstrating the capability of this system for monitoring G-Ins release in real-time. Then whether the release of RSM can be monitored by the change of fluorescence signal was investigated. As can be seen in Figure 4A and Figure S5, with increasing the concentration of glucose, the percent RSM released was increased. Approximately 25.5%, 39.6%, 56.6%, and 73.1% RSM was released within 400 min



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at glucose concentration of 10, 20, 40, 80 mM, respectively. In the same concentration range of glucose, the fluorescence intensities of RSM@MSN-ALC-BA-G-Ins have declined 4.2%, 6.5%, 10.5%, and 16.1% (Figure 3B). From this data, the plot of the fluorescence signal change versus the percentage of released RSM was well established (Figure 4B), which is in accordance with our proposed assumption and presents a great potential for real-time monitoring glucose-responsive double drugs release. To further mimic the release behaviors under the fluctuation of body’s blood glucose level, the repeated on/off effect of this system was investigated by stepwise treatments of RSM@MSN-ALC-BA-G-Ins alternatively with a glucose solution (80 mM) and a blank buffer solution. As can be seen in Figure S6, with the alternate addition of glucose and PBS, RSM@MSN-ALC-BA-G-Ins displayed a direct on/off effect, which suggesting the excellent control release performance of this system in response to glucose. After the meal, the insulin is secreted rise to 250-300 pM within 30 min, while the insulin levels in diabetic patients is only maintained at20~30pM. According to the release curve, 32.5% was released from our system at glucose concentration of 20 mM, which implied that only 0.325~0.39 µg mL-1 RSM@MSN-ALC-BA-G-Ins was needed to deliver 250-300 pM G-Ins. Also, 39.5% RSM was released from MSN, which works as an insulin sensitizer. So, we envision that the amount of released RSM and insulin be achieved the therapeutic dosage for potential in vivo applications. Fluorescence monitoring glucose-responsive drugs release in human serum.


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Soon afterwards, quantitative monitoring of drug release in human serum was carried out. MSN-ALC-BA-G-Ins was added to the glucose-spiked serums. The fluorescence intensity in different blood glucose concentrations levels were detected. As depicted in Figure 5A, with the increase of blood glucose concentration, the fluorescence signal at 570nm decreased gradually. The descended extents of fluorescence intensity was dependent on the blood glucose concentration, which was accordant with our expectation as dissociation of boronate ester would result in the decrease of the fluorescence. To clearly show the controlled release behavior of MSN-ALC-BA-G-Ins system, Rhodamine 6G (Rh6G) was also loading into MSN-ALC-BA-G-Ins as the guest molecules. Figure 5B shows the correlation between the percent Rh6G released from the MSN-ALC-BA-G-Ins and change of fluorescence intensity. These data clearly demonstrated that there was a positive correlation between the Rh6G released and change in fluorescence intensity. Biocompatibility of the system As is widely known, the biocompatibility of delivery systems is very important to further application. So MTT assay was applied to evaluate the cell viability. As can be seen in Figure 6A, MSN-ALC-BA-G-Ins did not induce obvious cytotoxicity when incubated with human hepatic L02 cells. The hemolysis assay was also applied to test the biocompatibility of MSN-ALC-BA-G-Ins because It is reported that MSNs can cause the damage of erythrocyte (RBCs) membrane. 42 The photographs of RBCs after incubation with MSN-ALC-BA-G-Ins samples are shown in Figure 6B. The diffusion of hemoglobin from damaged RBCs was not occurred even at high dose of



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MSN-ALC-BA-G-Ins, which clearly demonstrates that the MSN-ALC-BA-G-Ins particles has low hemolytic activity. Conclusions In summary, we have successfully fabricated an effective glucose-responsive MSN-based double delivery system with the ability of quantitative monitoring the release of insulin and rosiglitazone in real-time. The mechanism behind glucose-responsive release and monitoring is the competitive binding properties of glucose and ALC with BA. By measuring the change in the fluorescent signal of the fluorescent reporter alizarin complexone, tracking the release of insulin and rosiglitazone from the MSN could be well carried. The results showed that there have a good correlation between fluorescence changes of MSN-ALC-BA-G-Ins and the extent of drugs released in buffer and in human serum. This system can quantitatively monitor the release of the hypoglycemic agents even if the drug is nonfluorescent, which has a great potential in diabetes managment. Experimental section Materials. Alizarin-3-methyliminodiacetic acid (Alizarin complexone, ALC), insulin

from

bovine

pancreas,

benzene-1,4-diboronic

3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazo-lium 4-methoxybenzyloxycarbonyl

azide

were

bromid

purchased

from

(MTT),

acid, and

Sigma-Aldrich.

