Multifunctional Magnetized Porous Silica Covered with Poly(2

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Multifunctional Magnetized Porous Silica Covered with Poly(2-dimethylaminoethyl methacrylate) for pH Controllable Drug Release and Magnetic Resonance Imaging Ling Li, Cheng Zhang, Run Zhang, Zushun Xu, Zhi Ping Xu, and Andrew Keith Whittaker ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b01131 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 21, 2018

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ACS Applied Nano Materials

Multifunctional Magnetized Porous Silica Covered with Poly(2-dimethylaminoethyl methacrylate) for pH Controllable Drug Release and Magnetic Resonance Imaging Ling Li⊥†, Cheng Zhang⊥‡§, Run Zhang‡§, Zushun Xu†, Zhiping Xu*‡§, Andrew K. Whittaker*‡§ † Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry-of-Education Key Laboratory for the Synthesis and Application of Organic Function Molecules, Wuhan, Hubei University 430062, People’s Republic of China. ‡ Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, 4072, Australia. §ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, The University of Queensland, Brisbane, QLD, 4072, Australia.

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Abstract The

authors

describe

a

smart

magnetic-targeting

drug

carrier

γ-Fe2O3@SiO2-PDMAEMA that consists of γ-Fe2O3 and coated with porous silica, further modified by poly(2-dimethylaminoethyl methacrylate). In this research, the porous silica was firstly coated with poly(2-dimethyl -aminoethyl methacrylate). The material can be loaded with up to79 mg g⁻¹ of Doxorubicin (DOX) and 90 mg g⁻¹ of rhodamine B (RhoB) (acting as a model drug), which are both greatly larger than reported materials. Cytotoxicity experiments indicated the biocompatibility of γ-Fe2O3@SiO2-PDMAEMA, which is beneficial as drug carriers. DOX and RhoB were both released rapidly at pH 5.5 but the drug release strongly decreased at pH 7.4. The γ-Fe2O3@SiO2-PDMAEMA showed an excellent pH-triggered drug release. The possibility of the particles in magnetic resonance imaging (MRI) was discussed and the significant dose-dependent contrast enhancement in T2-weighted MRI suggested that γ-Fe2O3@SiO2-PDMAEMA can be potentially applied in T2 MRI. The fluorescence imaging of γ-Fe2O3@SiO2-PDMAEMA loaded with RhoB proved the particles can be taken up by cells. All the results exhibit a promising application in safe cancer therapy.

Key words: porous silica; PDMAEMA; pH-responsive; MRI; fluorescence; breast cancer

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1. Introduction A current major challenge for cancer treatment is the engineering of smart multifunctional drug carriers combined with targeting, high drug loading, stimuli-responsive and diagnostic imaging.1-3 How to improve the accuracy of diagnosis and treatment is a long-standing challenge. Magnetic resonance imaging (MRI) is a diagnostic technique for detailed observation of the internal structure of the body with excellent spatial resolution.4 Fe3O4 and γ-Fe2O3 nanoparticles have been widely used as drug carriers not only because they are biocompatible and low toxic to human body, but also because they can be potentially used for magnetic targeting and MRI.5-7 However, a major problem with the application in the biological field is that their stability is relatively poor.8 In order to improve the stability, the formation of core-shell drug carriers with Fe3O4 or γ-Fe2O3 as the core and functional materials as the shell can protect the core from the biological environment and enhance the stability. The properties of drug delivery such as high drug loading and controllable release can be therefore improved.9,10 Therefore, research interest in magnetic core-shell particles has been steadily increasing, with major research focused on MRI, magnetic targeting, effective drug loading and release 3. Among the multifunctional drug carriers with magnetic nature, mostly are used to carry one kind of drug, but the use of the same drug will result in the antitumor drug resistance.11,12 Synergistic effects of the multiple drugs can enhance the efficiency of anti-cancer, so drug carriers which can carry different drugs are demanded.13-15 There functional groups of different drugs are not the same, which results in the difficulty to design a shell materials to carry different drugs on basis of the chemical adsorption with drug molecules. Hence, shell materials to carry different drugs on basis of the physical adsorption can be designed. The porous materials can carry the different drugs whose molecular sizes are smaller than the pore size. Silica shells can be easily formed on the surface of SPIONs and importantly responsive molecules can be also attached to achieve control of drug release.16, 17 As we reported before, porous silica shells can greatly improve the drug loading because 3



