pH-triggered Charge-Reversal Mesoporous Silica Nanoparticles

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pH-triggered Charge-Reversal Mesoporous Silica Nanoparticles stabilized by Chitosan oligosaccharide/Carboxymethyl Chitosan hybrids for Effective Intracellular Delivery of Doxorubicin Lan Cui, Wentao Liu, Hao Liu, Qian Qin, Shuangxia Wu, Suqin He, Xinchang Pang, Chengshen Zhu, and peihong shen ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00830 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 4, 2019

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ACS Applied Bio Materials

Schematic illustration of DOX@MSNs-CSCMC

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pH-triggered Charge-Reversal Mesoporous Silica Nanoparticles stabilized by Chitosan oligosaccharide/Carboxymethyl Chitosan hybrids for Effective Intracellular Delivery of Doxorubicin Lan Cui,‡a, Wentao Liu,‡ *a Hao Liu, *a Qian Qin, a Shuangxia Wu,a Suqin He,a Xinchang Pang,a Chengshen Zhu*a and Peihong Shenb a

School of Material Science and Technology, Zhengzhou University, Zhengzhou,450001, China.

b

Department of Pathology, The Cancer Hospital of Henan (Affiliated Tumor Hospital of Zhengzhou University)

Zhengzhou, 450003, China. ‡ These authors contributed equally to this work.

ABSTRACT: Surface modification of mesoporous silica nanoparticles (MSNs) is a promising way to enhance therapeutic efficacy and minimize side effects of anti-cancer drugs. In this work, MSNs with reduced particle size and optimum pore diameter were obtained and catalyzed by ammonia/triethanolamine. In view of the negative–charged carboxymethyl chitosan (CMC) and positive–charged chitosan oligosaccharide (CS), the pH-triggered charge-reversal CS/CMC bilayer was designed as a stimuliresponsive switch for MSNs via the protonation and deprotonation effect. Results showed that MSNs-CS/CMC were core-shell and mesoporous in structure. Surface charge conversion and pH dependence was clearly observed in the doxorubicin hydrochloride (DOX) delivery. The intracellular uptake indicated that DOX@MSNsCS/CMC could be distributed in the cytoplasm of MCF-7 cells and exhibited lower toxicity, which would improve the stability and prolong the retention time than free DOX and unmodified DOX@MSNs at pH 7.4. Moreover, the cellular uptake and internalization of DOX@MSNs-CS/CMC were enhanced to promote drug delivery into cell nucleus at pH 6.5. The biocompatible and surface charge-reversible MSNsCS/CMC have the potential to prolong the retention time in bloodstream, facilitate the endosome escape and enrich the targeted anti-tumor strategy, providing an alternative platform for efficient drug delivery in breast cancer therapy. KEYWORDS: mesoporous silica, chitosan, doxorubicin hydrochloride, charge reversal, pH-sensitive, breast cancer cells.


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1. INTRODUCTION Breast cancer, one of the most common disease in women, has been the major issue to the human health throughout the world.1 Nowadays, chemotherapeutic therapy remains to be the main strategy for breast cancer treatments.2, 3 Whereas chemo-drugs, such as doxorubicin, paclitaxel and 5-fluorouracil, have been proved to cause side effects on normal cells. To date, various nanocarriers have been utilized to encapsulate chemo-drugs to improve the tumor specificity, but the biocompatibility, particle size, surface charge property, drug-loading capacity and drug release efficiency of nanoparticles have been the main obstacles to the biomedical application.4, 5 Mesoporous silica nanoparticles (MSNs) have been extensively researched in the chemotherapy drug delivery, due to the high specific surface area, tunable pore structure and easy surface functionalization.6-8 It has been reported that MSNs as the drug carriers presented higher drug loading efficiency.9 However, the dose-dependent toxicity remains a substantial barrier in biomedical application.10-13 Besides, nanocarriers should possess a long lifetime during the blood circulation and present intelligent drug delivery in vivo.14 Consequently, surface functionalization is necessary to improve the biocompatibility and cancer therapy efficacy. Inorganic and organic molecule,15-18 polymer,19-24 supramolecule

25, 26

are widely used as the sealing agents and bioactive

moieties for the surface modification. Compared with synthetic polymers, chitosan has elicited numerous attentions due to its excellent biocompatibility, biodegradability and nontoxicity.27-29 Chitosan oligosaccharide (CS), the hydrolytic product of chitosan, has been reported to show antioxidant and anti-inflammatory effects on the drug-induced renal failure. 30 In addition, researchers have indicated that "core-shell" structure could be constructed in chitosan-MSNs by electrostatic adsorption or chemical bond, which would be beneficial to improve the biocompatibility and uptake availability.23 Nevertheless, positively charged nanocarriers are severely aggregated and prone to interact with serum proteins and normal tissues in a nonspecific manner, which would cause rapid clearance via the phagocytosis of reticuloendothelial system during the blood circulation.31



Recently, charge reversible nanocarriers have been developed and show significant superiority in the drug delivery system.21 The surface charge of nanocarrier is expected to remain negative to improve the stability and prolong the retention time in bloodstream. On the basis of charge-reversal character, the surface charge and configuration could be changed to trigger drug delivery to the cytoplasm or extracellular space under the tumor tissues condition (pH 6.5–6.9). Moreover, due to the endocytosis of lysosomes and endosomes, the chemotherapy drugs would be intelligently transported to cellular nucleus in tumor tissues at pH 5.0–5.5.32, 33 In this study, charge-reversible and pH-responsive nanocarrier was designed via the self–assembly between the hydroxyl of MSNs and the protonated amino groups of CS, and the outmost layer was carboxymethyl-modified chitosan (CMC) due to its hydrophilic carboxyl group. Meanwhile, doxorubicin hydrochloride (DOX) was used as the model drug to evaluate the drug delivery and antitumor activity. The surface charge and configuration of MSNs-CS/CMC were investigated to evaluate the drug loading and release behavior in acidic conditions. The intracellular toxicity and cellular uptake and internalization were also evaluated in human breast cancer MCF-7 cells. The aim of this study is to focus on the effective intracellular drug delivery and favorable biocompatibility in breast cancer therapy.

