Article pubs.acs.org/ac
Tumor-Marker-Mediated “on-Demand” Drug Release and Real-Time Monitoring System Based on Multifunctional Mesoporous Silica Nanoparticles Xiang-Ling Li, Nan Hao, Hong-Yuan Chen, and Jing-Juan Xu* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China S Supporting Information *
ABSTRACT: “On-demand” drug release can maximize therapeutic efficacy for specific states of malignancies and minimize drug toxicity to healthy cells. Meanwhile, there is lack of a real-time monitoring platform to accurately investigate the amount of anticancer drugs released, especially nonfluorescent ones. So it is significant to integrate both issues in one ideal drug delivery system. To achieve this, here we present a novel stimuli-responsive controlled drug delivery system toward the tumor marker survivin mRNA, using a real-time monitoring approach based on the fluorescence resonance energy transfer (FRET) strategy to quantify the process of drug release. First, 7-amino-4-methlcoumarin (AMCA) dye terminated short oligonucleotide (FlareA) will hybridize with fluorescein isothiocyanate (FITC) labeled long oligonucleotide (S1F), which contains a recognition element to a specific RNA transcript, to form a FRET pair capped on the pores of mesoporous silica nanoparticles (MSNs). Following a target-recognition reaction, the target with a longer strand displaces the FlareA strand to form a longer and more stable duplex with S1F, which leads to the removal of the capped oligonucleotide from the MSNs and triggers the release of the entrapped cargo while FRET between AMCA and FITC is broken. The relevant change in donor and acceptor fluorescence signal can be used to monitor the unlocking and release event in real-time. Further investigations have also demonstrated that this release system possesses the capacity of modulating the extent of drug release according to the cell states, giving the platform an equally broad spectrum of applications in anticancer therapy.
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tissue,37,38 providing more exquisite control of cargo release at desired locations during drug delivery. Tumor markers could be excellent triggers for constructing “on-demand” drug release delivery systems,39−41 as tumor markers are always associated with malignancies.42,43 Despite these burgeoning developments, use of tumor markers as internal stimuli for the controlledrelease systems is still in its nascent stage. In addition to on-demand releasing, the ability to monitor drug release in real time is another important factor to regulate the spatiotemporal control over the drug delivery.44−48 Fluorescent models are widely used as entrapped guest in drug delivery to investigate the release process,45−47 which limits their potential use in monitoring actual drug molecules, as most current antineoplastic drugs are nonfluorescence. Therefore, it is necessary to develop a real-time monitoring system for investigating drug release in complex cellular microenvironments. Fluorescence resonance energy transfer (FRET) is a well-established energy transfer process between the energy donor and acceptor chromophores that is very
ince most of the commonly used antineoplastic drugs cannot discern between diseased and healthy cells and have serious side effects on healthy cells, an extensive amount of work has been done to develop site-specific stimuli-responsive controlled drug delivery systems. Among them, numerous organic,1−3 inorganic,4,5 and inorganic/organic hybrid6,7 materials have been used as drug delivery platform to transport anticancer drugs to a specific point in the body, thus mitigating harmful side effects of drug to healthy tissues. In this regard, mesoporous silica nanoparticles (MSNs) have attracted substantial attention as nanocarriers due to their unique porous structure, tunable pore size, large pore volume, high surface area, biocompatibility, and chemically modifiable surfaces.8−15 Diverse molecular valves that functionalize the pores of MSNs can work as gatekeepers and be removed by various external or internal stimuli, including light,16−19 pH,20−23 redox potential,24−28 temperature,29−31 and biomolecules.26,32−35 Despite the prominent potential that had been achieved in these drug delivery systems triggered by various external stimuli, low penetration in tissue still limits their performance.36 Most recently, using biomolecules as internal triggers to release entrapped guest in delivery systems is becoming a momentous field, as these stimuli always exhibit increased activity in cancer © XXXX American Chemical Society
Received: June 29, 2014 Accepted: September 17, 2014
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labeling on oligonucleotides for monitoring drug release in real time. Initially, FlareA hybridizes with part of S1F. The AMCA and FITC moieties labeling FlareA and S1F are in close proximity, creating a FRET pair. As FlareA just hybridized with one end of S1F, it still possesses the single-strand characteristic that can be flexibly adsorbed on the surface of APTS-modified MSNs with partially positively charged aminopropyl groups at neutral pH.33 The FRET-MSNs display a typical double emission peak at 450 and 520 nm due to partial transfer of energy from AMCA to FITC, when they are excited at the excitation wavelength of 353 nm. Nevertheless, in the presence of the target strand survivin mRNA, more stable hybridization between survivin mRNA and S1F will dissociate FlareA and cause the removal of the oligonucleotide cap from the MSNs, thereby triggering the unlocking and releasing process. Because of the dehybridization between S1F and FlareA, the FRET between AMCA and FITC is broken, and the system only displays the characteristic emission wavelength of AMCA at 450 nm. Due to the correlation between the fluorescence signal change and drug release, the release process can be monitored in real time. Moreover, the amount of drug released relies on the state of malignancies by utilizing the tumor marker survivin mRNA as an internal stimulus; the more survivin mRNA that is synthesized in the cancer cells, the greater the lethality induced by the anticancer drug to the cancerous cells.
sensitive to the changes in donor-to-acceptor separation distance in the range of 10−80 Å.49 This unique feature of FRET makes it a promising candidate for monitoring delicate pore unlocking and cargo release interactions in real time during the drug delivery process.50,51 Notably, despite these prominent achievements in delivery systems, most MSNs-based drug delivery carriers possess only one of the two features (ondemand release or real-time monitoring) and cannot function well under complex physiological conditions. In order to improve anticancer drug delivery performance, it would be advantageous to design a capped MSNs nanodevice that would have on-demand release and allow tracking and monitoring of the process of drug release. Such a combination, we believe, is extremely paramount, as it allows simultaneous imaging diagnosis and controllable drug delivery in one. To address the aforementioned issues, herein we designed a novel MSNs drug delivery system based on a targetrecognition-responsive FRET valve (henceforth referred to as FRET-MSNs). Survivin mRNA is used as the target and FRETpair-labeled nucleic acids are used as gates of the pores in MSNs. Survivin mRNA, which is responsible for survivin overexpression in all the most common human malignancies, is becoming a significant tumor marker.52,53 Therefore, survivin mRNA can work as a promising internal stimulus to permit drug release inside malignant cells more precisely and alleviate the undesired systemic toxicity. Thus, this system enables realtime monitoring of the target recognition controllable drug release occurring in cancer cells. As illustrated in Scheme 1, the
