Multistage pH-responsive mesoporous silica nanohybrids with charge reversal and intracellular release for efficient anticancer drug delivery

Journal of Colloid and Interface Science 555 (2019) 82–93

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Regular Article

Multistage pH-responsive mesoporous silica nanohybrids with charge reversal and intracellular release for efficient anticancer drug delivery Xiaozhe Yuan a, Shiyuan Peng a, Wenjing Lin b, Jufang Wang c, Lijuan Zhang a,⇑ a

School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, PR China School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510006, PR China c School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510640, PR China b

g r a p h i c a l a b s t r a c t

a r t i c l e

i n f o

Article history: Received 31 May 2019 Revised 22 July 2019 Accepted 23 July 2019 Available online 24 July 2019 Keywords: Charge-reversal Control release Morpholino Hydrazone

a b s t r a c t This study introduced multistage pH-responsive nanohybrids (MSN-hyd-MOP) based on mesoporous silica nanoparticles (MSNs) modified with polymers with charge-reversal property via an acid-labile hydrazone linker, which were applied as a drug delivery system loaded anticancer drugs. In this study, MSNhyd-MOP nanohybrids were completely investigated for their synthesis, pH response, drug release behavior, cytotoxicity capability and endocytic behavior. Responding to the acidic extracellular microenvironment of solid tumor (pH 6.5), MSN-hyd-MOP nanohybrids exhibited surface charge-reversal characteristic from negative (
10.2 mV, pH 7.4) to positive (16.6 mV, pH 6.5). The model drug doxorubicin (Dox) was efficiently loaded within the channels of MSN-hyd-MOP (encapsulation efficiency about 87%). The increased acidity in endo-/lysosome promote Dox-loaded MSN-hyd-MOP (MSN-hydMOP@Dox) release Dox quickly. In vitro study revealed the drug delivery system had good biocompatibility and could deliver the payload to tumor cells. Overall, the described nanohybrids can be used as a potential anticancer drug delivery system. Ó 2019 Published by Elsevier Inc.

1. Introduction In recent decades, mesoporous silica nanoparticles (MSNs) have been extensively studied for their potential applications in anticancer drug delivery systems [1,2]. Nanosized MSNs could utilize ⇑ Corresponding author. E-mail address: [email protected] (L. Zhang). https://doi.org/10.1016/j.jcis.2019.07.061 0021-9797/Ó 2019 Published by Elsevier Inc.

the advantages of nanosize to enrich in solid tumor through the enhanced permeability and retention (EPR) effect of solid tumors [3]. Compared with other nanoparticles, MSNs have several unique characteristics such as simple synthesis process, controllable channels, high thermal stability, good biocompatibility and easy surface modification [4,5]. The large surface areas and pore volumes of MSNs provide the possibility of encapsulating and delivering a large amount of drug to tumors [6]. The drug can be encapsulated

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in the channel simply by grafting pore blocking agent. The ideal drug carrier needs to prevent premature drug leakage before reaching the therapeutic target, and to release the drug rapidly when reaching the target. Therefore, the research of current MSN-based drug delivery systems is to achieve controlled drug release and the cell internalization of nanoparticles. As for MSN-based controlled release systems, diverse physicochemical signals specific to the tumor microenvironment were applied as stimuli to trigger drug delivery, such as pH [7–9], redox [10,11], enzyme [12,13], etc. Of these stimuli response, pH-stimuli response is one of the most widely studied at present. As reported, extracellular microenvironment of tumor is weakly acidic (pH < 6.5) because of the elevated metabolisms and rapid proliferation of tumor cells, and the environment in the endosomes/lysosomes is more acidic (pH 4.0–6.0), compared with the normal physiological pH of 7.3–7.4[14–16]. In addition, pH response has multiple strategies, including the change in electrostatic interactions [17], switch between hydrophobic and hydrophilic [18], cleavage of chemical bonds [18,19], etc. Because if the drugloading system releases drugs extracellularly, it causes inefficiency in killing drug-resistant cells [19]. Many researchers have developed various MSN-based controlled release systems that respond only to the acid environment of endosomes/lysosomes. Gan and coworkers reported a pH-sensitive MSNs ensemble (MCM-TAAFe3O4) which exhibited quick release at pH 5.0–6.0 [20]. 1,3,5triazaadamantane (TAA) linkers are intact under neutral/basic conditions, while rapidly hydrolyze into tri(aminomethyl)ethane when the pH is lower than 6.0. Chen et al. used benzoic-imine bond to anchored adamantine on MSN, and fabricated pH-operated mechanized MSN(MMSMs) by the supramolecular interaction between adamantine (AD) and b-cyclodextrin (b-CD) [21]. Because of the acid-triggered disintegration of benzoic-imine bonds, about 90% of payload was rapidly released at pH 5.5, analogous to the acid environment of late endosome [21]. Niedermayer’s group introduced a multifunctional MSN-based drug delivery system modified with a pH-responsive polymer poly(2-vinylpyridine) (PVP) [22]. The polymer PVP is hydrophobic at pH values around 5.5 or higher, which would switch into hydrophilic owing to the protonation of PVP in acidic environment of endo-/lysosome, which promotes drug diffusion. However, the above-mentioned delivery systems responding to intracellular environment relied on EPR effect to accumulate at tumor sites, and the cellular uptake of the nanocarrier by tumor cells was not considered. Poor tumor penetration and low cellular uptake may decrease anticancer efficacy [23]. In order to enhance the internalization of nanocarriers by tumor cells, the targeting ligands, such as folic acid [24], peptide [25], biotin [26], etc, were modified onto the surface of nanocarriers. These ligands specifically recognize receptors overexpressed by tumor cells, thereby promoting cellular internalization of nanocarriers. Lv et al. reported a pH-sensitive targeted mesoporous silica nanohybrid MSNs covered with chitosan-biotin (CTS-Biotin) conjugates [27]. Cheng et al. introduced poly(ethylene glycol)-folic acid onto dopamine-modified mesoporous silica to synthesize a targeted drug delivery system (MSNs@PDA-PEG-FA)[28]. However, normal cells also express a small number of identical receptors, which limits the targeting efficiency and anticancer efficiency of nanocarriers [29]. In addition to modifying the targeting ligand, introducing positively charged agents, such as cationic polymer, polypeptide, was also a means to enhance cellular internalization. This was demonstrated in a number of studies that cells were inclined to adsorb positive-charged nanoparticles, owing to strong electrostatic interactions with negatively charged cell membranes [30]. Hakeem’s group designed polyaspartic acid-anchored mesoporous silica nanoparticles [31]. Polyaspartic acid is positively charged, which contributes to the cellular uptake of nanoparticles.