of its high surface area.18 In addition, the porous structure of porous silica can be controlled and the surface of silica can be modified by functional group for responsive release, thus it can be used for carry different drugs with high loading efficiency and stimuli in response to drug release. In the application of drug carrier, effective drug release is demanded. The pH of the tumor site is reported to be acidic while that of normal tissue is weakly alkaline.19, 20 Thus, the approach of delivery of drugs into cancer cells can be obtained through pH responsive carriers. Poly(2-dimethylaminoethyl methacrylate) (PDMAEMA), which has a tertiary amino group, has been investigated as pH responsive attaching segments. The pH-responsive behavior arises from protonation and deprotonation of the tertiary amine across the pKa.21, 22 As a consequence of this property a series of pH-responsive core-shell composites based on PDMAEMA have been synthesized as controlled drug delivery systems.23-26 Among these reports about the drug carriers using magnetic PDMAEMA, mesoporous silica for the increase of drug loading has not been reported, and PDMAEMA has not been coated by the surface of the porous shell. Therefore, core-shell materials, with Fe3O4 or γ-Fe2O3 as core and PDMAEMA modified mesoporous silica as shell, have potential to be used as a multi-functioned drug carrier for high loading efficiency, MRI, magnetic targeting and pH responsive release. Doxorubicin (DOX) is a chemotherapy medication used to treat cancer. Rhodamine B (RhoB) is a kind of dye which is widely used in biomedicine and biotechnology. DOX and RhoB are two kinds of drug models which represent different drugs with different sizes, which can be selected to prove the possibility of drug loading for different drugs. In addition, rhodamine B can help to prove whether the drug carrier can enter into the cells through the fluorescent imaging.27,28 Expired by this, we design and synthesis the pH-responsive magnetic particles using γ-Fe2O3 as the magnetic core, porous SiO2 as the shell modified by PDMAEMA as the pH-responsive segments (γ-Fe2O3@SiO2-PDMAEMA) (Scheme 1). The successful synthesis of the nanoparticles were confirmed by TEM, FTIR and TGA etc. The γ-Fe2O3@SiO2-PDMAEMA can be acted as both magnetically-targeted drug carrier and MRI contrast agent. More important, the γ-Fe2O3@SiO2-PDMAEMA particles 4


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have a high loading of drug, which can be released on a change in pH value. DOX and RhoB are used to discuss the properties of drug loading and pH controllable release.

Scheme 1. A schematic illustration of the synthesis of γ-Fe2O3@SiO2-PDMAEMA nanoparticles. 2. Experimental Section 2.1 Chemicals and Materials The chemicals and Materials were listed in the ESI. 2.2 Preparation of γ-Fe2O3@ SiO2-PDMAEMA The magnetic γ-Fe2O3 and γ-Fe2O3@SiO2 nanoparticles were prepared according to the literature.26 The detailed information was listed in the ESI. After the γ-Fe2O3@ SiO2 particles were obtained, they were redispersed in ethanol to form the ethanol solution of γ-Fe2O3@ SiO2

for use. 4 mL this solution was taken, and 0.6 mL

NH3·H2O and 0.4mL APTES were with stirring at 50 ℃ for 24h, then the γ-Fe2O3@SiO2-NH2 particles were achieved. After the γ-Fe2O3@SiO2-NH2 particles were washed with ethanol absolute repeatedly and dispersed in 4 mL ethanol absolute, 300 mg PDMAEMA and 19.6 mg EDC were added and the reaction system was being stirred for 24 h. Finally, the γ-Fe2O3@SiO2-PDMAEMA particles were obtained, which were washed with absolute ethanol repeatedly and redispersed in ethanol for use.