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EXPERIMENTAL SECTION Reagents, Materials, Apparatus, and Statistical Analysis. Refer to the Supporting Information. Preparations of MSNs, DOX-Loaded MSNs, FRETMSNs, and FRET-DOX-Loaded MSNs Complexes. The MSNs were synthesized in a typical synthesis procedure.41 Briefly, N-cetyltrimethylammonium bromide (CTAB, 1.00 g, 2.74 × 10−3 mol) was first dissolved in 480 mL of deionized water, and then NaOH (aq) (2.00 M, 3.50 mL) was added to CTAB solution under vigorous stirring, followed by adjusting the solution temperature to 95 °C. After the solution became clear, 5 mL of tetraethyl orthosilicate was added dropwise to the surfactant solution; hence, the mixture was allowed to stir for 3 h to give rise to white precipitates, and then the synthesized materials were centrifuged and washed thoroughly with deionized water and ethanol. To remove the surfactant template, the final products were calcined at 550 °C for 5 h after being dried for 12 h at 60 °C in a vacuum. In the loading process, calcined MSNs (0.1 g) were dispersed in ethanol (20 mL). Then the drug doxorubicin hydrochloride (DOX) (0.2 mM) was added and the mixture was stirred for 24 h at 36 °C with the aim of achieving maximum loading in the pores of the MSNs scaffolding. After this procedure, an excess of APTS (0.374 mL) was added and the mixture stirred for 5.5 h. The nanoparticles were then centrifuged (8000 rpm, 10 min), washed six times with methanol, and dried under vacuum to obtain the intermediate product MSNs-NH2. The capping process by oligonucleotide is done according to the method of Climent et al.33 Briefly, the oligonucleotide FlareA hybridizes with S1F in equal concentration to form FRET-pair-labeled nucleic acids for the capping process. Then 600 μg of intermediate MSNs-NH2 was suspended in 600 μL of hybridization buffer (20 mM Tris-HCl, 37.5 mM MgCl2 at pH 7.5) containing the nucleic acids in concentrations of 3 × 10−5 M and each suspension was stirred at 37 °C, respectively. After 1 h, the mixture was centrifuged at 8000 rpm for 10 min, and
Scheme 1. Schematic Representation of Target-RecognitionResponsive FRET-MSNs Release System for Cancer Therapy
real-time monitoring of the drug controllable release platform is comprised of four components: (i) 3-aminopropyltriethoxysilane (APTS) functionalized MSNs as the drug carriers, (ii) 7amino-4-methlcoumarin (AMCA) dye terminated short oligonucleotide (FlareA) hybridized with a part of antisense oligonucleotide [fluorescein isothiocyanate (FITC) labeled, S1F] as cap to entrap the drugs within the MSNs, (iii) survivin mRNA hybridized with S1F as the target-recognition-responsive trigger to release the entrapped drug molecules on demand, and (iv) the FRET donor−acceptor pair of AMCA and FITC B
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Target-Recognition-Responsive Behavior and Drug Release in Vitro. The AMCA moiety in the FRET pair can be excited at an excitation wavelength of 353 nm, resulting in an emission wavelength in the range of 410−490 nm. When the FRET pair is intact, they displayed dual emission peaks at 450 and 520 nm upon excitation at 353 nm (Figure 2A), as the
then the pellet (FRET-DOX-loaded MSNs) was collected carefully, dispersed in hybridization buffer, and stored at 4 °C for further use. Besides, the complexes FRET-MSNs or DOXloaded MSNs were formed by the above procedure just without the loading process or capping process, respectively. Cell Culture and Preparation. Acute myeloblastic leukemia (HL-60) cells and normal liver (LO2) cells were cultured in DMEM supplemented with 10% FBS and 100 IU mL−1 penicillin−streptomycin. The cells were maintained at 37 °C in a humidified atmosphere (95% air and 5% CO2). In the delivery experiment, HL-60 cells and LO2 cells were seeded in 24-well plates. After 24 h of plating, culture media were exchanged with fresh serum-free basal media (500 μL) containing MSNs, FRET-MSNs, DOX-loaded MSNs, or FRET-DOX-loaded MSNs complexes, respectively. After 6 h, media were exchanged with fresh culture media. Fluorescence measurements and fluorescence confocal microscopy imaging were performed over a period of time (0−24 h) after transfection.
Figure 2. (A) Emission spectra of FRET-MSNs. (B) Fluorescence intensity change in blue (450 nm) and green (520 nm) emission wavelengths upon addition of different S2 concentrations to FRETMSNs system: (a) 0 M, (b) 1 × 10−7 M, (c) 1 × 10−6 M, (d) 5 × 10−6 M, and (e) 1 × 10−5 M. Excitation wavelength was 353 nm.
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RESULTS AND DISCUSSION Characterization of FRET-MSNs. As shown in transmission electron microscopy (TEM) images (Figure 1A), the
AMCA moiety can act as a photon donor for the FITC, which can be excited maximally at 490 nm. The target-recognitionresponsive property of the FRET-MSNs was investigated by observing the changes in donor and acceptor fluorescence signal through addition of synthesized survivin mRNA (S2), which mimicked the intracellular environment. As shown in Figure 2B, increasing the concentrations of S2 [(0−1) × 10−5 M] in the buffered solution of FRET-MSNs led to a decrease in the green fluorescence (520 nm) and an increase in the blue fluorescence (450 nm). This strongly indicated that, in the presence of a target strand, the target strand can displace FlareA to hybridize with S1F, leading to the removal of the FRET acceptor, FITC. These results demonstrated that the FRETpair-labeled nucleic acids can be adsorbed onto the surface of the MSNs through electrostatic attraction and the targetrecognition reaction of the FRET-MSNs can result in a concomitant fluorescence signal change. To monitor the drug release from the pores in vitro, the drug doxorubicin hydrochloride (DOX) was chosen as model cargo, which was loaded into the pores of MSNs by mixing aqueous buffered solutions of MSNs and DOX for 24 h. The results indicated that the encapsulation efficiency (EE) of 80% could be achieved (Figure S-3, Supporting Information). The delivery profile of cargo in the presence and absence of the target oligonucleotide was monitored through UV−vis measurement of the released model cargo. FRET-DOX-loaded MSNs were dispersed in hybridization buffer (20 mM Tris-HCl, 37.5 mM MgCl2 at pH 7.5), and the released DOX in the absence of target oligonucleotide S2 was first monitored. As can be seen in Figure 3A (curve a), a less than 4% leakage of cargo over a period of 24 h indicated that the FRET-MSNs system had a stable capping capability in the absence of target. The presence of the target strand S2 would induce the hybridization between S2 and S1F, which could open the pores and release the cargo. When a low concentration of target strand S2 was added, the release percent sharply increased to 16.8% at 24 h (curve b). Furthermore, a higher concentration of target would lead to a more significant release, and almost 70% entrapped drug could be released with a target concentration up to 10−5 M (curve e). Meanwhile, the release percent was also changing with time, due to more target strand hybridizing with S1F and inducing
Figure 1. (A) TEM image of MSNs and HRTEM images of MSNs (inset a) and FRET-DOX-loaded MSNs (inset b). (B) Low-angle XRD pattern of MSNs, and the insets are BET and BJH assays of MSNs.
synthesized MSNs had an average diameter of ∼100 nm. The highly ordered lattice array demonstrated the ordered, welldefined mesoporous structure of the obtained MSNs, which was further substantiated by X-ray diffraction (XRD) and N2 adsorption isotherms tests (Figure 1B). The N2 adsorption− desorption isotherm demonstrated that the obtained MSNs have a Burnauer−Emmett−Teller (BET) surface area of 639 m2 g−1 and a narrow Barrett−Joyner−Halenda (BJH) pore-size distribution (average pore diameter = 2.3 nm, Figure 1B, inset). The surface of MSNs was characterized by FT-IR (Figure S-1A, Supporting Information). To obtain MSNs-NH2, the surface of MSNs was functionalized with APTS. The FTIR spectrum of MSNs-NH2 showed a primary amine characteristic absorption band at 1560 cm−1. The process was also characterized by ζpotential analysis and dynamic light scattering (DLS) characterization (Figure S-1B−D, Supporting Information). The obtained MSNs-NH2 nanocomplex showed a positive ζpotential. When FRET-pair-labeled nucleic acids adsorbed on the nanocomplex MSNs-NH2, the ζ-potential fell to 7.8 mV as the adsorped nucleic acids contained more negative phosphate groups. Meanwhile, the diameter of FRET-MSNs significantly increased from 110 ± 6 to 120 ± 5 nm, which indicated a successful capping process as well. Additionally, the length of anti-RNA oligonucleotide (S1F) was optimized to find the most suitable nucleic acids cap of MSNs (Figure S-2, Supporting Information). C
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Figure 3. (A) Release of DOX from the FRET-DOX-loaded MSNs following treatment with different concentrations of oligonucleotide S2: (a) 0 M, (b) 1 × 10−7 M, (c) 1 × 10−6 M, (d) 5 × 10−6 M, (e) 1 × 10−5 M. (B) DOX released from the FRET-DOX-loaded MSNs system in the presence of oligonucleotides S2, S3, and S4 at concentrations of 5 × 10−6 M.