2. EXPERIMENTAL SECTION 2.1 Materials Tetraethyl orthosilicate (TEOS), ammonia, triethanolamine, cetyltrimethyl ammonium bromide (CTAB), chitosan (molecular weight of 200, 500 and 1000 kDa, deacetylation degree of 85%), sodium tripolyphosphate (TPP) and doxorubicin hydrochloride (DOX) were obtained from Aladdin Chemistry Co., Ltd. (Shanghai, China). chitosan oligosaccharide (molecular weight of 3 and 10 kDa, deacetylation degree of 80%) was purchased from Yuhuan Marine Biochemistry Co., Ltd. (Zhejiang, China). All reagents and solvents were of analytical grade. The human breast cancer cells (MCF-7 cells) were cultured in Eagle's minimal essential medium (EMEM)


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containing 10% FBS, 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). 2.2 Design and Characterization of MSNs MSNs were derived from tetraethyl orthosilicate (TEOS) through sol-gel process with some changes.17 MSNs with high specific surface area and optimum pore diameter were catalyzed by ammonia/triethanolamine. Triethanolamine has been reported to be competitively involved in the hydrolysis of TEOS and inhibit the subsequent polycondensation.34 Triethanolamine was mixed with ammonia (2 mL) at the volume of 0, 0.2, 1 and 2 mL, respectively. Cetyltrimethyl ammonium bromide (CTAB, 3.6 g) was dissolved in 250 mL of distilled water containing ethanol (20 mL) and ammonia/triethanolamine mixture at 60 °C and magnetically stirred for 1 h. Subsequently, 5 mL of TEOS was dropwise added and magnetically stirred for another 24 h. The products were collected by centrifugation and refluxed with 120 mL of ethanol solution containing hydrochloric acid (1 mL, 37%) for 3 h. The reflux procedure was repeated three times to completely remove the template CTAB. The as-synthesized materials (named as MSNs-1, MSNs-2, MSNs-3 and MSNs-4) were washed with ethanol and distilled water, then lyophilized under high vacuum. The morphologies of nanoparticles were observed by transmission electron microscopy (TEM, Hitachi-HT7700, Hitachi Ltd., Japan) and field emission scanning electron microscope (FESEM, JSM-7001F, JOEL Ltd., Japan). The size distribution and zeta potential were measured by dynamic light scattering (DLS, Zetasizer NanoZS90, Malvern Instruments Limited, UK). The structures of nanoparticles were characterized by Fourier Transform infrared spectroscopy (FT-IR, VERTEX70 spectrometer, Bruker Corporation, German) and X-ray photoelectron spectroscopy (XPS, Axis Supra, Kratos Ltd., UK). 2.3 Preparation of CS/CMC Polyelectrolyte Complexes Carboxymethyl chitosan (CMC, molecular weight of 200 kDa, deacetylation degree of 85%, substitution degree of 92% as shown in Figure S1) was synthesized as



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described by Chen et al.35 Subsequently, CS/CMC polyelectrolyte nanoparticles were prepared by ionic crosslinking method. The specific operation was employed as follows: Chitosan was dissolved in 2% acetic acid solution. Sodium tripolyphosphate (TPP, 0.5 mg/mL, 2 mL) was dropwise added to the chitosan solution (CS, 1 mg/mL, 3 mL) and magnetically stirred for 0.5 h. Subsequently, carboxymethyl chitosan (CMC, 1 mg/mL, 4 mL) was blended with the mixture under constant stirring for 1 h. 2.4 Construction of DOX@MSNs-CS/CMC DOX was loaded into the MSNs channels by capillary action. The concentration of DOX was examined by ultraviolet spectrophotometer at 481 nm (UV-721G, Jingke Co., Ltd., Shanghai, China). The drug loading method of MSNs-CS/CMC was designed as follows: 10 mg of MSNs were dissolved in 10 mL of phosphate buffer solution (PBS) at pH 7.4. DOX (0.5 mg/mL) was added to the solution and stirred for 48 hours under dark condition. Then sodium tripolyphosphate (TPP, 0.25 mg/mL) was dropwise injected and magnetically stirred for 0.5 h. Subsequently, CS (1mg/mL) was mixed with the suspension and stirred for 0.5 h. Then CMC (1mg/mL) was added and magnetically stirred for another 2 h. The encapsulation efficiency and loading efficiency were calculated based on formula (1) and (2), respectively: Encapsulation efficiency (%) = Loading efficiency (%) =

weight of DOX in nanoparticles total weight of DOX

weight of DOX in nanoparticles weight of nanoparticles

×100% (1)

×100%

(2)

2.5 Drug Release of DOX@MSNs-CS/CMC The drug release of DOX@MSNs-CS/CMC was determined by dialysis in vitro. The drug-loaded nanoparticles were added into the dialysis bag (a cellulose membrane, molecular weight cut-off of 500 Da) and placed in 25 mL of phosphate buffer solution at different pH value. Subsequently, the nanoparticles were kept under constant shaking for 100 rpm at 37 °C. Samples were collected at appropriate intervals, meanwhile, a corresponding equal volume of fresh buffer was replenished into the tube. The absorbance of DOX was determined by ultraviolet spectrophotometer at 481 nm, the cumulative release rate was calculated according to the standard curve.


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2.6 In Vitro Cytotoxicity of DOX@MSNs-CS/CMC The CCK-8 assay was applied to quantify the cytotoxicity of free DOX, DOX@MSNs and DOX@MSNs-CS/CMC against MCF-7 cells. MSNs and MSNsCS/CMC were also investigated to evaluate the cytotoxicity of blank nanocarriers. The cells without treatment were chosen as the blank control. MCF-7 cells were seeded in 96-well plates at the density of 1×104 cells/well and cultured to grow for 24 h. Subsequently, the culture media containing DOX loaded nanoparticles were transferred to the 96-well plates for another 24 and 48 h. The DOX loaded samples were added at the DOX concentrations ranging from 0 to 100 μg/mL. Afterwards, the culture media were totally removed and cells were washed with PBS. Then fresh media containing CCK-8 solution were appended to each well. After incubated for 3 h, the samples were measured by the multimode plate reader (EnSpire, PerkinElmer, USA) at 450 nm. 2.7 Analysis of Intracellular Drug Release Confocal laser scanning microscope (CLSM) was applied to investigate intracellular uptake of the charge-reversible DOX@MSNs-CS/CMC. MCF-7 cells were seeded into a glass-bottom dishes at the density of 1×105 cells/dish and cultured to grow at 37 °C for 24 h. Subsequently, culture media containing DOX@MSNs and DOX@MSNs-CS/CMC at equivalent DOX concentration (5 μg/mL) were transferred to the glass-bottom dishes for another 24 h. Then the cells were washed three times with PBS and fixed with paraformaldehyde at 4 °C for 15 min, the cell nucleus were stained with Hoechst for 5 min. The internalization of nanoparticles in MCF-7 cells were observed by Nikon eclipse Ti confocal microscope under the excitation of 401 nm (blue fluorescence) and 561 nm (red fluorescence). 2.8 Quantitative Evaluation of Intracellular Uptake by Flow Cytometry Analysis Flow cytometry was used for quantitative analysis of DOX red fluorescence in MCF-7 cells. MCF-7 cells were cultured in 6-well plates at the density of 2×105 cells /well for 24 h. The cells without treatment were chosen as the blank control. DOXloaded nanoparticles at the equivalent DOX concentration (5 μg/mL) were added and