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2.3 Characterization and Cell viability The instruments , characterization methods and cell viability method were all listed in the ESI. 2.4 MRI Different amount of γ-Fe2O3@SiO2-PDMAEMA were dispersed in water to form series solutions. The concentrations of iron in the solutions were in the range of 0-1.0 mmol L-1. The solutions were put into series of 600 μL tubes, which were placed in a 9.4 T small animal MRI scanner (BrukerbioSpec 94/30 USR) for phantom images and relaxivity analysis. The measured relaxation rates (1/T1 and 1/T2) were plotted against the concentration of Fe, and from the slopes, the relaxivities (r1 and r2) were achieved. 2.5 Loading and release of DOX and rhodamine B After washed by phosphate buffer saline (pH 5.5), at room temperature, each 10 mg γ-Fe2O3@SiO2-PDMAEMA was added to 10 mL DOX solution (100 mg⋅ L-1) and RhoB solution (100 mg⋅ L-1), respectively and shaken for 24 h under dark conditions. After centrifugation, the drug loaded γ-Fe2O3@SiO2-PDMAEMA was washed with water and dried in a vacuum drier. The drug loading capacities for DOX and RhoB were separately calculated on basis of the absorbance of the solution at 480nm and 554 nm, respectively. The drug loading can be calculated according the following Equation: Drug loading =V(Cinitial drug - Cdrug in supernatant)/m drug carrier. Here, the concentration can be determined by UV-vis spectroscopy, V is the volume of the drug solution, m is the mass of the γ-Fe2O3@SiO2-PDMAEMA. For

the

example

of

the

DOX

release,

2

mg

of

DOX-loaded

γ-Fe2O3@SiO2-PDMAEMA was sealed in a semipermeable membrane and placed in 10 mL of PBS (pH 5.5 and pH 7.4). Both samples were placed at 37oC in the dark condition and gently oscillated. At intervals, 1 mL solution was taken and measured for absorbance at 480 nm and replaced with the same volume of fresh PBS. The experiments of the release of RhoB is the same as the release of DOX, and the concentration of the RhoB was determined at 554 nm. 2.6 Fluorescence cell imaging 6


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Imaging was conducted to ascertain if the γ-Fe2O3@SiO2-PDMAEMA were internalized by the MCF-7 cells. Cells were seeded onto coverslips and 1 mg⋅ mL-1 of γ-Fe2O3@SiO2-PDMAEMA loading with RhoB was added and incubated for 6 hours. The cells were fixed in 4% paraformaldehyde (PFA) for 15 minutes and then rinsed in PBS for 3 × 5 minutes. Subsequently, ActinRed™ 555 reagent (Thermo Fisher) which selectively labels F-actin was added and incubated with the fixed cells and for 30 minutes, then rinsed in PBS for 3× 5 minutes and fixed with DAPI in Vectashield. ActinRed™ 555 dye is excited at 540 nm and

the maximum emission

was observed at 565 nm. Confocal microscope (Zeiss LSM 710) was used to acquire the images and and ZEN software (Zeiss) was used to analyze . 27,28 2.7

MRI of MCF-7 cells

Cell MRI experiments were performed on 106 MCF-7 cells, which were incubated with γ-Fe2O3 @SiO2-PDMAEMA in a 50 μL tube containing 6% gelatin for 24 hours. The concentrations of γ-Fe2O3 @SiO2-PDMAEMA were 0, 30, 60, 120 and 480 µm⋅ mL-1, respectively. MRI Images were obtained on the MRI scanner with the following parameters: FOV=5.8 cm, MTX=256, TR=1000 ms and TE=20 ms. 3. Results and discussion 3.1 Characterization of γ-Fe2O3@SiO2-PDMAEMA

Figure 1. FTIR spectra (a) and expansions of the spectra in the range of 3300 cm-1 to 2500 cm-1 (b) of PDMAEMA, γ-Fe2O3@SiO2-NH2 and γ-Fe2O3@SiO2-PDMAEMA.

