Preparation of a Novel Thiol Surface Modifier and Fe 3 O 4 Drug Loading Agent as well as Releasing under pH-Sensitivity

In this paper, in order to take advantage of the combination between magnetic nano-Fe 3 O 4 and surface modi ﬁ er, a pH-sensitive drug delivery system that could e ﬀ ectively deliver doxorubicin (DOX) to tumor tissue was constructed. The novel drug delivery system named Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) was prepared through three steps. The ﬁ rst step, a surface modi ﬁ er with the thiol group, thiohydrazide-iminopropyltriethoxysilane surface modi ﬁ er (named TIPTS), was synthesized for the ﬁ rst time. The second step, Fe 3 O 4 -TIPTS was synthesized by treating nano-Fe 3 O 4 with TIPTS. The last step, Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) was synthesized in the presence of the Fe 3 O 4 -TIPTS, polyethyleneimine (PEI), and polyethylene glycol (PEG) by mercapto-initiated radical polymerization. Among them, magnetic nanoparticles (MNPs) were used as magnetically responsive carriers, PEG was the surface-modifying compound, and PEI was the drug loading site which primary amine reacts with doxorubicin (DOX). Targeted nanoparticles were considerably stabilize in various physiological solutions and exhibited pH-sensitive performance in drug release. Thence, Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) is a promising nanocarrier for targeting tumor therapy.


Introduction
In the last few decades, the incidence and mortality of malignancy increased year by year, and it has become the leading cause of death in humans. Chemotherapy [1] was the most commonly used clinical treatment for cancers. However, traditional anticancer drug formulations were nonspecificity [2] for tumors; especially when used in large doses, severe side effects were often caused [3]. That is why the development of efficient delivery systems with the ability to improve in vivo distribution and significant controlled sustained release behavior is required. One innovative technological approach to solve this problem is nanotechnology which focuses on the transfer of nano-sized biocompatible devices into the cells [4]. Among different types of nanomaterials, Fe 3 O 4 nanoparticles is one kind of MNPs that have shown great promise as novel delivery systems and theranostics for personalized medicine due to their shape controllability and large specific surface area. And most importantly, their unique optical, electrical, and superparamagnetic properties give potential imaging development, targeted delivery, and synergistic drug therapy, suitable for drug delivery in cells [5]. Naked Fe 3 O 4 NPs are easy to aggregate and oxidize and thus were often coated by hydrophilic materials and biocompatible polymers for targeted drug delivery [6][7][8].
The mercaptosilane surface modifier [9] is a particular kind of organosilicon compounds. The mercaptosilane surface modifier contains both a mercapto group reactive with an organic substance and a silicon functional group reactive with an inorganic substance. In view of this special molecular structure, a mercaptosilane surface modifier could be used as a "molecular bridge" [10] between organic substance and inorganic substance to prepare composite materials having excellent performances.
The surface coating [11] controls the absorption of particles by different cell types and affects biocompatibility, as well as the distribution of nanoparticles in the tissues of the organism [12][13][14], although many scientists use cationic bonds [15] to graft polymers onto the surface of nanoparticles as a drug carrier now. However, in the case of a pharmaceutical carrier obtained in this manner, cationic binding is extremely easily deactivated in physiological medium environment, resulting in poor stability. For this shortcoming, we use mercapto (-SH) [16] and polyethylene glycol (PEG) [17] propose for particle coating by free radical bonding, which can significantly improve the stability of nanoparticles in physiological medium environment, prolong the circulation time in the body, and improve the targeted delivery efficiency. PEG [18] in particular is considered to be a very promising material that protects the nanoparticles from the immune system, promotes a longer circulation time, and inhibits removal by the reticuloendothelial system. Although the application of polyethyleneimine (PEI) is plagued by their toxicity concerns, modification of PEI with PEG can address some of these concerns, improve the transfection efficiency, and enhance the systemic duration [19] at the same time.
Doxorubicin (DOX) is the most widely used chemotherapeutic drug. Although it has been standardized as an anticancer drug and has potential diverse toxicities, the clinical use of DOX is restricted [20]. In order to minimize the side effects, an efficient strategy is using nanoparticles as carriers for DOX delivery [21][22][23]. The novel drug delivery system in my manuscript is named as Fe 3 O 4 -TIPTS-g-(PEI-co-PEG). PEI and PEG were grafted Fe 3 O 4 through TIPTS, which may load DOX to improve selective cytotoxicity of the drug to targeted cells and reduce the systemic toxicity to normal cells.
In normal tissues, the extracellular pH is relatively basic (pH = 7:4), whereas in tumor tissues, the pH is close to endosomes (pH = 5:0 − 6:0) or lysosomes (pH = 4:5 − 5:0) [24]. This difference provides a new idea for cancer treatment, which is to build a pH-sensitive drug delivery system. In the present paper, the -NH 2 group belonging to PEI of Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) reacts with the -C=O group of DOX, and the resulting bond is the hydrazone bond. The hydrazone bond is kept stable in physiological condition; once the pH value decreases to 4.0-6.0, the hydrazone bond becomes unstable and then releases massive drugs [25,26]. This pH-triggered delivery system will improve the efficacy of DOX while decreasing its cytotoxicity toward healthy cells (Scheme 1).  Briefly, 25 ml methylbenzene and 1 g Fe 3 O 4 nanoparticles were stirred at room temperature for 30 min. This was followed by the addition of 4 g TIPTS [27] (preparation of a lab-made novel thiol-containing silane coupling agent TIPTS was described in reference 39) and further stirring until dissolution was complete. Under purified N 2 atmosphere, this solution was heated to 65°C in a water bath, stirring for 8 h. Finally, the resulting product was filtered, washed with distilled methylbenzene for three times, and dried under vacuum for 24 h. This part of the experiment process is shown in Scheme 3.