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However, positive-charged nanoparticles have higher affinity with protein in the blood, which leads to short blood circulation halftime and side effect on normal cells [32,33]. In order to solve this contradiction, the charge-reversal drug delivery system that triggered by weakly acidic extracellular environment of tumor cells is considered as a promising strategy to improve the stability of nanocarriers and enhance the internalization of nanocarriers by tumor cells. Wu et al. reported mesoporous silica nanoparticles modified with a cationic polymer (low molecular weight polyethyleneimine, LPEI) and negatively charged bovine serum albumin [34]. The surface charge of mesoporous nanohybrids showed negative charge at pH 7.4 and positive charge at pH 6.5 because of the protonation effect of LPEI. Yao’s groups introduced charge-reversal mesoporous silica nanoparticles which coated with PEGylated tetraphenylporphyrin zinc (Zn-Por-CA-PEG) [35]. The pH sensitive cis-aconitic anhydride (CA) could be broken down and the amino group of the surface of Zn-Por would be positively charged at tumor extracellular. The drug delivery system combining the two functions of intracellular release and internalization is an effective strategy to improve therapeutic efficiency. But the MSN-based drug delivery systems above mentioned can only achieve a single function through pH response. However, there are a few reports about pH-responsive MSN-based drug delivery systems which can achieve both of two functions. Therefore, we fabricated a multistage pH responsive charge-reversal mesoporous silica nanohybrids (MSN-hyd-MOP), which could achieve charge-conversion triggered by the pH values of extracellular environment and release their payloads in lysosome/endosome of tumor cells (Scheme 1). The polymer morpholin-4-yl-acetyl-poly(ethylene glycol)-b-poly(lactic acid) (MOP) was employed as a gatekeeper to seal nanopore, which was grafted to the MSNs surface by using acid-sensitive hydrazone bonds. And the charge of the polymer MOP could be readily switched from negative to positive through the deprotonation–protonation of the morpholine groups upon exposure to the acidic extracellular environment of the tumor, which would facilitate cell internalization [36,37]. Furthermore, by the degradation of hydrazone bonds at endosomal/lysosomal pH (4–6) [19,38], the grafted MOP was removed, and then the loaded-drug can be quickly released from the nanohybrids. In present study, the synthesis of MSN-hyd-MOP is demonstrated. The size, morphology, and structure of MSN-hyd-MOP were characterized. And pH stimuli-responsive charge-reversal and controlled release properties were investigated. We also discussed the release mechanism of MSN-hyd-MOP@Dox at different pH. Furthermore, the cytotoxicity capability and endocytic behavior of nanohybrids were evaluated in vitro in the HepG2 (hepatocellular carcinoma) liver hepatocellular cells. We hope that this nanohybrids can efficiently deliver anticancer drug to tumor cells, thus enhancing the therapeutic efficiency.

2. Experimental section 2.1. Materials Hexadecyl trimethyl ammonium bromide (CTAB, 99%), pyruvic acid (99%), tetraethyl orthosilicate (TEOS, 99%), triethylamine (TEA, 99%), D,L-lactide (99%), N-(3-dimethylaminopropyl)-N’-ethyl carbodiimide hydrochloride (EDC, AR), (3-aminopropyl) triethoxysilane (APTES, 99%), N-hydroxysuccinimide (NHS, 99%), morpholin-4-yl-acetic acid hydrochloride (Morp, 98%), polymer (ethylene glycol) (PEG, 2000 Da), methoxy poly(ethylene glycol) (MeO-PEG-OH, 1900 Da), N,N’-diisopropylcarbodiimide (DIC, AR), 4-dimethylaminopyridine (DMAP, 99%), hydrazine monohydrate (99%) and 4-Nitrophenyl chloroformate (NPC, 99%) were purchased

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Scheme 1. Schematic illustration of MSN-hyd-MOP loaded with Dox for charge reversal and controlling release.

from J&K Scientific Ltd.. Stannous octanoate (Sn(Oct)2, AR) was acquired from Alfa Aesar. The follow reagents required further purification before use: dichloromethane (DCM, AR), methylbenzene (AR) and N,N-dimethylformamide (DMF, AR). Dulbecco’s modified eagle medium (DMEM), 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Hoechst 33324, penicillin and streptomycin were purchased from Invitrogen. Fetal bovine serum (FBS) was obtained from Zhejiang Tianhang Biotechnology Co. Ltd.. HepG2 cells were purchased from the American Type Culture Collection (ATCC).