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Figure 1 (a) displays FTIR spectra of PDMAEMA, γ-Fe2O3@SiO2-NH2, and γ-Fe2O3@SiO2-PDMAEMA. The strong peak of PDMAEM at 1731 cm-1 is attributed to

the

carbonyl

stretching

vibration,

which

cannot

be

observed

in

γ-Fe2O3@SiO2-PDMAEMA. It can be explained by the interaction between the carbonyl group and the amino group of γ-Fe2O3@SiO2-NH2. Fig. 1 (b) shows the expansions of the spectra in the range from 3300 cm-1 to 2500 cm-1. The spectra of γ-Fe2O3@SiO2-NH2 and γ-Fe2O3@SiO2-PDMAEMA are very similar since the structure

of

γ-Fe2O3@SiO2-PDMAEMA

predominantly

retained

after

the

modification of γ-Fe2O3@SiO2-NH2. In the meantime, new characteristic peaks are observed

between

3250

cm-1

and

2500

cm-1

in

the

γ-Fe2O3@SiO2-PDMAEMA (Fig. 1 (b)). The peaks at 2940 cm-1

spectrum

of

are attributed to

the stretching of methyl and methylene, and the peak at 2820 cm-1 is attributed to the stretching of N(CH3)2 group, proving the the presence of PDMAEMA. It can be concluded that γ-Fe2O3@SiO2-PDMAEMA was prepared successfully. The intensities of the peaks between 3250 cm-1 and 2500 cm-1 in the γ-Fe2O3@SiO2-PDMAEMA are weak due to the small amount of PDMAEMA on the particle surface. Figure S1 displays the TGA decomposition curves of γ-Fe2O3@SiO2-NH2 and γ-Fe2O3@SiO2-PDMAEMA. The onset of decomposition of the γ-Fe2O3@SiO2-NH2 occurred at about 200 oC, and was due to the loss of -NH2. The decomposition of the γ-Fe2O3@SiO2-PDMAEMA commences at approximately 260 oC with a dramatic loss of weight at temperatures above 450 oC, consisting with the results reported for the decomposition of PDMAEMA .29 The TGA results indicate that the silica can be successfully coated by PDMAEMA with a content of ~ 5 wt%. The porous nature of the γ-Fe2O3@SiO2 and γ-Fe2O3@SiO2-PDMAEMA can be confirmed by analysis of the N2 adsorption-desorption isotherms, as shown in Figure S2. The curves both show an IV type isotherm according to the IUPAC classification. The relative pressures of 0.4-0.6 -Fe2O3@SiO2 indicates a uniform mesoporous structure. The average pore size of γ-Fe2O3@SiO2 is 3 nm and the average pore size of γ-Fe2O3@SiO2-PDMAEMA is 12 nm. The specific surface area of γ-Fe2O3@SiO2 is measured to be 718 m2/g by the BET method, while the pore volume is 0.886 cm3/g. 8


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In comparison, the specific surface are of γ-Fe2O3@SiO2-PDMAEMA is measured to be 447 m2/g by the BET method, while the pore volume is 0.708 cm3/g. The specific surface area decreases a lot and the pore volume changes a little, indicating the porous silica is successfully coated with PDMAEMA and has little effect on the structure of the porous silica, which can be used for drug loading.

Figure 2. TEM images of γ-Fe2O3 (a, b), γ-Fe2O3@SiO2 (c, d), γ-Fe2O3@SiO2-NH2 (e, f) and γ-Fe2O3@SiO2-PDMAEMA (g, h). As shown in Figure 2, TEM images confirms the size of γ-Fe2O3, γ-Fe2O3@SiO2, γ-Fe2O3@SiO2-NH2 and γ-Fe2O3@SiO2-PDMAEMA. It is clear that the γ-Fe2O3 particles were uniform in size and the average diameter is about 20 nm. The γ-Fe2O3@SiO2 particles show a distinct core-shell structure and the average diameter is about 60 nm. It can be seen that the outer shell possessed a porous structure. The γ-Fe2O3@SiO2-NH2 particles retain a similar core-shell structure, however the surface structure showed subtle differences, which may be due to the surface being modified by -NH2 group. The γ-Fe2O3@SiO2-PDMAEMA particles tend to aggregate, which can be explained by the amphiphilic properties of PDMAEMA.30 The size of the agglomerate through the aggregation of γ-Fe2O3@SiO2-PDMAEMA is approximately 200 nm. The size of the different particles can be further proved by the distribution of particle

9



size in ethanol, as shown in Figure 3 (a). It is obvious that the size of γ-Fe2O3 particles is about 25 nm, the size of γ-Fe2O3@SiO2 is about 70 nm, and that of γ-Fe2O3@SiO2-PDMAEMA is about 200 nm. The results are accordance with the results from TEM.

