English

• 0.   Introduction

  From the reports published on the International Agency for Research on Cancer (IARC), approximately 10 million new tumor cases arise and no fewer than 6 million patients die from the disease per year^[1]. A main challenge in clinical treatment of tumor is how to accurately and high-efficiently deliver a nanocarrier which carries therapeutic agent to a tumor site to achieve ideal therapeutic effect. A drug delivery system is a means which enables the introduction of therapeutic drugs in the body and improves its effectiveness and safety by controlling the releasing rate, the releasing site and adjusting the releasing time. Drug delivery system is an interface between the patient and the drug^[2]. In a bid to overcome in vivo adverse pharmacokinetic characteristics and non-specific distribution of most conventional chemotherapeutic agents, many advances in nanocarriers have been used in cancer targeted therapies^[3-4].

  Studies have been conducted on stimuliresponsive microgels and applied in many fields^[5-8]. For their resemblance of physicochemical properties to those of the living tissues, these materials show good biocompatibilities, high water content, low interfacial tension, etc. N-isopropylacrylamide (NIPAM) is the most commonly and successfully researched materials among temperature-sensitive polymers. The lower critical solution temperature (LCST) of thermal-responsive nanogels based on NIPAM is about 32.0 ℃^[9-10]. This temperature is close to that of the physiological human body. Furthermore, as the surrounding temperature increases, the structure of the polymer microgel undergoes a phase conversion from a swollen structure to collapse aggregation, which provides an opportunity for loading or releasing small molecules, potentially having expectable applications in tumor treatment^[9-10]. Due to their reversible transition between dispersion and flocculation with the function of temperature, temperature variation can adjust the loading and releasing rate by PNIPAM-based nanocarriers. In addition, the LCST can adjust by adding comonomer into PNIPAM. Hydrophilic comonomer tends to increase the LCST and hydrophobic comonomer lowers the LCST, which provides an opportunity to embed or release small molecule, thus enabling drug delivery for tumor therapy^[11].

  In the last few years, organic and inorganic nanocomposites have become a hot spot in material science research. Attapulgite (ATP) is a hydrous magnesiumrich aluminosilicate clay mineral with chain layer structure, unique physical and chemical properties, including relatively permeability, good chemical stability and so on^[12-13]. Magnetic iron oxide nanoparticles have attracted great attention in biomedical field owing to their good biocompatibility, low toxicity and high stability in physiological environment. Magnetic nanoparticles have unique characteristics and have been widely used in drug targeting and delivery for diagnosis and treatment^[14]. It's easy to separate Fe₃O₄ nanoparticles with strong paramagnetism by an external magnetic field and this property is widely used in magnetic sepa- ration and presented excellent recyclability^[15-17].

  Folic acid (FA) is a kind of necessary vitamin necessary for a single-carbon transfer reaction. Since FA is important for the nucleotide biosynthesis sequences, it's consumed in large quantities through cell proliferation. Two membrane- associated proteins were used in normal cells. The former is the main approach in charge of metabolizing physiological folic acid present in almost whole cells. Most of them are found in degenerated epithelial cells and activated macrophages. They preferentially bound and internalized oxidized folate through receptor - mediated endocytosis. Though low concentrations of the reduced folate carrier may be sufficient to meet the requirements of most normal cells, folate receptor (FR) is often overexpressed in tumor cells, which may allow malignant cells to successfully contend for the vitamin when the supply is limited^{[15, 18]}.

  Doxorubicin hydrochloride (DOX) has a wide range of biochemical effects in the body. It inhibits the synthesis of nucleic acids by embedding itself in DNA. This mechanism may eventually lead to the death of cancer cells and thus has anti- cancer effects. It has a significant influence on the chemotherapy of acute leukemia, lymphoma and other solid tumors. DOX is a satisfactory anticancer drug.