2.2. Measurement The structures of polymer were determined by 1H NMR (AVANCE III 400, Bruker, Switzerland) at 250 MHz using deuterated chloroform (CDCl3) or deuterated dimethyl sulfoxide (d6DMSO) as the solvent and tetramethylsilane (TMS) as the internal reference. The number average molecular weight (Mn) and polydispersity index (PDI) of polymer were measured by gel permeation chromatography (GPC) adopting an Agilent 1200 series GPC system equipped with an RI detectorTHF was used as the mobile phase with a flow rate of 1 mLmin
1. The morphology of nanoparticles was observed under transmission electron microscopy (TEM, JEM-2100F, JEOL) at an accelerating voltage of 80 kV and scanning electron microscopy (SEM, HITACHI UHR FE-SEM SU8200, Japan). Briefly, a drop of nanoparticle suspension was placed on a copper grid or aluminum foil at room temperature. The zeta potential and size distribution of nanohybrids were examined by Malvern Zetasizer Nano S instrument at 25 °C. The nanoparticles were dispersed in phosphate buffer solution (pH 7.4 or 6.5, 0.02 M) at a concentration of 0.5 mgmL
1. Small angle x-ray diffraction (SAXS, polycrystall X-ray diffraction, Rigaku SmartLab SE) was used to character the pore channel structure of nanoparticles. Surface area and pore size were determined by N2 adsorption-desorption isotherms (Micromeritics Tristar 3000 pore). Prior to testing, the nanoparticles were degassed at 150 °C for 7 h under vacuum, and then the samples were tested under the temperature of liquid nitrogen. The pore size distribution curves were obtained via analysis of the desorption portion of the isotherms using the BJH

(Barrett-Joyner-Halenda) method. The surface-modified groups of MSNs were characterized by FT-IR (Nicolet Nexus-6700, Nicolet). The samples were mixed with the highly pure potassium bromide and then compressed, and the spectrum of the wave number 400– 4000 cm
1 was collected. And superconducting Fourier transform nuclear magnetic resonance spectrometer (NMR, AVANCE III HD 400, Bruker) was also used to character whether the polymer was successfully grafted onto MSNs. The number of modified groups present on the surface of MSNs was quantitatively measured by thermogravimetric analysis instrument (TGA, Pyrisis 1 TGA) under nitrogen flow at a heating rate of 100 °C min
1 up to 800 °C. Confocal laser scanning microscopy (CLSM) images were obtain by a Leica TCS SP5 II microscope (Germany). 2.3. Synthesis of a hydrazine terminated morpholin-4-yl-acetyl-poly (ethylene glycol)-b-poly(lactic acid) (MOP-NHNH2)

2.3.1. Synthesis of morpholin-4-yl-acetyl-poly(ethylene glycol) (MorpPEG-OH) Briefly, PEG (4.0 g, 2 mmol), Morp (0.343 g, 2 mmol), DMAP (13 mg, 0.01 mmol) and TEA (273 lL, 2 mmol) were blended in 20 mL of dichloromethane (DCM) under argon atmosphere. Subsequently, a solution of DIC (465 lL, 3 mmol) in DCM was added dropwise into the mixture. The resulting mixture was heated to 40 °C for 24 h and was concentrated by rotary evaporation to remove most of the solvent after the reaction was completed. The obtained concentrate was dropped into ice-cold diethyl ether to get the residue. The residue was dissolved in DCM and precipitated in ice-cold diethyl ether again. This purification process was repeated twice to remove impurities. The precipitation was vacuum-dried at 30 °C for 24 h to obtain Morp-PEG-OH. 2.3.2. Synthesis of morpholin-4-yl-acetyl-poly(ethylene glycol)-b-poly (lactic acid) (MOP) Morp-PEG-OH (1.2 g, 0.6 mmol) and D,L-lactide (3.98 g, 28 mmol) were melting at 80 °C for 2 h under vacuum. After cooling down to ambient temperature, the reaction flask was evacuated again and flushed with argon. 4 mL of methylbenzene was

