Intelligent Drug Delivery System Based on Mesoporous Silica

Dec 16, 2015 - Intelligent Drug Delivery System Based on Mesoporous Silica Nanoparticles Coated with an Ultra-pH-Sensitive Gatekeeper and Poly(ethylen...
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Intelligent Drug Delivery System Based on Mesoporous Silica Nanoparticles Coated with an Ultra-pH-Sensitive Gatekeeper and Poly(ethylene glycol) Tianchan Chen,†,# Wei Wu,†,‡,# Hong Xiao,§,# Yanxiao Chen,*,∥ Min Chen,⊥ and Jianshu Li*,† †

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, and ∥College of Chemical Engineering, Sichuan University, Chengdu, 610065, China § Department of Anesthesiology, West China Hospital, Sichuan University, Chengdu, 610041, China ⊥ Department of Materials Science and State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai, 200433, China ‡ Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory For Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400030, China S Supporting Information *

ABSTRACT: Mesoporous silica nanoparticles (MSNs) exhibit significant advantages for efficient drug/gene delivery but it is hard for simple MSNs to deliver the loaded drug to the target sites of disease. Considering that there are some wellknown pH differences in the body, it is a useful strategy to modify the exterior surface of MSNs with stimuli-responsive gatekeepers to realize open−close transformation of their mesopores. In this work, multifunctional pH-sensitive MSNs were designed with mixed polymeric coatings, that is, poly(ethylene glycol) (PEG) as a dispersity-enhancer and poly(2-(pentamethyleneimino)ethyl methacrylate) (PPEMA) as an ultra-pH-sensitive gatekeeper. Enhanced dispersity, high drug loading capacity, long-circulation time, pH-triggered targeting, and better cellular uptake of the multifunctional MSNs make them potential candidates for pH-sensitive drug delivery such as tumor therapy.

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mesopores would be a feasible and desirable method. Stimuliresponsive gatekeeper materials can be inorganic or organic nanoparticles,3 supramolecular (pseudo) rotaxanes,4 and stimuli-sensitive polymers.5 So far, the widely investigated stimuli include magnetism,3 temperature,5b pH,5c,6 redox,5e and photochemical changes.7 Among them, pH-sensitivity is extensively applied for cancer therapy because of the specific pH condition of tumor tissues.1a,8 It is well-known that two types of ubiquitous pH differences exist in the body: the cancer extracellular matrix has a slightly acidic pH value of 6.5, and intracellular endo/ lysosomes have much lower pH values of 5.0 as compared with the normal tissues and blood having a pH value of 7.4.9 For example, by taking advantage of these pH differences, Bae et al. developed a core−shell mixed micelle based on two different block copolymer components, which could expose the cell interacting ligand (biotin) on the surface under slightly acidic environmental conditions of various solid tumors.10 Recently,

n recent decades, the use of mesoporous silica nanoparticles (MSNs) as potential scaffolds for efficient drug/gene delivery has been prevalent because of their significant advantages, such as high drug loading capacity, good biocompatibility, low apparent cytotoxicity, uniform size, high surface areas, excellent stability, and easily tailorable surface properties.1 However, there are still some problems to be resolved before MSNs are used for clinical applications. For instance, since most of the anticancer drugs (e.g., chemotherapy drugs) have serious side effects, it is of paramount importance to improve the controllability of drug release to meet the actual needs of tumor therapy.1b However, it is hard for simple MSNs to deliver the loaded drug to the target sites of disease, which not only reduces the therapeutic efficiency but also increases the toxic side effects. Besides, the poor dispersity of MSNs in water seriously limits their potential in vivo biomedical applications.2 Therefore, designing multifunctional MSNs with good dispersity, disease-targeting property and controllable drug release properties will significantly improve their efficiency for disease therapy. To this end, the strategy of developing stimuli-responsive gatekeepers on the exterior surface of MSNs with open-close transformation of their © XXXX American Chemical Society

Received: October 27, 2015 Accepted: December 15, 2015

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DOI: 10.1021/acsmacrolett.5b00765 ACS Macro Lett. 2016, 5, 55−58

