Dual Drug Delivery System Based on Biodegradable Organosilica

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Dual Drug Delivery System Based on Biodegradable Organosilica Core-Shell Architectures Jiang-Lan Li, Ying-Jia Cheng, Chi Zhang, Han Cheng, Jun Feng, Ren-Xi Zhuo, Xuan Zeng, and Xian-Zheng Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17949 • Publication Date (Web): 19 Jan 2018 Downloaded from http://pubs.acs.org on January 19, 2018

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ACS Applied Materials & Interfaces

Dual Drug Delivery System Based on Biodegradable Organosilica Core-Shell Architectures

Jiang-Lan Li, Ying-Jia Cheng, Chi Zhang, Han Cheng, Jun Feng, Ren-Xi Zhuo, Xuan Zeng* and Xian-Zheng Zhang*

Key Laboratory of Biomedical Polymers of Ministry of Education, Institute for Advanced Studies (IAS), Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China

*

Corresponding author. Tel.: + 86 27 6875 5993; Fax: + 86 27 6875 4509

E-mail addresses: [email protected] (X. Zeng), [email protected] (X.Z. Zhang)

1

ACS Paragon Plus Environment

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ABSTRACT: To overcome drug resistance, efficient cancer therapeutic strategies using combination of small-molecule drugs and macromolecule drugs are highly desired. However, due to their significant differences in molecular weight and size, it is difficult to load them simultaneously in one vector and to release them individually. Here, a biodegradable organosilica based core/shell-structured nanocapsule was designed and used as a dual stimuli-responsive drug vector to solve this problem. Biodegradable organosilica shell coated outside the macromolecule model drug “core” would be disrupted by high glutathione (GSH) levels inside tumor cells, resulting in the escape of the entrapped drugs. Small-molecule drugs capping on the surface of organosilica shell via pH-responsive imine bonds can be cut and released in the acidic lysosomal environment. Transmission electron microscopy has shown that the framework of the organosilica shell was dissolved and degraded after 8 h incubation with 5 mM GSH. Confocal imaging confirmed that small-molecule and macromolecular drugs were individually released from the nanoparticles due to the pH or redox triggered degradation under the tumor microenvironment, and thus led to the strong fluorescence recovery in the cytoplasm. As expected, these biodegradable organosilica nanoparticles could not release drugs into normal cells, but specifically release them into tumor cells, owning to their tumor-triggered targeting capability. This system will serve as an efficient shuttle for multi-drug delivery, and also provide a potential strategy to overcome drug resistance. KEYWORDS: cancer therapy, biodegradable organosilica, multi-drug delivery, stimuli-responsive, tumor-triggered targeting 2


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INTRODUCTION Using drug delivery system is one of the most important approaches to improve the cure effect in current cancer treatment.1-3 Significantly different from traditional chemotherapy, drug delivery systems offer many advantages, such as improved therapeutic effects, less drug dose, lower toxicity and minimized side effects.4-8 Stimuli-responsive

controlled-release

systems, especially the tumor-triggered

targeting (TTT) systems, can effectively and accurately trigger the drug release at the tumor sites.9-15 Although great progress has been made over the past decade to further develop the innovative capabilities of drug carriers, there is still a lack of efficient vector to overcome the big challenge of multi-drug resistance (MDR).16 As P-glycoprotein will be overexpressed in cancer cells after single-agent chemotherapy and will lead to MDR17, the “cocktail therapy” plan (combined drug treatment) has been chosen to avoid such defenses of the cancer cell and achieve synergistic anti-tumor effects.18 Considerable efforts have been made in this field

19-21

, but

co-delivery of small-molecule drug (chemotherapeutic agents, like DOX) and macromolecular drug (like gene, antibody) has not been solved in a satisfactory way. Therefore, well-organized multi-drug carriers with individual release behavior of each drug are highly desired. Silica-based nanoparticles with modifiable interface, controllable mesoporous structure, varied functionality and high drug loading have been widely used as the promising and versatile platform for cancer therapy and diognosis.22-30 Despite that these silica carriers have been applied in animal models, systematic bio-safety and 3



