Charge-Reversal APTES-Modified Mesoporous Silica Nanoparticles

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Charge-Reversal APTES-modified Mesoporous Silica Nanoparticles with High Drug Loading and Release Controllability Yifeng Wang, Yi Sun, Jine Wang, Yang Yang, Yulin Li, Yuan Yuan, and Changsheng Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05370 • Publication Date (Web): 17 Jun 2016 Downloaded from http://pubs.acs.org on June 20, 2016

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ACS Applied Materials & Interfaces

Charge-Reversal

APTES-modified

Mesoporous

Silica

Nanoparticles with High Drug Loading and Release Controllability Yifeng Wang#,†,a, Yi Sun#,†, Jine Wang†, Yang Yang†, Yulin Li,*,†, § Yuan Yuan*,† and Changsheng Liu*,†

†

Key Laboratory for Ultrafine Materials of Ministry of Education, The State Key

Laboratory of Bioreactor Engineering, and Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, People’s Republic of China. §

Shanghai Collaborative Innovation Center for Biomanufacturing, East China

University of Science and Technology, Shanghai 200237, People’s Republic of China. ABSTRACT: In this study, we demonstrate a facile strategy (DL-SF) to develop MSN-based nanosystems through drug loading (DL, using doxorubicin as a model followed by surface functionalization (SF) of mesoporous silica nanoparticles (MSN) via aqueous (3-aminopropyl)triethoxysilane (APTES) silylation. For comparison, a reverse functionalization process (i.e, SF-DL) was also studied. The pre-DL process allows for an efficient encapsulation (the encapsulation efficiency is ~75%) of an anticancer drug (doxorubicin, DOX) inside MSN and the post-SF enables an in-situ formation of APTES outer layer to restrict DOX leakage under physiological conditions. This method makes it possible to tune DOX release rate by increasing

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APTES decoration density through variation of APTES concentration. However, the SF-DL approach results in a rapid decrease of drug loading capacity at an increase of APTES concentration due to the formation of APTES outer layer hampers the inner-permeability of DOX drug, resulting in a burst release similar to undecorated MSN. The resulting DOX-loaded DL-SF MSN present slightly negative-charged surface under physiological conditions and become positively charged under extracellular microenvironment of solid tumor due to the protonation effect under acidic conditions. These merits benefit their maintenance of long-term stability in blood circulation, high cellular uptake by a kind of skin carcinoma cells and an enhanced intracellular drug release behavior, showing their potential in delivery of many drugs beyond anticancer chemotherapeutics.

KEYWORDS: mesoporous silica nanocarriers, charge-reversal, doxorubicin, drug delivery, pH responsive

INTRODUCTION Nowadays cancer is a kind of popular pathological disorder to cause global mortality, which poses great challenge for its successful therapy.1 In with surgical tumor removal, small anticancer drugs, such as doxorubicin paclitaxel have been used for treating different types of cancers.2, 3, 5 However, the rapid diffusion of small anticancer drugs as well as existence of multidrug resistance from cancer cells often results in drug leakage in normal tissues, low


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drug permeability through dense-collagen structure of tumors, and drug expelling problems even delivered into tumors, thus producing high toxicity and limited anticancer efficacy.

2, 6-14

Recently, various kinds of nanosystems

have been designed to administer small molecular anticancer drugs, aiming to increase their solubility and stability in biological buffer, and targetability to cancer location.2,

4,

15-17

Among them, nanoformulations encapsulating

chemotherapeutics with a size of ~100 nm have been used for treatment of cancers at a clinical (trial) level.18-20 Such nanocarriers with negative-charged surface can carry drugs through improved blood circulation and accumulate around tumoral tissues.15-17, 21, 22 However, such negative nanoparticles (NPs) with a big nanosize have poor permeability across the dense collagen matrix of the interstitial space into tumor blood vessels and tumor depth, as well as low cell uptake ability, resulting in a limited antitumor efficacy.23, 24 It is known that negative-charged NPs tend to have a prolonged circulation time because too positive NPs tend to interact with negative proteins existent in the blood to cause particle aggregation.25 However, in acidic tumor tissues (pH 6.5), positive-charged nanocarriers were reported to have higher capacity to be internalized into tumor cells through their affinity with negative surface of cell membranes.26,

27

In this case, it is preferential to design a kind of

charge-reversal nanocarriers which have negative-charged surface under normal physiological conditions, while they can resume their positive-charged surface under acidic microenvironment of solid tumors.28