Synthesis of Fe 3 O 4 -TIPTS-g-(PEI-co-PEG)
. Fe 3 O 4 -TIPTS (1.77 g) was dissolved in 50 ml methylbenzene and stirred at room temperature for 30 min. Followed by the addition of 4.425 g PEI dissolved in 10 ml ethanol and 10 g PEG dissolved in 20 ml methylbenzene. This solution was heated to 55°C in a water bath, continuous flow of nitrogen into the stream, stirring for 8 h. Finally, the resulting product was filtered, washed with distilled water for three times, and dried under vacuum for 24 h. This part of the experiment process is shown in Scheme 4.

Drug Loading.
To load DOX on modified MNPs, 20 mg dry Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) was dispersed in 8 ml DMSO; 3 mg DOX was added and allowed to react with the nanoparticles for 24 h in the dark. The resulted products were collected by magnetic decantation and washed twice with deionized water. The DOX-loaded Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) were freeze-dried and stored in the dark at 4°C. The amount of unbound DOX was quantified using a UV-Vis spectrophotometer at 420 nm.

Results and Discussion
3.1.1. FTIR Analysis. FTIR spectra of products are shown in Figure 1. From these curves, the peak could be seen at 589 cm -1 attributed to the stretching vibration of the Fe-O group, the peak could be seen at 1039 cm -1 attributed to the stretching vibration of the C-H group, the peak could be seen at 1171 cm -1 attributed to the C-C group, the peak could be seen at 1642 cm -1 attributed to the C-OH group, the peak could be seen at 2571 cm -1 attributed to the -SH group, and the peak could be seen at 2856 cm -1 and 2922 cm -1 attributed to the stretching vibration of the -CH 2 group.