Figure 4. Confocal fluorescence images of HL-60 cells after incubation with FRET-MSNs nanocarriers for 6 h at 37 °C (A) and 4 °C (B). Images from left to right: channel 1 represents cell stained by Hoechst 33342, and channel 2 represents the fluorescence intensity of the FRET pair in HL-60 cells treated with FRET-MSNs, with bright-field and merged images also shown.
greater drug release. The UV/vis absorbance spectra of the released drug are specifically shown in Figure S-4 (Supporting Information) after the nanocarriers interacted with target strand for 24 h. The release profiles of cargo in the presence of target strand depicted an increase in the percentage of DOX released and the percent was proportionally dependent on the target concentration. For the purpose of investigating the selectivity in the target recognition responsive opening protocol, model cargo released from FRET-MSNs was tested in the presence of other oligonucleotides (S3, a single-base mismatch sequence to S2, and S4, a two-base mismatch sequence to S2). As shown in Figure 3B, less than 15% drug release was observed with S3 or S4, indicating that the nontarget strands induced a poor uncapping and releasing process. The results revealed that only the presence of S2 induced a remarkable release of entrapped cargo, which strongly suggested that the FRET-MSNs delivery system has high selectivity in the opening protocol (Figure 3B). Observing on-Demand Drug Release in Cancer Cells Using FRET-MSNs. Before observing on-demand drug release in cancer cells, we investigated the intracellular uptake pathway and the intracellular localization of FRET-MSNs nanocarriers in mammalian cells. Acute myeloblastic leukemia (HL-60) cells were incubated with FRET-MSNs nanocarriers at 4 and 37 °C. After the transfection process, cells were stained with the bisbenzimidazole dye Hoechst 33342 at 37 °C for 10 min. Then these cells were washed with PBS solution and imaged by confocal fluorescence microscopy immediately. As Hoechst 33342 binds to the minor groove of double-stranded DNA, this dye stained nuclei blue54 to highlight the cell nucleus region (channel 1, Figure 4). A large amount of sky-blue spots emitted from the FRET pairs in HL-60 cells at 37 °C was observed by confocal fluorescence microscopy when nanocarriers just entered in the cells (channel 2, Figure 4A), confirming that vast FRET-MSNs systems could be ingested by the cells. Meanwhile, the sky-blue spot revealed that the FRET pair capped on MSNs, which own both blue and green dual emission, was intact. Additionally, the merged image (Figure 4A) and the z-stack images (Figure S-5 and Video S-1, Supporting Information) also revealed that these nanocarriers were internalized by cells and mostly escaped from endosome/ lysosome into the cytoplasm. Compared with the cells incubated at 37 °C, almost no fluorescence of the FRETMSNs system could be observed in the cells incubated at 4 °C (Figure 4B), suggesting that few nanocarriers could be internalized by cells at this temperature. The mean fluorescence intensity of internalized FRET-MSNs systems at 37 °C was
almost 40 times that at 4 °C. All the results demonstrated the efficient uptake of nanocarriers at 37 °C and that the uptake mechanism of FRET-MSNs systems is an energy-dependent endocytosis.55 As survivin mRNA is overexpressed in many malignancies while tightly controlled in terminally differentiated adult tissues, normal liver (LO2) cells were chosen as control cells for further confirming the capacity of on-demand drug release in the FRET-MSNs system. To investigate whether FRET-DOXloaded MSNs nanocarriers could serve as target-responsive delivery platform with real-time monitoring of drug release, the fluorescence intensity change was observed in HL-60 cells or LO2 cells after being incubated with these carriers for a period of time (0−24 h). As shown in Figure 5A (panel a), at time t =
Figure 5. (A) Laser confocal microscopy measurement of the fluorescence signal of the FRET pair in HL-60 (a) and LO2 (b) cells cultured with FRET-DOX-loaded MSNs at different times. (B) The magnification of HL-60 (a) and LO2 (b) cells after treatment with FRET-DOX-loaded MSNs for 24 h. Images from left to right: channel 1 represents the fluorescence intensity of the FRET pair, channel 2 represents the fluorescence of the drug DOX, and channel 3 represents cell stained by Hoechst 33342, with bright-field and merged images shown. The magnifying power is 40×. D
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0 h, vast sky-blue spots were visible in the cytoplasm region, indicating that most FRET pairs were still intact and have dual emission fluorescence. Meanwhile, as can be seen in Figure S-6 (Supporting Information), the high red fluorescence of the drug DOX was colocalization with these sky-blue spots when the delivery systems just entered in the cells, which indicating that FRET-DOX-loaded MSNs nanocarriers were still in their locked stage and the entrapped drug still gathered in the nanaocarriers. When incubated with FRET-DOX-loaded MSNs for a period of time, a significant increase in the blue fluorescence intensity and a corresponding decrease in the green fluorescence intensity were observed in these target cells. At approximately 24 h, negligible green fluorescence with extremely bright blue fluorescence could be observed by microscopy. This was consistent with the results of in vitro detection (Figure 2B), as the target-responsive reaction would lead to the dissociation of the FRET pair, thereby inducing the recovery of the blue fluorescence intensity. As expected, the entrapped drug released concurrent with the fluorescence signal change. As DOX could be inserted into the double-helix of DNA to damage DNA, higher and higher red fluorescence intensity of released DOX in the nuclei could be detected in these target cells with prolonged incubation time (Figure S-6A, panel a, marked with a circle, Supporting Information). In the magnified image, we can clearly observe that vast purple spots, which were the merger of the released DOX fluorescence with the nuclei dye Hoechst 33342 fluorescence accumulated in the nuclei, and a mass of apoptotic bodies appeared in cells after incubating with the drug delivery system for 24 h (Figure 5B, panel a). Additionally, little fluorescent signals emitted from FRET-MSNs demonstrated a colocalization with the red fluorescence from Dox (formed white spots), indicating that most drug was released and not entrapped in these nanocarriers. Nevertheless, the control cells treated with the same process produced different final results. Negligible fluorescence change in the FRET pairs coupled with no fluorescence of released drug with prolonged time could be observed in control cells (Figures 5A and S-6, panel b, Supporting Information). Meanwhile, the magnified image distinctly presented that all the drug was still entrapped in the nanocarriers and presented a high colocalization with the delivery system in the cytoplasm region of control cells (merge channel, panel b, Figure 5B). Because the drug release happened only upon the removal of the FRET-pair-labeled cap based on target recognition, the correlation between the fluorescence signal change (R = F520/ F450) and the mean fluorescence of released drug was tested. In HL-60 cells, the mean fluorescence intensity of released drug in nucleus gradually increased with prolonged incubation time, and the intensity at 24 h was 20 times that at 0 h, indicating that more and more drug was released from the nanocarriers and gathered in the cell nucleus (Figure 6, inset a). Simultaneously, a gradual increase in the blue emission intensity (F450) and a corresponding decrease (F520) in the green emission intensity led the fluorescence signal change R to gradually reduce to zero during the releasing process. In contrast, in the control cells, negligible DOX release was accompanied by a slight R change. All this revealed that greater drug release coincided with a lower R (Figure 6, black curve). This outcome indicated that the FRET-MSNs system can not only monitor the release of fluorescent drug but also of nonfluorescent cargo based on the R change. These results demonstrated that this drug release system has the capability of
Figure 6. Correlation between the mean fluorescence intensity of released DOX and fluorescence signal change R of the FRET pair at different time points for HL-60 cells and LO2 cells (inset b) incubated with FRET-DOX-loaded MSNs nanocarriers. Inset a is the detailed mean fluorescence of released DOX at different time points. Thirty cells were used to obtain the mean fluorescence intensity of FRET pairs and DOX.