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Figure 1. Schematic representation on how the process of DOX loading (DL) and surface functionalization (SF) affects the loading/release properties of mesoporous silica nanocarriers (MSN). Upper: Drug can be effectively loaded into bare MSN with well interconnected porous structure, which can be protected by surface functionalization with 3-(aminopropyl) triethoxysilane (APTES) through its silylation on MSN surface. The surface charge of the nanocarriers and their drug release properties can be tuned by adjustment of APTES graft degree, which can be further accelerated under acidic microenvironment of both extracellular tumor and endo/lysosomal compartments. Lower: if MSN surface is firstly decorated with APTES before drug loading, the outer layer may limit the drug diffusion into inner part of MSN loading capacity, and drug can be only adsorbed onto nanocarrier surface, which may give a burst release under physiological conditions. As one of two limited most promising inorganic nano-biomaterials (one is gold NPs), silica NPs (i.e., Cornell Dot) have been approved by Food and Drug Administration at a clinical-trial level, due to their unique advantages, including tunable

size

dimension,

adjustable

porous

structure

and

hierarchical

architecture, surface functional availability, excellent biocompatibility and biodegradability.29 Although various kinds of nanogated MSN nanocarriers have been investigated, most of them are involved in a complicated process, which

limited

their

drug

loading

capacity

and the

following

drug

controllability, together with high cost for further potential applications.30 The


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point-by-point grafting of functional groups onto MSN surface at solvent atmosphere can be used to increase drug loading capacity in their mesoporous architecture, but drug release sustainability cannot be easily achieved due to the loose surface structure.1 For instance, Bariana reported that 60% of hydrophobic drug was rapidly released from MSN in the beginning 2 h,31 and Gao’s study indicated that a complete release occurred in 12 h from amino-modified MSN.32 Different from silylation in organic solvent in the absence of water, aqueous silylation may allow for formation of functional outer layer onto MSN through condensation process,33 which can be used for adjustment of drug loading and/or release properties. However, conventional modification focused only on whether the amino can be grafted, ignoring how the grafting/loading process themselves affect the loading/release properties and surface conditions of nanocarriers and how the therapeutic release property can be mediated by changing amino graft degree. Herein, we propose that, if drug was firstly loaded

into

MSN

with

mesoporous

functionalization

through

silylation

(APTES),

loading

capacity

drug

structure,

of could

followed

3-(aminopropyl) not

be

by

surface

triethoxysilane

negatively

affected.

Furthermore, the release rate of the payloads inside MSN might be tuned by adjustment of the

post APTES graft degree. Otherwise, if surface

functionalization of MSN happens before drug loading, the additional surface decoration may hamper drug loading process and completely change the



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following release property (Figure 1). Furthermore, since APTES surface modification would introduce amino groups on to MSN, which can be protonated under acidic conditions. Therefore, the variation of APTES concentration may be useful to adjust surface charge of MSN, which can present negative-charged surface under physiological conditions, while become positively-charged state after their entry into acidic extracellular tumor environment (pH 6.5).34 In order to check this hypothesis in this study, APTES was grafted on MSN via its silylation on MSN in an environment-friendly approach (in aqueous solution). It was found that loading-grafting process is beneficial not only to achieving high drug loading capacity, but also to maintaining a sustainable drug delivery property, more advantageous than the grafting-loading process. This study may enlighten new ideas on fabrication of biocompatible drug delivery nanosystems with high efficacy.

MATERIALS AND METHODS Materials and Cells. Tetraethyl orthosilicate (TEOS), N-cetyltrimethylammonium bromide (CTAB) and triethylamine (TEA) was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd, China. (3-Aminopropyl) triethoxysilane (APTES) was obtained from Aladdin Co. Ltd, China. Doxorubicin (DOX) was ordered from Dalian MeiLun

Biology

Technology

Co.,

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium

Ltd, bromide

China. (MTT)

and

4',6-diamidino-2-phenylindole (DAPI) were purchased from Life Technology, USA. Ethanol was gotten from Shanghai Taitanchem Co., Ltd, China. All chemicals