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added into the mixture above. And then the mixture was heated to 120 °C, followed by adding stannous octoate (10 lL, 0.03 mmol). The reaction was heated to 140 °C and stirred for 12 h. After that, the product was precipitated in cold diethyl ether. This process was repeated serveral times to remove impurities. The residue dried at 30 °C under vacuum for 24 h to get MOP. And the polymer MEP was synthesized in the same way, replacing Morp-PEG-OH with MeO-PEG-OH. 2.3.3. Synthesis of MOP-NHNH2 The polymer MOP (Mn = 6886, 0.5 mmol, 3.4 g) and TEA (0.5 mmol, 70 lL) were mixed in DCM (5 mL) under argon atmosphere. And then a solution of NPC (1.5 mmol, 302.3 mg) in DCM (5 mL) was added dropwise into the mixture above in a dropwise manner at 0 °C. After 1 h of reaction in ice bath, the reaction was continued at room temperature for 24 h. The solid was collected via precipitation in cool diethyl ether and filtration. This purification process was repeated in several times. The activated polymer (MOP-NPC) was obtained by vacuum-dried at 30 °C for 24 h. MOP-NPC (0.3 mmol, 2.1 g) was dissolved in 5 mL of DMF under argon atmosphere. Then hydrazine monohydrate (3 mmol, 138 lL) was added dropwise into the solution. The reaction was left at ambient temperature and stirred for 6 h. The product was obtained by dialysis of mixture in deionized water with dialysis bags (MWCO: 3500 Da). The final hydrazine terminated polymer (MOP-NHNH2) was obtained via lyophilization. The preparation of MEP-NHNH2 required only the replacement of MOP with MEP (Mn = 7030, 0.5 mmol, 3.5 g). 2.4. Synthesis of mesoporous silica nanoparticles modifying ketone groups (MSN-PA) Mesoporous silica nanoparticles were procured by a basecatalyzed sol-gel process. Briefly, CTAB (0.2 g) acting as template was dissolved in a mixture of distilled water (96 mL) and 0.2 M NaOH (0.7 mL), and stirred vigorously for 30 min at 80 °C. Then TEOS (1.0 mL) was added dropwise to the above mixture. The final mixture was stirred for additional 2 h. The precipitation was collected by centrifugation (5600 rpm, 20 min), washed for several times with ethanol and deionized water. The products (MSN@CTAB) dried in vacuo at 40 °C for 24 h. In order to avoid the functionalization in the internal surface of the pores, the template (CTAB) was removed after grafting the amino group. MSN@CTAB (0.5 g) was dispersed in anhydrous toluene (20 mL) by ultrasonication. After adding APTES (0.5 mL), the resulting mixture was heated to 110 °C and refluxed for 24 h. The precipitation was obtained by centrifugation at 5600 rpm for 15 min and washed several times with deionized water and ethanol. To obtain products without surfactant templates, the precipitation was suspended in a mixture of HCl (37.5%) and methanol (1:16 v/v). And the suspension was refluxed at 80 °C for 24 h. MSN-NH2 nanoparticles were acquired by centrifugation and vacuum drying at 40 °C for 24 h. Pyruvic acid (0.6 mL) was added to water solution (30 mL) contain EDC (25 mg) and NHS (12 mg). After 2 h of reaction, 5 mL of the suspension which was 0.5 g MSN-NH2 nanoparticles in deionized water was add into the mixture. The mixture was reacted at room temperature for 24 h. The products were obtained by centrifugation and washed for three times with deionized water and ethanol. Then MSN-PA was obtained after drying under vacuum at 30 °C.

suspension. The mixture was stirred in the dark at ambient temperature for 24 h. Then a few drops of acetic acid were added into the mixture. Then the polymer MOP-NHNH2 (50 mg) was dissolved in 1 mL of THF, which added to the above mixture dropwise. The reaction mixture was stirred at room temperature for another 24 h. The products were collected by centrifugation and washed with PBS buffer (pH 7.4) until the supernatant was almost colorless. The drug-loaded nanohybrids (MSN-hyd-MOP@Dox) were obtained by drying in vacuum at 30 °C for 24 h. The preparation of blank particles (MSN-hyd-MOP) was similar to the above steps, eliminating the step of drug loading. The supernatant of the above process was collected. The absorbance of supernatant at 480 nm was measured by a UV–vis spectrophotometer (UV-2450, Shimadzu, Japan). The concentration of Dox in supernatant was calculated from the absorbanceconcentration standard curve of Dox in PBS solution (Fig S1). The amount of Dox loaded in nanohybrids was equal to the amount of Dox in feed minus the amount of Dox in the supernatant, thereby calculating the loading content (LC) and encapsulation efficiency (EE) of nanohybrids. 2.6. In vitro release studies The in vitro release behavior of MSN-hyd-MOP@Dox was investigated in solutions at pH values of 7.4, 6.5, 5.0 and 1.0. Briefly, 25 mg of MSN-hyd-MOP@Dox were suspended in 40 mL of Na2HPO4-NaH2PO4 (PBS, pH 7.4 or 6.5), HOAc-NaOAc (pH 5.0) and HCl (pH 1.0) solutions, which were maintained at 37.2 °C under sustained shaking at 170 rpm. 4 mL of suspensions was taken out at different time intervals and separated by centrifugation. And the precipitation and the same volume fresh solutions were added to the original suspension. Supernatants were removed, whose absorbance at 480 nm was monitored by UV–vis spectrophotometer. The cumulative release of Dox was calculated by the formula (1). Each experiment was repeated three times and averaged.