  Herein, we synthesized FA-Fe₃O₄/ATP-P(NIPAM- AAM) based on emulsion polymerization. Due to the large surface area of ATP, the drug loading efficient is improved. The LCST of FA-Fe₃O₄/ATP-P(NIPAM- AAM) is well tuned by adjusting the composition of the two monomers before polymerization to adapt to human body temperature. Combining FA to the microgels through chemical bonds can increase the targeting ability. DOX is a model anticancer drug to carry out the in vitro test to verify the performance of the FA-Fe₃O₄/ATP-P(NIPAM-AAM) in identification and destruction cancer cells (Scheme 1).

  Scheme1

  

  Scheme1.  Schematic diagram of synthesis and targeting process of FA-Fe₃O₄/ATP-P(NIPAM-AAM)

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  1.   Experimental

  1.1   Materials

  N, N' - methylenebisacrylamide (MBA, 98%), N - isopropyl acrylamide (NIPAM, 99%), ammonium persulfate (APS, 98%) and sodium hyposulfite (SPS, 98%) were purchased from Acros Organics Co., Ltd. DMSO, toluene, ferric acetylacetonate (Fe(C₅H₇O₂)₃), triethylene glycol, N-hydroxysuccinimide (NHS, 98%), γ - methacryloxypropyl trimethoxysilane (KH-570), N-(3- dimethylaminopropyl)-N'-ethylcarbodllmidehydrochioricia (EDC, 98%), dialysis bag (M_W: 8~14 kD) and folic acid (98%) were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Attapulgite was purchased from Hongjin Attapulgite Instruments (Xuyi, China). Sodium dodecyl sulfate (SDS) was purchased from Alpha Chemical Industry Co., Ltd. (3-4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was obtained from Sigma - Aldrich (St. Louis, MO, USA). The fetal bovine serum (FBS) was from Corning (Medford, MA, USA). Dulbecco's modified eagle's medium (DMEM basic), penicillin - streptomycin solution and trypsin-EDTA (0.25%) were purchased from InvitrogenGibco (Carlsbad, CA, USA).

  1.2   Instrumentation and procedures

  The morphologies and the particle size of the obtained samples were characterized by transmission electron microscopy (TEM, JEOL JEM-200CX, 120 kV) and scanning electron microscopy (SEM, JEOL JSM-6700F, 10 kV). The ultraviolet- visible (UV-Vis) spectroscopy was acquired with AVATAR- 360 FT-IR spectrophotometer from Nicolet Corporation. The magnetic properties of Fe₃O₄/ATP were characterized by vibrating sample magnetometer (VSM, AGFM, Iran) at 25 ℃. The crystal structure of the material was analyzed by an X - ray diffractometer with Cu Kα (_λ =0.154 06 nm, X'pert-PRO). The tube voltage was 40 kV and the tube current was 60 mA. The scanning range was 5°~ 90°, and the scanning rate was 5 (°)·min^-1.The MTT as-say was probed using a spectrophotometric plate reader (Elx808, Biotek, USA) at a wavelength of 570 nm in vitro uptake images and obtained by LSM700 laser con- focal microscope (nikon A1). The DLS was character- ized by Malvern particle size analyzer (Zetasizer Nano ZS) and the model of freeze dryer was FD-1A-50 from Beijing medical laboratory instrument Co. LTD.

  1.3   Preparation of Fe₃O₄/ATP

  First, 0.2 g of dried ATP and 1.000 g of Fe (C₅H₇O₂)₃ were mixed evenly in 30 mL of triethylene glycol under the ultrasonic condition. The mixture was heated to 270 ℃ for 2 h^[19-20]. After cooling to room temperature, the prepared precipitate was washed with EtOH/deionized water for several times and dried in the vacuum oven over night at 50 ℃.