Journal of Nanomaterials
From curve (a), curve (b) and curve (c), the peak at 2856 cm -1 and 2922 cm -1 could only be seen at curve (b) and curve (c), not at curve (a), because TIPTS and copolymer could make nano-Fe 3 O 4 organized. The peak at 2571 cm -1 could only be seen at curve (b), because the -SH group was decomposed to obtain free radicals for grafting two polymers on Fe 3 O 4 -TIPTS. The peak at 1642 cm -1 could only be seen at curve (b), because the C-OH group belongs to PEI, which further indicated that polymers were successful to be grafted on Fe 3 O 4 -TIPTS.
3.1.2. Particle Size Analysis. The particle size spectra for Fe 3 O 4 , Fe 3 O 4 -TIPTS and Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) are shown in Figure 2. The results showed that the diameter size of Fe 3 O 4 was 39.6 nm, the diameter size of Fe 3 O 4 -TIPTS was 47.6 nm, and the diameter size of Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) was 112.8 nm. It indicated that the diameter size of the latter one is gradually larger than the diameter size of the previous one, because TIPTS by lab-made could modify Fe 3 O 4 in a smooth way. Moreover, TIPTS could also obtain free radicals for grafting PEI and PEG onto the surface of Fe 3 O 4 -TIPTS. And then, the diameter size results of all products were between 20 and 150 nm, which is beneficial to the absorption of endothelial reticular system and recognition of phagocytic cells.    3.1.5. VSM Analysis. Neither the remanence nor the coercivity was observed in the three hysteresis curves; therefore, the magnetization results shown in Figure 7 suggested that Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) was indeed superparamagnetic and had a strong magnetic response. They exhibited superparamagnetism with the saturation magnetization (Ms) values of 68.23, 63.58, and 55.22 emu/g at 25°C, respectively. It indicated that the polymerization did not affect the magnetic properties of the superparamagnetic nanoparticles because the structure of the Fe 3 O 4 nanoparticles remained in the polymerization procedure. Therefore, the DOXloaded Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) can be easily controlled by an external magnetic field to accurately deliver DOX to the target area. Furthermore, the decrease in the saturation magnetization of the Fe 3 O 4 -TIPTS and Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) nanoparticles compared with the Fe 3 O 4 was ascribed to the TIPTS and the copolymer of PEI and PEG ingredients grafted.   Figure 8(a), the Fe 3 O 4 synthesized by the method in this paper presented a uniform particle size, and each nano-microsphere is basically in an independent state. Figure 8(b) shows the higher magnification image of Fe 3 O 4 -TIPTS; it could be seen that TIPTS (a silane surface modifier with thiols group) was grafted on the surface of Fe 3 O 4 . Figure 8(c) shows the higher magnification image of Fe 3 O 4 -TIPTS-g-(PEI-co-PEG). Under the action of a mercapto group, branching and cluster polymers were formed by PEI and PEG grafted onto Fe 3 O 4 . And then, the particle size of Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) was uneven due to the difference in the amount of the graft polymer.  Figure 9. From these curves, the peak could be seen at 558 cm -1 attributed to the stretching vibration of the Fe-O group, the peak could be seen at 2851 cm -1 and 2920 cm -1 attributed to the stretching vibration of the -CH 2 group, the peak at 3332 cm -1 could be attributed to the O-H groups of PEG and DOX. The peaks at 1617 cm -1 could be attributed to N-H bending. The peaks at 1280 cm -1 could be attributed to C-N stretching modes. The peaks at 1408 cm -1 could be attributed to quinine. The peaks at 1,285 cm -1 could be attributed to anthracycline. The peaks at 1730 cm -1 could be attributed to 13-carbonyl    Figure 10. In vitro release studies of DOX over time were studied by monitoring the absorbance at 482 nm. In vitro release of DOX from Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) was simulated at 37°C. Standard curve was calculated at pH 5.5. The relationship between the absorption value (Abs) and the concentration is derived according to

XRD Analysis. The XRD spectra for the products for
Thus, the standard curve of vitro release of DOX was y = 25:5216x + 0:00887 R 2 = 0:99926 The result of pH sensitive about vitro release of DOX is shown in Figure 11. It indicated that DOX onto Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) was relatively stable at blood pH and more effectively released its payload at pH = 4:5 than pH = 5:5 or pH = 7:4. The functionalized particles slowly released DOX over 80 h at 37°C under pH 4.5 (lysosomes),

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Journal of Nanomaterials 5.5 (endosomes), and 7.4(normal tissues) PBS solutions, which was both time-and pH-dependent; the cumulative dissolution profiles of nanoparticles are shown in Figure 11. It indicated that only 21.06% of drug was released from Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) at pH 7.4, separately, over the process of 80 h, while at pH 5.5, it demonstrated higher release satisfied with 75.68% and at pH = 4:5 up to 80.24%. The result indicated that nanoparticles under acidic conditions showed higher DOX release rates at endosomal pH (4.5-5.5) as compared with normal tissues pH (7.4). This phenomenon could be attributed to the fact that after placing Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) in acidic PBS, the C=N bond between DOX and Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) is attacked by H + , releasing DOX. While from pH 5.5 to 4.5, the release rate of DOX was also increased slightly. This phenomenon was due to the protonation of the DOX amino group, which could gave DOX a positive charge to enhance its solubility in acidic conditions; accordingly, a faster drug release was caused.

Conclusion
In summary, our research results have synthesized a DOXloaded pH-sensitive magnetic system for targeted drug delivery. Nano-Fe 3 O 4 was modified by the mercaptosilane surface modifier TIPTS, and block copolymer poly(ethylene glycolco-ethyleneimine) grafted Fe 3 O 4 to obtain Fe 3 O 4 -TIPTS-g-(PEI-co-PEG). The nano-Fe 3 O 4 was a core of Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) which possesses the targeted function. DOX was bonded with Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) by a hydrazone bond. At different pH, the hydrazone bond could act as the switch to control the release of the drug encapsulated, so the potential of DOX-loaded Fe 3 O 4 -TIPTS-g-(PEI-co-PEG) as the carrier for pH-sensitive drug release is demonstrated. In vitro, DOX was released more readily at pH 4.5, which 80.24% DOX was released within 80 h. Therefore, the results demonstrate the versatility of the DOX-loaded magnetic nanoparticles as a potential antitumor drug delivery system.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
No potential conflict of interest was reported by the authors.