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underwent no further treatment before use. A human epithelial carcinoma cell line cells) was ordered from Chinese Academy of Sciences, Shanghai, China. Human normal oral epithelial cell line (NOK cells) was obtained at our lab. Preparation, Characterization and Drug Loading of MSN-DOX-APTES (MDA) Nanoparticles. Mesoporous silica nanoparticles (MSN) were prepared using an emulsion method. Briefly, 2.18 g hexadecyl trimethyl ammonium bromide (CTAB) 0.08 g triethanolamine (TEA) was dissolved in 20 mL distilled water under stirring at 95 °C. After 1 h stirring, tetraethyl orthosilicate (TEOS, 1.5 mL) was dropped into the above mixture. After 1 h reaction, the materials were centrifuged and washed with ethanol thrice for purification. The precipitate underwent further reflux for 2 d with a solution of hydrochloric acid in ethanol (10%, v/v) at 78 °C to remove the remaining CTAB, followed by lyophilization to obtain pure MSN. 2 mg DOX was dissolved in 1 mL distilled water, followed by mixing with 10 mg MSN suspensions in 19 mL PBS (pH 7.4) under 400 rpm stirring for 24 h. The was centrifuged at 10,000 rpm for 10 min and washed with water (this process was performed thrice). The obtained DOX-loaded MSN was abbreviated as “MD”. For surface modification, a certain amount (5-30 µL) of APTES aqueous solution (100 µL/mL) was poured into 5mL of aqueous suspension (2 mg/mL) of MSN or “MD” samples, which were stirred under 400 rpm (24 h, 50 °C). The suspension was centrifuged/washed thrice. The resulting precipitate was re-suspended and abbreviated as “MA” or “MDA”, respectively. The loading of DOX into MA samples were performed in a way similar to “MD” preparation process. The obtained DOX-loaded



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MA samples were abbreviated as “MAD”. The supernatant were measured by a UV spectrophotometer (SpectraMax M2, Molecular Devices, USA) at DOX absorption wavelength 480 nm for drug loading amount evaluation. The surface charges of the nanoparticles in PBS at different pH values were analyzed on a Nano ZS Zetasizer (Malvern Instruments, UK) via Dynamic Light Scattering (DLS) technique. The microstructure of MSN before and after modification was investigated on a Fourier transform infrared (FTIR) spectrometer (Nicolet 5700, Thermo Electron, USA) by tracking their transmission profile in the 500-4000 cm–1 range at room temperature.

MSN and MA samples were suspended in ethanol, followed by drop on a copper-gridded substrate to be air-dried, which were then observed on a JEOL JEM-2100 transmission electron microscope (TEM, Japan) under 120 kV voltage. Nitrogen sorption isotherms of the samples before and after surface modification were

measured

on

a

TriStar Ⅱ

Micromeritics

instrument

(USA).

Brunauer–Emmett–Teller (BET) method was used for determining surface area, and Barret–Joyner–Hallenda (BJH) model for analysis of porous properties. Evaluation of Drug Delivery Properties In Vitro. 1mL aqueous solutions of MD, MDA and MAD NPs with 100 µg DOX were dialyzed against 9 mL PBS buffer using 3,500 Da MWCO dialysis membrane (Shanghai Yuan Ju biological technology Co., Ltd., Shanghai, China), respectively. At a specific time, 0.1 mL of the solution was


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into a SpectraMax M2 microplate reader (Molecular Devices, USA), and the fluorescent intensity (λex = 480 nm, λem = 580 nm) of DOX was recorded. The cumulative release (Cr) was quantitatively calculated based on the following formula: Cr = 100 * Wt/Wtot

(1)

where Wt stands for the released drug amount at time t, while Wtot is the initial drug encapsulated in the NPs which were tested for release study. In Vitro Bioactivity. KB cells were cultured at 37 °C in 25 cm2 plates containing Dulbecco’s Modified Eagle Medium (DMEM) and 10% Fetal Bovine Serum (FBS), which were placed an incubator with constant humidity under 5% CO2 atmosphere. NOK cells were cultured under the same condition as above in 5 cm2 plates in Keratinocyte Serum Free Medium (K-SFM) containing 10% FBS and 1% penicillin. Afterward, the cells were incubated until achievement of about 80% confluence, and then trypsinized for cell collection, which were resuspended in DMEM medium for further use. MTT assay was used for analysis of bioactivity of the samples on KB cells and cells. Briefly, for KB cells, 100 µL DMEM solution containing 5,000 cells was put a well of 96-well plate and incubated for 1 d, after which the DMEM solution was substituted with 200 µL fresh one containing DOX, MD, MDA and MAD NPs with equivalent DOX concentrations, followed by continuous incubation for 1, 2, 3, 4 and d at 37 ºC before the MTT assay. NOK cells were treated with the samples under condition, just using K-SFM solution as cell medium instead of DMEM and