Figure 3. The distribution of particle sizes of different particles in ethanol (10 mg⋅ mL-1) (a) and the γ-Fe2O3@SiO2-PDMAEMA in solution (10 mg⋅ mL-1) in different pH environments (b). The distribution of particle sizes is determined by DLS of solutions having different values of pH in order to evaluate the pH-responsive performance of the γ-Fe2O3@SiO2-PDMAEMA. Figure 3 (b) clearly shows that the particle size decreases with increasing pH, mainly resulted from the deprotonation of the tertiary amino groups at higher pH and hence loss of charge on the polymer. The reason is that PDMAEMA has a pKa of ~7.4-7.8.31 In acid environment, because of the protonation of PDMAEMA, the chain will stretch and the particle size will increase. Therefore, PDMAEMA can be used to seal the surface of porous silica at high pH to keep the drug from being released or released in small amounts. At low pH, the coated PDMAEMA begins to swell so that the sealed channel is opened, causing the drug to release. As a result, so the drug can be triggered by the pH release through the different protonation of PDMAEMA in different pH environment. In order to help prove each coating of nanoparticles, the zeta potential of different

10


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particles is measured. The zeta potential of γ-Fe2O3@SiO2 , γ-Fe2O3@SiO2-NH2 and γ-Fe2O3@SiO2-PDMAEMA were −28.9 mV, 22.5 mV, and 31.8 mV, respectively. It indicates that the γ-Fe2O3@p-silica particles carry a negative charge, indicating the abundant -OH groups of γ-Fe2O3@p-silica. The γ-Fe2O3@SiO2-NH2 carry a positive charge, proving the successful amination through the electrostatic interaction. It is clear that after the coating of PDMAEMA, the positive charge increases, which results from the protonation of nitrogen atom of dimethylamino group in the synthesis process. The reaction mechanism of PDMAEMA and γ-Fe2O3@SiO2-NH2 is based on the hydroxylamine condensation between the -COOH of PDMAEMA and -NH2 of γ-Fe2O3@SiO2-NH2, as shown in Figure S3. The reaction between PDMAEMA and γ-Fe2O3@SiO2-NH2 is done through standard EDCI/DMAP esterification between the -COOH from the polymer ((propionic acid)yl butyl trithiocarbonate, PABTC as RAFT agent) and the -NH2 from the γ-Fe2O3@SiO2-NH2 .

Figure 4. Superparamagnetic behavior of γ-Fe2O3@SiO2-PDMAEMA. The superparamagnetic nature of γ-Fe2O3@SiO2-PDMAEMA is confirmed

by VSM,

and the magnetization curve is shown in Figure 4. The saturation magnetization of γ-Fe2O3@SiO2-PDMAEMA is 13.5 emu⋅ g-1, indicating the magnetic nature. The inserted figure is the photograph demonstrating the magnetic separation of the

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γ-Fe2O3@SiO2-PDMAEMA before and after using a magnetic field. It can be seen that both γ-Fe2O3@SiO2-PDMAEMA can be easily separated through exposure to a magnetic force. All the results prove the γ-Fe2O3@SiO2-PDMAEMA particles are magnetic, and therefore there is potential for targeted delivery of drug molecules to a specific site through the application of a magnetic field5. 3.2 Cytotoxicity of γ-Fe2O3@SiO2-PDMAEMA MTS

method

was

used

to

investigate

the

cell

cytotoxicity

γ-Fe2O3@SiO2-PDMAEMA to both 3T3 cells and MCF-7 cells.

of

the

It can be observed

from Figure 5 that the γ-Fe2O3@SiO2-PDMAEMA shows no obvious cytotoxicity to both 3T3 cells and MCF-7 cells. The cell viability both remains at 95% even for concentrations of γ-Fe2O3@SiO2-PDMAEMA as high as 1200 μg⋅ mL-1. It is clear that the γ-Fe2O3@SiO2-PDMAEMA nanoparticles don’t dramatically affect the viability of the cells.