incubated for 24 and 48 h. The culture media were collected and the cells were washed twice with PBS. Then the cells were digested with trypsin for 2 min. Subsequently, MCF-7 cells were centrifuged, washed and resuspended in 0.25 mL of PBS to analyze the fluorescence intensity by flow cytometer (BD LSRFortessa, USA). 2.9 Statistical Analysis Experiments were carried out at least three times and the data were expressed as mean ± standard deviation. Statistical analysis was determined by student’s t−test and one-way analysis of variance (ANOVA) with SPSS 21.0 software. *P < 0.05 and **P < 0.01 indicate statistical significance and extremely statistical significance, respectively.

3. RESULTS AND DISCUSSION 3.1 Preparation and Drug Loading Property of MSNs MSNs synthesized by sol-gel method were spherical with ordered mesoporous structure. The TEM images (Figure 1A–D) show that the obtained MSNs (MSNs-1, MSNs-2, MSNs-3, MSNs-4) have different particle sizes (100, 60, 40 and 20 nm) due to the complexing impact of triethanolamine/ammonia mixture on the hydrolysis rate and polycondensation degree of unisilicate. 34 Triethanolamine exhibited much stronger competition and acted as a growth inhibitor for MSNs, owing to the lower alkalinity (pKa of triethanolamine is 7.74, pKa of ammonia is 9.55) in aqueous media and the existence of a large number of dihydroxyethyl. Therefore, with the increase of triethanolamine volume, MSNs are prone to form smaller-sized particles. The size of nanoparticles plays an important role in blood circulation time and tumor accumulation. Nanoparticles smaller than 20 nm are reported for deep tumor penetration, but they are easily removed from blood circulation. In contrast, nanoparticles of about 100 nm have longer blood circulation and better tumor accumulation due to the enhanced permeability and retention effect.40 The obtained MSNs with smaller-size are suitable for further investigation. Furthermore, typical IV isotherm and H1 hysteresis loop were observed in the N2


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adsorption-desorption curves as shown in Figure 1E–F. The results suggest that the obtained MSNs exhibit hexagonal mesoporous structure, which is in accordance with the TEM image.34 Based on nitrogen adsorption isotherms, the surface area and pore size distribution was calculated by the Brunauer–Emmet–Teller (BET) method and the Barrett–Joyner–Halenda (BJH) method, respectively.34 MSNs-2 exhibited the higher surface area and optimum pore diameter and their adsorption capacity of N2 was rapidly increased in the low-pressure region (P/P0 = 0.2–0.4), where a large number of pores were formed at around 2−5 nm as shown in Table S1. While MSNs-3 and MSNs-4 exhibited a notable H4 type hysteresis loop and larger pore diameter at the relative pressure range of 0.8–0.9, which demonstrates a narrow slit-lick.24 The results indicate that MSNs-2 with ordered mesoporous structure and relatively narrow distribution of pore size are optimum to deliver small-sized doxorubicin hydrochloride (DOX, 1.5 nm). 24

MSNs-2 was selected as the drug carrier to encapsulate DOX via capillary action. The complexes can be formed by electrostatic interaction with the positively charged DOX (pKa=8.3) and negatively charged MSNs at pH 7.4.36 As shown in Figure 1G, the zeta potentials of MSNs were about −1, −12 and −14 mV at pH 5.5, 6.5 and 7.4, respectively. Therefore, the electrostatic interaction of hydroxyl groups in MSNs and amino groups in DOX were enhanced at pH 7.4, improving drug loading capacity of MSNs. Besides, the zeta potentials of DOX@MSNs were measured to be 12 ± 2 mV. Figure 1H shows that the encapsulation efficiency of DOX@MSNs was significantly increased with the mass ratio of MSNs/DOX. The drug loading efficiency was correspondingly reduced from 32% to 17%, nevertheless, the encapsulation efficiency of DOX@MSNs was significantly increased from 50% to 91%, when the mass ratio of MSNs/DOX increased from 1:1 to 4:1. The adsorption capacity of MSNs reached a plateau, which was attributed to the distribution of internal pores. The optimum encapsulation efficiency and drug loading efficiency are about 68 ± 4% and 25 ± 1%, respectively, when the MSNs/DOX mass ratio is 2:1.



Figure 1. Characterizations of mesoporous silica nanoparticles (MSNs). (A–D) TEM images of MSNs-1, MSNs-2, MSNs-3, MSNs-4. (E–F) Nitrogen sorption isotherm and pore size distribution curve of MSNs. (G) Zeta potentials


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of MSNs-2 at different pH value. (H) Effect of MSNs/DOX mass ratio on encapsulation efficiency

3.2 Drug Loading Analysis of Charge-Reversal CS/CMC Nanoparticles Researchers have indicated that MSNs capped by chitosan are beneficial to improve the biocompatibility and uptake availability,23 nevertheless, the nanocarriers are positively charged and prone to interact with serum proteins and normal tissues in a nonspecific manner, causing rapid

clearance via the phagocytosis

of

reticuloendothelial system during the blood circulation. Herein, charge reversal property was realized to enhance internalization via the protonated amino groups of CS and the deprotonated carboxyl of CMC, respectively, which has a fixed charge–reversal point around pH 6.5 as shown in Figure 2A. CS/CMC nanoparticles were prepared via ionic crosslinking interaction between the deprotonated carboxyl and protonated amino groups. Due to the electrostatic interaction, the nanoparticles were spherical in shape and maintained favorable stability. Figure 2B shows the FTIR spectra analysis of CS/CMC nanoparticles. The absorption bands at 1552 cm–1 and 1637 cm–1 were the characteristic peaks of amino groups in CS, absorption band at 1410 cm–1 was characteristic of CMC (–COO–). The appearance of new peaks in 1693 cm–1and 1234 cm–1 were attributed to the amide of carboxyl and amino groups condensation, moreover, the intensity of hydroxyl group at 3420 cm–1 decreased, indicating the electrostatic interactions between CS and CMC. The morphologies of CS/CMC nanoparticles were investigated and shown in Figure 2C, D. The nanoparticles were spherical in shape and uniformly distributed at different pH value. The electrostatic repulsion was weakened during drying, which led to the congregate structure in slightly acidic environment. Considering the swelling in solution (Figure 2E), the particle sizes measured by DLS were larger than that observed in SEM or TEM images. Due to the protonated amino groups of chitosan, the nanoparticles could present positive charges in the acidic condition (Figure 3F). Whereas the exposure of CMC endowed the nanoparticles with negative charges in neutral or basic condition. The results indicate that nanoparticles are smaller than 100 nm and possess excellent stability. The CS/CMC hybrids present reversible character