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further applications would be hampered by their metabolic defect.31 Most of them are resistant to degradation in vivo and are retained for a relatively long-term (more than 3 months) due to high-density silicate frameworks. The silica nanoparticles are aggregated in liver, kidney, lungs, spleen and bladder, and cause body damage.32 Therefore, various studies have focused on the this issue, and degradable hybrid organosilica materials have been developed for this purpose.33 For example, Quesada and co-workers34 synthesized the redox-responsive periodic mesoporous organosilica (PMO) nanoparticles with PLGA cores, which could be degraded in 100 mM dithiotreitol environments. Croissant et al.35 also synthesized disulfide based PMO carriers, which could be degraded in 6 µM mercaptoethanol. Based on these studies, GSH concentration of cancer cells, which is significantly higher than normal cells, can lead to rapid degradation of organosilica in cancer cells and tumor specific drug release. Those degradable silica nanovectors would be more convenient and feasible in clinical applications. We

previously

microcapsules

for

developed cancer

dual

cocktail

stimuli-responsive therapy.20

This

multi-drug platform

delivery

could

load

bio-macromolecular drug and small-molecule drug individually, and showed dual stimuli-response (MMP or UV irradiation) and separately controllable release abilities. However, compared with external stimuli, internal stimuli from the tumor microenvironment would be more accurate and would be considered as tumor-triggered targeting switches. In this study, a versatile small-/macromolecule dual drug delivery system based on biodegradable organosilica core-shell architecture 4


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was designed and synthesized as a more efficient and convenient strategy. In order to investigate the encapsulation capability and release behavior of the dual drug delivery system, rhodamine-modified-chitosan (CS-RhB) was chosen as the macromolecular model drug, and aminated fluorescein was used as the small-molecule model drug. Cervical cancer cells (HeLa) and normal cells (COS7) were used in this study to confirm the tumor specificity of the nanoparticles. Biodegradable organosilica shell was coated outside the macromolecular model drug “core”. As illustrated in Scheme 1, once the nanocapsule was internalized, small-molecule drugs capping on the surface of organosilica shell with pH-responsive imine bonds would be cut and released in acidic lysosomal environment. After that, the degradation of the shell would be triggered by high levels of GSH in tumor cells, resulting in the release of the entrapped macromolecule model drugs.

5



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Scheme 1. (A) Schematic illustration of the preparation of dual drug-loaded biodegradable organosilica nanoparticles; (B) Schematic illustration of the acid-triggered release of small-molecule model drug, the GSH-triggered degradation and release of macromolecular model drug. MATERIALS AND METHODS Materials. Triethoxysilylbutyraldehyde (TESBA), bis[3-(triethoxysilyl)propyl] disulfide (BTESPDS), 1,2-bis(triethoxysilyl)ethylene (BTESE) were purchased from Gelest Co. and used without purification. Chitosan (CS, Mw=4×105 Da), rhodamine B (RhB) were provided by Sigma-Aldrich Co. and used as recived. Fluorescein, sodium tripolyphosphate (TPP), N-cetyltrimethylammonium bromide (CTAB), 6


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dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), sodium hydroxide (NaOH), sodium chloride (NaCl), methanol solution (MeOH), hydrochloric acid (HCl), hydrofluoric acid (HF), acetic acid (AcOH), sodium acetate (AcONa), sodium dihydrogen

phosphate

dihydrate

(NaH2PO4•2H2O),

disodium

phosphate

dodecahydrate (Na2HPO4•12H2O), triethylamine (TEA), petroleum ether (PE), ethyl acetate (EA), methanol, ethanol were obtained from Shanghai Reagent Chemical Co. and used directly. N,N-Dimethylformamide (DMF) and 1,6-hexanediamine was received from Shanghai Reagent Chemical Co. and distilled before use. Equipment. The Fourier transform infrared spectrum (FTIR) was measured by a Perkin-Elmer spectrophotometer (USA) using potassium bromide (KBr) pellets. Nitrogen adsorption-desorption isotherms were performed with a micromeritics instrument (ASAP2020). The surface areas and pore sizes distribution of materials were tested by Brunauer Emmett Teller (BET) and Barett Joyner Halenda (BJH) measurements. Small angle X-ray diffraction (SAXRD) analysis was carried out on an X' Pert Pro diffractometer (PAN alytical). The graph of high performance liquid chromatography (HPLC) was detected by liquid chromatography (LC-6AD, Shimadazu). The fluorescence results were provided by fluorescence spectrometer (LS-55, Perkin Elmer). The hydrogen nuclear magnetic resonance (1H-NMR) spectra were recorded by Mercury Vx-300. The ζ-potentials and hydrodynamic sizes were measured on a Malvern Zetasizer Nano-ZS ZEN3600 (UK). The morphologies of nanoparticles were observed by transmission electron microscopy (TEM, JEM-2100 microscope). 7