Figure 5. Relative cell viabilities of 3T3 and MCF-7 cells cultured in solutions of γ-Fe2O3@SiO2-PDMAEMA. Error bars represent standard deviations obtained from three measurements. 3.3 MRI In order to demonstrate MR imaging, T1 and T2-weighted MR images are obtained at 9.4 T for aqueous solutions of the γ-Fe2O3@SiO2-PDMAEMA at different concentrations. Furthermore the longitudinal (r1) and transversal relaxivity (r2) are 12


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also measured at this field strength. Figure 6 (a) shows that both T1- and T2-weighted MRI images exhibit obvious concentration-dependent contrast enhancement. As shown in Figure 6 (b), the relaxivities r1and r2 are 0.5 mM-1s-1 and 157.7 mM-1s-1, respectively. The figure shows that r2>>r1, which means that the nanoparticles will act predominantly as T2 rather than T1 contrast agents.32 Reports of the relaxivity r2 of iron oxide particles at 9.4 T are sparse, however Smolensky et al.

reported the r2 of

hexade cyltetramethylammonium bromide (CTAB) coated iron oxide particles ranging from 20-200 mM-1s-1 at different field strengths in the range of 20 MHz (0.5 T) to 500 MHz (11.7 T).33 The large r2 value of the nanoparticles in the current study can be explained by that the water molecules are easily accessible to the magnetic core by the PDMAEMA modified mesoporous silica shell.

Figure 6. MRI images of the γ-Fe2O3@SiO2-PDMAEMA in aqueous solution at different concentrations at 9.4 T (a). T1 and T2 relaxivity plots for aqueous solutions of the γ-Fe2O3@SiO2-PDMAEMA nanoparticles at 9.4 T (b). 3.4 Loading and release of DOX and RhoB The level of drug loading achieved with γ-Fe2O3@SiO2-PDMAEMA is significantly higher than in previous reports for loading into nanoparticles. To be more specific, the 13



DOX loading is 79 mg⋅ g-1 and the RhoB loading is 90.0 mg⋅ g-1 in our study, which are much higher than other reported drug carrier.34, 35 This demonstrates that excellent drug-loading efficiency of the γ-Fe2O3@SiO2-PDMAEMA can be achieved. It further proves that the porous silica can help to improve the drug loading. In order to prove the stability of the γ-Fe2O3@SiO2-PDMAEMA particles as drug carriers, the distribution of particle sizes and the TEM images after loading DOX and RhoB are shown in Figure S4. It is clear that the dimensions of DOX and RhoB are different. In addition, after loading DOX or RhoB, there is no obvious change in the particle size. It indicates that stability of the γ-Fe2O3@SiO2-PDMAEMA in water solution. The stability of the γ-Fe2O3@SiO2-PDMAEMA can be further prove by the TEM images of the γ-Fe2O3@SiO2-PDMAEMA after drug loading, as shown in Fig. Figure S4 (c1, c2) and Figure S4 (d1, d2). Compared with the the TEM image of γ-Fe2O3@SiO2-PDMAEMA before drug loading, the γ-Fe2O3@SiO2-PDMAEMA particle show the similar aggregation with the similar size. It not only proves the stability of γ-Fe2O3@SiO2-PDMAEMA as drug carrier, but also shows the ability for drug loading for drugs with different sizes. The drug release experiments are carried out in PBS in different pH conditions (7.4 and 5.5) at 37 ℃, as shown in Figure 7. The rates and extents of released DOX and RhoB are both strongly pH-dependent. The cumulative DOX release reaches 60% after 6 h at pH 7.4. The cumulative RhoB release reaches 15 % after 10 h at pH 7.4 but reaches 80% in the pH 5.5 environment. In the pH 7.4 environment, the coated PDMAEMA seals the channels of the porous silica, thus little drugs can be released. In an environment of pH 5.5, chain extension occurs due to protonation of the tertiary amino group of PDMAEMA, therefore, the channel of the porous silica is opened and the drugs can be released. In addition, the release of RhoB is faster and reaches 80% only after 4 h. It can be explained that the size of RhoB is smaller than that of DOX, and RhoB is more easily released from the porous channel after the channels are opened. Therefore, the porous silica can load different drugs and can achieve the pH release through the coating of PDMAEMA.