on the surface charge, which would endow the biocompatible CS/CMC shell with appropriate stability during the blood circulation (pH 7.4). The sizes of nanoparticles have predominant effects on blood circulation time and tumor accumulation, CS/CMC nanoparticles should be controlled at smaller sizes by changing the operational conditions. CS and CMC had the similar trends on the particle size (Figure S2A,B). The diameters of nanoparticles were rapidly increased with the excessive addition of CS and CMC. The nanoparticles were relatively smaller than 200 nm when the TPP concentration was adjusted to 0.25 mg/mL (Figure S2C). However, due to the gradually weakened protonation, the zeta potentials of CS/CMC particles were significantly decreased from 45 to 30 mV with further addition of TPP.37, 38 In view of the viscosity and intermolecular entanglement, Figure S2D reveals that the particle size was much larger when the molecular weight was increased to 1000 kDa. Moreover, the CS/CMC nanoparticles were rapidly formed by ionic crosslinking and electrostatic interaction in the first 1 h, and then the size reached a plateau after 10 h (Figure S2E). Due to the maldistribution of TPP, the particles had narrow distributions when the rotation rate was less than 500 rpm (Figure S2F). Considering the results, the optimum particle size and zeta potential of CS/CMC nanoparticles are about 140 nm and 25 mV, respectively, when chitosan (3 kDa) is added at an appropriate weight ratio of chitosan/TPP/CMC (3: 0.5: 4) and magnetically stirred at 500 rpm for 1 h. The molecular weight of CS, mass ratio of CS/DOX, reaction time and temperature have predominant effects on the encapsulation efficiency of DOX@CS/CMC nanoparticles. As presented in the Figure 3A, in view of the viscosity and intermolecular entanglement, the encapsulation efficiency was slightly declined with the molecular weight of chitosan. The encapsulation efficiency was slightly increased from 52% to 61% with the mass ratio of chitosan/DOX up to 30:1 (Figure 3B). Moreover, the high temperature was beneficial to the interaction between TPP and DOX (Figure 3C). Meanwhile, the reaction time had a negligible effect on the encapsulation efficiency (Figure 3D), the trend was similar to the particle size. The appropriate encapsulation and loading efficiency are about 78% and 6%, respectively, when TPP and DOX is pre-reacted at 80 °C, meanwhile, CS (3 kDa) is added at


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CS/DOX mass ratio of 15:1 and CMC is blended with the mixture and stirred for 4 h.

Figure 2. Schematic illustration, characterizations and effects of operating conditions on particle sizes and zeta potentials of CS/CMC nanoparticles. (A) Schematic illustration and (B) Infrared spectra of CS/CMC nanoparticles. Effects of (C) TEM and (D) SEM images of CS/CMC nanoparticles at different pH value. Effect of pH value on the (E) particle size and (F) zeta potential.



Figure 3. Effects of operating conditions on encapsulation and loading efficiency of CS/CMC nanoparticles. (A) Molecular weight of CS. (B) Mass ratio of CS to DOX. (C) Pre-reaction temperature of TPP and DOX. (D) Reaction time.

3.3 Preparation of Charge-Reversal DOX@MSNs-CS/CMC The schematic illustration of DOX@MSNs-CS/CMC is shown in Scheme 1. The positively charged DOX was encapsulated in MSNs by electrostatic interaction at pH 7.4. In view of the exposure of carboxyl groups in carboxymethyl chitosan (Figure S1, Substitution degree of 92%, Isoelectric points of 2.2, the percentage of free amino groups is 56%), deprotonated amino groups in chitosan (pKa 6.5) and amino groups in DOX (pKa=8.3), the charge reversal is related to the molar ratio of amino groups and carboxyl groups. Herein, the charge reversal of DOX@MSNs-CS/CMC were reversed to negative charge by protonation and deprotonation effect at pH 6.5, when the weight ratio of doxorubicin hydrochloride, chitosan and carboxymethyl chitosan was 0.2:3:4. The pH-responsive and core-shell MSNs-CS/CMC were successfully prepared (Scheme 1A). The surface charge reversal from negative (pH 7.4) to positive charge (pH 6.5) is clearly observed, which significantly promotes the cellular uptake and


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stimulates the DOX delivery to nucleus (Scheme 1B). The FTIR spectra analysis is presented in Figure 4A, two noticeable adsorption bands at around 1080 and 800 cm-1 were assigned to asymmetric and symmetric adsorption of Si-O-Si. The adsorption band at 1700 cm-1 was corresponded to C=O stretching vibration peak within DOX@MSNs. DOX@MSNs-CS/CMC exhibited absorption bands at around 1653 and 1524 cm-1, which were ascribed to the stretching vibrations of C-N and N-H. The results indicate that MSNs are successfully capped by the CS/CMC hybrids. Compared with DOX@MSNs, the average diameter of DOX@MSNs-CS/CMC measured by DLS was slightly increased to 200 ± 20 nm with narrow size distribution (Figure 4B). Figure 4C–D show that spherical and core-shell structure could be clearly observed in DOX@MSNs-CS/CMC and the particle size were slightly increased to 150 nm. The encapsulation efficiencies of nanocarriers were summarized in Table 1. Compared with MSNs nanocarriers, the DOX utilization ratio was greatly improved in the MSNs-CS/CMC nanocarriers, the encapsulation efficiencies were increased from 68% to 79%. The discrepancies resulted from the hydrogen bond and ionic crosslinking interaction between DOX and MSNs-CS/CMC hybrids. The surface structure and interaction of DOX-loaded nanoparticles were further evaluated by X-ray photoelectron spectroscopy (XPS) (Figure 5). Compared with MSNs, the intensities of Si2p and Si2s peaks of DOX@MSNs at 103.1 and 152.5 eV were decreased, confirming that DOX was incorporated on the surface of MSNs. Moreover, the significant decreases of Si2p and Si2s peaks and the appearance of Na1s peaks demonstrated the existence of CS/CMC shell. Additionally, the Si2p peaks of DOX@MSNs-CS/CMC at 101.9 and 103.2 eV were ascribed to Si-C and Si-O bonds, respectively. The C1s peaks at 284.7, 286.2, 287.9 and 288.4 eV were ascribed to C-N, C-C, C=O and -COO- bonds, respectively. 41 The N1s peaks at 399.5 and 402.2 eV were ascribed to N-C and N-H bonds, respectively. The results verify that the incorporation of DOX and CS/CMC shell with MSNs is successful.