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(TEM) was used to characterize MSNs’ morphology. It showed that MSNs have a spherical shape with a uniform size of 50 nm. Further analysis by the Brunauer−Emmett−Teller (BET) method revealed that the MSNs’ specific surface area is 840 m2/g and average pore size is 5.78 nm. These results (Figure S1) indicate that the MSNs are suitable for further modification to provide high drug loading capacity. The pH-sensitive polymer (PPEMA-Br) was prepared by atom transfer radical polymerization (ATRP).11 The molecular weight (MW) and polydispersity index (PDI) of PPEMA were measured via GPC determination as 6493 and 1.32, respectively (Figure S2). Meanwhile, the bromoacetyl bromide (BAB) was used to modify PEG-OH (MW 2000) to obtain PEG-Br.13 The successful syntheses of PPEMA-Br and PEG-Br were confirmed by 1H NMR (Figures S2 and S3). Then PPEMA-Br and PEGBr were further grafted onto the surface of MSNs to form the mixed coatings (Figure S4). In this work, in order to obtain optimum MSN@PPEMA/PEG with high pH-sensitivity (provided by PPEMA) and good dispersity/circulation (provided by PEG), a series of products with different polymer feeding ratios, that is, PEG/(PEG + PPEMA) = 10, 20, 30, and 40% were investigated, and named as MSN@P/P-1, MSN@P/ P-2, MSN@P/P-3, and MSN@P/P-4, respectively (Table S1). Among them, the unmodified MSNs and MSN@P/P-1 exhibited poor dispersity and remained aggregated in water even after a long time of sonication treatment. Whereas, MSN@P/P-3 and MSN@P/P-4 both exhibited good dispersity in water (Figure S5). Hence, it is clear that a higher PEG percentage in the mixed coating will be beneficial to improve the dispersity of MSNs. The structure of MSN@PPEMA/PEG nanoparticles was also monitored by infrared radiation (IR) spectrum and thermogravimetric analysis (TGA). The IR spectra provided direct identification of chemical groups in MSN@PPEMA/PEG as PEG has a characteristic peak at 1180 cm−1, and PPEMA has a characteristic peak at 1360 cm−1 (Figure S6). TGA is also used to estimate the weight loss of modified MSNs (Figure S7). For example, the TGA curve of MSN@P/P-3 showed 13% weight losses when the samples were heated from 25 to 250 °C, which can be ascribed to the desorption of physically adsorbed water. An additional weight loss that occurred between 250 and 320 °C could be due to the removal of PEG shells and that in the temperature range from 320 to 600 °C could be attributed to the removal of PPEMA shells. The above results demonstrated that PEG and PPEMA chains are successfully grafted on the MSNs surface. By above data, the graft densities of PEG and PPEMA chains on MSN@P/P-3 can be estimated to be 0.0021 and 0.0019 mmol/m2, respectively. The hydrodynamic diameter (Dh) and size distribution of the samples in water, and their zeta potential at different pH values, were measured by dynamic light scattering (DLS, Figure 1). As can be seen, the Dh of the modified MSNs ranged from 85 to 115 nm (Figure 1A). Considering that the PEG chains are hydrophilic, a hydration layer is arising from these PEG chains on the surface of MSN@PEG/PPEMA nanoparticles.5f As compared to the other grafted nanoparticles, MSN@P/P-3 and MSN@P/P-4 have more PEG chains, which can induce more chain−chain repulsions and chain−chain interactions, thus, resulting in a larger particle diameters distribution. The MSN@ PEG/PPEMA is stable and no aggregation between pH of 6.5− 7.4. We also found that the hydrodynamic diameter of MSN@ P/P-3 at pH 6.5 is larger than at pH 7.4 (Figure S8) due to the protonation of PPEMA, which can transform the surface to be

Gao et al. reported a series of smart micellar nanoparticles with tunable and ultra-pH-sensitivity, which can be activated at acidic tumor pH values or in specific intracellular organelles upon uptake in targeted cells.11 Meanwhile, pH-responsive poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA) brushes have also been anchored on MSN surfaces to form a controlled drug release system.5d However, to the best of our knowledge, multifunctional MSNs coated with both pHsensitive gatekeeper and dispersity-enhancer have rarely been reported so far. In this work, multifunctional pH-sensitive MSNs were prepared with mixed polymeric coatings, showing pHresponsive targeting ability, high drug loading capacity, longcirculation time, excellent stability and better cellular uptake. The system has a MSN inner core, and poly(2(pentamethyleneimino)ethyl methacrylate) (PPEMA) and poly(ethylene glycol) (PEG) are grafted onto the exterior surface of MSN to obtain the final product (MSN@PPEMA/ PEG). The PEG component can improve the dispersity and blood circulation time of this system.2 After the loading of model drug (doxorubicin hydrochloride, DOX·HCl), PPEMA shows hydrophobicity at neutral pH during blood circulation, causing the PPEMA chains to collapse on the MSNs surface and block their mesopores, thus encapsulating DOX·HCl compactly within the mesoporous structure to prevent premature drug loss. Once MSN@PPEMA/PEG is accumulated into the tumor tissue by enhanced permeability and retention (EPR) effect, PPEMA responds to the slight acidic pH of tumor extracellular matrix and subsequently becomes protonated to transform as hydrophilic and positively charged. This enables the nanoparticles to not only adhere to the target tumor cell through electrostatic interaction but also gradually open the previously blocked mesopores for drug release (Scheme 1). Furthermore, while in the more acidic conditions, Scheme 1. Schematic Illustration of Multifunctional MSN@ PPEMA/PEG with Close−Open Transformation for pHSensitive Drug Delivery