N-cetyltrimethylammonium bromide (CTAB) and 3-aminopropyltriethoxysilane (APTES)

were

bought

from

Alfa

Aesar

(Tianjing,

China).

N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride crystalline (EDAC)


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was obtained from Shenggong Bioengineering Ltd. Company (Shanghai, China). D-gluconic acid sodium salt was purchased from Aladdin Reagent Corporation (Shanghai, China). N-hydroxysuccinimide Sodium (NHS) was obtained from Shanghai Reagent Corporation (Shanghai, China). Sodium hydroxide (NaOH), HCl solution (37%), and tetraethylorthosilicate (TEOS) were obtained from Xilong Reagent Company (Guangdong, China). Rosiglitazone maleate (RSM) was purchased from Melonepharma Ltd. Company (Dalian, China). Other reagents and solvents were provided by Dingguo Reagent Company (Beijing, China). All the chemical reagents in this experiment were analytical grade and used without further purification. Characterization. The sizes and morphologies of as-made MSN-ALC and MSN-ALC-BA-G-Ins were carried out with transmission electron microscopy (TEM,

JEOL 3010 microscope with an accelerating voltage of 100 kV) and scanning electron microscope (SEM, Hitachi S-4800 microscope). The crystallographic information of MSN-ALC was characterized with Small angle powder X-ray diffraction pattern (XRD), which was obtained in a Scintag XDS-2000 powder diffractometer. N2 adsorption–desorption isotherm and Brunauer–Emmett–Teller (BET) surface area were got at 77 K on a Micromeritics ASAP 2010 sorptometer. The surface charge distribution of the synthesized samples were determined by a Malvern ZetaSizer Nano instrument. The cytotoxicity of nanoparticles was investigated by MTT assay, which was operated on a Benchmark Plus, Biorad Instruments Inc, Japan. The fluorescence data were obtained from Hitachi F-7000 FL Spectrophotometer. The UV-vis absorption data were measured by using Shimadzu UV-2600 spectrometer.



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Synthesis of surface amino-functionalized mesoporous silica nanoparticles (MSN-NH2). For the synthesis of mesoporous silica nanoparticles (MSN) as reported in the literature,38 1.00 g CTAB surfactants and 3.5 mL NaOH solution (2.00 M) were added in a 500 mL round-bottom flask and dissolved with 480 mL deionized water. When the solution temperature was heated to 80 °C, 5.0 mL TEOS solution was added dropwise. After the mixture under stirring for 2 h, a white precipitate was produced. Then, the abtained crude product was centrifuged (8000 r/min, 10 min), washed thoroughly with water and ethanol. For amination of MSN, 1.0 g as-made nanoparticles were suspended in 100 mL anhydrous toluene, 1.0 mL APTES was added. The mixture was refluxed for 20 h and centrifuged, washed thoroughly with ethanol and water. To remove the structure-directing agent (CTAB), 0.7 g as-prepared samples were redispersed in a solution of ethanol (70 mL) and HCl (0.7 mL, 37.2%). After ethanol extraction for 16 h, the resulting white precipitate was separated by centrifugation and washing to obtain MSN-NH2. Synthesis of surface ALC-functionalized mesoporous silica nanoparticles (MSN-ALC). 400 mg purified MSN-NH2 was dispersed in 20 mL DMSO buffer. 116 mg ALC was reacted with 0.24 g NHS and 2.4 g EDAC in 5 mL PBS buffer (0.3 M NaCl, 10 mM phosphate, pH = 7.4), stirring for 40 min to activite carboxyl groups of ALC. The mole ratio of ALC: EDAC: NHS is 1: 7: 42. After reaction, the color of solution was changed from dark purple to orange pink. Then the reaction mixture was poured into MSN-NH2 solution and stirred under room-temperature. After 24h incubation, a visible colour change from white to purple was observed. The resulting