Scheme 1. Schematic illustration of DOX@MSNs-CS/CMC. (A) The synthesis procedure of DOX@MSNsCS/CMC. (B) The charge-conversion and extracellular DOX Release of DOX@MSNs-CS/CMC by pH Trigger.


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Figure 4. Characterizations of DOX@MSNs-CS/CMC. (A) Infrared spectra and (B) Particle size distribution of DOX@MSNs-CS/CMC. (C-D) TEM images of DOX@MSNs and DOX@MSNs-CS/CMC. Table1. Drug loading performance of MSNs-CS/CMC

Weight ratios(mg) MSNs

CS

CMC

pH DOX

Zeta potential

Encapsulation

Loading

(mV)

efficiency

efficiency

(%)

(%)

10

－

－

5

7.4

12 ± 2

68 ± 4

25 ± 1

－

15

20

5

7.4

−14 ± 3

52 ± 4

9±1

10

15

20

5

7.4

−16 ± 2

79 ± 2

22 ± 1



Figure 5. XPS characterizations of MSNs, DOX@MSNs, DOX@MSNs-CS/CMC. (A) Surveys of all tested peaks. (B) Si2p XPS spectra, (C) C1s XPS spectra and (D) N1s XPS spectra of DOX@MSNs-CS/CMC.

3.4 pH Triggered Charge Reversal and Drug Release of DOX@MSNs-CS/CMC DOX release was depended on the electrostatic interaction of DOX, MSNs and CS/CMC at different pH value. Figure 6A shows that the surface charges of the nanoparticles have significant effects on the release process. The surface charge of DOX@MSNs-CS/CMC was reversed from −16 to 11 mV at pH 6.5. Afterwards, it was changed to 15 mV at pH 5.5. Moreover, the particle size was smaller than 200 nm at pH 5.5. (Figure 6B). As shown in Figure 6C, the cumulative release of DOX from DOX@MSNs was approximated to 7% at pH 7.4 after 200 h. The release rate was gradually improved in the acidic condition after 48 h (release rate was higher than 15% at pH 5.5). In contrast, pH sensitivity was clearly observed in the DOX release from DOX@MSNs-CS/CMC (Figure 6D). The cumulative release rate of DOX was much lower than 6% at pH 7.4 after 200 h. While the release rate of DOX@MSNs-CS/CMC


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was significantly improved in the acidic condition. The drug release rate could significantly reach 25% at pH 5.5 after 48 h, four times higher than the release at pH 7.4. Afterwards, the drug release could reach a plateau after 120 h, the release amount was higher than 50%. The release process of DOX@MSNs-CS/CMC can be divided into three stages at pH 5.5. Burst release of DOX was observed in the first 24 h. Subsequently, sustained release of DOX was observed after 24 h. The electrostatic repulsion between shell and positively charged drug promoted the DOX release when the pH value was adjusted to 5.5. Then the drug release could reach a plateau after 120 h, which is attributed to the gradual dissolution and degradation of chitosan nanoparticles. The results confirm that the DOX is encapsulated in the core MSNs and shell CS/CMC. The DOX@MSNsCS/CMC with tunable release capacity would provide a platform for the avoidance of frequent drug administration in breast cancer treatment.

Figure 6. The Drug release and charge reversal of DOX@MSNs-CS/CMC at different pH value. (A) Zeta potential and (B) particle size of DOX@MSNs-CS/CMC at different pH value. (C–D) Drug release from DOX@MSNs and DOX@MSNs-CS/CMC at different pH value.



3.5 Biocompatibility and Cytotoxicity of MSNs-CS/CMC The intrinsic biocompatibility and cytotoxicity of free DOX, DOX@MSNsCS/CMC and blank nanocarriers were investigated in human breast cancer MCF-7 cells. Figure 7A shows the cytotoxicity of nanoparticles after 24 h. Due to the favorable biocompatibility of chitosan shell, the cell viability of blank MSNs-CS/CMC were around 90% and significantly higher than MSNs. Both free DOX and DOX-loaded nanoparticles displayed a time and dosage dependent cytotoxicity toward MCF-7 cells. Compared with free DOX and DOX@MSNs, DOX@MSNs-CS/CMC still exhibited lower toxicity when the DOX concentration was added up to 100 μg/mL. Similar trends were observed in Figure 7B, when the incubation time was prolonged to 48 h. It is mainly attributed to the gradual release of DOX from DOX@MSNs-CS/CMC, which would facilitate the effective drug transportation to cell nucleus during the blood circulation. Meanwhile, significant cytotoxicity of DOX@MSNs-CS/CMC were clearly observed in tumor acidic microenvironment than that in normal tissues (Figure 7C–D and Table S2). The half maximal inhibitory concentration (IC50) of DOX@MSNsCS/CMC was 13 μg/mL and the IC50 of free DOX was 25 μg/mL, when the nanoparticles were incubated at pH 6.5 for 24 h. Similar trends were observed when the incubation time was prolonged to 48 h. It may be attributed to the surface chargereversal character that trigger the DOX release and further improve cellular uptake in the cytoplasm and extracellular tumor tissues at pH 6.5, which would promote the transformation of DOX to cellular nucleus in the lysosomes and endosomes of tumor tissues at pH 5.0−5.5. The results demonstrate that the pH-responsive DOX@MSNsCS/CMC may minimize side effects of chemotherapeutic drugs. Moreover, it has good practical application prospects in breast cancer treatment.


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Figure 7. In vitro viability of MCF-7 cells incubated with free DOX, DOX@MSNs and DOX@MSNs-CS/CMC. (A-B) In vitro viability of MCF-7 cells incubated with drug loaded nanoparticles at pH 7.4 for 24 and 48 h. (C-D) In vitro viability of MCF-7 cells incubated with drug loaded nanoparticles at pH 6.5 for 24 and 48 h. Statistical analysis was determined by student’s t−test and one-way analysis of variance (ANOVA) with SPSS 21.0 software. *P < 0.05, **P < 0.01 and **P < 0.001 indicate statistical significance and extremely statistical significance, respectively.