Cumulative releaseð%Þ ¼

Ve

Pn
1

Ci þ V 0Cn  100% mDox

i¼1

ð1Þ

where mDox is the weight of Dox in nanohybrids; Ve is the volume of supernatant removed from suspension (4 mL); V0 is the total volume of the suspension (40 mL); Ci is the concentration of Dox in the ith supernatant; n is the number of samples. 2.7. In vitro cellular uptake The cellular uptake behavior of nanohybrids was detected by Confocal laser microscopy (CLSM). HepG2 cells were inoculated in 8 well plates at a seeding density of 5  104 cells/well and cultured for 24 h before the experiment. The culture medium was replaced with fresh culture medium containing MSN-hydMOP@Dox or free Dox with the same Dox concentration (50 mg/ L), and then incubated at 37 °C for another 1 h or 4 h. After taking out the medium, the cells were washed with PBS solution in triplicate. Paraformaldehyde (4% w/w) in PBS was added and maintained for 30 min to fixing cells. And then the treated cells were washed with PBS solution several times and stained with Hoechst 33,342 for 15 min. The fluorescence was observed with CLSM, where the red and blue fluorescence belonged to Dox and the nuclei stained with Hoechst 33342, respectively. 2.8. In vitro cytotoxicity

2.5. Synthesis of MSN-hyd-MOP/MSN-hyd-MOP@Dox Briefly, MSN-PA (100 mg) was dispersed in 15 mL of PBS buffer (0.1 M, pH 7.4). 20 mg of DoxHCl was dissolved in the above

HepG2 cells were cultured in DMEM with 10%(v/v) FBS, 100 mg/ mL streptomycin and 100 lL/mL penicillin. The antitumor activity against HepG2 cells of nanohybrids was assessed by MTT assay.

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HepG2 cells were incubated in 96-well plates at a seeding density of 1  104 cells/well for 24 h before the experiment. Then the culture medium was replaced with fresh culture medium at different concentrations of MSN-hyd-MOP, Dox MSN-hyd-MOP@Dox and MSN-hyd-MEP@Dox. After 24 h or 48 h of culture, 180 lL of fresh culture medium and 20 lL of MTT solution (5 g/L in PBS solution) instead of the medium for another 4 h culture. Subsequently, the medium was taken out and added 200 lL of DMSO to dissolve the purple formazan crystals. The absorbance at 490 nm of each well was measured by a microplate reader (SpectraMax M5, Molecular Devices). The cell viability is based on the following equation:

Cell viabilityð%Þ ¼

Asample
Ablank  100% Acontrol
Ablank

ð2Þ

where Ablank and Acontrol are the absorbance at 490 nm of cell-free well plates and cell-containing well plates. Asample is the absorbance at 490 nm in the plate containing drugs or nanohybrids. Each experiment was repeated 6 times and averaged. 3. Results and discussion 3.1. Synthesis and characterization of multistage pH-responsive mesoporous silica nanohybrids Nanohybrids (MSN-hyd-MOP) were composed of mesoporous silica nanoparticle as core and pH-stimulated responsive polymers as shell. The synthetic route is illustrated in Scheme 2. Firstly, the polymer morpholine-4-acetyl-polyethylene glycol-b-polylactide (MOP) was synthesized by esterification and ring-open polymerization (ROP). And the polymer MOP was activated by 4nitrophenyl chloroformate and derivatized to its hydrazine derivative (MOP-NHNH2) [39]. Secondly, hexagonal mesoporous silica nanoparticles were synthesized, and then modified with ketone

groups (MSN-PA). Finally, the final nanohybrids were obtained by that MOP-NHNH2 and MSN-PA formed hydrazone bonds. The chemical structure and molecular weight of the MOPNHNH2 and its intermediate products were determined by 1H NMR and GPC. As can be seen from Fig. 1A(a), the obvious 1H signal at 4.3 ppm (f) was assigned to the ACH2A linked by an ester bond formed by esterification. In Fig. 1A(b), the characteristic peaks at 5.18 ppm (h) and 1.56 ppm (i) were the signals of ACHA and ACH3 in MOP, indicating the successful synthesis of the polymer MOP [37]. According to the peak area of the proton peak of PEG2000 in 1H NMR spectrum, the molecule weight of the polymer MOP was calculated to be 7024, which was consistent with the molecular weight (Mn = 6886, PDI = 1.29) measured by GPC (Fig S2). In the 1H NMR spectra of activated MOP (MOP-NPC) (Fig. 1A (c) and Fig. 1B(a)), the 1H signal at 8.31 ppm, 8.14 ppm (k) and 6.97 ppm, 7.54 ppm (j) were assigned to the proton of phenyl. In Fig. 1B(b), the characteristic peaks of phenyl disappeared, which indicated that the polymer MOP-NPC was completely converted to MOP-NHNH2 [40]. As SEM image and TEM image shown in Fig. 2A and B, MSN-NH2 was a spherical nanoparticle with the diameter of 150–200 nm, which was similar to the particle size of MSN-NH2 determined by particle analysis (Fig. 2D). And the particle size was theoretically appropriate for cellular internalization and enrichment in tumor because of EPR effect [41]. A thin polymer layer on MSNs could be observed in Fig. 2C, which suggested the polymer were successfully grafted onto MSN. In addition, the parallel strips on MSN-NH2 suggested the ordered channel structures. And the mesoporous structure wasn’t damaged by the functionalization process, as the parallel fringes were observed from MSN-hydMOP. The SAXS pattern of MSN-NH2 showed three obvious reflection peaks to the (1 0 0), (1 1 0) and (2 0 0) planes of the typical MCM-41 fragment (Fig. 3A), which was consistent with the observation from TEM image. After functionalization of ketone

Scheme 2. Schematic description of the synthesis of MSN-hyd-MOP.

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Fig. 1. (A) 1H NMR spectra of Morp-PEG-OH, MOP and MOP-NPC in CDCl3. (B) 1H NMR spectra of MOP-NPC and MOP-NHNH2 in d6-DMSO.