releasing drug on-demand and monitoring the procedure in real time. Cell viability was observed after both cell lines (HL-60 and LO2 cells) were cultured with different concentrations of MSNs, DOX-loaded MSNs, and FRET-DOX-loaded MSNs composites for 24 h (Figure 7). Without the specific nucleic
Figure 7. Cytotoxicity assays of HL-60 cells (a) and LO2 cells (b) incubated with MSNs, DOX-loaded MSNs, and FRET-DOX-loaded MSNs.
acids cap, a large amount of DOX was released in both target and control cells (Figure S-7, Supporting Information) and there was no significant difference (P > 0.05) in cell viability of HL-60 and LO2 cells (Figure 7b). While incubated with FRETDOX-loaded MSNs composites, highly significant differences in cell viability were shown between target and control cells (P < 0.001). FRET-DOX-loaded MSNs composites showed significant lethality to just HL-60 cells (cell viability = 28.6 ± 4%, mean ± SD) compared with the control cells (cell viability >72%), revealing that the current smart nanodevices yielded slight side effects for normal cells and the specificity of tumor marker stimuli for controllable drug release. With the aim of observing the on-demand release capability of the FRET-MSNs delivery cargo based on different states of malignancies, antisense oligonucleotides (S1) and synthetic survivin mRNA mimics (S2) were transfected into HL-60 cells by lipofectamine 3000 according to the manufacturer’s E
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instructions for up- and down- regulation of the concentration of the internal stimulus survivin mRNA.52 As shown in Figures 8 and S-8 (Supporting Information), less drug release coupled
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ASSOCIATED CONTENT
S Supporting Information *
A summary of reagents and materials, apparatus, and statistical analysis; the experiment process for the cell viability assay; characterization of MSN, MSN-NH2, and FRET-MSNs; optimization of the FRET-MSNs system; encapsulation efficiency calculation; location analysis of drug delivery systems; laser confocal microscopy measurement of cells treated with different nanocarriers; and Video S-1. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +86-25-83597294. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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Figure 8. Laser confocal microscopy measurement of HL-60 cells treated with transfection of antisense oligonucleotides (S1) or synthetic survivin mRNA mimics (S2), for conditions a−d, transfected S1 (5 × 10−7 M), S1 (0 M), S2 (5 × 10−7 M), and S2 (1 × 10−6 M), respectively, and then incubating these cells with FRET-DOX-loaded MSNs for 24 h. FRET field represents the fluorescence signal of the FRET pair in HL-60 cells, and the DOX field represents the merged image of the drug fluorescence field, Hoechst 33342 fluorescence field, and bright field of HL-60 cells.
ACKNOWLEDGMENTS This work was supported by the 973 Program (Grant 2012CB932600, 2013CB933800), the National Natural Science Foundation (Grants 21327902, 21025522), and the National Natural Science Funds for Creative Research Groups (Grant 21121091).
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REFERENCES
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with slight fluorescence signal change (R) in condition a (transfected S1) was observed by microscopy compared to condition b (without transfection), indicating that less targetrecognition reaction induced less drug release, as antisense oligonucletides transfected into cells would reduce the concentration of internal stimulus.52 On the contrary, a vast amount of DOX was released and accumulated in the nuclei coupled with a significant R signal change in conditions c and d (transfected S2), indicating that a higher concentration of internal stimulus hybridized with the cap system and induced more release of entrapped cargo. The mean fluorescence intensity of released drug in condition d was nearly 2.5 times that in condition a, revealing that the delivery system can achieve the on-demand drug release. All these results demonstrated that the smart drug delivery system can control drug release based on each cell specific state and monitor the controllable release process in real time, which can maximize the desired therapeutical effects.
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CONCLUSIONS In this study, we present a smart drug delivery system based on integration of FRET-based real-time monitoring system and on-demand drug release in mesoporous silica nanoparticles. Compared to conventional drug delivery systems, this new nanocarrier possesses greater potential in anticancer therapy. The system has an equally broad spectrum in monitoring the cargo release, even if the drug is nonfluorescent, making it a promising candidate for investigating the relevance between drug amount and therapeutic efficacy. The significant tumor marker survivin mRNA as internal stimulus endows the drug release with more selectivity and accuracy in cancer cells, which can greatly minimize side effects while maximizing desired effects. With other tumor markers coupled with corresponding FRET systems, such a platform could be extended and serve as a general basis to develop site-specific drug delivery in cancer therapy. F
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(24) Zhu, Y.; Liu, H.; Li, F.; Ruan, Q.; Wang, H.; Fujiwara, M.; Wang, L.; Lu, G. Q. J. Am. Chem. Soc. 2010, 132, 1450−1451. (25) Mortera, R.; Vivero-Escoto, J.; Slowing, I. I.; Garrone, E.; Onida, B.; Lin, V. S. Y. Chem. Commun. 2009, 3219−3221. (26) Zhao, Y.; Trewyn, B. G.; Slowing, I. I.; Lin, V. S. Y. J. Am. Chem. Soc. 2009, 131, 8398−8400. (27) Liu, R.; Zhao, X.; Wu, T.; Feng, P. J. Am. Chem. Soc. 2008, 130, 14418−14419. (28) Nguyen, T. D.; Liu, Y.; Saha, S.; Leung, K. C. F.; Stoddart, J. F.; Zink, J. I. J. Am. Chem. Soc. 2007, 129, 626−634. (29) Liu, C.; Guo, J.; Yang, W.; Hu, J.; Wang, C.; Fu, S. J. Mater. Chem. 2009, 19, 4764−4770. (30) Ma, M.; Chen, H.; Chen, Y.; Wang, X.; Chen, F.; Cui, X.; Shi, J. Biomaterials 2012, 33, 989−998. (31) Tian, B.-S.; Yang, C. J. Nanosci. Nanotechnol. 2011, 11, 1871− 1879. (32) Park, C.; Kim, H.; Kim, S.; Kim, C. J. Am. Chem. Soc. 2009, 131, 16614−16615. (33) Climent, E.; Martinez-Manez, R.; Sancenon, F.; Marcos, M. D.; Soto, J.; Maquieira, A.; Amoros, P. Angew. Chem., Int. Ed. 2010, 49, 7281−7283. (34) Singh, N.; Karambelkar, A.; Gu, L.; Lin, K.; Miller, J. S.; Chen, C. S.; Sailor, M. J.; Bhatia, S. N. J. Am. Chem. Soc. 2011, 133, 19582− 19585. (35) Popat, A.; Ross, B. P.; Liu, J.; Jambhrunkar, S.; Kleitz, F.; Qiao, S. Z. Angew. Chem., Int. Ed. 2012, 51, 12486−12489. (36) Argyo, C.; Weiss, V.; Braeuchle, C.; Bein, T. Chem. Mater. 2014, 26, 435−451. (37) Mas, N.; Agostini, A.; Mondragon, L.; Bernardos, A.; Sancenon, F.; Dolores Marcos, M.; Martinez-Manez, R.; Costero, A. M.; Gil, S.; Merino-Sanjuan, M.; Amoros, P.; Orzaez, M.; Perez-Paya, E. Chem. Eur. J. 2013, 19, 1346−1356. (38) Coll, C.; Mondragon, L.; Martinez-Manez, R.; Sancenon, F.; Dolores Marcos, M.; Soto, J.; Amoros, P.; Perez-Paya, E. Angew. Chem., Int. Ed. 2011, 50, 2138−2140. (39) Li, L.-L.; Xie, M.; Wang, J.; Li, X.; Wang, C.; Yuan, Q.; Pang, D.W.; Lu, Y.; Tan, W. Chem. Commun. 2013, 49, 5823−5825. (40) Zhang, P.; Cheng, F.; Zhou, R.; Cao, J.; Li, J.; Burda, C.; Min, Q.; Zhu, J.-J. Angew. Chem, Int. Ed. 2014, 53, 2371−2375. (41) Lai, J.; Shah, B. P.; Garfunkel, E.; Lee, K.-B. ACS Nano 2013, 7, 2741−2750. (42) Wu, J.; Fu, Z.; Yan, F.; Ju, H. TrAC, Trends Anal. Chem. 2007, 26, 679−688. (43) Meyer, T.; Rustin, G. J. S. Br. J. Cancer 2000, 82, 1535−1538. (44) Jana, A.; Devi, K. S. P.; Maiti, T. K.; Singh, N. D. P. J. Am. Chem. Soc. 2012, 134, 7656−7659. (45) Ock, K.; Jeon, W. I.; Ganbold, E. O.; Kim, M.; Park, J.; Seo, J. H.; Cho, K.; Joo, S.