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In Vitro Cell Culture. Cervical cancer cells (HeLa) and African green monkey SV40-transformed kidney fibroblast cells (COS7) were purchased from Cell Bank of Chinese

Academy

of

Sciences

(Shanghai,

China).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were provided by Invitrogen. Synthesis and Characterization of CS-RhB. Briefly, 0.479 g RhB, 0.247 g DCC and 0.138 g NHS were dissolved in 10mL anhydrous N,N-dimethylformamide (DMF), and stirred for 24 h in dark. The resulting cloudy solution was filtered. Then 0.805 g chitosan (CS) was dissolved in 80 mL of deionized water with HCl (0.5 mL). The filtered solution was added dropwise to the CS solution, and then stirred at 50 °C in the dark for 48 h. Excess NaOH solution was added, and the CS-RhB was precipitated. The CS-RhB was collected by centrifugation, purified by dialysis against deionized water (MWCO 14,000 Da) for 48 h and then lyophilized. The grafting degree was measured by 1H-NMR using CDCl3 as solvent. Synthesis and Characterization of Fluorescein-hexanediamine (FL-C6-NH2). The synthesis process of FL-C6-NH2 was similar to that of CS-RhB. Fluorescein (1.66 g), DCC (1.24 g), and NHS (0.69 g) were dissolved in anhydrous DMF (20 mL). The mixture was stirred for 24 h in dark. The cloudy solution was filtered and evaporated under vacuum. The resulting solid (FL-NHS) was purified by column chromatograph, and the structure was identified by 1H-NMR. 1,6-hexanediamine (0.3 g) was dissolved in anhydrous DMF (7 mL) followed by addition of TEA (0.5 mL) and FL-NHS (0.85 g, dissolved in 10 mL of anhydrous DMF). The mixed solution was stirred for 24 h at 8


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room temperature, and further evaporated in vacuum. The resulting FL-C6-NH2 was purified by column chromatograph. The purity of FL-C6-NH2 was confirmed by HPLC, with C18 reversed phase column and UV-vis detector at 220 nm, from methanol at 3.0 mL / min for 20 min. Synthesis and Characterization of CS-RhB/TPP (CRP) Nanoparticles (CRPN). The CRPN with spherical shape were self-assembled by electrostatic interactions between negatively charged TPP and polycationic CS-RhB. In brief, CS-RhB (300 mg) and TPP (60 mg) were dissolved in 1000 mL of deionized water, and the pH value of the solution was adjusted to 5.5. The solution was stirred at 50 °C in the dark for 1 h. Synthesis and Characterization of CRP@Biodegradable Organosilica Nanoparticles (CRP@dOSN). CTAB (1.13 g) was added to the solution of CRPN and stirred for 1 h. Then 0.5 mL of BTESPDS and 0.5 mL of BTESE were added, and reacted at 50 °C in the dark for 10 h. After that, 0.2 mL of TESBA was added to the reaction mixture and further reacted for 2 h. The resulting CRP@dOSN was centrifugated and washed with ABS (pH=5.0, 10mM) three times. Synthesis and Characterization of FL-C6-NH2 Modified CRP@dOSN (CRP@dOSN-FL). The FL-C6-NH2 was covalent bonded to the CRP@dOSN by imine bond. The CRP@dOSN nanoparticles (50 mg) were dispersed in 100 mL of deionized water. Then, 20 mL of ethanol with 10 mg FL-C6-NH2 and 1 mL of TEA was added. The reaction mixture was stirred for 24 h at room temperature. CRP@dOSN-FL with CTAB was collected by centrifugation, and washed with water 9



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three times. Then, the nanoparticles were dispersed in 40 mL of NaCl / MeOH (1% wt). The mixture was stirred for 6 h and centrifugated. This process was repeated three times to remove CTAB. The final product CRP@dOSN-FL was washed with water, collected by centrifugation, and lyophilized. Synthesis and Characterization of CRP@Nondegradable Organosilica Nanoparticles