Fig. 2. (A) SEM image of MSN-NH2 nanoparticles. TEM images of (B) MSN-NH2 and (C) MSN-hyd-MOP. (D) Particle size distributions of MSN-NH2.

groups (MSN-PA), a weak peak to the (1 0 0) meant MSN-PA still possessed mesoporous structure. But it could be observed from the SAXS spectrogram of MSN-hyd-MOP that all peaks

disappeared, which suggested the polymer sealed the pore channel of MSN. Furthermore, the N2 adsorption–desorption isotherm of MSN-NH2 and MSN-PA demonstrated a type IV curve in Fig. 3B,

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Fig. 3. (A) SAXS patterns, (B) N2- adsorption-desorption isotherm curves, (C) BJH pore size distribution plots and (D) FT-IR spectra of MSN-NH2, MSN-PA and MSN-hyd-MOP.

which further confirmed the presence of mesoporous structure. The MSN-NH2 possessed relatively high specific surface areas of 802.61 m2/g. And MSN-NH2 exhibited a comparatively narrow pore distribution of pore size centered at 2.3 nm (Fig. 3C). The specific surface area of MSN-PA was relatively reduced (524.49 m2/g), but the pore size (2.3 nm) was still suitable to load Dox [42]. The specific surface area of nanoparticles reduced to 66.08 m2/g after grafting of the polymer, confirming the polymer could completely block the pore of MSN. The FT-IR spectra was performed to provide direct identification of functionalized groups on the MSNs surfaces, as shown in Fig. 3D. The adsorption peak at 2941 cm
1 corresponded to the stretching vibration of CAH, and the peaks at 1509 cm
1 and 695 cm
1 were ascribed to the symmetrical bending vibration and bending vibration of ANH2, indicating the existence of the amino groups on MSNs (MSN-NH2). After the reaction between ANH2 and PA, typical stretching vibration of C@O and ACH3 at 1765 cm
1 and 1399 cm
1 appeared. Successful synthesis of MSN-hyd-MOP was confirmed by the characteristic peaks of C@O shifted from 1765 cm
1 to 1756 cm
1 and the new peaks at 1462 cm
1 belong the shear vibration peak of ACHA in MOP. Other absorption peak at 1631 cm
1 was attributed to C@N from hydrazone bonds [43]. The polymer capping layer of MSNs was further confirmed by 13 C NMR and TGA. In the 13C NMR spectrum of MSN-PA (Fig. 4A), the peaks at 43 ppm, 21 ppm, 9 ppm and 175 ppm were ascribed to the three kinds of methylene carbons from APTES molecules and the ketone groups of PA [44]. The peaks of C@O was shifted from 175 to 170 ppm shown in the 13C NMR spectrum of MSNhyd-MOP, which could be attributed to the carbons of ester group and carbonyl acetamide in MSN-hyd-MOP. And there are two new

peaks at 19 ppm and 70 ppm could be assigned to methyl carbon and methylene carbons from MOP. The results of TGA in Fig. 4B showed the weighting loss of MSN-NH2, MSN-PA and MSN-hydMOP were 13.4%, 15.4% and 33.7% at 800 °C, respectively, demonstrating the percentage of PA and MOP on the nanoparticles were 2.0% and 18.3%. In order to investigate the charge reversal performance of MSNhyd-MOP nanohybrids, the surface charge of MSN-hyd-MOP in 0.02 M PBS buffer solution of pH 7.4 and 6.5 was characterized by Malvern Zetasizer Nano S instrument. At pH 7.4, MSN-hydMOP were negatively charged. When the pH value was dropped to 6.5, the zeta potential increase from
10.2 mV to 16.6 mV, indicating MSN-hyd-MOP has a charge reversal capability at the pH condition of extracellular environment. 3.2. Drug loading and release The anticancer drug Dox was used as a model drug molecule. The LC and EE of MSN-hyd-MOP were individually about 12% and 87%, when the mass ratio of MSN-PA to Dox was 5:1. The pH-triggered controlled drug release behavior of MSN-PA@Dox and MSN-hyd-MOP@Dox was evaluated by measuring the cumulative release by UV–vis spectrum in various pH buffer (pH 7.4, 6.5 and 5.0), as shown in Fig. 5. At pH 7.4 and 6.5, the cumulative release of MSN-hyd-MOP@Dox were 7.4 ± 0.6% and 13.7 ± 0.9%, respectively, which much less than that of MSN-PA@Dox (58.2 ± 1.0% at pH 7.4 and 58.9 ± 0.8% at pH 6.5). This could be attributed to the polymer sealed pores of MSNs (Fig. 3), which suggested MSN-hyd-MOP effectively suppressed drug release at pH 7.4 and 6.5. But the released amount of Dox remarkably increased

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Fig. 4. (A)

13

89

C NMR spectra of MSN-PA and MSN-hyd-MOP. (B) TGA curves of MSN-NH2, MSN-PA and MSN-hyd-MOP.