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cells for 2 d before the MTT assay. MSN and MA NPs without DOX drug were also evaluated as controls to check if NPs themselves are cytotoxic. MTT assay was performed according to a general procedure: in brief, cells were treated with 30 µL MTT solution per well for 4 h at 37 °C, and then cell medium was replaced with 200 DMSO to dissolve formazan crystals. The solution was spectrophotometrically analyzed by tracking its 492 nm absorbance, which was used for calculation of cell viability based on the equation of ODtest/ODcontrol×100%. To check the effect of the DOX-loaded NPs on drug internalization, cells treated with the samples were observed on an A1R Nikon Confocal Laser Scanning Microscopy (CLSM, Japan). In this case, 40,000 KB cells were cultured in Φ20 mm cell culture dish containing 2 mL DMEM solution for 1 d. Afterwards, the old DMEM solution was renewed with 2 mL fresh one containing of free DOX, MD, MDA NPs (all samples contained 0.5 µM DOX), followed by 4 and 24 h incubation, respectively. After wash with PBS thrice, the cells underwent 15 min fixation treatment with 2.5% glutaraldehyde as well as and 15 min DAPI (2 µg/mL) nucleus staining at ambient atmosphere, followed by CLSM imaging. For quantitative analysis of intracellular drug accumulation, 150,000 KB cells were cultured in DMEM at each well of a 6-well plate for 1 d, followed by replacement of cell medium with fresh DMEM (2 mL, as control), and the DMEM solutions of DOX, MD and MDA_0.43 at the equivalent DOX concentration (0.5 µM). After 4 h incubation, KB cells were then trypsinized and washed by PBS solution thrice, followed by analysis by a flow cytometer (BD, USA).


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Statistical Analysis. Statistical significance was analyzed by the software Origin 8.0. Statistical significance was adopted at a value of P < 0.05. Each experiment was performed thrice (n = 3).

RESULTS AND DISCUSSION Surface Modification, Drug Loading and Characterization. Sol-gel chemistry was used for fabrication of mesoporous silica nanoparticles (MSN) via an emulsion approach. MSN were first decorated with amino groups through grafting APTES onto their surface in aqueous solution. MSN samples before and after APTES modification were characterized by a Fourier transform infrared (FTIR) spectrometer. All the MSN-based samples presented clear Si–O–Si asymmetric stretching peaks at 1085 and 801 cm−1 (Figure 2a). Compared to original MSN, APTES-treated ones gave a new absorption at 2924 cm−1 probably coming from –CH2 stretching vibration, suggesting the successful functionalization of APTES on MSN’s surface.35 The ζ-potentials of the nanoparticles were further measured (Figure 2b). introduction of APTES (from 0 to 2.56 µM) onto MSN induced an increase of the ζ-potential from -11.0 ± 0.3 (MSN) to 8.1 ± 0.6 mV (MA_2.56), reconfirming the successful grafting of cationic APTES on MSN’s surface. These results indicate that APTES modification in aqueous solution is an effective way to decorate MSN with amine groups to obtain nanocarriers with adjustable surface charge. In order to check the morphological properties of the nanoparticles, MSN and APTES-modified MSN (MA_2.56) were observed by transmission electron microscope. As shown in Figure 3, both MSN and MA_2.56 owned a nanosize of ~50 nm in round shape, indicating the



surface modification did not cause a significant effect on the particle dimension.

a)

MSN

MA_0.43

MA_2.56

Zeta Potential, mV

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2924 -CH2-

3600 3200 2800 2400 2000 1600 1200 800 400

Wavelength Number,

cm-1

10 8 6 4 2 0 -2 -4 -6 -8 -10 -12

b)