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material was purified by centrifugation and washing with PBS buffer to yield MSN-ALC. Synthesis of gluconic acid modified insulin (G-Ins). According to the method using 4-methoxybenzyloxycarbonyl (MeO-Z) protective group as reported in the literature, G-Ins was prepared.39 Briefly, 200 mg bovine insulin and 31mg MeO-Z were weighed and dissolved in 9 ml NHD solution (1.0 M, NaHCO3: H2O: DMF = 2:3:4). The mixed solution was stirred under room-temperature for 2.5 h. Then, 5 ml of 50 % -acetic acid was added. The abtained crude product was extracted by evaporation to yield MeO-Z protected insulin. 43.6 mg D-gluconic acid sodium salt was reacted with 11.5 mg NHS, and 17.2 mg EDAC in 7.5 mL DMSO for 1h at 25°C. Then 150 mg of MeO-Z-protected insulin in 18 mL NHD was added into this mixture. After 2h, 100 mg anisole in 80 mL of trifluoroacetic acid was added. The mixed

solution incubated in an ice bath for 1 h to remove the MeO-Z groups. The gluconic acid-functionalized insulin was further purified by dialysis to remove unreacted gluconic acid. RSM loading into MSN-ALC and capping with G-Ins. The purified MSN-ALC (100

mg)

was

redispersed

in

10

mL

DMSO

solution

consisting

of

benzene-1,4-diboronic acid (20 mg) and stirred at room temperature for 3 h. After collected by centrifugation, RSM (5 mM) in PBS buffer was added kept for stirring overnight. Finally, 100 mg G-Ins in 5 mL PBS solution was added and allowed to proceed under stirring for another 24 h. To remove physisorbed, uncapped RSM and uncoated G-Ins from the exterior surface of the material, the abtained product was



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washed with PBS for several times. The resulting precipitate dried under vacuum to yield RSM loaded MSN-ALC-BA-G-Ins (RSM@MSN-ALC-BA-Ins) . Monitoring glucose-responsive release of G-Ins and RSM. Glucose triggered G-Ins

and

RSM

release

was

achieved

through

dispersing

the

RSM@MSN-ALC-BA-Ins in PBS with different glucose concentration with shaking. Samples were centrifuged at every 5 min (10000 rpm). The suspensions were collected for analysis and replaced with fresh PBS. UV-vis measurements at the wavelength of 320 nm was used to measure release profiles of RSM molecules from the pores to aqueous solution. The released G-Ins amount was determined through the Bradford method via centrifuging the suspension at defined time intervals and measuring the absorbance of the upper clear solution at a UV-vis spectrometer at 595 nm. Time-dependent release of G-Ins and RSM from the RSM@MSN-ALC-BA-Ins was studied at 37 °C. All measurements were carried out at least three times. The G-Ins release study by stepwise glucose treatment was performed following the above method except that the glucose buffer solution (80 mM) and non-glucose buffer solution were introduced every 1 h alternatively. The release behaviors was monitored through the fluorescent changes of ALC on the surface of the nanoparticles. The fluorescence intensities were measured under 460 nm excitation. A typical set of fluorescence spectra reflected the large fluorescence changes upon glucose concentrations. Investigate the monitoring capability of MSN-ALC-BA-G-Ins system in blood serum. In order to mimic the real blood environment in vitro, monitoring drug release


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from MSN-ALC-BA-G-Ins in human serum was carried out. Firstly, glucose at various concentrations was added into the human serum, which treated with 10 mg mL−1 glucose oxidase to exhaust the intrinsic glucose. Then, the glucose triggered drug release and monitoring was carried out using the above method. In order to clearly show the monitoring capability of MSN-ALC-BA-G-Ins system, Rhodamine 6G (Rh6G) was also selected as the guest molecules, by mixing MSN-ALC and Rh6G for 24 h. The correlation between the percent Rh6G released from the MSN-ALC-BA-G-Ins and change of fluorescence intensity was investigated by the above method. In vitro cytotoxicity. To evaluate the cell viability, L02 cells were seeded into 96-well plates at 1×104 cells per well and cultured in RPMI 1640 media containing 10% fetal calf serum in a humidified atmosphere for 12 h. Then the culture medium was replaced with fresh culture medium, and MSN-ALC-BA-G-Ins at different concentration

were

added

in

each

well.