(CRP@ndOSN)

and

FL-C6-NH2

Modified

CRP@ndOSN

(CRP@ndOSN-FL). The synthesis and characterization methods of CRP@ndOSN and CRP@ndOSN-FL are similar to those of CRP@dOSN and CRP@dOSN-FL, except using 1mL BTESE instead of 0.5 mL of BTESPDS and 0.5 mL of BTESE. The resulting products were named as CRP@ndOSN, CRP@ndOSN-FL with CTAB, CRP@ndOSN-FL, respectively. Acid-Triggered Release Assay. The acid-responsive release experiment of fluorescein was carried out in different buffer solution PBS (pH=7.4, 10 mM), and in ABS (pH=5.0, 10 mM). For example, 1 mg CRP@dOSN-FL was dispersed in media at a concentration of 1 mg/mL. The above solution was encapsuled in a dialysis bag (MWCO 14000 Da) and then drowned in 10 mL media (same to the media inside the bag), shaking at 37 °C (120 rpm). At predetermined time intermission, all the released solution was replaced with an equal volume of fresh media, and the performance of fluorescein release was measured at 540 nm by spectrofluorophotometer with the excitation wavelength of 488 nm. 1 mg CRP@dOSN-FL was dissolved completely in 0.2 mL HF, and diluted by 5 mL PBS. The pH of the reaction mixture was adjusted to 7.4 by adding NaOH solution. The content of fluorescent was measured at 540 nm by 10


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spectrofluorophotometer. Three independent experiments were performed to ensure accuracy, and the mean values were used as results. The total fluorescein release was calculated as follow: Total fluorescein release (%) = (Mt / M∞) × 100, where Mt is the amount of fluorescein released from the CRP@dOSN-FL and M∞ is the amount of fluorescein in the CRP@dOSN-FL. GSH-triggered Release Assay. The GSH-responsive release experiment of CS-RhB was carried out in PBS (pH=5.0, 10 mM) with or without GSH (5mM). For example, 1 mg CRP@dOSN-FL was dispersed in two different media (3 mL) at a concentration of 0.33 mg/mL, and then incubated with shaking (120 rpm) at 37 °C. At predetermined time interval, the solution was centrifugated. The fluorescence intensities of the supernatant were measured at 580 nm by spectrofluorophotometer with the excitation wavelength of 543 nm. Then the solution was added back and redispersed. 1 mg CRP@dOSN-FL was dissolved completely in 0.2 mL HF, and diluted by 5 mL ABS. Then the pH of the solution was adjusted to 5.0 by adding NaOH solution, and the content of CS-RhB was measured at 580 nm by spectrofluorophotometer. Three independent experiments were performed to ensure accuracy, and the mean values were used as results. The total CS-RhB release was calculated as follow: Total CS-RhB release (%) = (Mt / M∞) × 100, where Mt is the amount of CS-RhB in the supernate (released), and M∞ is the amount of CS-RhB in the CRP@dOSN-FL. The changing of morphology of the degradable process was observed by TEM. The CRP@ndOSN-FL was treated in the same condition and observed by TEM. 11



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In Vitro Cytotoxicity. The MTT assay was applied to evaluate the in vitro cytotoxicity of CRP@dOSN-FL. Briefly, HeLa cells and COS7 cells were seeded in 96-well plates (8 × 103 cells/well), and incubated in 100 µL DMEM containing 10% FBS and 1% penicillin/streptomycin for 24 h. Then, CRP@dOSN-FL/DMEM solution with different concentration was added to the plate and incubated for a further 48 h. 20 µL MTT solution (5 mg/mL) was added to each well and incubated for another 4 h. Afterward, the medium was removed and replaced with 150 µL DMSO. The optical density (OD) values of alive cell in the plate were provided from a microplate reader model 550 (BIO-RAD, USA) at 570 nm. The final data was determined by the mean value of eight replicates for each sample. The cytotoxicity was computed as follows: Cell Viability (%) = (ODsample / ODcontrol) × 100. ODcontrol is the value of the media without CRP@dOSN-FL, and ODsample is the value of the media with CRP@dOSN-FL. Intracellular Uptake. In brief, COS7 and HeLa cells were seeded in a glass-bottom dish at a density of 1 × 105 cells per well for 24 h. Then, cells were incubated with CRP@dOSN-FL in the cell culture media for another 4 h. Then the medium was removed, and cells were washed with PBS three times. They were further incubated in fresh DMEM medium without CRP@dOSN-FL for predetermined time, and washed with PBS three times. Cell nucleus were dyed by blue molecular probe (Hoechst 33258) for 15 min. Cells were washed with PBS three times, and isopyknic fresh DMEM solution was added. Then all cells were observed by confocal laser scanning microscopy (CLSM, Nikon C1-si TE2000, BD Laser). 12