-W.; Lee, S. Y. Anal. Chem. 2012, 84, 2172−2178. (46) Santra, S.; Kaittanis, C.; Santiesteban, O. J.; Perez, J. M. J. Am. Chem. Soc. 2011, 133, 16680−16688. (47) Weinstain, R.; Segal, E.; Satchi-Fainaro, R.; Shabat, D. Chem. Commun. 2010, 46, 553−555. (48) Lee, M. H.; Kim, J. Y.; Han, J. H.; Bhuniya, S.; Sessler, J. L.; Kang, C.; Kim, J. S. J. Am. Chem. Soc. 2012, 134, 12668−12674. (49) Ha, T.; Enderle, T.; Ogletree, D. F.; Chemla, D. S.; Selvin, P. R.; Weiss, S. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 6264−6268. (50) Bagalkot, V.; Zhang, L.; Levy-Nissenbaum, E.; Jon, S.; Kantoff, P. W.; Langer, R.; Farokhzad, O. C. Nano Lett. 2007, 7, 3065−3070. (51) Ho, Y.-P.; Chen, H. H.; Leong, K. W.; Wang, T.-H. J. Controlled Release 2006, 116, 83−89. (52) Olie, R. A.; Simoes-Wust, A. P.; Baumann, B.; Leech, S. H.; Fabbro, D.; Stahel, R. A.; Zangemeister-Wittke, U. Cancer Res. 2000, 60, 2805−2809. (53) Altieri, D. C. Oncogene 2003, 22, 8581−8589. (54) Reixach, N.; Deechongkit, S.; Jiang, X.; Kelly, J. W.; Buxbaum, J. N. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 2817−2822. (55) Lu, J.; Liong, M.; Sherman, S.; Xia, T.; Kovochich, M.; Nel, A. E.; Zink, J. I.; Tamanoi, F. Nanobiotechnology 2007, 3, 89−95.
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Tumor-Marker-Mediated “on-Demand” Drug Release and Real-Time Monitoring System Based on Multifunctional Mesoporous Silica Nanoparticles Xiang-Ling Li, Nan Hao, Hong-Yuan Chen, and Jing-Juan Xu* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China S Supporting Information *
ABSTRACT: “On-demand” drug release can maximize therapeutic efficacy for specific states of malignancies and minimize drug toxicity to healthy cells. Meanwhile, there is lack of a real-time monitoring platform to accurately investigate the amount of anticancer drugs released, especially nonfluorescent ones. So it is significant to integrate both issues in one ideal drug delivery system. To achieve this, here we present a novel stimuli-responsive controlled drug delivery system toward the tumor marker survivin mRNA, using a real-time monitoring approach based on the fluorescence resonance energy transfer (FRET) strategy to quantify the process of drug release. First, 7-amino-4-methlcoumarin (AMCA) dye terminated short oligonucleotide (FlareA) will hybridize with fluorescein isothiocyanate (FITC) labeled long oligonucleotide (S1F), which contains a recognition element to a specific RNA transcript, to form a FRET pair capped on the pores of mesoporous silica nanoparticles (MSNs). Following a target-recognition reaction, the target with a longer strand displaces the FlareA strand to form a longer and more stable duplex with S1F, which leads to the removal of the capped oligonucleotide from the MSNs and triggers the release of the entrapped cargo while FRET between AMCA and FITC is broken. The relevant change in donor and acceptor fluorescence signal can be used to monitor the unlocking and release event in real-time. Further investigations have also demonstrated that this release system possesses the capacity of modulating the extent of drug release according to the cell states, giving the platform an equally broad spectrum of applications in anticancer therapy.
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tissue,37,38 providing more exquisite control of cargo release at desired locations during drug delivery. Tumor markers could be excellent triggers for constructing “on-demand” drug release delivery systems,39−41 as tumor markers are always associated with malignancies.42,43 Despite these burgeoning developments, use of tumor markers as internal stimuli for the controlledrelease systems is still in its nascent stage. In addition to on-demand releasing, the ability to monitor drug release in real time is another important factor to regulate the spatiotemporal control over the drug delivery.44−48 Fluorescent models are widely used as entrapped guest in drug delivery to investigate the release process,45−47 which limits their potential use in monitoring actual drug molecules, as most current antineoplastic drugs are nonfluorescence. Therefore, it is necessary to develop a real-time monitoring system for investigating drug release in complex cellular microenvironments. Fluorescence resonance energy transfer (FRET) is a well-established energy transfer process between the energy donor and acceptor chromophores that is very
ince most of the commonly used antineoplastic drugs cannot discern between diseased and healthy cells and have serious side effects on healthy cells, an extensive amount of work has been done to develop site-specific stimuli-responsive controlled drug delivery systems. Among them, numerous organic,1−3 inorganic,4,5 and inorganic/organic hybrid6,7 materials have been used as drug delivery platform to transport anticancer drugs to a specific point in the body, thus mitigating harmful side effects of drug to healthy tissues. In this regard, mesoporous silica nanoparticles (MSNs) have attracted substantial attention as nanocarriers due to their unique porous structure, tunable pore size, large pore volume, high surface area, biocompatibility, and chemically modifiable surfaces.8−15 Diverse molecular valves that functionalize the pores of MSNs can work as gatekeepers and be removed by various external or internal stimuli, including light,16−19 pH,20−23 redox potential,24−28 temperature,29−31 and biomolecules.26,32−35 Despite the prominent potential that had been achieved in these drug delivery systems triggered by various external stimuli, low penetration in tissue still limits their performance.36 Most recently, using biomolecules as internal triggers to release entrapped guest in delivery systems is becoming a momentous field, as these stimuli always exhibit increased activity in cancer © XXXX American Chemical Society
Received: June 29, 2014 Accepted: September 17, 2014
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labeling on oligonucleotides for monitoring drug release in real time. Initially, FlareA hybridizes with part of S1F. The AMCA and FITC moieties labeling FlareA and S1F are in close proximity, creating a FRET pair. As FlareA just hybridized with one end of S1F, it still possesses the single-strand characteristic that can be flexibly adsorbed on the surface of APTS-modified MSNs with partially positively charged aminopropyl groups at neutral pH.33 The FRET-MSNs display a typical double emission peak at 450 and 520 nm due to partial transfer of energy from AMCA to FITC, when they are excited at the excitation wavelength of 353 nm. Nevertheless, in the presence of the target strand survivin mRNA, more stable hybridization between survivin mRNA and S1F will dissociate FlareA and cause the removal of the oligonucleotide cap from the MSNs, thereby triggering the unlocking and releasing process. Because of the dehybridization between S1F and FlareA, the FRET between AMCA and FITC is broken, and the system only displays the characteristic emission wavelength of AMCA at 450 nm. Due to the correlation between the fluorescence signal change and drug release, the release process can be monitored in real time. Moreover, the amount of drug released relies on the state of malignancies by utilizing the tumor marker survivin mRNA as an internal stimulus; the more survivin mRNA that is synthesized in the cancer cells, the greater the lethality induced by the anticancer drug to the cancerous cells.