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Figure

7.

DOX

release

(a)

and

RhoB

release

(b)

from

the

γ-Fe2O3@SiO2-PDMAEMA nanoparticles in PBS with different pH values (7.4 and 5.5) at 37 ℃. Error bars represent standard deviations obtained from three measurements. For the potential application of these pH responsive nanoparticles in vivo, a faster release will occur once the tumor cells ingest the nanoparticles by endocytosis, the pH of the endocytic region ranges from 4.5 to 6.5, well below the normal physiological pH.

Figure 8. Ortho projection of 3D confocal fluorescence images, The nuclei are stained with DAPI (blue), NPs (green) and actin is stained red, 40X magnification. Excitation wavelengths are 358 nm, 492 nm and 540 nm, respectively.

15



In order to confirm the internalization of the γ-Fe2O3@SiO2-PDMAEMA nanoparticles with MCF-7 cells, confocal fluorescent microscopy images are conducted and shown in Figure 8. The boundaries of the cells are labelled with ActinRedTM 555 reagent and shown in red. The ortho projection of the 3D image displays in Figure 8 clearly shows that the nanoparticles have been taken up and stay within the cells after incubation.36 In order to confirm the cell imaging of the γ-Fe2O3@SiO2-PDMAEMA nanoparticles with MCF-7 cells, 1H MRI images are conducted and shown in Figure 9 (a). It can be seen that the MRI images in cells show concentration-dependent contrast enhancement, and the MRI signals enhance with increasing particles concentration, as shown in Figure 9 (c).37 It proves that γ-Fe2O3@SiO2-PDMAEMA can be successfully used as a T2-MRI contrast in cells. From Figure 9 (b), it reveals the concentration- dependent enhancement in green fluorescence. It further proves that the RohB loaded γ-Fe2O3@SiO2-PDMAEMA can be taken up and stay in cells.

Figure 9. MRI detection of MCF-7 cells with different incubation concentration of the γ-Fe2O3@SiO2-PDMAEMA nanoparticles. (a) 1H MR and (b) fluorescence images of gelatin samples containing 106 MCF-7 cells. (c) MRI signal enhancement as a function of incubation concentration. The error bars represent the standard deviation of 3 times 4. Conclusions In summary, γ-Fe2O3@SiO2-PDMAEMA nanoparticles have been successfully prepared with a view to improving the efficiency of drug loading and controlled release of basic drugs. The experimental results confirm the formation of magnetic γ-Fe2O3@SiO2-PDMAEMA nanoparticles with high drug loading for DOX and RohB, 16


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and clearly demonstrates pH-dependent release of the drug. Furthermore, MRI images indicate the γ-Fe2O3@SiO2-PDMAEMA

can be used for T2 -MRI to detect MCF-7

cells. Cytotoxicity tests against 3T3 cells and MCF-7 cells demonstrate that the nanoparticles

are

with

high

biocompatibility.

As

a

conclusion,

these

γ-Fe2O3@SiO2-PDMAEMA nanoparticles can be potentially used for T2 magnetic resonance imaging and pH controllable drug release. The high drug loading achieved in our study is mainly due to the high adsorption capacity of the porous silica. Supporting Information In this supporting information, chemicals and materials, detailed preparation of γ-Fe2O3@ SiO2-PDMAEMAEDS, characterization method, cell viability experiment, TGA data, N2 adsorption-desorption isotherms, the reaction mechanism, the particle size and TEM images after drug loading are introduced. Author information ⊥These authors contributed equally to this work. *Corresponding Author Zhiping Xu: [email protected]; Andrew K. Whittaker: [email protected] ORCID Ling Li: 0000-0002-2046-3462 Cheng Zhang: 0000-0002-2722-7497 Run Zhang: 0000-0002-0943-824X Zushun Xu: 0000-0001-7314-170X Zhiping Xu: 0000-0001-6070-5035 Andrew K. Whittaker: 0000-0002-1948-8355 Acknowledgments This work was supported by Natural Science Foundation of Hubei province (No.2017CFB530), Wuhan Morning Light Plan of Youth Science and Technology (No.2017050304010282) and the National Natural Science Foundation of China (No.51302071). We acknowledge funding from the Australian Research Council (DP110 104299 (AKW)). This research was supported by the ARC Centre of Excellence in Convergent Bio-Nano Science and Technology (CE140100036). C.Z. 17