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In Vitro Flow Cytometry. HeLa and COS 7 cells were seeded in 6-well dishes (5 × 104 cells per well), and cultured in the DMEM containing 10% FBS for 24 h under a humidified 5 % CO2 atmosphere, respectively. Then, the medium was replaced by DMEM containing CRP@dOSN-FL nanoparticles and incubated for 4 h. After that, the medium was removed and cleaned with PBS three times. Fresh DMEM was added to each well and cells were further incubated for different times. The cells were digested by trypsin and harvested by centrifugation, and then studied on a flow cytometer (BD FACSAria TM III, USA). HeLa cells and COS7 cells without any treatment served as the blank control, named as PBS group. Statistics Analysis. Statistical significance was analyzed by a three-sample Student's text. Statistical significance was inferred at a value of p < 0.05.

RESULTS AND DISCUSSION

Preparation and Characterization of CRP@dOSN-FL. In this study, our aim is to provide an efficient small-/macromolecule dual drug delivery system based on biodegradable organosilica core-shell architecture for cancer cocktail therapy. So we focused on the encapsulation capability and release behavior of this system. Utilizing the model drugs could clearly and intuitively present the drug release behavior of the nanoparticles under the tumor microenvironment. The synthesis processes of materials were illustrated in the Scheme 2. Briefly, fluorescein was aminated by 1,6-hexanediamine, resulting to FL-C6-NH2. CS-RhB was assembled with TPP to form the CRPN core. The core was coated with biodegradable organosilica shell and functionalized with aldehyde groups, obtained the CRP@dOSN. Then, FL-C6-NH2 13



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was covalently bonded to the surface of CRP@dOSN, and the resultant nanoparticles was CRP@dOSN-FL with CTAB. After the CTAB was removed, the final product CRP@dOSN-FL was obtained.

Scheme 2. Synthesis processes of FL-C6-NH2 (A) and CRP@dOSN-FL (B).

The structures of small-molecular compounds (FL-NHS, CS-RhB and FL-C6-NH2) were confirmed by 1H NMR spectrum (Figure S1), and these results were in line with our expectation. FL-NHS: 8.00 (1H), 7.74 (2H), 7.21 (1H), 6.67 (2H), 6.54 (4H), 2.67 (4H). FL-C6-NH2: 8.03 (1H), 7.67 (2H), 7.22 (1H), 6.83 (2H), 6.65 (2H), 6.58 (2H), 3.10 (2H), 2.63 (2H), 1.91 (4H), 1.21 (4H). CS-RHB: 3.72 (104H), 3.54 (104H), 2.97 (60H), 1.85 (12H). The RhB-substitution degree of chitosan calculated from the 1H 14


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NMR result was 2 %. The purity of FL-C6-NH2, which was measured by HPLC, was above 99.8% (Figure S2). Since the mean diameters of CRPNs (Figure 1A) and CRP@dOSN-FL (Figure 1B) observed in the TEM images were about 25 nm and 35 nm respectively, it revealed that the thickness of biodegradable silica shell was about 5 nm. Non-degradable control groups were also synthesized using the equal volume of BTESE instead of BTESPDS. Like the biodegradable group, products were named as CRP@ndOSN, CRP@ndOSN-FL with CTAB, CRP@ndOSN-FL, respectively. Their hydrodynamic sizes were about 1 times larger than TEM results (Table S1). The TEM images and the hydrodynamic particle sizes showed that these nanoparticles were well dispersed and not aggregated. The particle size of the biodegradable organosilica nanocapsule was based on the diameter of CS-RhB core and the thickness of organosilica shell, which could be tuned by reaction parameters, such as pH, temperature and time.

Figure 1. (A) TEM image of CRPN (negative stained); (B) CRP@dOSN-FL.

Fourier transform infrared spectrum (FTIR) (Figure S3A) and ζ-potentials (Figure S4A) were used to confirm the functionalization procedure of nanoparticles. A mild peak at 2800 cm-1 appeared in the FTIR spectrum of CRP@dOSN-FL with CTAB 15