sensitive to the changes in donor-to-acceptor separation distance in the range of 10−80 Å.49 This unique feature of FRET makes it a promising candidate for monitoring delicate pore unlocking and cargo release interactions in real time during the drug delivery process.50,51 Notably, despite these prominent achievements in delivery systems, most MSNs-based drug delivery carriers possess only one of the two features (ondemand release or real-time monitoring) and cannot function well under complex physiological conditions. In order to improve anticancer drug delivery performance, it would be advantageous to design a capped MSNs nanodevice that would have on-demand release and allow tracking and monitoring of the process of drug release. Such a combination, we believe, is extremely paramount, as it allows simultaneous imaging diagnosis and controllable drug delivery in one. To address the aforementioned issues, herein we designed a novel MSNs drug delivery system based on a targetrecognition-responsive FRET valve (henceforth referred to as FRET-MSNs). Survivin mRNA is used as the target and FRETpair-labeled nucleic acids are used as gates of the pores in MSNs. Survivin mRNA, which is responsible for survivin overexpression in all the most common human malignancies, is becoming a significant tumor marker.52,53 Therefore, survivin mRNA can work as a promising internal stimulus to permit drug release inside malignant cells more precisely and alleviate the undesired systemic toxicity. Thus, this system enables realtime monitoring of the target recognition controllable drug release occurring in cancer cells. As illustrated in Scheme 1, the
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EXPERIMENTAL SECTION Reagents, Materials, Apparatus, and Statistical Analysis. Refer to the Supporting Information. Preparations of MSNs, DOX-Loaded MSNs, FRETMSNs, and FRET-DOX-Loaded MSNs Complexes. The MSNs were synthesized in a typical synthesis procedure.41 Briefly, N-cetyltrimethylammonium bromide (CTAB, 1.00 g, 2.74 × 10−3 mol) was first dissolved in 480 mL of deionized water, and then NaOH (aq) (2.00 M, 3.50 mL) was added to CTAB solution under vigorous stirring, followed by adjusting the solution temperature to 95 °C. After the solution became clear, 5 mL of tetraethyl orthosilicate was added dropwise to the surfactant solution; hence, the mixture was allowed to stir for 3 h to give rise to white precipitates, and then the synthesized materials were centrifuged and washed thoroughly with deionized water and ethanol. To remove the surfactant template, the final products were calcined at 550 °C for 5 h after being dried for 12 h at 60 °C in a vacuum. In the loading process, calcined MSNs (0.1 g) were dispersed in ethanol (20 mL). Then the drug doxorubicin hydrochloride (DOX) (0.2 mM) was added and the mixture was stirred for 24 h at 36 °C with the aim of achieving maximum loading in the pores of the MSNs scaffolding. After this procedure, an excess of APTS (0.374 mL) was added and the mixture stirred for 5.5 h. The nanoparticles were then centrifuged (8000 rpm, 10 min), washed six times with methanol, and dried under vacuum to obtain the intermediate product MSNs-NH2. The capping process by oligonucleotide is done according to the method of Climent et al.33 Briefly, the oligonucleotide FlareA hybridizes with S1F in equal concentration to form FRET-pair-labeled nucleic acids for the capping process. Then 600 μg of intermediate MSNs-NH2 was suspended in 600 μL of hybridization buffer (20 mM Tris-HCl, 37.5 mM MgCl2 at pH 7.5) containing the nucleic acids in concentrations of 3 × 10−5 M and each suspension was stirred at 37 °C, respectively. After 1 h, the mixture was centrifuged at 8000 rpm for 10 min, and
Scheme 1. Schematic Representation of Target-RecognitionResponsive FRET-MSNs Release System for Cancer Therapy
real-time monitoring of the drug controllable release platform is comprised of four components: (i) 3-aminopropyltriethoxysilane (APTS) functionalized MSNs as the drug carriers, (ii) 7amino-4-methlcoumarin (AMCA) dye terminated short oligonucleotide (FlareA) hybridized with a part of antisense oligonucleotide [fluorescein isothiocyanate (FITC) labeled, S1F] as cap to entrap the drugs within the MSNs, (iii) survivin mRNA hybridized with S1F as the target-recognition-responsive trigger to release the entrapped drug molecules on demand, and (iv) the FRET donor−acceptor pair of AMCA and FITC B
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Target-Recognition-Responsive Behavior and Drug Release in Vitro. The AMCA moiety in the FRET pair can be excited at an excitation wavelength of 353 nm, resulting in an emission wavelength in the range of 410−490 nm. When the FRET pair is intact, they displayed dual emission peaks at 450 and 520 nm upon excitation at 353 nm (Figure 2A), as the
then the pellet (FRET-DOX-loaded MSNs) was collected carefully, dispersed in hybridization buffer, and stored at 4 °C for further use. Besides, the complexes FRET-MSNs or DOXloaded MSNs were formed by the above procedure just without the loading process or capping process, respectively. Cell Culture and Preparation. Acute myeloblastic leukemia (HL-60) cells and normal liver (LO2) cells were cultured in DMEM supplemented with 10% FBS and 100 IU mL−1 penicillin−streptomycin. The cells were maintained at 37 °C in a humidified atmosphere (95% air and 5% CO2). In the delivery experiment, HL-60 cells and LO2 cells were seeded in 24-well plates. After 24 h of plating, culture media were exchanged with fresh serum-free basal media (500 μL) containing MSNs, FRET-MSNs, DOX-loaded MSNs, or FRET-DOX-loaded MSNs complexes, respectively. After 6 h, media were exchanged with fresh culture media. Fluorescence measurements and fluorescence confocal microscopy imaging were performed over a period of time (0−24 h) after transfection.
Figure 2. (A) Emission spectra of FRET-MSNs. (B) Fluorescence intensity change in blue (450 nm) and green (520 nm) emission wavelengths upon addition of different S2 concentrations to FRETMSNs system: (a) 0 M, (b) 1 × 10−7 M, (c) 1 × 10−6 M, (d) 5 × 10−6 M, and (e) 1 × 10−5 M. Excitation wavelength was 353 nm.