  1.4   Surface modification of Fe₃O₄/ATP

  Silane coupling reagent KH-570 was used as surface modifier. The vinyl group of KH - 570 was introduced onto the surface of Fe₃O₄/ATP to copolymerize with NIPAM^[21]. 1.0 mL of deionized water and 1.0 g of Fe₃O₄/ATP were dispersed in 100 mL of methylbenzene. Then, 3.0 mL of KH-570 was added into the mixture. In a 250 mL three-necked flask, the mixture was ultrasonificated for 40 min before stirred at 45~50 ℃ for 4 h. Finally, KH-570 modified Fe₃O₄/ATP was separated with a magnetic bar and sequentially washed with toluene, EtOH and deionized water. The KH-570- Fe₃O₄/ATP was dried at 65 ℃ in a vacuum oven.

  1.5   Preparation of Fe₃O₄/ATP-P(NIPAM-AAM)

  The KH-570-Fe₃O₄/ATP was dispersed into deionized water to obtain a Fe₃O₄/ATP dispersion with a concentration of 0.04 g·mL^-1. 0.4 g of NIPAM, the formulated amount of AAM (mass ratio of NIPAM/ AAM was 1:14), 5 mg of MBA and 2 mg of SDS were added in 2 mL of Fe₃O₄/ATP dispersion and then further diluted with 30 mL of deionized water^[22-23]. After the deoxygeneration of above solution was carried out with nitrogen for 30 min, the temperature was raised to 70 ℃. 1 mL of APS (3 mg·mL^-1) and 1 mL of SPS (2.5 mg·mL^-1) were added into the solution with a syringe. The nitrogen atmosphere was maintained in the whole process. After being stirred for 6 h with a magnetic stirrer, the solution was cooled down to the room tempera- ture and then dialyzed with the deionized water for 7 d to remove unreacted monomers and impurities to obtain Fe₃O₄/ATP-P(NIPAM-AAM). The product was frozen by a refrigerator and then stored in a freeze dryer before use.

  1.6   Preparation of FA - Fe₃O₄/ATP - P(NIPAM - AAM)

  First, 0.034 mmol·L^-1 of N-hydroxysuccinimide (NHS), 1.3 mg of 1 - ethyl- 3 - (3 - dimethylaminopropyl) carbodiimide (EDC) and 1.3 mg of FA were dissolved in DMSO. 20 mg of Fe₃O₄/ATP - P(NIPAM - AAM) was dispersed in 20 mL of PBS (pH 7.4) and adjust the pH value to 4.5~4.7. The above FA solution was added to the Fe₃O₄/ATP-P(NIPAM-AAM) solution and stirred at room temperature for 16 h in the dark^[24-25]. After adjusting the pH value to 9.0 to end the reaction, reagents were dialyzed in PBS solution (pH 7.4) and deionized water (molecular weight cutoff of 14 kD) for 6 d. The resultant FA-Fe₃O₄/ATP-P(NIPAM-AAM) was dried for the following uses.

  1.7   In vitro study

  1.7.1   In vitro drug loading and releasing

  20 mg of Fe₃O₄/ATP-P(NIPAM-AAM) was dispersed in 20 mL of deionized water containing 5 mg of DOX. The mixture was stored in refrigerator at 4 ℃ to make the nanogel fully inflated^[26-27]. The drug loading proceeded for 48 h.

  The dialysis of above solution was conducted in deionized water for 4 h, whilst water was altered per half hour. The concentration of drug in outer water was measured by UV-visible spectrophotometer, thereby the drug loading rate was calculated. DOX absorbance was measured per half hour from 26 to 50 ℃ and the temperature gradient was 2 ℃^[26-27].

  The DOX loading and releasing amount was determined by UV-visible spectrophotometer at the wavelength of 225 nm.

  1.7.2   Cell culture

  Human liver cancer cells (HepG2) were purchased from American Type Culture Collection (ATCC). HepG2 cells were cultured in DMEM medium containing 10% (V/V) FBS and 1% (V/V) antibiotics at 37 ℃ in a HERA cell 150 incubator (Thermo Fisher Scientific Inc., USA).