Fig. 5. The Dox release curves of MSN-hyd-MOP@Dox (A) and MSN-PA@Dox (B).

to 43.4 ± 1.4% at pH 5.0, more than 6 times that at pH 7.4, indicating MSN-hyd-MOP was sensitive to endosomal/lysosomal pH. The phenomenon could be interpreted as that the pores of MSNs were exposed as cleavage of hydrazone bonds at pH 5.0, resulting in a rapid release of payload. In this respect, the nanocarriers could effectively reduce drug leakage in normal physiological conditions and extracellular environment of tumor cells but obviously increase intracellular drug release, which will be certainly conducive to effective cancer therapy. In order to determine the kinetics and mechanisms of drug release from the systems, the release data were individually analyzed with zero order, first order, Higuchi and Korsmeyer-Pappas models, as shown in Table 1. The value of the correlation coefficient (R2) was used to describe the degree of fit. According to the results, the comparatively low value of R2 of the Higuchi (0.812 < R2 < 0.876), zero order (0.568 < R2 < 0.662) and first order (0.343 < R2 < 0.493) models suggested poor curve fitting of these models. And the experimental data were most suitable for Korsmeyer-Pappas model (shown in Eq. (3)) with highest linearity (R2 > 0.95).



log

Mt M1



¼ nlogðtÞ þ logk

ð3Þ

where Mt and M1 are the release quantity of drug at time t and infinite time, k is the kinetic constant and n is an exponent. The value of exponent n could be used to characterize diverse release mecha-

nisms [45,46]. In case of spherical matrix, when n value is 0.43, corresponds to Fickian diffusion, while 0.43 < n < 0.85 represents anomalous transport mechanism, n value is equal to 0.85 indicates the case-Ⅱ transport. The release process was divided into two stages at different pH values, and Korsmeyer-Peppas equations were given in Fig S5B, S5C and S5D. In the first 1.5 h, anomalous transport can be used to describe the release behaviors at pH 7.4 (n = 0.538) and pH 6.5 (n = 0.514), which indicated the release processes were controlled by the diffusion of Dox and the swelling of the grafting polymers. In the case of pH 5.0, the value of n was lower than 0.43 in 1.5 h, representing the diffusion of Dox through the pore was a dominant controlling factor. Due to most of hydrazone bonds were damaged and most of polymers were removed. After 1.5 h, the drug release was primarily controlled by the diffusion of Dox and the erosion of nanohybrids owing to the grafting polymers attained a steady swelling state. The k values at pH 5.0 were obviously higher than those at pH 7.4 and 6.5, which should be due to the removal of polymer shell. This is in corresponding to the results that the fastest release rate and the highest release amount of Dox at pH 5.0. It is worth to noting that the hydrolysis of hydrazone bond is a reversible process [47]. And hydrazone bonds were only partially cleavage at pH 5.0 for 24 h, which may be the reason for relatively low release at pH 5.0. Therefore, one additional drug release investigation was carried out. We treated MSN-hyd-MOP@Dox in pH 1.0 solution to accelerate the cleavage of hydrazone bonds. After the

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Table 1 Model parameters obtained by fitting release data of Dox from MSN-hyd-MOP at different pH values. Model

Parameter

Period

Korsmeyer-Peppas model

n

0–1.5 h 2–24 h 0–1.5 h 2–24 h 0–1.5 h 2–24 h

k R2 Higuchi’s model Zero order model First order model

k R2 k R2 k R2

treatment with pH 1.0 solution for 24 h (Fig S5A), there was 62% of Dox released from the nanohybrids, which suggested the drugloaded nanohybrids could release more drugs with the increasing degree of cleavage of the hydrazone bonds. The results of in vitro drug release experiment imply the MSN-hyd-MOP drug delivery system is suitable of cancer therapy. 3.3. Cellular uptake The internalization and intracellular release of MSN-hydMOP@Dox into HepG2 cells were studied by CLSM. It can be observed from Fig. 6, a relatively weak red fluorescence of Dox could be observed in HepG2 cells after incubation with MSNhyd-MOP@Dox for a short time (1 h), which suggested a low accumulation of nanohybrid in the cytoplasm. When the co-culture time of HepG2 cells and MSN-hyd-MOP@Dox was extended to 4 h, the red fluorescence intensity increased obviously and overlapped with the blue fluorescence. This phenomenon was pronounced that the Dox in nanohybrids was released and reached

pH 7.4

6.5

5.0

0.538 0.189
1.445
1.371 0.992 0.973 0.013 0.816 0.002 0.577 0.019 0.393

0.514 0.192
1.178
1.105 0.999 0.950 0.024 0.812 0.004 0.568 0.019 0.398

0.377 0.193
0.655
0.621 0.990 0.987 0.066 0.876 0.011 0.662 0.016 0.493

the nucleus in 4 h. However, it was also observed that the red fluorescence intensity in cells cultured with free Dox for 4 h was stronger than that of MSN-hyd-MOP@Dox. Because Dox is a small molecule drug, which rapidly enters cells through diffusion process and then reaches the nucleus to interact with DNA [48]. Hence, MSN-hyd-MOP@Dox could be effectively internalized by HepG2 cells, followed by the cleavage of hydrazone bonds in the endosomes/lysosomes and the release of drug payload.

3.4. Cytotoxicity study For a drug delivery, the cytotoxicity and biocompatibility are important factors in evaluating its potential for application. The cytotoxicity of drug-free nanocarriers against HepG2 cells was evaluated by MTT assays to eliminate any nonspecific effect (Fig S6). MSN-hyd-MOP nanocarriers didn’t significantly inhibit cell growth and proliferation (cell viability >90%) after 48 h incubation even at the MSN-hyd-MOP concentration of 200 mg/L. This clearly

Fig. 6. CLSM images of HepG2 cells co-cultured with Dox and MSN-hyd-MOP@Dox for 1 and 4 h (Scale bars: 20 lm).