After

the

incubation

with

MSN-ALC-BA-G-Ins for another 48 h, MTT solution (20 µL, 5 mg mL−1) was added and then statically cultivatied for 4 h. Subsequently, the MTT medium was replaced with DMSO (150 µL). The optical density (OD) values at 570 nm were detected by amicroplate reader. The cell viability was calculated as follows: Viability (%) =

ODtreated − ODblank ×100% ODcontrol − ODblank

[1]

where ODtreated was obtained from the cells treated by MSN-ALC-BA-G-Ins, ODblank was obtained from the well plates without treatments by cells or MSN-ALC-BA-G-Ins, ODcontrol was obtained from the cells without any treatment.



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AUTHOR INFORMATION Corresponding Author *Phone: +86-731-88823930. Fax: +86-731-88823930. E-mail: [email protected]; [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported in part by the National Natural Science Foundation of China (21190044, 21322509, 21305035, 21305038, and 21221003). Supporting Information The additional supporting data such as FTIR spectra, zeta potential, TGA, the loading of RSM; spectra of released RSM; the repeated on/off releases of RSM; standard curve of the Bradford method for G-Ins quantitation. This material is available free of charge via the Internet at http://pubs.acs.org.

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Scheme 1. Schematic illustration of real-time monitoring of glucose-responsive double drugs release using alizarin complexone modified multifunctional mesoporous silica nanoparticles.



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Figure 1. (A) high-resolution TEM images of MSN-ALC; (B) SEM images of MSN-ALC; (C) high-resolution TEM images of MSN-ALC-BA-G-Ins; (D) powder X-ray diffraction pattern of MSN-ALC.


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Figure 2. (A) Schematic diagram indicating the synthesis of MSN-ALC-BA-G-Ins and following treatment of MSN-ALC-BA-G-Ins with glucose. Inset figures show the corresponding change in color of MSN-ALC-BA-G-Ins under a UV lamp. (B) Change in fluorescence signal with the capping of MSN-ALC and treating with 500 mM glucose; (C) selectivity of the MSN-ALC-G-Ins for glucose. The concentration of saccharide triggers is 500 mM.



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Figure 3. (A) Scheme showing the correlation between the released RSM and G-Ins from MSN-ALC-BA-G-Ins and the change in the fluorescence signal (FL) with increasing concentrations of glucose; (B) Change in fluorescence signal following treatment with increasing concentrations of glucose; (C) Percent G-Ins released at different time points following treatment with increasing concentrations of glucose; (D) Change in fluorescence signal (F0-Fx)/F0 at different time points following treatment of RSM/MSN-ALC-BA-G-Ins with increasing concentrations of glucose; (E) Correlation between percent G-Ins released and percent of the fluorescence change. (a = 80 mM glucose, b = 40 mM glucose, c = 20 mM glucose, d = 10 mM glucose, and e = no glucose).


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Figure 4.(A) Percent RSM released from the RSM@MSN-ALC-G-Ins at different time following treatment with increasing concentrations of glucose; (B) Correlation between percent RSM released and percent of the fluorescence change. (a = 80 mM glucose, b = 40 mM glucose, c = 20 mM glucose, d = 10 mM glucose, and e = no glucose).



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Figure 5. (A) Change in fluorescence signal following treatment with increasing concentrations of glucose in human serum; (B) Correlation between percent Rh6G released from Rh6G loaded MSN-ALC-BA-G-Ins and percent of the fluorescence change in human serum.


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Figure 6. (A) In vitro cytotoxicity assay for L02 cells obtained by plotting the cell viability percentage against the concentration of MSN-ALC-BA-G-Ins; (B) photographs

of

RBCs

incubated

with

MSN-ALC-BA-G-Ins

at

different

concentrations ranging from 20 to 640 µg mL−1 for 3h. The presence of red hemoglobin in the supernatant indicates damaged RBCs. D.I. water (+) and PBS (-) are used as positive and negative control, respectively.



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Table of Contents


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Alizarin Complexone Functionalized Mesoporous Silica

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