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RESULTS AND DISCUSSION Characterization of FRET-MSNs. As shown in transmission electron microscopy (TEM) images (Figure 1A), the
AMCA moiety can act as a photon donor for the FITC, which can be excited maximally at 490 nm. The target-recognitionresponsive property of the FRET-MSNs was investigated by observing the changes in donor and acceptor fluorescence signal through addition of synthesized survivin mRNA (S2), which mimicked the intracellular environment. As shown in Figure 2B, increasing the concentrations of S2 [(0−1) × 10−5 M] in the buffered solution of FRET-MSNs led to a decrease in the green fluorescence (520 nm) and an increase in the blue fluorescence (450 nm). This strongly indicated that, in the presence of a target strand, the target strand can displace FlareA to hybridize with S1F, leading to the removal of the FRET acceptor, FITC. These results demonstrated that the FRETpair-labeled nucleic acids can be adsorbed onto the surface of the MSNs through electrostatic attraction and the targetrecognition reaction of the FRET-MSNs can result in a concomitant fluorescence signal change. To monitor the drug release from the pores in vitro, the drug doxorubicin hydrochloride (DOX) was chosen as model cargo, which was loaded into the pores of MSNs by mixing aqueous buffered solutions of MSNs and DOX for 24 h. The results indicated that the encapsulation efficiency (EE) of 80% could be achieved (Figure S-3, Supporting Information). The delivery profile of cargo in the presence and absence of the target oligonucleotide was monitored through UV−vis measurement of the released model cargo. FRET-DOX-loaded MSNs were dispersed in hybridization buffer (20 mM Tris-HCl, 37.5 mM MgCl2 at pH 7.5), and the released DOX in the absence of target oligonucleotide S2 was first monitored. As can be seen in Figure 3A (curve a), a less than 4% leakage of cargo over a period of 24 h indicated that the FRET-MSNs system had a stable capping capability in the absence of target. The presence of the target strand S2 would induce the hybridization between S2 and S1F, which could open the pores and release the cargo. When a low concentration of target strand S2 was added, the release percent sharply increased to 16.8% at 24 h (curve b). Furthermore, a higher concentration of target would lead to a more significant release, and almost 70% entrapped drug could be released with a target concentration up to 10−5 M (curve e). Meanwhile, the release percent was also changing with time, due to more target strand hybridizing with S1F and inducing
Figure 1. (A) TEM image of MSNs and HRTEM images of MSNs (inset a) and FRET-DOX-loaded MSNs (inset b). (B) Low-angle XRD pattern of MSNs, and the insets are BET and BJH assays of MSNs.
synthesized MSNs had an average diameter of ∼100 nm. The highly ordered lattice array demonstrated the ordered, welldefined mesoporous structure of the obtained MSNs, which was further substantiated by X-ray diffraction (XRD) and N2 adsorption isotherms tests (Figure 1B). The N2 adsorption− desorption isotherm demonstrated that the obtained MSNs have a Burnauer−Emmett−Teller (BET) surface area of 639 m2 g−1 and a narrow Barrett−Joyner−Halenda (BJH) pore-size distribution (average pore diameter = 2.3 nm, Figure 1B, inset). The surface of MSNs was characterized by FT-IR (Figure S-1A, Supporting Information). To obtain MSNs-NH2, the surface of MSNs was functionalized with APTS. The FTIR spectrum of MSNs-NH2 showed a primary amine characteristic absorption band at 1560 cm−1. The process was also characterized by ζpotential analysis and dynamic light scattering (DLS) characterization (Figure S-1B−D, Supporting Information). The obtained MSNs-NH2 nanocomplex showed a positive ζpotential. When FRET-pair-labeled nucleic acids adsorbed on the nanocomplex MSNs-NH2, the ζ-potential fell to 7.8 mV as the adsorped nucleic acids contained more negative phosphate groups. Meanwhile, the diameter of FRET-MSNs significantly increased from 110 ± 6 to 120 ± 5 nm, which indicated a successful capping process as well. Additionally, the length of anti-RNA oligonucleotide (S1F) was optimized to find the most suitable nucleic acids cap of MSNs (Figure S-2, Supporting Information). C
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Figure 3. (A) Release of DOX from the FRET-DOX-loaded MSNs following treatment with different concentrations of oligonucleotide S2: (a) 0 M, (b) 1 × 10−7 M, (c) 1 × 10−6 M, (d) 5 × 10−6 M, (e) 1 × 10−5 M. (B) DOX released from the FRET-DOX-loaded MSNs system in the presence of oligonucleotides S2, S3, and S4 at concentrations of 5 × 10−6 M.
Figure 4. Confocal fluorescence images of HL-60 cells after incubation with FRET-MSNs nanocarriers for 6 h at 37 °C (A) and 4 °C (B). Images from left to right: channel 1 represents cell stained by Hoechst 33342, and channel 2 represents the fluorescence intensity of the FRET pair in HL-60 cells treated with FRET-MSNs, with bright-field and merged images also shown.
greater drug release. The UV/vis absorbance spectra of the released drug are specifically shown in Figure S-4 (Supporting Information) after the nanocarriers interacted with target strand for 24 h. The release profiles of cargo in the presence of target strand depicted an increase in the percentage of DOX released and the percent was proportionally dependent on the target concentration. For the purpose of investigating the selectivity in the target recognition responsive opening protocol, model cargo released from FRET-MSNs was tested in the presence of other oligonucleotides (S3, a single-base mismatch sequence to S2, and S4, a two-base mismatch sequence to S2). As shown in Figure 3B, less than 15% drug release was observed with S3 or S4, indicating that the nontarget strands induced a poor uncapping and releasing process. The results revealed that only the presence of S2 induced a remarkable release of entrapped cargo, which strongly suggested that the FRET-MSNs delivery system has high selectivity in the opening protocol (Figure 3B). Observing on-Demand Drug Release in Cancer Cells Using FRET-MSNs. Before observing on-demand drug release in cancer cells, we investigated the intracellular uptake pathway and the intracellular localization of FRET-MSNs nanocarriers in mammalian cells. Acute myeloblastic leukemia (HL-60) cells were incubated with FRET-MSNs nanocarriers at 4 and 37 °C. After the transfection process, cells were stained with the bisbenzimidazole dye Hoechst 33342 at 37 °C for 10 min. Then these cells were washed with PBS solution and imaged by confocal fluorescence microscopy immediately. As Hoechst 33342 binds to the minor groove of double-stranded DNA, this dye stained nuclei blue54 to highlight the cell nucleus region (channel 1, Figure 4). A large amount of sky-blue spots emitted from the FRET pairs in HL-60 cells at 37 °C was observed by confocal fluorescence microscopy when nanocarriers just entered in the cells (channel 2, Figure 4A), confirming that vast FRET-MSNs systems could be ingested by the cells. Meanwhile, the sky-blue spot revealed that the FRET pair capped on MSNs, which own both blue and green dual emission, was intact. Additionally, the merged image (Figure 4A) and the z-stack images (Figure S-5 and Video S-1, Supporting Information) also revealed that these nanocarriers were internalized by cells and mostly escaped from endosome/ lysosome into the cytoplasm. Compared with the cells incubated at 37 °C, almost no fluorescence of the FRETMSNs system could be observed in the cells incubated at 4 °C (Figure 4B), suggesting that few nanocarriers could be internalized by cells at this temperature. The mean fluorescence intensity of internalized FRET-MSNs systems at 37 °C was
almost 40 times that at 4 °C. All the results demonstrated the efficient uptake of nanocarriers at 37 °C and that the uptake mechanism of FRET-MSNs systems is an energy-dependent endocytosis.55 As survivin mRNA is overexpressed in many malignancies while tightly controlled in terminally differentiated adult tissues, normal liver (LO2) cells were chosen as control cells for further confirming the capacity of on-demand drug release in the FRET-MSNs system. To investigate whether FRET-DOXloaded MSNs nanocarriers could serve as target-responsive delivery platform with real-time monitoring of drug release, the fluorescence intensity change was observed in HL-60 cells or LO2 cells after being incubated with these carriers for a period of time (0−24 h). As shown in Figure 5A (panel a), at time t =
Figure 5. (A) Laser confocal microscopy measurement of the fluorescence signal of the FRET pair in HL-60 (a) and LO2 (b) cells cultured with FRET-DOX-loaded MSNs at different times. (B) The magnification of HL-60 (a) and LO2 (b) cells after treatment with FRET-DOX-loaded MSNs for 24 h. Images from left to right: channel 1 represents the fluorescence intensity of the FRET pair, channel 2 represents the fluorescence of the drug DOX, and channel 3 represents cell stained by Hoechst 33342, with bright-field and merged images shown. The magnifying power is 40×. D