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Fig. 7. HepG2 cells viability exposed to drug or drug-loaded nanohybrids for (A) 24 h and (B) 48 h (*denote p < 0.05 the comparison between cells treated with the different concentration at the same time, # denote p < 0.05 the comparison between cells treated with different samples of the same concentration of drug(compared with MSN-hydMEP@Dox).)

confirmed that the synthesized MSN-hyd-MOP were biocompatible and possessed negligible cytotoxicity. Then, the dose-dependent cytotoxicity of MSN-hyd-MOP@Dox against HepG2 cells was further investigated. As shown in Fig. 7A, after incubating HepG2 cells for 24 h, free Dox showed higher cytotoxicity than MSN-hyd-MOP@Dox. As the dosage of Dox increased to 20 mg/L, the cell viability of HepG2 cells treated with free Dox (blue) and drug-loaded particles (red) decreased to 4.8 ± 0.9% and 43.2. ± 2.0%, respectively. The IC50 of free Dox (2.17 mg/L) was smaller than that of MSN-hyd-MOP@Dox (3.36 mg/L). The result was consistent with the observation from CLSM images that free Dox could diffuse into nucleus faster than MSN-hyd-MOP@Dox. When the co-culture time was up to 48 h, the difference between cytotoxicity of MSN-hyd-MOP@Dox and that of free Dox significantly reduced (Fig. 7B). It was explained that acidic excretion from tumor cells in 24 h incubation was not enough to achieve charge reversal of MSN-hyd-MOP@Dox, leading to only a small number of MSN-hyd-MOP@Dox entering the cells through endocytosis [49]. And the acidity of the culture solution was enhanced by the accumulation of excreta from cancer cells after incubation for 48 h, which reversed the surface charge of MSN-hyd-MOP@Dox, leading to the nanohybrids being easier to enter the cells. There are more MSN-hyd-MOP@Dox entered to cell and more Dox was transmitted to the cytoplasm triggered by the acidic condition of endosomes/lysosomes. The cytotoxicity of MSN-hyd-MOP@Dox was still lower than that of free Dox, because the maximum drug release of drug-loaded particles was 62.1 ± 1.5%. Generally, positively charged nanoparticles could be easily internalized by tumor cells, and further improving anticancer efficiency. Therefore, comparative experiments were carried out on drug-loaded particles with charge-reversing groups (MSN-hydMOP@Dox) and without charge-reversing groups (MSN-hydMEP@Dox) (Fig. 7B). When the drug concentration increased from 0.1 mg/L to 20 mg/L, the HepG2 cells viability for MSN-hydMOP@Dox (48 h) decreased from 87.4 ± 4.2% to 18.1 ± 4%, while that for MSN-hyd-MEP@Dox (48 h) decreased from 99.0 ± 2.2% to 39.6. ± 2.5%. And the IC50 of MSN-hyd-MOP@Dox (0.74 mg/L) was lower than the IC50 of MSN-hyd-MEP@Dox (2.41 mg/L), further confirming the charge-reversal property of morpholino groups can effectively enhance the internalization efficiency of the nanohybrids. These results pronounced that the constructed MSN-hyd-MOP has a good biocompatibility and Dox-loaded

nanocarriers efficiency.

showed

the

superior

anticancer

therapeutic

4. Conclusion Compared to previous MSN-based drug delivery system with single pH-responsive function, MSN-hyd-MOP nanohybrids in our study have a multi-stage pH response to both extracellular and endo-/lysosomal pH environment to simultaneously achieve charge conversion and controlled drug release. The results showed that MSN-hyd-MOP nanohybrids responded to the extracellular pH of tumor, resulting in the zeta potential of nanohybrids increased from
10.2 mV (pH 7.4) to 16.6 mV (pH 6.5). The cytotoxicity experiments further confirmed the charge reversal property could enhance the cytotoxicity of drug-loaded nanohybrids due to improving cell internalization. MSN-hyd-MOP@Dox reduced drug leakage in the pH of normal physiological environment (pH 7.4) or tumor cell environment (pH 6.5), and quickly released Dox in endo-/lysosomal pH (pH 5.0), because of the cleavage of hydrazone bonds. In addition, we use Korsmeyer-Pappas model to explain the kinetics and mechanisms of drug release from the systems in detail. Until today, only a small amount of research on MSNbased drug delivery system with both charge reversal and intracellular release through multi-stage pH response. Thus, this study has important instruction significance for improving drug delivery efficiency of MSN-based drug carriers. However, the cumulative release at pH 5.0 was relatively low, which is a similar problem in the reported drug delivery systems using hydrazide bonds [47,50]. Subsequent studies should further explore the effects of different channel types or different ‘‘gatekeepers” of MSNs on drug release for controlling drug release. Declaration of Competing Interest There are no conflicts to declare. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. 21776101 and No. 21808039), the Fundamental Research Funds for the Central Universities, China (No. 2019PY29) and the Research Fund Program of Guang-

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dong Provincial Key Lab of Green Chemical Product Technology (GC201807).

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