  1.7.3   In vitro cytotoxicity

  The MTT assay was used to determine the cytotoxicity of FA - Fe₃O₄/ATP - P(NIPAM - AAM) of HepG2 according to the previous literature^[28]. Briefly, cells were seeded in 96-well plate at a density of 2×10⁵ cells per well. After incubation overnight, cells were treated with different concentrations of samples. After 48 h of incubation, the medium of MTT solution was added. The cell survival rate was calculated according to the following formula:

  $ {\rm{Cell}}{\kern 1pt} {\kern 1pt} {\kern 1pt} {\rm{viability = }}\frac{{{\rm{O}}{{\rm{D}}_{{\rm{sample group}}}} - {\rm{O}}{{\rm{D}}_{{\rm{blank group}}}}}}{{{\rm{O}}{{\rm{D}}_{{\rm{negative control}}}} - {\rm{O}}{{\rm{D}}_{{\rm{blank group}}}}}} \times 100\% $

  Where, OD_{sample group} is the absorbance of sample group; OD_{negative group} is the absorbance of control group; OD_{negative group} is the absorbance of blank group.

  1.7.4   In vitro cellular uptake

  HepG2 cells were seeded in 96 - well plates at a density of 3×10⁵ cells per well and cultured incubator for an additional 16~20 h. The cells were treated with 200 μg·mL^-1 of FA-Fe₃O₄/ATP-P(NIPAM-AAM) for indicated time points. Then the medium was removed. The cells were washed in following way: trice with PBS, fixed with 4% paraformaldehyde for 15 min, trice with PBS. Finally, cells were observed under a LSM700 laser confocal microscope after stained with an anti - fade mounting medium with DAPI.

  2.   Results and discussion

  2.1   Qualitative characterization of Fe₃O₄/ATP

  Fig. 1A shows the XRD patterns of Fe₃O₄, Fe₃O₄/ ATP, and ATP. Six characteristic peaks (30.1°, 35.44°, 43.08°, 53.58°, 57.16°, and 62.78°) corresponding to the Fe₃O₄ nanoparticles (NPs) were also obtained for the Fe₃O₄/ATP, revealing that the Fe₃O₄ NPs were bonded to the ATP. What's more, the peak at 26.52° is well consistent to the primary diffraction plane of the ATP. The results indicated that the Fe 3O4/ATP were successfully synthesized^[21].

  图 1

  

  Figure 1.  (A) XRD patterns of (a) ATP, (b) Fe₃O₄ and (c) Fe₃O₄/ATP; (B) VSM patterns of (a) Fe₃O₄ and (b) Fe₃O₄/ATP

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  Fig. 1B exhibits the magnetic hysteresis loops of Fe₃O₄ and Fe₃O₄/ATP obtained with the VSM for magnetic fields from -5 to 5 kOe. The maximum values of magnetization saturation of the Fe₃O₄ and Fe₃O₄/ATP were 9.73 and 6.16 emu·g^-1. Compared with Fe₃O₄, the saturation magnetization of Fe₃O₄/ATP was reduced.The difference in saturation magnetization was attributed to the non-magnetic ATP clay particles^[21].

  2.2   Morphology characterization of FA-Fe₃O₄/ ATP-P(NIPAM-AAM)

  The TEM images depicted in Fig. 2a and 2b show the morphology and structure of Fe₃O₄/ATP and FA - Fe₃O₄/ATP-P(NIPAM-AAM). It is found that there is a gray layer on the edge of Fe₃O₄/ATP. This result indi-cates that the coating of temperature responsive poly- mer on the Fe₃O₄/ATP is successful.

  图 2

  

  Figure 2.  TEM images of (a) Fe₃O₄/ATP and (b) Fe₃O₄/ATP-P(NIPAM-AAM); SEM images of (c) Fe₃O₄/ATP and (d) Fe₃O₄/ATP-P(NIPAM-AAM)

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  The surface morphology of the Fe₃O₄/ATP and FA- Fe₃O₄/ATP-P(NIPAM-AAM) was probed by SEM. Fig. 2c and 2d are the SEM images of the Fe₃O₄/ATP and FA-Fe₃O₄/ATP-P(NIPAM-AAM) respectively. As a result, the irregular surface of Fe₃O₄/ATP implies that a layer of P(NIPAM-AAM) gel was coated on the surface of FA-Fe₃O₄/ATP particles.