3.6 Intracellular Uptake of DOX@MSNs-CS/CMC The intracellular uptake of free DOX, DOX@MSNs and DOX@MSNs-CS/CMC in human breast cancer MCF-7cells was evaluated by CLSM technique. Hoechst was used as an indicator to label the nucleus. Figure 8A shows that strong red fluorescence was observed in MCF-7 cells after incubated with free DOX and DOX@MSNs for 24 h. With further incubation of 48 h (Figure 8B), the fluorescence intensity inside the cells increased and the accumulation of DOX in cell nucleus became more evident. In comparison, when MCF-7 cells were treated with DOX@MSNs-CS/CMC, weaker DOX fluorescence was observed in the cytoplasm and only negligible DOX fluorescence was accumulated within the perinuclear region. Moreover, due to the



surface charge-reversal character of MSNs-CS/CMC, DOX was burst into release from the disrupted nanoparticles when the extracellular microenvironment was adjusted from neutral media to slightly acidic media at pH 6.5 (Figure 8C–D). The character of pHsensitive nanoparticles would facilitate the endosomal escape and trigger the diffusion to perinuclear region via enhancing the penetration effect or interaction with endosomal membrane during intracellular uptake.39 The results indicate that DOX is firmly encapsulated in the MSNs-CS/CMC, which may require long period to locate in the nucleus of MCF-7 cells as compared with free DOX. The intracellular drug release from DOX@MSNs-CS/CMC were in line with higher anti-tumor activity in the acidic microenvironment, confirming that the nanoparticles with pH-responsive and chargereversible character may extensively shorten the time for translocating from cytoplasm to nucleus.

Figure 8. CLSM images of MCF-7 cells incubated with free DOX, DOX@MSNs and DOX@MSNs-CS/CMC. (AB) CLSM images of MCF-7 cells at pH 7.4 for 24 and 48 h. (C-D) CLSM images of MCF-7 cells at pH 6.5 for 24 and 48 h. (DOX: 5 μg/mL, Scale bar: 20 μm)


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3.7 Quantitative Evaluating of Intracellular Uptake The similar phenomena were also observed in the flow cytometry analysis. The mean fluorescence intensity (MFI) of DOX in DOX@MSNs-CS/CMC was much stronger in breast cancer MCF-7 cells, when the medium was adjusted to acidic microenvironment (Figure 9A–B). Figure 9C shows the fluorescence intensity of MCF7 cells treated by DOX loaded nanoparticles for 24 h. Since free DOX has no selectivity, the MFI of DOX@MSNs was approximated to 0.6-fold higher than free DOX at pH 6.5. Moreover, the cells treated by DOX@MSNs-CS/CMC displayed the stronger fluorescence intensity, the MFI was significantly increased from 0.3-fold to 1.3-fold higher than free DOX at pH 6.5. When the cells were treated for 48 h, the similar trends were observed in Figure 9D. The MFI of DOX@MSNs-CS/CMC was approximated to 3.7-fold higher than free DOX at pH 6.5. These results demonstrate that the pHresponsive and charge reversal DOX@MSNs-CS/CMC have superior DOX delivery efficacy, moreover, the tumor targeting release can be observed via the surface modification of MSNs–based nanoparticles.

Figure 9. Flow cytometry analysis of MCF-7 cells treated by DOX, DOX@MSNs and DOX@MSNs-CS/CMC. (AB) Flow cytometry measurement of internalized DOX signals in MCF-7 cells. (C-D) The mean fluorescence



intensity (MFI) of MCF-7 cells after treated by different samples for 24 and 48 h. (DOX:5 μg/mL). Statistical analysis was determined by student’s t−test and one-way analysis of variance (ANOVA) with SPSS 21.0 software. *P < 0.05, **P < 0.01 and **P < 0.001 indicate statistical significance and extremely statistical significance, respectively.

4. CONCLUSIONS MSNs-CS/CMC with core-shell structure, pH-responsibility and charge-reversible character are successfully prepared using ionic crosslinking and self-assembly methods. DOX@MSNs-CS/CMC with core-shell structure have been investigated to evade the premature release and exhibit sustained release and lower toxicity in MCF−7 cells, which would improve the biocompatibility, minimize the side effects and prolong the lifetime during blood circulation at pH 7.4. Moreover, surface charge reversal from negative (pH 7.4) to positive charge (tumor acidic microenvironment at pH 6.5) is clearly observed in the DOX@MSNs-CS/CMC, which significantly promotes the cellular uptake, stimulates the DOX delivery to nucleus and further triggers the apoptosis of MCF-7 cells in tumor extracellular microenvironment. The MSNs modified by charge-reversible CS/CMC hybrids could be served as promising drug delivery vehicles to improve the anticancer efficiency and biocompatibility in breast cancer therapy.


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ASSOCIATED CONTENT Supporting Information Specific surface area, pore volume and pore size of mesoporous silica nanoparticles; values of IC50 in DOX loaded nanoparticles at different pH values; substitution degree analysis of carboxymethyl group in carboxymethyl chitosan; effects of concentration, molecular weight, reaction time and rotation rate on the particle sizes and zeta potentials of CS/CMC nanoparticles.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (Chengshen Zhu). *E-mail: [email protected] (Wentao Liu). ORCID: 0000-0001-8402-7486 *E-mail: [email protected] (Hao Liu). ORCID: 0000-0002-7785-7075

Conflicts of interest The authors declare that no competing interest exists.

ACKNOWLEDGEMENTS The financial support of this work by the National Natural Science Foundation of China (Grant No. U1504527) is gratefully acknowledged.

REFERENCES (1) Cardoso, F.; Harbeck, N.; Barrios, C. H.; Bergh, J.; Cortés, J.; Saghir, N. E.; Francis, P. A.; Hudis, C. A.; Ohno, S.; Partridge, A. H; Sledge, G.W.; Smith, I.E.; Gelmon, K.A. Research Needs in Breast Cancer. Ann. Oncol. 2017, 28 (2), 208-217. (2) Sparano, J. A.; Gray, R. J.; Makower, D. F.; Pritchard, K. I.; Albain, K.S.; Hayes, D. F.; Geyer, C. E. Jr.; Dees, E. C.; Goetz, M. P.; Olson, J. A. Jr.; Lively, T.; Badve, S. S.; Saphner, T. J.; Wagner, L. I.; Whelan, T. J.; Ellis, M. J.; Paik, S.; Wood, W. C.; Ravdin, P. M.; Keane, M. M.; Gomez Moreno, H. L.; Reddy, P. S.; Goggins, T. F.; Mayer, I. A.; Brufsky, A. M.; Toppmeyer, D. L.; Kaklamani, V. G.; Berenberg, J. L.; Abrams, J.;