Page 18 of 32

acknowledges the University of Queensland for his Early Career Researcher Grant (UQECR1720289). The authors acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland. This work was performed in part at the Queensland node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano- and micro-fabrication facilities for Australia’s researchers. References (1) Syamchand, S.S.; Sony, G. Multifunctional Hydroxyapatite Nanoparticles for Drug Delivery and Multimodal Molecular Imaging. Microchim Acta 2015, 182, 1567-1589. (2) Yang, Y. Upconversion Nanophosphors for Use in Bioimaging, Therapy, Drug Delivery and Bioassays. Microchimica Acta 2014, 181, 263-294. (3) Fang, J.H.; Chiu, T.L.; Huang, W.C.; Lai, Y.H.; S.Y.

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TOC

Multifunctional magnetized porous silica covered with poly(2-dimethylaminoethyl methacrylate) for pH-responsive drug delivery and magnetic resonance imaging

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Scheme 1. A schematic illustration of the synthesis of γ-Fe2O3@SiO2-PDMAEMA nanoparticles. Figure 1. to

FTIR spectra (a) and expansions of the spectra in the range of 3300 cm-1

2500

cm-1

(b)

of

PDMAEMA,

γ-Fe2O3@SiO2-NH2

and

γ-Fe2O3@SiO2-PDMAEMA. Figure 2. TEM images of γ-Fe2O3 (a, b), γ-Fe2O3@SiO2 (c, d), γ-Fe2O3@SiO2-NH2 (e, f) and γ-Fe2O3@SiO2-PDMAEMA (g, h). Figure 3. The distribution of particle sizes of different particles in ethanol (10 mg⋅ mL-1) (a) and the γ-Fe2O3@SiO2-PDMAEMA in solution (10 mg⋅ mL-1) in different pH environments (b). Figure 4. Superparamagnetic behavior of γ-Fe2O3@SiO2-PDMAEMA Figure 5. Relative cell viabilities of 3T3 and MCF-7 cells cultured in solutions of γ-Fe2O3@SiO2-PDMAEMA. Error bars represent standard deviations obtained from three measurements. Figure 6. MRI images of the γ-Fe2O3@SiO2-PDMAEMA in aqueous solution at different concentrations at 9.4 T (a). T1 and T2 relaxivity plots for aqueous solutions of the γ-Fe2O3@SiO2-PDMAEMA nanoparticles at 9.4 T (b). Figure

7.

DOX

release

(a)

and

RhoB

release

(b)

from

the

γ-Fe2O3@SiO2-PDMAEMA nanoparticles in PBS with different pH values (7.4 and 5.5) at 37 ℃. Error bars represent standard deviations obtained from three measurements. Figure 8. Ortho projection of 3D confocal fluorescence images, The nuclei are stained with DAPI (blue), NPs (green) and actin is stained red, 40X magnification. Excitation wavelengths are 358 nm, 492 nm and 540 nm, respectively. Figure 9. MRI detection of MCF-7 cells with different incubation concentration of the γ-Fe2O3@SiO2-PDMAEMA nanoparticles. (a) 1H MR and (b) fluorescence images of gelatin samples containing 106 MCF-7 cells. (c) MRI signal enhancement


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as a function of incubation concentration. The error bars represent the standard deviation of 3 times



Scheme 1.


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Figure 1.



Figure 2 .


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Figure 3 .

Figure 4 .



Figure 5 .


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Figure 6 .

Figure 7 . .



Figure 8 .

Figure 9 .


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Multifunctional Magnetized Porous Silica Covered with Poly(2

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