  2.3   Qualitative characterization of FA-Fe₃O₄/ATP-P(NIPAM-AAM)

  Fig. 3A presents the IR spectra of ATP, Fe₃O₄, Fe₃O₄/ATP, KH-570-Fe₃O₄/ATP, P(NIPAM-AAM) and Fe₃O₄/ATP-P(NIPAM-AAM), respectively. The curve of ATP shows an apparent peak at 1 035 cm^-1, corresponding to the stretching vibration peak of Si—O of attapulgite clay. The stretching vibration peak observed in the curve of Fe 3O4 was at 580 cm^-1 according to the Fe—O of Fe₃O₄. The curve c in Fig. 3A presents the characteristic absorption peaks of Fe₃O₄ and ATP. Compared with Fe₃O₄/ATP, the stretching vibration peaks of —CH3 and —CH2 appeared in the curve of KH- 570 -Fe₃O₄/ATP, which are at about 2 876 and 2 930 cm^-1, indicating the successful modification of KH- 570. For P(NIPAM-AAM), the peak at 1 385 and 1 456 cm^-1 are C—H stretching vibration peaks on isopropyl, 1 547 cm^-1 is the bending vibration peak of —NH, and 2 876, 2 930 and 2 973 cm^-1 are the stretching vibration peaks of —CH3 and —CH2, 1 644 cm^-1 is the stretching vibration peak of the C＝O double bond. 1 035 cm^-1 is the bending vibration peak of Si—O. Compared with P(NIPAM-AAM), there are characteristic absorption peaks of Si-O and Fe-O for Fe₃O₄/ATP-P (NIPAM-AAM), indicating the successful grafting of Fe₃O₄/ATP-P(NIPAM-AAM)^[19].

  图 3

  

  Figure 3.  (A) IR spectra of (a) ATP, (b) Fe₃O₄, (c) Fe₃O₄/ATP, (d) KH-570-Fe₃O₄/ATP, (e) P(NIPAM-AAM) amd (f) Fe₃O₄/ATP-P(NIPAM-AAM); (B) TG curves of (a) ATP, (b)Fe₃O₄/ATP, (c) KH-570-Fe₃O₄/ATP and (d) Fe₃O₄/ATP-P(NIPAM-AAM); (C) UV-Vis spectra of (a) FA, (b) Fe₃O₄/ATP-P(NIPAM-AAM) and (c) FA-Fe₃O₄/ATP-P(NIPAM-AAM); (D) DLS curve of FA-Fe₃O₄/ATP-P(NIPAM-AAM)

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  Fig. 3B shows the TG curves of ATP, Fe₃O₄/ATP, KH-570-Fe₃O₄/ATP and Fe₃O₄/ATP-P(NIPAM-AAM). From the picture, we can see that compared to KH-570/ Fe₃O₄/ATP, the weight loss of Fe₃O₄/ATP-P(NIPAM- AAM) increased, indicating that P(NIPAM-AAM) has been coated onto KH-570-Fe₃O₄/ATP successfully.

  Fig. 3C shows the UV - Vis spectra of FA, Fe₃O₄/ ATP-P(NIPAM-AAM) and FA-Fe₃O₄/ATP-P(NIPAM- AAM). The absorption peak of FA was 225 nm and the absorption peak of Fe₃O₄/ATP-P(NIPAM-AAM) was 280 nm. From the above results, the FA-Fe₃O₄/ATP- P (NIPAM-AAM) was synthesized successfully.

  As shown in Fig. 3D, the thermo-sensitive profile of FA-Fe₃O₄/ATP-P(NIPAM-AAM) was characterized in the temperature range of 27.0~49.0 ℃ by DLS monitoring hydrodynamic diameter (D_h). It is clear that compared with the LCST (32.0 ℃) of PNIPAM microgel，the LCST of FA-Fe₃O₄/ATP-P(NIPAM-AAM) was around 38.5 ℃ due to the hydrophilic group of acrylamide. The temperature is close to that of human body and it's expected to be applied to the study of targeted sustained release cancer drugs.