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belongs to the various methylene groups in CTAB. After removing the CTAB, the peak belonging to the -CH2- in the organosilica framework becomes sharper in the FTIR spectrum of CRP@dOSN-FL. On the other hand, mild peaks at 1400 cm-1 appeared in CRP@dOSN and CRP@dOSN-FL belong to the δCH of the CTAB, but only a sharp peak could be found in CRP@dOSN-FL. The ζ-potentials of CRPN and CRP@dOSN were around 16.8 ± 1.2 mV and 15.3 ± 0.9 mV respectively. It indicated that some residual of CS-RhB had been react with the aldehyde groups, and thus reduced the positive surface charge of naked ionized –NH3+. After adequately reacted with FL-C6-NH2, the value was decreased to -3.6 ± 0.2 mV, which was attributed to substitution of CS-RhB by FL-C6-NH2. The hydroxyl group in fluorescein and Si-OH was partially ionized with negative charge. The ζ-potential was decreased to -6.0 ± 0.6 mV after the elimination of positively charged CTAB, indicating that the templates were successfully removed. The zeta potentials of CRP@dOSN, CRP@dOSN-FL with CTAB and CRP@dOSN-FL in PBS were -0.7 ± 0.9 mV, -22.9 ± 4.0 mV, -26.6 ± 0.9 mV, respectively, lower than deionized water (Figure S4B). FTIR (Figure S3B) and ζ-potentials (Figure S4A) of the non-degradable control group were also measured, and the results were similar to the biodegradable group. Fluorescence spectrum was used to confirm the drug loading ratio of the CRP@dOSN-FL. After the organosilica shell of CRP@dOSN-FL was completely dissolved by HF, fluorescence intensities were measured by spectrofluorophotometer (calculated and corrected by standard curves of FL-C6-NH2 and CS-RhB). Results indicated the contents of FL-C6-NH2 and CS-RhB were 1.8 wt % and 20.3 wt %, respectively. 16


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Traditional mesoporous silica nanoparticles (MCM-41 was used as control) usually got a peak at about 2 degree (2 Theta) in SAXRD curve,9 however, no peak was found in the SAXRD curves of CRP@dOSN-FL (Figure S5), which means that it was

a

non-mesoporous

scaffold.36

Moreover,

the

surface

properties

of

CRP@dOSN-FL were calculated using the nitrogen adsorption-desorption isotherms curves. The BET surface area (SBET) was 29.08 m2/g, and the BET pore volume (Vp) was 0.048 cm3/g. As for MCM-41 control, the SBET was about 999 m2/g, and the Vp was about 1.03 cm3/g.3 With a significant magnitude difference, it could be concluded that a thin and dense silica shell was coated on the CRPN core with a non-mesoporous structure. This architecture could physically prevent the escape of encapsulated macromolecules. Dual Stimuli Responsive Drug Release. Acid-responsive drug release behavior of FL-C6-NH2 was investigated in vitro (Figure 2A). In the acid condition (ABS, pH=5.0, 10mM), the release percentage reached 20 % after 4 h, and 28 % after 24 h. on the contrary, the drug release percentage was about 4 % after 24 h in the neutral conditions (PBS, pH=7.4, 10mM). It indicated that this drug delivery system could release the small-molecule model drug under acid conditions (which was responsible for metabolism in tumor cells), and was expected to have the tumor-triggered targeting property. As small-molecule model drug was barely released and stable in normal physiology environment (pH=7.4), the release process could be controlled individually.

17



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Figure 2. (A) In vitro FL-C6-NH2 release behavior of CRP@dOSN-FL in PBS (10 mM, pH=7.4) and ABS (10 mM, pH=5.0) at 37 °C; (B) In vitro CS-RhB release behavior of CRP@dOSN-FL in ABS (10 mM, pH=5.0) with/without 5 mM GSH.

GSH-responsive release behavior of CS-RhB was also studied. Since particle sizes of CS-RhB and FL-C6-NH2 were significantly different, the test method of GSH-responsive release experiments was different from acid-responsive release experiments. Small molecules (FL-C6-NH2) could be dialyzed, however, the macro-molecule should be separated by centrifugation. Moreover, solubilities of CS-RhB were different in acidic and neutral media. In acid media (ABS, pH=5.0), CS-RhB was ionized and solute in the centrifugated solution. The results were showed in Figure 2B. At t=0, the release percentage was 6.8 %, it was because the nanoparticles could not be precipitated completely by centrifugation. In the ABS solution with 5 mM GSH, the value was increased quickly, reached to 41 % after 24 h. Then the release process was slowed down, after 48 h, the percentage was 47 %. In the ABS solution without GSH, the release percentage was constant, stayed at 7.3 % even after 48h. It revealed that cleavage of the reduction-sensitive organosilica shell 18