0

MA

0.5

1

1.5

2

2.5

3

APTES Content, µM

Figure 2. (a) FTIR spectra of MSN and APTES-modified MSN (MA_0.43 and MA_2.56); (b) The change of ζ-potential (in PBS with pH 7.4) of APTES-modified MSN (MA) treated with an increase of APTES concentrations from 0 to 2.56 µM. Nitrogen adsorption-desorption characterization results were listed in Table 1. The increase of APTES concentration from 0 to 2.56 µM resulted in decrease of both surface area (from 511 to 194 m2/g) and pore volume (from 0.72 to 0.41 cm3/g). MA_0.43 presented 73% surface area and 86% pore volume of MSN, indicating surface modification with low APTES concentration below 0.43 µM could still maintain the main mesoporous structure of MSN. The further increasing APTES concentration led to a rapid reduction in surface area as well as pore volumetric value. These results suggest that the larger APTES concentration results in a higher degree in MSN’s surface, which may lead to higher coverage of mesopores, especially for the ones with smaller sizes. The above results also indicate that modification of MSN surface with APTES of different concentrations through an aqueous co-condensation method can be used to effectively adjust the surface and mesoporous structure of MSN. We propose that variation of loading-grafting process might be useful to meditate their drug loading capacity and the following release controllability.


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a)

b)

Figure 3. TEM photographs of (A) MSN and (B) MA_2.56. Table 1. Changes in surface area and pore volume of modified MSN samples as a function of APTES concentration. 0

0.43

0.86

1.71

2.56

2

511

371

254

196

194

Pore Volume (cm /g)

0.72

0.62

0.48

0.42

0.41

APTES Content (µM) BET Surface Area (m /g) 3

Table 2. Encapsulation efficiency of DOX in MDA and MAD samples as a function APTES concentration. Sample

EE, % a

Sample

EE, % a

MDA_0 MDA_0.43 MDA_0.86 MDA_1.71 MDA_2.56

76.0 ± 1.2 73.1 ± 3.4 74.0 ± 2.6 69.0 ± 4.5 71.6 ± 2.3

MAD_0 MAD_0.43 MAD_0.86 MAD_1.71 MAD_2.56

75.9 ± 1.2 43.4 ± 3.7 36.4 ± 4.9 17.6 ± 1.3 18.7 ± 1.3

a

Encapsulation efficiency (EE) = 100*We/W0, W0 stands for the initial DOX amount for drug encapsulation, and We means DOX amount which has been successfully loaded. Because doxorubicin has fluorescent property,36-39 it was used to be loaded into the modified MSN for their drug delivery study. It can be seen from Table 2, when concentration was varied from 0 to 2.56 µM, the encapsulation efficiency (EE) of system was rapidly decreased from 76 ± 1 to 18 ± 1%, while the EE of MDA systems nearly kept constant high encapsulation efficiency (~75%). FTIR spectra in Figure 4 indicate that MAD assumed an overlaid profile of MSN and DOX, where DOX



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shielded the intensity of the peak around 1085 cm−1 of siloxane asymmetric stretching of MSN. This suggests that DOX was loaded on the surface of MSN. As comparison, MDA had a FTIR curve similar to MSN, probably because DOX was loaded inside interior of MDA instead of covering on their surface. As such, this makes us propose that, if the drug is first loaded into MSN with mesoporous structure, drug can be more effectively encapsulated into the mesoporous pores inside MSN, which can be well protected by decoration of MSN with APTES outer layer. However, if MSN was first functionalized with APTES, the grafted APTES layer may form an additional barrier hamper the penetration of DOX in the inner part of MSN. The loading of DOX on the NPs probably come from mainly through electrostatic interactions with MSN outer surface, resulting in a significant decrease in loading ability.

Figure 4. Fourier transform infrared spectra of DOX, MSN, MAD_0.43 and MDA_0.43.


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Figure 5. The colloidal stability of MD and MDA_0.43 in PBS buffer (pH 7.4) at 0, 2, 4 and 24 h, respectively.

Nanomedicines which are used in vivo should keep a long-term colloidal stability, so the state of the dispersion of DOX-loaded MSN before and after APTES decoration was photographed with soaking time increase in PBS (pH 7.4). It can be seen from Figure 5, obvious precipitation of MD samples occurred after 4 h soaking in PBS buffer, while MDA_0.43 were able to maintain the stability after 24 h soaking, indicating that APTES surface modification improved the colloidal stability of MSN.