  2.4   In vitro assay

  2.4.1   In vitro drug loading and releasing

  Fig. 4 exhibits the DOX releasing amount spectra of P(NIPAM - AAM) and Fe₃O₄/ATP - P(NIPAM - AAM) from 26.0 to 28.0 ℃. In Fig 4a, the increase of releasing amount of DOX was obvious. It may be ascribed to the free DOX which was unloaded on P(NIPAM - AAM) microgel. The shape of P(NIPAM-AAM) and Fe₃O₄/ ATP-P(NIPAM-AAM) curves was similar between 26.0~30.0 ℃, namely two- stage releases. There was a sharp hopping on curve of Fe₃O₄/ATP-P(NIPAM-AAM) at around 38.5 ℃ due to the swell - shrink of nanogel corresponding to the analysis of DLS. Compared with P(NIPAM-AAM), the loading efficiency of Fe₃O₄/ATP- P(NIPAM-AAM) rose from 62% to 79% and the releasing efficiency increased from 36% to 56%. The advantage of ATP is apparent to raise the adsorption capacity and the releasing amount.

  图 4

  

  Figure 4.  Plots of releasing amount of (a) P(NIPAM-AAM) and (b) Fe₃O₄/ATP-P(NIPAM-AAM)

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  2.4.2   In vitro cytotoxicity

  To evaluate the safety of FA-Fe₃O₄/ATP-P(NIPAM - AAM), HepG2 cells were treated with 0, 6.25, 12.5, 25, 50, and 100 μg·mL^-1 DOX, 0, 31.65, 63.29, 126.58, 253.16, and 506.32 μg·mL^-1 FA-Fe₃O₄/ATP- P(NIPAM-AAM) and 0, 25.4, 50.79, 101.58, 203.16, and 406.32 μg·mL^-1 DOX loaded FA-Fe₃O₄/ATP- P(NIPAM-AAM), respectively. MTT was used to detect cell viability. As shown in Fig. 5a, after treatment with different concentrations of FA-Fe₃O₄/ATP-P(NIPAM-AAM) for 48 h, no significant cytotoxicity was observed on HepG2 cells. The cell viability was above 1.0, indicating that FA-Fe₃O₄/ATP-P(NIPAM-AAM) promoted the growth, rather than inhibited, the cell viability. The result indicates that the synthesized FA-Fe₃O₄/ATP- P(NIPAM-AAM) polymer is safe and can be utilized as a media for drug delivery in biomedical field.

  图 5

  

  Figure 5.  MTT test of (a) FA-Fe₃O₄/ATP-P(NIPAM-AAM), (b) free DOX and (c) DOX loaded FA-Fe₃O₄/ATP- P(NIPAM-AAM)

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  From Fig. 5b and 5c, a dose-dependent inhibitory effect of DOX and DOX loaded FA-Fe 3O4/ATP-P(NIPAM - AAM) was observed on HepG2 cells. The cytotoxicity increased as the concentration increased. However, the cytotoxicity of free DOX was weaker than that of DOX loaded FA-Fe₃O₄/ATP-P(NIPAM-AAM). This is mainly attributed to the targeting ability of FA. The IC₅₀ (the concentration at which cell proliferation is half inhibited) values of free DOX and DOX loaded FA-Fe₃O₄/ATP-P(NIPAM-AAM) were 46 and 19 μg· mL-1 respectively. The inhibitory effect on cancer cells of DOX loaded FA-Fe₃O₄/ATP-P(NIPAM-AAM) was stronger than that of free DOX.