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0 h, vast sky-blue spots were visible in the cytoplasm region, indicating that most FRET pairs were still intact and have dual emission fluorescence. Meanwhile, as can be seen in Figure S-6 (Supporting Information), the high red fluorescence of the drug DOX was colocalization with these sky-blue spots when the delivery systems just entered in the cells, which indicating that FRET-DOX-loaded MSNs nanocarriers were still in their locked stage and the entrapped drug still gathered in the nanaocarriers. When incubated with FRET-DOX-loaded MSNs for a period of time, a significant increase in the blue fluorescence intensity and a corresponding decrease in the green fluorescence intensity were observed in these target cells. At approximately 24 h, negligible green fluorescence with extremely bright blue fluorescence could be observed by microscopy. This was consistent with the results of in vitro detection (Figure 2B), as the target-responsive reaction would lead to the dissociation of the FRET pair, thereby inducing the recovery of the blue fluorescence intensity. As expected, the entrapped drug released concurrent with the fluorescence signal change. As DOX could be inserted into the double-helix of DNA to damage DNA, higher and higher red fluorescence intensity of released DOX in the nuclei could be detected in these target cells with prolonged incubation time (Figure S-6A, panel a, marked with a circle, Supporting Information). In the magnified image, we can clearly observe that vast purple spots, which were the merger of the released DOX fluorescence with the nuclei dye Hoechst 33342 fluorescence accumulated in the nuclei, and a mass of apoptotic bodies appeared in cells after incubating with the drug delivery system for 24 h (Figure 5B, panel a). Additionally, little fluorescent signals emitted from FRET-MSNs demonstrated a colocalization with the red fluorescence from Dox (formed white spots), indicating that most drug was released and not entrapped in these nanocarriers. Nevertheless, the control cells treated with the same process produced different final results. Negligible fluorescence change in the FRET pairs coupled with no fluorescence of released drug with prolonged time could be observed in control cells (Figures 5A and S-6, panel b, Supporting Information). Meanwhile, the magnified image distinctly presented that all the drug was still entrapped in the nanocarriers and presented a high colocalization with the delivery system in the cytoplasm region of control cells (merge channel, panel b, Figure 5B). Because the drug release happened only upon the removal of the FRET-pair-labeled cap based on target recognition, the correlation between the fluorescence signal change (R = F520/ F450) and the mean fluorescence of released drug was tested. In HL-60 cells, the mean fluorescence intensity of released drug in nucleus gradually increased with prolonged incubation time, and the intensity at 24 h was 20 times that at 0 h, indicating that more and more drug was released from the nanocarriers and gathered in the cell nucleus (Figure 6, inset a). Simultaneously, a gradual increase in the blue emission intensity (F450) and a corresponding decrease (F520) in the green emission intensity led the fluorescence signal change R to gradually reduce to zero during the releasing process. In contrast, in the control cells, negligible DOX release was accompanied by a slight R change. All this revealed that greater drug release coincided with a lower R (Figure 6, black curve). This outcome indicated that the FRET-MSNs system can not only monitor the release of fluorescent drug but also of nonfluorescent cargo based on the R change. These results demonstrated that this drug release system has the capability of
Figure 6. Correlation between the mean fluorescence intensity of released DOX and fluorescence signal change R of the FRET pair at different time points for HL-60 cells and LO2 cells (inset b) incubated with FRET-DOX-loaded MSNs nanocarriers. Inset a is the detailed mean fluorescence of released DOX at different time points. Thirty cells were used to obtain the mean fluorescence intensity of FRET pairs and DOX.
releasing drug on-demand and monitoring the procedure in real time. Cell viability was observed after both cell lines (HL-60 and LO2 cells) were cultured with different concentrations of MSNs, DOX-loaded MSNs, and FRET-DOX-loaded MSNs composites for 24 h (Figure 7). Without the specific nucleic
Figure 7. Cytotoxicity assays of HL-60 cells (a) and LO2 cells (b) incubated with MSNs, DOX-loaded MSNs, and FRET-DOX-loaded MSNs.
acids cap, a large amount of DOX was released in both target and control cells (Figure S-7, Supporting Information) and there was no significant difference (P > 0.05) in cell viability of HL-60 and LO2 cells (Figure 7b). While incubated with FRETDOX-loaded MSNs composites, highly significant differences in cell viability were shown between target and control cells (P < 0.001). FRET-DOX-loaded MSNs composites showed significant lethality to just HL-60 cells (cell viability = 28.6 ± 4%, mean ± SD) compared with the control cells (cell viability >72%), revealing that the current smart nanodevices yielded slight side effects for normal cells and the specificity of tumor marker stimuli for controllable drug release. With the aim of observing the on-demand release capability of the FRET-MSNs delivery cargo based on different states of malignancies, antisense oligonucleotides (S1) and synthetic survivin mRNA mimics (S2) were transfected into HL-60 cells by lipofectamine 3000 according to the manufacturer’s E
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instructions for up- and down- regulation of the concentration of the internal stimulus survivin mRNA.52 As shown in Figures 8 and S-8 (Supporting Information), less drug release coupled
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ASSOCIATED CONTENT
S Supporting Information *
A summary of reagents and materials, apparatus, and statistical analysis; the experiment process for the cell viability assay; characterization of MSN, MSN-NH2, and FRET-MSNs; optimization of the FRET-MSNs system; encapsulation efficiency calculation; location analysis of drug delivery systems; laser confocal microscopy measurement of cells treated with different nanocarriers; and Video S-1. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +86-25-83597294. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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Figure 8. Laser confocal microscopy measurement of HL-60 cells treated with transfection of antisense oligonucleotides (S1) or synthetic survivin mRNA mimics (S2), for conditions a−d, transfected S1 (5 × 10−7 M), S1 (0 M), S2 (5 × 10−7 M), and S2 (1 × 10−6 M), respectively, and then incubating these cells with FRET-DOX-loaded MSNs for 24 h. FRET field represents the fluorescence signal of the FRET pair in HL-60 cells, and the DOX field represents the merged image of the drug fluorescence field, Hoechst 33342 fluorescence field, and bright field of HL-60 cells.
ACKNOWLEDGMENTS This work was supported by the 973 Program (Grant 2012CB932600, 2013CB933800), the National Natural Science Foundation (Grants 21327902, 21025522), and the National Natural Science Funds for Creative Research Groups (Grant 21121091).
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with slight fluorescence signal change (R) in condition a (transfected S1) was observed by microscopy compared to condition b (without transfection), indicating that less targetrecognition reaction induced less drug release, as antisense oligonucletides transfected into cells would reduce the concentration of internal stimulus.52 On the contrary, a vast amount of DOX was released and accumulated in the nuclei coupled with a significant R signal change in conditions c and d (transfected S2), indicating that a higher concentration of internal stimulus hybridized with the cap system and induced more release of entrapped cargo. The mean fluorescence intensity of released drug in condition d was nearly 2.5 times that in condition a, revealing that the delivery system can achieve the on-demand drug release. All these results demonstrated that the smart drug delivery system can control drug release based on each cell specific state and monitor the controllable release process in real time, which can maximize the desired therapeutical effects.
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CONCLUSIONS In this study, we present a smart drug delivery system based on integration of FRET-based real-time monitoring system and on-demand drug release in mesoporous silica nanoparticles. Compared to conventional drug delivery systems, this new nanocarrier possesses greater potential in anticancer therapy. The system has an equally broad spectrum in monitoring the cargo release, even if the drug is nonfluorescent, making it a promising candidate for investigating the relevance between drug amount and therapeutic efficacy. The significant tumor marker survivin mRNA as internal stimulus endows the drug release with more selectivity and accuracy in cancer cells, which can greatly minimize side effects while maximizing desired effects. With other tumor markers coupled with corresponding FRET systems, such a platform could be extended and serve as a general basis to develop site-specific drug delivery in cancer therapy. F
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