10 MD

Zeta Potential, mV

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MDA_0.43

5 0

7.4 -5

6.5

5 pH Value

-10 -15

Figure 6. Zeta potential of MD and MDA samples in PBS buffer at different pH values (7.4, 6.5 and 5.0). The DOX-loaded NPs were then investigated concerning their surface charges in PBS with pH values mimicking normal physiological (pH 7.4), tumoral extracellular microenvironments (pH 6.5) as well as endo-lysosomal conditions (pH 5.0). Both MD and MDA presented negative surface of -11.4 ± 0.4 and -4.8 ± 0.8 mV at pH 7.4, respectively (Figure 6). However, when pH value decreased to 6.5 mimicking extracellular microenvironment of solid tumor, MDA had a charge-reversal transition from negative value to positive one (0.9 ± 0.3 mV), while MD did not have such reversion sensitivity. It has to be mentioned that MSN themselves also present an increasing charges with pH decrease probably due to the existence of the higher concentrations at more acidic conditions. Since the amine groups tend to be it will be helpful to increase the surface charge of the nanoparticles as a whole. That is why adjustment of APTES concentrations caused a charge-reversal transition at pH This means that MDA can maintain slight negative surface charge in the blood to prolong circulation period for their accumulation around solid tumor. Upon reaching near tumor, the acidic environment may induce their transformation into slight


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charge state to enhance their uptake ability by cancer cells, resulting in a high drug accumulation intracellularly.26,27 Drug Release Study. In order to optimize drug bioactivity, it is desirable that the encapsulated drug should maintain a limited drug leakage during blood circulation and quicker release upon special stimuli existent in tumor location.12,34 It can be seen from Figure 7a, MDA_0.43 presented only around 10% DOX release in the first 24 h at normal physiological situation (pH 7.4), while about 40% release happened for both MD and MAD_0.43 systems. Furthermore, after 24 h, the former continues to give an increasing drug release, but the release for the latter systems finished after 8 h. These results indicate that, compared to the grafting-loading approach, the loading-grafting process is not only beneficial to achievement of a high drug loading efficiency but also to maintenance of a sustainable release behavior. The difference in the release behaviors probably attributed to that if the drug is first loaded into MSN, followed by capping with APTES, the formed APTES outer layers may limit the diffusion rate of the encapsulated DOX drug from the NPs, leading to a more sustainable release. On the other hand, if MSN was first functionalized with APTES, the modification may limit drug loading inside MSN. DOX drug may be mainly loaded on the NPs’ surface through surface-drug complexation via their electrostatic interactions, which are weak drug-matrix bonding, together with the DOX short diffusion pathway, resulting in a rapid DOX escape from the NPs (Figure 7a).



60

MD_7.4 MAD_0.43_7.4 MDA_0.43_7.4

a)

50 40 30 20 10 0

Cumulative Release, %


50 MDA_0.43_7.4 MDA_0.43_6.5 MDA_0.43_5.0

b)

40 30 20 10 0

0

8

16

24

32

40

48

0

20

40

80

MD_5.0 MDA_0.86_5.0 MDA_2.56_5.0

c)

60

80

100 120 140 160

Time, h

Time, h


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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MDA_0.43_5.0 MDA_1.71_5.0

60 40 20 0 0

20

40

60

80 100 120 140 160

Time, h

Figure 7. DOX release behaviors in PBS buffer at 37 °C from (a) MD, MAD_0.43 and MDA_0.43 (pH 7.4), (b) MDA_0.43 at pH 7.4, pH 6.5 as well as pH 5.0, (c) MD, MDA_0.43, MDA_0.86, MDA_1.71, MDA_2.56 at pH 5.0.

The materials were then evaluated concerning their release properties at normal pH 7.4 and acidic pH (pH 6.5 mimicking microenvironments of tumoral tissues, and pH mimicking endo/lysosomal conditions).40,41 The pH responsiveness of MDA_0.43 samples can be indicated by their acidic-triggered release rate (e.g., up to 48 h, only ± 0% DOX was release at pH 7.4, while 15 ± 0% and 25 ± 1% DOX release occurred 6.5 and 5.0) (Figure 7b). These results suggest that much amount of DOX may be preserved in the reservoir of the MDA samples during their circulation in the blood with physiological pH (7.4), while, upon their arrival at tumoral tissues (pH 6.5) or internalization into cells (pH 5.0), more drug will be released under triggering from acidic signal in these spaces. Therefore, the pH sensitivity of MDA samples is