that is, in the intracellular endo/lysosome compartments, DOX·HCl (positively charged) can undergo burst release, due to both the open-status of MSNs and charge repulsion from the positively charged PPEMA, to efficiently induce the death of tumor cells. In addition, these multifunctional MSNs also exhibit relatively low cytotoxicity, which makes them promising for potential clinical applications. In order to synthesize MSN@PPEMA/PEG, the uniform MSNs were first prepared by using tetraethyl orthosilicate (TEOS) as silica source and cetyltrimethylammonium bromide (CTAB) as structure directing agent according to the method reported by Shi et al.12 Transmission electron microscopy 56

DOI: 10.1021/acsmacrolett.5b00765 ACS Macro Lett. 2016, 5, 55−58

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Then DOX·HCl was used as a model to study the drug loading efficiency and release behavior of MSN@P/P-3. DOX· HCl is loaded into the MSNs core through both physical encapsulation and electrostatic interaction (DOX·HCl is positively charged and MSNs is negatively charged). The DOX·HCl-loaded system showed relatively high drug loading ability, that is, loading efficiency (LE) of 13.1% and encapsulation efficiency (EE) of 65.2%. Subsequently, in order to further study the pH-sensitivity of MSN@PPEMA/ PEG, the in vitro drug release profiles are investigated at different pH phosphate buffer solutions (PBS, 0.01 M, pH 7.4, 6.5, and 5.0; Figure 3A). The result shows that due to its

Figure 1. Size distribution of MSN, MSN@P/P-1, MSN@P/P-2, MSN@P/P-3, and MSN@P/P-4 in buffer solution at pH 7.4 (A); Zeta potential of MSN@P/P-2, MSN@P/P-3, MSN@P/P-4 at different pH values (B).

more hydrophilic. Meanwhile, unmodified MSNs always had negative zeta potential in the tested pH range. It is noted that all of MSN@P/P-2, MSN@P/P-3, and MSN@P/P-4 exhibit obvious pH sensitivity and a charge conversion between pH 7.4 and 6.5 (Figure 1B). The zeta potential of these MSN@ PPEMA/PEG nanoparticles continuously increase from negative to positive along with the decrease of pH value. In particular, the nanoparticles display negative surface charge at pH 7.4, whereas they are positively charged at pH 6.5. The degree of pH sensitivity should depend on the PPEMA ratio in the mixed coating since MSN@P/P-4 (with more PEG but less PPEMA) showed weaker charge reversal capability, that is, zeta potential changed from −20.1 to 59.1 from pH 7.4 to 6.5, as compared with that of MSN@P/P-3 (zeta potential changed from −22.6 to 69.4 from pH 7.4 to 6.5). Thus, considering a balance between high pH-sensitivity and good dispersity/ circulation, we chose MSN@P/P-3 as the optimum sample for further studies. The TEM image of MSN@P/P-3 is shown in Figure 2A. Compared with the image of unmodified MSNs (Figure S1),

Figure 3. Release profiles of DOX·HCl from MSN@P/P-3 at different pH values (A). Cytotoxicity assay of samples on HeLa cells against the concentration of DOX·HCl (B).