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would be triggered by tumor-relevant GSH concentration, resulting in the shell-shedding of CRP@dOSN-FL and the release of macromolecule model drug CS-RhB. In contrast, the structure of organosilica shell remained stable without GSH addition. Therefore, small-molecule drugs (fluorescein) and macromolecular drugs (CS-RhB) could be loaded on the surface of organosilica shell and the central core respectively, and their release behaviors could be controlled separately in response to different stimuli (acid or GSH). GSH-Induced Degradation. The morphological changes of materials during degraded process could be monitored by TEM (Figure 3A). In the group which had been treated by ABS solution (10 mM, pH=5.0) with/without 5 mM GSH, the particle become larger and looser after 4 h, the core was increased to about 40 nm and the shell was about 10 nm (Figure 3A1). After 8 h, the diameter of core was increased to 50 nm, with a 50 nm thick loose shell outside. Obviously estimated, the framework of the shell was dissolved and damaged (Figure 3A2). As time goes on, the organosilica shell was completely destroyed and disintegrated, finally turned into fragments after 24 h (Figure 3A3). But in the control group with ABS only, compact packing nanoparticles were observed even after 24 h, with some solids on the surface, which could be formed by sodium acetate (Figure 3B1). The asynchronous result in the release experiment and TEM could be caused by the flexible structure of the CS-RhB. It could be escaped from the core through small pores on the surface, without the completely disintegration of the shell. The degradation process of control group CRP@ndOSN-FL was test in the same condition with CRP@dOSN-FL. After 24 h, 19



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TEM image showed that the CRP@ndOSN-FL maintained the core-shell structure without any measurable difference (Figure 3B2).

Figure 3. (A) TEM image of degraded process of CRP@dOSN-FL in ABS (10 mM, pH=5.0) and GSH (5 mM): (A1) 4h, (A2) 8h, (A3) 24h; (B) Control group: (B1) CRP@dOSN-FL in ABS without GSH, 24h, (B2) CRP@ndOSN-FL in ABS and GSH, 24h.

In Vitro Cytotoxicity. In vitro cytotoxicity of drug vector is crucial for its further application in vivo or in clinical. To further evaluate the biocompatibility of the CRP@dOSN-FL in vitro, MTT assay was used to assess cell viability after cells were treated with CRP@dOSN-FL. As shown in Figure 4, the cell viability of both tumor cells and normal cells treated with CRP@dOSN-FL decreased slightly with increasing of CRP@dOSN-FL concentration. Even at a high CRP@dOSN-FL concentration of 200 mg/L (CS-RhB concentration was 40 mg/L), the cell viability was over 80% in HeLa and COS7 cell lines. The results demonstrated that the drug delivery system CRP@dOSN-FL had good biocompatibility and was adaptable for in vivo drug 20


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delivery.

Figure 4. Cell viability of COS7 and HeLa cells treated with CRP@dOSN-FL after 48 h incubation.

Fluorescence Recovery. The core of organosilica nanocapsule was fabricated via electrostatic interaction between TPP and the macromolecular drug CS-RhB, and the distance between RhB groups were short enough to cause self-quenching. The fluorescence quenching of CS-RhB could be fully recovered after drug release, so that the degradation process of organosilica nanocapsule could be detected according to the change of fluorescence intensity. To confirm the fluorescence recovery of CS-RhB was indeed induced by the degradation of organosilica nanocapsule, the fluorescence spectra of CS-RhB in CRP@dOSN-FL nanoparticles were measured before/after the HF treatment. HF could react with Si-O-Si bonds, resulting in Si-F bonds, which led to the complete degradation of organosilica frameworks and the release of CS-RhB. As shown in Figure 5, the fluorescence intensity of CRP@dOSN-FL solution treated with HF was three times higher than that of the untreated CRP@dOSN-FL solution, 21



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attributed to the degradation of the silica shell. The result proved that the fluorescence intensity of the CS-RhB could be greatly enhanced after it had been released.