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to increase drug delivery selectivity to tumors (high bioactivity) while reducing DOX release at normal spaces (less toxicity).11 Since variation of APTES concentration has been shown to exert a great effect on adjustment of surface area and mesoporous structure, we proposed this behavior would be also used to mediate DOX release controllability. To check this idea, the DOX release profile of MDA decorated with different APTES amount in PBS at pH 5.0 (mimicking endo-lysosomal compartment) were evaluated. As shown in Figure 7c, the increasing APTES concentration actually promote the sustainability of DOX release from MDA NPs (e.g., during the first 24 h period, cumulative DOX release from the modified MDA treated with the APTES concentrations of 0, 0.43 and 2.56 µM was 42 ± 0, 17 ± 3 and 10 ± 2%). This property can be employed for development of nanocarriers with different release speed, depending on specific purpose in biomedical applications.13,27 Biological study In Vitro. The biocompatibility of nanocarriers is a necessity for their entrance into the assembly line for biomedical applications.42,43 Therefore, the cytotoxicity of MSN and APTES-modified MSN in the absence of DOX was first quantitatively evaluated by culture with both KB cells (epithelial cancer cell model) and NOK cells (epithelial normal cell model) through MTT assay. Under 48 h incubation , similar to MSN, the MA samples maintained a high cell viability (~80%) to both KB cancer cells and normal cells till usage of high-concentrated nanoparticle solution (50 µg·mL−1), suggesting their good biocompatibility as a drug delivery platform (Figure 8). It has to



be noted that APTES-treated MSN still showed slight higher cytotoxicity to cells especially for samples modified with higher APTES amount at bigger nanoparticle concentrations, probably associated with their more positive charges than pure MSN.5 120

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CTR 33.3 µg/mL

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Figure 8. Cytotoxicity of pure MSN and APTES-treated MSN (MA_0.43 and MA_2.56) under 48 h treatment with (a) KB cells and (b) NOK cells (± standard deviation, n = 3). 120

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Figure 9. Cytotoxicity of KB cells treated with free DOX, MD, MDA_0.43, MDA_0.86, MDA_1.71, MDA_2.56 (with equivalent DOX concentration) under 48 h treatment period (± standard deviation, n = 3). KB cells and NOK cells were used for evaluation of bioactivity of DOX drug DOX drug released from MD and MDA nanoparticles with DOX amount from 0.5 to 3.0 µM. It can be seen from Figure 9, compared with free DOX drug, MD, MDA_0.86, MDA_1.71 samples displayed an accelerated antiproliferation toward KB


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cells at higher equivalent DOX concentrations. The increase of APTES concentration led to a decreased cytotoxicity, which may be related to the lower release rate and release efficiency in the same release period, which compared to MD system. It is possible that a further increase of APTES concentration may completely cover the surface of MSN, which can completely block the release of drug encapsulated. In this case, the modified MSN can act as a reservoir for imaging agent beyond anticancer drugs for bioimaging applications. This trend can be seen from the very low cytotoxicity for MDA_2.56 system with limited DOX release at 48 h (Figure 10a). Interestingly, although MDA_0.43 samples killed more KB cells, they displayed more biocompatibility for normal NOK cells, which may be attributable to a limited DOX neutral physiological conditions for normal cells (Figure 10 and Figure 7b).

a)

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Figure 10. Cytotoxicity of DOX, MD, MDA_0.43 and MDA_2.56 at the equivalent DOX amount under 48 h treatment with (a) KB cells and (b) NOK cells (± standard deviation, n = 3). As mentioned above, MDA_0.43, compared to MD and MAD_0.43 samples, presented more sustainable drug delivery ability, which may be beneficial to achievement of high bioactivity in the long period. For evaluation of the proposed



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cell viability with increasing culture time for which KB cells were treated with the samples was checked via MTT analysis. cell viability after 72 h treatment with MDA_0.43 with 0.5 µM DOX was obviously higher (89 ± 13%) as compared with the ones treated with DOX (36 ± 8%) and MD (53 ± 5%) (Figure 11). However, at the 96 h-culture time, MDA_0.43 gave an anticancer cytotoxicity (55 ± 16%) equivalent to that of MD (41 ± 9%). As mentioned above, the corresponding DOX-free samples biocompatible, and thus the cytotoxicity should be attributed to DOX released from MDA. It has to be noted that even free DOX and MDA_0.43 exhibited a higher cytotoxicity in vitro, their burst release behavior is not proper for in vivo application because most of drug during circulation may be released in the blood or in the RES systems, thus probably resulting in big side effects. The above results clearly suggest that the MDA_0.43 nanocarriers with ability to maintain bioactivity in a long period time are expected to act as a kind of effective nanoplatform for drug delivery. It is of great importance if nanomecines are helpful to enhance intracellular drug accumulation for improvement of its bioactivity.44 The cell uptake ability of the NPs was evaluated by tracking the status of DOX inside KB cells via fluorescence microscopy. As can been seen from Figure 12, 4 h treatment of cell with MDA_0.43 resulted in a higher intracellular reddish color than those treated with free DOX as well as MD samples, suggesting more DOX accumulation occurring for the former instead of the latter. MDA_0.43 are able to present slightly negative ζ-potential (-4.8 ± 0.8 mV) at physiological conditions (pH 7.4) and turn into slightly positive-charged state (0.9 ± 0.3 mV)