  2.4.3   In vitro cellular uptake

  For further research on the in vitro targeting of liver cancer cells by FA-Fe₃O₄/ATP-P(NIPAM-AAM), DOX was chosen as model drugs to identify the cellular uptake of microgels. A qualitative test of cellular uptake was performed using confocal laser scanning microscope (CLSM) of HepG2 cells after 3 or 6 h of exposure. It should be noted that the blue fluorescent signal was the nucleus of DAPI negative staining and the surrounding red fluorescent signal was the intake of DOX. As shown in Fig. 6, DOX loaded FA-Fe₃O₄/ATP-P(NIPAM-AAM) was swallowed intracellularly by HepG2 cells after incubating HepG2 cells with DOX loaded FA-Fe₃O₄/ATP-P(NIPAM-AAM) for 3 h. A red fluorescent signal was observed arising from the cytoplasmic matrix. CLSM test's results showed that when the time is prolonged to 6 h, the red fluorescent signal in the cells of the free DOX group was almost unchanged when compared with the HepG2 cells incubated for 3 h. In contrast, the red fluorescent signal of FA- Fe₃O₄/ATP - P(NIPAM - AAM) - DOX was significantly enhanced relative to the group incubated for 6 h. It can verify its targeting ability.

  图 6

  

  Figure 6.  In vitro cellular uptake assay of free DOX and DOX loaded FA-Fe₃O₄/ATP-P(NIPAM-AAM) (NPs/DOX)

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  3.   Conclusions

  In summary, magnetic and temperature - sensitive nanoparticle microgels FA-Fe₃O₄/ATP-P(NIPAM-AAM) with targeting were synthesized by emulsion polymerization method. The phase transition temperature rose from 32.0 to 38.5 ℃, which is slightly higher than that of human body and suitable for cell experiment in vitro. In vitro cell test results show that the FA-Fe₃O₄/ATP-P(NIPAM-AAM) exhibits good biocompatibility. At the same time, the microgels with targeting ability can effectively prolong the time of drug release and action. In vitro drug release experiments show that ATP can increase drug loading and drug release. This material is expected to accurately release anticancer drugs to tumor cells so as to reduce side effects, which is expanding the application of targeted tumor therapy.

  
  
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  Scheme1  Schematic diagram of synthesis and targeting process of FA-Fe₃O₄/ATP-P(NIPAM-AAM)

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  Figure 1  (A) XRD patterns of (a) ATP, (b) Fe₃O₄ and (c) Fe₃O₄/ATP; (B) VSM patterns of (a) Fe₃O₄ and (b) Fe₃O₄/ATP

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  Figure 2  TEM images of (a) Fe₃O₄/ATP and (b) Fe₃O₄/ATP-P(NIPAM-AAM); SEM images of (c) Fe₃O₄/ATP and (d) Fe₃O₄/ATP-P(NIPAM-AAM)

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  Figure 3  (A) IR spectra of (a) ATP, (b) Fe₃O₄, (c) Fe₃O₄/ATP, (d) KH-570-Fe₃O₄/ATP, (e) P(NIPAM-AAM) amd (f) Fe₃O₄/ATP-P(NIPAM-AAM); (B) TG curves of (a) ATP, (b)Fe₃O₄/ATP, (c) KH-570-Fe₃O₄/ATP and (d) Fe₃O₄/ATP-P(NIPAM-AAM); (C) UV-Vis spectra of (a) FA, (b) Fe₃O₄/ATP-P(NIPAM-AAM) and (c) FA-Fe₃O₄/ATP-P(NIPAM-AAM); (D) DLS curve of FA-Fe₃O₄/ATP-P(NIPAM-AAM)

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  Figure 4  Plots of releasing amount of (a) P(NIPAM-AAM) and (b) Fe₃O₄/ATP-P(NIPAM-AAM)

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  Figure 5  MTT test of (a) FA-Fe₃O₄/ATP-P(NIPAM-AAM), (b) free DOX and (c) DOX loaded FA-Fe₃O₄/ATP- P(NIPAM-AAM)

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  Figure 6  In vitro cellular uptake assay of free DOX and DOX loaded FA-Fe₃O₄/ATP-P(NIPAM-AAM) (NPs/DOX)

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