Figure 5. Fluorescence intensity of CRP@dOSN-FL before and after completely degraded by HF. Cellular Uptake and Intracellular Drug Release. To study the differential intracellular drug release behaviors of CRP@dOSN-FL in tumor cells and normal cells, confocal laser scanning microscopy (CLSM) was used to monitor changes in fluorescence intensity during endocytosis. After 4 h endocytotic uptake of CRP@dOSN-FL, cells were washed several times in PBS to remove excess nanoparticles outside the membranes. Under this condition, the increased red fluorescence was mainly attributed to the intracellular release of CS-RhB. As shown in Figure 6B, C, D, there was no observable changes in the fluorescence areas and intensities of small-molecule drugs (fluorescein, green) and macromolecular drugs (CS-RhB, red) in COS7 cells after incubated for 4 h, 8 h and 24 h. On the contrary, fluorescence areas were greatly broadened and intensities were strongly enhanced in

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HeLa cells after incubated for 8 h (Figure 6F2-H2) and 24 h (Figure 6F3-H3). The bright fluorescent spots around nucleus were the aggregation of drugs. These changes indicated that small-molecule drugs and macromolecular drugs successfully individually escaped from the shell-core structure due to the acid or GSH triggered degradation of CRP@dOSN-FL, resulting in the fluorescent diffusion/aggregation in cytoplasm and the significant flourescence recovery. As expected, these biodegradable organosilica nanoparticles could not release dual drugs into normal cells, but specifically release them into tumor cells, attributing to the tumor-triggered targeting capability of vectors.

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Figure 6. CLSM images of the CRP@dOSN-FL (50 mg/L) incubated with COS7 (A, B, C, D) and HeLa cells (E, F, G, H) for 4 h (1), 8 h (2) and 24 h (3) treatment. Blue fluorescence images (A1~3, E1~3); red fluorescence images (B1~3, F1~3); green fluorescence images (C1~3, G1~3); overlap of confocal fluorescence images (D1~3, H1~3). Size bar: 20 µm.

Flow Cytometry Analysis. In order to further confirm the drug release behaviors 24


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of the pH/redox dual stimuli-responsive biodegradable nanovectors, flow cytometry analysis was utilized to assess the cell uptakes of fluorescent model drugs, and the mean fluorescence intensities (MFI) values were also quantitatively measured. As shown in Figure 7A (green fluorescence) and 7B (red fluorescence), the flow cytometric profiles clearly demonstrated the gradually increases in fluorescence intensities of CRP@dOSN-FL treated HeLa cells after different incubation times, attributed to the release of model drugs FL-C6-NH2 and CS-RhB. On the contrary, there were no obvious changes either in the green fluorescence intensities (Figure 7C) or the red fluorescence intensities (Figure 7D) of COS7 cells incubated under the same conditions. Meanwhile, Figure 7E (green fluorescence) and 7F (red fluorescence) illustrated the comparison of the MFIs in HeLa cells and COS7 cells. The MFIs belonged to HeLa cells after 24 h incubation with CRP@dOSN-FL were about 10-fold higher than that of COS7 cells. All these results indicated that tumor cells could significantly enhance dual-drug release of CRP@dOSN-FL rather than normal cells, attributed to the tumor specific microenvironments (acidic, high GSH level). Therefore, the change fluorescence intensities of CRP@dOSN-FL exhibited obvious tumor cell selectivity, which was consistent with the in vitro drug release studies.

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Figure 7. Flow cytometry analysis (A-D) and MFI (E, F) of HeLa cells (A, B) and COS7 cells (C, D) incubated with CRP@dOSN-FL for 4 h, 8 h incubation and 24 h. Cells treated with PBS were used as negative controls.

CONCLUSIONS In summary, a dual drug delivery system based on biodegradable organosilica

core-shell architectures was developed for cancer cocktail therapy. Small-molecule drugs (fluorescein) and macromolecular drugs (CS-RhB) were loaded on the surface of organosilica shell and the central core, respectively. Their release behaviors could 26


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be controlled separately in response to different stimuli (acid or GSH). This drug delivery system could serve as an efficient shuttle for tumor-triggered targeting delivery and also provide a potential strategy to overcome drug resistance.

ASSOCIATED CONTENT

Supporting Information. 1H-NMR of CS-RhB, FL-NHS and FL-C6-NH2. HPLC results of FL-C6-NH2. FTIR, ζ-potentials of CRPN, CRP@dOSN, CRP@dOSN-FL with CTAB, CRP@dOSN-FL and nondegradable control groups. SAXRD. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author * Tel. & Fax: 86-27-68754509. E-mail address: [email protected] Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

The study was sponsored by the National Nature Science Foundation of China (51233003, 51303137, 51573142), the Chinese Postdoctoral Science Foundation (2014T70729, 2016M590708), the National Science and Technology Major Project (2016YFC1100703), and the MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Zhejiang University (2015MSF004).

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The table of contents Graphic

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Dual Drug Delivery System Based on Biodegradable Organosilica

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