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under acidic extracellular tumor microenvironments (pH 6.5) (Figure 6). This charge-reversal property will entice cells to endocytose them through strong electrostatic interactions, while reducing membrane trapping of protonated DOX.5 More importantly, after 24 h, MDA_0.43 sample enabled to maintain a high-intensified reddish color intracellularly than those of free DOX and MD samples. The enhanced long-term DOX accumulation probably comes from the improved ability of the MDA nanoparticles taken up by cancer cells, together with their pH sensitivity which may accelerate DOX release from endo-lysosome into cytosol and nucleus for a long period of time (Figure 7).45

120

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1d

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100 80 60 40 20 0 X DO

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0.4

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. 43 _0

Figure 11. Cytotoxicity of KB cells under 24h-, 48h-, 72 h-, and 96 h- treatment with free DOX and the release media of MD, MDA_0.43 and MAD_0.43 samples with 0.5 µM DOX.



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Figure 12. Bright field and fluorescence microscopy photographs of KB cells after 4 and 24 h culture with DOX (0.5 µM), MD and MDA_0.43 with 0.5 µM DOX, respectively.


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To quantify DOX internalization ability, DOX accumulation inside cells was treated with the solution of MD, MDA_0.43 at equivalent DOX concentration (0.5 µM) and then analysed by flow cytometry. As shown in Figure 13, MDA presented a higher DOX accumulation than both DOX and MD. The higher intracellular DOX accumulation of MDA samples are in line with the results in Figure 12, which may be associated with their transition from negative charge into positive one when transferred from normal conditions to acidic tumor microenvironments.5

Figure 13. Quantification of DOX cellular uptake via flow cytometry assay upon 4 h treatment of KB cells with 0.5 µM DOX drug, as well as MD and MDA_0.43 nanoparticles containing 0.5 µM DOX. (± standard deviation, n = 3, * p < 0.05). CONCLUSIONS Charge-reversal MSN-based nanosystems (MDA) with high drug encapsulation efficiency and controllable drug delivery ability through drug loading (DL)/surface functionalization (SF) process via an aqueous APTES silylation technique. The process allows for effective loading of drug (doxorubicin, DOX) into MSN, and for



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adjustment of release controllability via the following formation of APTES outer through its post silylation. MDA displayed a ~10% of DOX release till 7 days, while corresponding DOX-loaded original MSN and SF-DL MSN (MAD) samples give a burst release (~45%) in 8 h under physiological conditions. Furthermore, this allows for adjustment of DOX release rate by variation of APTES concentrations. MDA samples present an improved uptake ability by cancer cells through their charge-reversal surface smartness under extracelluar microenvironments of solid and display a long-term anticancer cytotoxicity through their acidic-accelerated intracellular release property. Their combinative advantages including charge reversal sensitivity, high loading capacity, pH-sensitive release sustainability, and long-term therapeutic effects, endow them with potentials for delivery of a variety of therapeutic agents beyond DOX anticancer drug. It is also believed that the method of DL-SF stepwise functionalization can act as a versatile approach to modification of various kinds of mesoporous materials for biomedical applications.

ACKNOWLEDGEMENTS We thank the financial support from the funding from Shanghai Municipal Natural Science Foundation (15ZR1408500). The Key Program of National Natural Science Foundation of China (31330028) and 111 Project (Grant No: B14018) were also acknowledged for their financial granting.

AUTHOR INFORMATION Corresponding Author


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Key Laboratory for Ultrafine Materials of Ministry of Education, The State Key Laboratory of Bioreactor Engineering, Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, People’s Republic of China. E-mail address: [email protected] (Yulin Li); [email protected] (Yuan Yuan); [email protected] (Changsheng Liu) Author Contributions #

These authors equally contributed to this work.

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Clathrin-Mediated Endocytic Pathway. Biochem. Biophys. Res. Comun. 2007, 353, 26-32.


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