Sledge, G. W. Jr. Adjuvant Chemotherapy Guided by a 21-Gene Expression Assay in Breast Cancer. N. Engl. J. Med. 2018, 379 (2), 111-121. (3) Brancato, V.; Gioiella, F.; Imparato, G.; Guarnieri, D.; Urciuolo, F.; Netti, P. A. 3D Breast Cancer Microtissue Reveals the Role of Tumor Microenvironment on the Transport and Efficacy of Free-Doxorubicin in Vitro. Acta Biomater. 2018, 75, 200-212. (4) Qin, B.; Liu, L.; Wu, X.; Liang, F.; Hou, T.; Pan, Y.; Song, S. mPEGylated Solanesol Micelles as Redox-Responsive Nanocarriers with Synergistic Anticancer Effect. Acta Biomater. 2017, 64, 211-222. (5) Tarn, D.; Ashley, C. E.; Xue, M.; Carnes, E.C.; Zink, J. I.; Brinker, C. J. Mesoporous Silica Nanoparticle Nanocarriers: Biofunctionality and Biocompatibility. Acc. Chem. Res. 2013, 46 (3), 792-801. (6) Cheng, Y.; Zhang, A. Q.; Hu, J. J.; He, F.; Zeng, X.; Zhang, X. Z. Multifunctional Peptide-Amphiphile End-Capped Mesoporous Silica Nanoparticles for Tumor Targeting Drug Delivery. ACS Appl. Mater. Interfaces 2017, 9 (3),2093-2103. (7) Slowing, I. I.; Trewyn, B. G.; Giri, S.; Lin, S. Y. J. Mesoporous Silica Nanoparticles for Drug Delivery and Biosensing Applications. Adv. Funct. Mater. 2007, 17 (8), 12251236. (8) Hadipour Moghaddam, S. P.; Yazdimamaghani, M.; Ghandehari, H. J. GlutathioneSensitive Hollow Mesoporous Silica Nanoparticles for Controlled Drug Delivery. J. Control. Release 2018, 282, 62-75. (9) Vallet-Regi, M.; Rámila, A.; Real, R. P. D.; Pérez-Pariente, J. A New Property of MCM-41: Drug Delivery System. Chem. Mater. 2001, 13 (2), 308-311. (10) Xu, C.; Chen, F.; Valdovinos, H. F.; Jiang, D.; Goel, S.; Yu, B.; Sun, H.; Barnhart, T. E.; Moon, J. J.; Cai, W. Bacteria-like Mesoporous Silica-coated Gold Nanorods for Positron Emission Tomography and Photoacoustic Imaging-Guided ChemoPhotothermal Combined Therapy. Biomaterials 2018, 165, 56-65. (11) Chou, C. C.; Chen, W.; Hung, Y.; Mou, C. Y. Molecular Elucidation of Biological Response to Mesoporous Silica Nanoparticles in Vitro and in Vivo. ACS Appl. Mater. Interfaces 2017, 9 (27), 22235-22251. (12) Chen, W. H.; Luo, G. F.; Leim Q.; Cao, F. Y.; Fan, J. X.; Qiu, W. X.; Jia, H. Z.;


Page 26 of 30

Page 27 of 30 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


Hong, S.; Fang, F.; Zeng, X.; Zhuo, R. X.; Zhang, X. Z. Rational Design of Multifunctional Magnetic Mesoporous Silica Nanoparticle for Tumor-Targeted Magnetic Resonance Imaging and Precise Therapy. Biomaterials 2016, 76 (10), 87-101. (13) Kim, I. Y.; Joachim, E.; Choi, H.; Kim, K. Medicine, Toxicity of Silica Nanoparticles Depends on Size, Dose, and Cell type. Nanomedicine 2015, 11 (6), 14071416. (14) Gurka, M. K.; Pender, D.; Chuong, P.; Fouts, B.; Sobelov, A.; Mcnally, M.; Mezera, M.; Woo, S.; Mcnally, L. R. Identification of Pancreatic Tumors in Vivo with LigandTargeted, pH Responsive Mesoporous Silica Nanoparticles by Multispectral Optoacoustic Tomography. J. Control. Release 2016, 231, 60-67. (15) Lai, C. Y.; Trewyn, B. G.; Jeftinija, D. M.; Jeftinija, K.; Xu, S.; Jeftinija, S.; Lin, V. S. A Mesoporous Silica Nanosphere-based Carrier System with Chemically Removable CdS Nanoparticle Caps for Stimuli-Responsive Controlled Release of Neurotransmitters and Drug Molecules. J. Am. Chem. Soc. 2003, 125 (15), 4451-4459. (16) Feng, W.; Nie, W.; He, C.; Zhou, X.; Chen, L.; Qiu, K.; Wang, W.; Yin, Z. Effect of pH-Responsive Alginate/Chitosan Multilayers Coating on Delivery Efficiency, Cellular Uptake and Biodistribution of Mesoporous Silica Nanoparticles based Nanocarriers. ACS Appl. Mater. Interfaces. 2014, 6 (11), 8447-8460. (17) Tang, Y.; Hu, H.; Zhang, M. G.; Song, J.; Nie, L.; Wang, S.; Niu, G.; Huang, P.; Lu, G.; Chen, X. An Aptamer-Targeting Photoresponsive Drug Delivery System using "off-on" Graphene Oxide Wrapped Mesoporous Silica Nanoparticles. Nanoscale 2015, 7 (14), 6304-6310. (18) Zhao, Y. L.; Li, Z.; Kabehie, S.; Botros, Y. Y.; Stoddart, J. F.; Zink, J. I. pHOperated Nanopistons on the Surfaces of Mesoporous Silica Nanoparticles. J. Am. Chem. Soc. 2010, 132 (37), 13016-13025. (19) Chen, C.; Pu, F.; Huang, Z.; Liu, Z.; Ren, J.; Qu, X. Stimuli-Responsive Controlled-Release System using Quadruplex DNA-Capped Silica Nanocontainers. Nucleic Acids Res. 2011, 39 (4), 1638-1644. (20) Han, L.; Tang, C.; Yin, C. Dual-Targeting and pH/Redox-Responsive MultiLayered Nanocomplexes for Smart Co-Delivery of Doxorubicin and siRNA.