particular core−shell structure, the cumulative release of DOX· HCl from MSN@P/P-3 is less than 15% at pH 7.4 within 40 h. While, about 68 and 84% of DOX·HCl has been released from MSN@P/P-3 at pH 6.5 and 5.0 in a same time range (most of them are released actually within the initial 3 h), respectively. Therefore, this pH-responsive nanoparticles can reduce premature drug loss during the blood circulation and enhance drug release in low pH environments such as in tumor tissues and in endo/lysosomes, which could reduce the toxic side effects for normal cell and significantly improve the drug delivery efficiency for disease therapy. In order to confirm the feasibility of this system, standard CCK-8 assay was carried out to evaluate the cytotoxicity (Figure 3B). MSN@P/P-3 did not show obvious cytotoxicity even at a concentration up to 83 μg/mL, which demonstrates its good biocompatibility for drug delivery. Meanwhile, the drug-loaded MSN@P/P-3@DOX·HCl nanoparticles remarkably inhibited the growth of HeLa cells at the same concentration range. The half maximal inhibitory concentration (IC50) of MSN@P/P-3@DOX·HCl was calculated to be 21.749 μg/mL, indicating its high therapeutic efficiency. Meanwhile, at the equivalent doses (21.749 μg/mL), the inhibitory concentration of free DOX·HCl was determined to be 61%. The cellular uptake and intracellular drug release behaviors of this system in HeLa cells were visually monitored by confocal laser scanning microscopy (CLSM, Figure 4). HeLa cells were incubated with free DOX·HCl and MSN@P/P-3@DOX·HCl, separately. Moreover, in order to better imitate the tumor cell endocytosis at the slight acidic condition of tumor extracellular matrix, the culture media of pH 6.5 was employed. Lysotracker Green DND-26 indicator was used to stain the acidic compartment. As shown in Figure 4A−C, the red fluorescence signals derived from DOX·HCl were detected in cell nuclei, revealing that DOX·HCl can enter into the cells and accumulate in the nucleis.14 Comparatively, Figure 4D−F

Figure 2. TEM images of MSN@P/P-3 (A), the scale bar is 50 nm; Zeta potential of MSN and MSN@P/P-3 at different pH values (B).

MSN@P/P-3 showed an enlarged average size since the MSN core is surrounded by a polymeric shell. In addition, the particle size of MSN@P/P-3 was determined as 70−80 nm, which could be easily endocytosed by cells. The diameter measured by TEM is slightly smaller than that determined by DLS, because the DLS result provides the hydrated hydrodynamic diameter but TEM shows the size in a dried state. The pH-induced charge conversion property of MSN@P/P3 was further studied in a wide pH range from 8.0 to 5.0 (Figure 2B). The surface charge of MSN@P/P-3 changed from negative to positive with the decrease in pH values, which proved its ultra-pH-sensitivity due to the protonation of PPEMA chains at a relatively low pH environment. Further, we found that the charge reversal happens at pH 7.0, which is consistent with the previous report.11 57

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and State Key Laboratory of Polymer Materials Engineering (sklpme2014-3-01) are gratefully acknowledged.

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Figure 4. Confocal images of HeLa cells incubated with free DOX· HCl (A−C) and MSN@P/P-3@DOX·HCl (D−F) for 2 h at the same drug concentration (10 μg/mL) at pH 6.5; The scale bars correspond to 20 μm in all the images: green, Lysotracker Green DND-26; red, DOX·HCl.

indicates that after HeLa cells were incubated with MSN@P/P3@DOX·HCl for 2 h, much more red fluorescence signals were clearly detected in cell nuclei. These results showed that the drug-loaded system can release DOX·HCl in response to the slight acid tumor environment. Moreover, due to the protonation of PPEMA, these positively charged MSN@P/P3 can also enhance the escape from endo/lysosome compartments, and then burst release DOX·HCl mainly into the nucleis of tumor cells and induces their apoptosis. These results are consistent with the in vitro pH-sensitive drug release studies. In conclusion, we prepared an intelligent stimuli-responsive drug delivery system based on MSNs modified with mixed coatings, which include PPEMA as an ultra-pH-responsive gatekeeper and PEG as dispersity-enhancer. The obtained MSN@PPEMA/PEG system showed improved dispersity, high drug loading capacity, pH-triggered surface charge reversal and controllable drug release profile, as well as better cellular uptake. These results indicate that MSN@PPEMA/PEG is a promising drug delivery system for pH-sensitive disease therapy such as antitumor applications.

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REFERENCES

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.5b00765. Detailed synthese procedures, characterization methods, instrument data, and the spectra that were referred to in the main text (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions #

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (21534008, 51322303, and 51573110) 58

DOI: 10.1021/acsmacrolett.5b00765 ACS Macro Lett. 2016, 5, 55−58