Biomaterials 2015, 60, 42-52. (21) Han, L.; Zhao, J.; Zhang, X.; Cao, W.; Hu, X.; Zou, G.; Duan, X.; Liang, X. J. Enhanced siRNA delivery and Silencing Gold-Chitosan Nanosystem with Surface Charge-Reversal Polymer Assembly and Good Biocompatibility. ACS Nano 2012, 6 (8), 7340-7351. (22) Kang, X. J.; Cheng, Z. Y.; Yang, D. M.; Ma, P. A.; Shang, M. M.; Peng, C.; Dai, Y. L.; Lin, J. Design and Synthesis of Multifunctional Drug Carriers Based on Luminescent Rattle-Type Mesoporous Silica Microspheres with a Thermosensitive Hydrogel as a Controlled Switch. Adv. Funct. Mater. 2012, 22 (7), 1470-1481. (23) Zhao, R.; Li, T.; Zheng, G.; Jiang, K.; Fan, L.; Shao, J. Simultaneous Inhibition of Growth and Metastasis of Hepatocellular Carcinoma by Co-Delivery of Ursolic Acid and Sorafenib using Lactobionic Acid Modified and pH-Sensitive Chitosan-Conjugated Mesoporous Silica Nanocomplex. Biomaterials 2017, 143 (2), 1-16. (24) Zhu, X.; Wang, C. Q. pH and Redox-Operated Nanovalve for Size-Selective Cargo Delivery on Hollow Mesoporous Silica Spheres. J. Colloid Interface Sci. 2016, 480, 39-48. (25) Liu, J.; Li, Q. L.; Zhang, J. X.; Huang, L.; Qi, C.; Xu, L. M.; Liu, X. X.; Wang, G. B.; Wang, L.; Wang, Z. Safe and Effective Reversal of Cancer Multidrug Resistance using Sericin-Coated Mesoporous Silica Nanoparticles for Lysosome-Targeting Delivery in Mice. Small 2016, 13 (9), 1602567. (26) Schlossbauer, A.; Kecht, J.; Bein, T. Biotin-Avidin as a Protease-Responsive Cap System for Controlled Guest Release from Colloidal Mesoporous Silica. Angew Chem. Int. Ed. Engl. 2010, 121 (17), 3138-3141. (27) Mathews, P. D.; Fernandes Patta , A. C. M.; Gonçalves, J. V.; Gama, G. D.S.; Garcia, I. T. S.; Mertins, O. Targeted Drug Delivery and Treatment of Endoparasites with Biocompatible Particles of pH-Responsive Structure. Biomacromolecules 2018, 19 (2), 499-510. (28) Pramod, P. S.; Ruchira, S.; Manickam, J. Dual Stimuli Polysaccharide Nanovesicles for Conjugated and Physically Loaded Doxorubicin Delivery in Breast Cancer Cells. Nanoscale 2015, 7 (15), 6636-6652.


Page 28 of 30

Page 29 of 30 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


(29) Li, J. L.; Cheng, Y. J.; Zhang, C.; Cheng, H.; Feng, J.; Zhuo, R. X.; Zeng, X.; Zhang, X. Z. Dual Drug Delivery System Based on Biodegradable Organosilica CoreShell Architectures. ACS Appl. Mater. Interfaces 2018, 15 (1), 5287-5295. (30) Ge, Z.; Liu, S. Functional Block Copolymer Assemblies Responsive to Tumor and Intracellular Microenvironments for Site-Specific Drug Delivery and Enhanced Imaging Performance. Chem Soc Rev. 2013, 42 (17), 7289-7325. (31) Feng, T. ; Ai, X. Z.; An, G. H.; Yang, P. P.; Zhao, Y. L. Charge-Convertible Carbon Dots for Imaging-Guided Drug Delivery with Enhanced in Vivo Cancer Therapeutic Efficiency. ACS Nano 2016, 10 (4), 4410-4420. (32) Wang, J.; Xu, M.; Cheng, X.; Kong, M.; Liu, Y.; Feng, C.; Chen, X. Positive/Negative Surface Charge of Chitosan Based Nanogels and Its Potential Influence on Oral Insulin Delivery. Carbohydr. Polym. 2016, 136, 867-874. (33) Zhao, X.; Wei, Z.; Zhao, Z.; Miao, Y.; Qiu, Y.; Yang, W.; Jia, X.; Liu, Z.; Hou, H. Design and Development of Graphene Oxide Nanoparticle/Chitosan Hybrids Showing pH-Sensitive Surface Charge-Reversible Ability for Efficient Intracellular Doxorubicin Delivery. ACS Appl. Mater. Interfaces 2018, 10 (7), 6608-6617. (34) Mo, J.; He, L.; Ma, B.; Chen, T. Tailoring Particle Size of Mesoporous Silica Nanosystem To Antagonize Glioblastoma and Overcome Blood–Brain Barrier. ACS Appl. Mater. Interfaces 2016, 8 (11), 6811-6825. (35) Chen, X. G.; Park, H. J. Chemical Characteristics of Carboxymethyl Chitosans Related to the Preparation Conditions. Carbohydr. Polym. 2003, 53 (4), 355-359. (36) Slowing, I. I.; Vivero-Escoto, J. L.; Wu, C. W.; Lin, V. S. Mesoporous Silica Nanoparticles as Controlled Release Drug Delivery and Gene Transfection Carriers. Adv. Drug Deliv. Rev. 2008, 60 (11), 1278-1288. (37) Moheini, A.; Cimmino, A.; Poggetto, G. D.; Biase, M. D.; Evidente, A.; Masi, M.; Lavermicocca, P.; Valerio, F.; Leone, A.; Santagata, G.; Malinconico, M. Effect of pH and TPP Concentration on Chemico-Physical Properties, Release Kinetics and Antifungal Activity of Chitosan-TPP-Ungeremine Microbeads. Carbohydr. Polym. 2018, 195, 631-641. (38) Wu, J.; Wang, Y.; Yang, H.; Liu, X.; Lu, Z. Preparation and Biological Activity



Studies of Resveratrol Loaded Ionically Cross-Linked Chitosan-TPP Nanoparticles. Carbohydr. Polym. 2017, 175, 170-177. (39) Wu, L.; Zou, Y.; Deng, C.; Cheng, R.; Meng, F.; Zhong, Z. Intracellular Release of Doxorubicin from Core-crosslinked Polypeptide Micelles Triggered by Both pH and Reduction Conditions. Biomaterials 2013, 34 (21), 5262-5272. (40) Sun, Q. H.; Zhou, Z. X.; Qiu, N. S.; Shen, Y. Q. Rational Design of Cancer Nanomedicine: Nanoproperty Integration and Synchronization. Adv. Mater. 2017, 29 (14), 1606628. (41) Cheng, W.; Nie, J.; Xu, L.; Liang, C.; Peng, Y.; Liu, G.; Wang, T.; Mei, L.; Huang, L.; Zeng, X. pH-Sensitive Delivery Vehicle Based on Folic Acid-Conjugated Polydopamine-Modified Mesoporous Silica Nanoparticles for Targeted Cancer Therapy. ACS Appl. Mater. Interfaces 2017, 9 (22), 18462-18473.


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pH-triggered Charge-Reversal Mesoporous Silica Nanoparticles

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