Preparation and Properties of Janus Heparin-Loaded Ammoniated

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C: Physical Processes in Nanomaterials and Nanostructures

Preparation and Properties of Janus Heparin-Loaded Ammoniated-Hollow Mesoporous Silica Nanomotors Sisheng Hu, Shuibin Shao, Huan Chen, Jiajia Sun, Jing Zhai, Hanyu Zheng, Mimi Wan, Yuhong Liu, Chun Mao, and Jing Zhao J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b02079 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 14, 2018

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Preparation and Properties of Janus Heparin-loaded Ammoniated-Hollow Mesoporous Silica Nanomotors Sisheng Hu,† Shuibin Shao,‡ Huan Chen,‡ Jiajia Sun,‡ Jing Zhai,‡ Hanyu Zheng, Mimi Wan,‡ Yuhong Liu,‡ Chun Mao*,‡ and Jing Zhao*,†

†

State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,

Institute of Chemistry and BioMedical Sciences, Nanjing University, Nanjing 210093, China.

‡

National and Local Joint Engineering Research Center of Biomedical Functional

Materials, Jiangsu Key Laboratory of Biofunctional Materials, School of Chemistry and Materials Science, Nanjing Normal University, Nanjing, 210023, China.

KEYWORDS: hollow mesoporous silica, heparin, platinum decorating, nanomotors

ABSTRACT:

In

this

case,

the

development

of

Janus

heparin-loaded

ammoniated-hollow mesoporous silica (H-A-HMS) nanomotors was reported. Firstly, A-HMS nanoparticles were prepared by combination of hard template (Fe3O4 regarded as core), soft template (cetyltrimethylammonium bromide (CTAB) regarded as mesoporous template agent) and ammoniated process. Then, the heparin was loaded into A-HMS nanoparticles by a simple soaking process, and Janus H-A-HMS


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nanomotors were fabricated by platinum (Pt) decorating onto the partial surface of H-A-HMS nanoparticles, which the bubble-propelled motion was obtain by the help of Pt catalytic decomposition of hydrogen peroxide (H2O2) into oxygen (gas) and water. Finally, results of performance test indicated this H-A-HMS nanomotors we proposed had good blood compatibility, non-cytotoxicity, high load and controlled release of heparin, and autonomous motion ability. They will bring great potential for more applications of heparin in the biomedical field.

■ INTRODUCTION Currently, inspired by the life movement in nature, material scientists have developed many artificial motor systems at the macro-, micro-/nanometer and molecular levels by multiple approaches.1-5 Based on their capability of converting chemical or external energy into mechanical movement, the artificial motors hold huge potential for applications in various field include micro-/nanosurgery, drug delivery, environmental remediation, sensing, and assisted fertilization.6-12 Specifically they are often used to treat disease. Research shows that artificial motors have infinite potential in biomedical fields, such as drug targeting transportation, blood clot removal, atherosclerosis treatment, wound cleaning, parasite elimination and renal stone crushing.13-19 In the field of drug delivery based on artificial motors, the diversity of the motion engines and loaded drugs and the versatile elegant materials structures offer a promising route and abundant source for the design and fabrication of artificial motor with multiple functions of drug delivery and release.20,21 Heparin is called glycosaminoglycans which is widely used as therapeutic agents in



biomedical fields due to its lots of crucial biological functions since its discovery in 1916.22-27 To date, it has been extensively used in clinical practice especially for the prevention and treatment of deep vein thrombosis, arterial thromboembolism, and pulmonary embolism.28-30 Thrombus is a blood coagulant made up of fibrin. Once the thrombus is formed in the blood vessel, it can become more and more tightened as time goes by, and the attachment to the blood vessel wall is also more and more solid. The thrombus in this state is hard to dissolve. Therefore, in order to dissolve the thrombus and achieve the goal of vascular recanalization, effective thrombolytic drugs must be used early in the formation of thrombus. The commonly used clinical anticoagulant drugs, such as aspirin, warfarin, heparin sodium, are not thrombolytic drugs, have anti-clotting effect and help prevent thrombus formation and expansion.31,32 Besides, more interesting will be to see how heparin contributes to the anti-tumor and anti-inflammatory diseases.33-43 Alur et al. reported the in vitro anti-tumor effects of low molecular weight heparins on HepG2 hepatocellular carcinoma and MIA PaCa-2 cancer cells including the cancer cell cycle arrest, migration, gene expression, apoptosis, invasion, viability, and colony formation.33 Schnoor et al. systematically reviewed preclinical and clinical studies of heparin and its low molecular weight derivatives as anti-tumor agents in glioblastoma multiforme, which is the most common primary brain tumor. The experimental results showed that heparin has some influence on glioblastoma multiforme such as the interdiction of assimilating of extracellular vesicles, and the disincentive of angiogenesis and neoplasm growth in vitro and in vivo.37 Zenerino and co-workers depicted a new


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molecular

mechanism

about

low

molecular

weight

heparins

exerts

its

anti-inflammatory effect on pre-eclamptic placentae.43 It will bring a broader space for the biomedical application of heparin. In this case, based on the concept of nanomotor, drug controlled release of mesoporous structure and the biomedical action of heparin, we put forward a design concept of Janus heparin-loaded ammoniated-hollow mesoporous silica (H-A-HMS) nanomotors. First, the ammoniated-hollow mesoporous silica (A-HMS) nanoparticles were prepared by combination of hard template (Fe3O4 regarded as core), soft template (cetyltrimethylammonium bromide (CTAB) regarded as mesoporous template agent) and ammoniated process. The mesoporous silica was used for drug release.44,45 Then, the heparin was loaded into A-HMS nanoparticles by a simple soaking process, and Janus H-A-HMS nanomotors were fabricated by platinum (Pt) decorating onto the partial surface of H-A-HMS nanoparticles, which the bubble-propelled motion was obtain by the help of catalytic decomposition of hydrogen peroxide (H2O2) into oxygen (gas) and water.46 The properties include blood compatibility, cytotoxicity, heparin load and release, and autonomous motion ability of the H-A-HMS nanomotors we proposed were investigated.

■ EXPERIMENTAL SECTION

Materials. Heparin was bought from Sigma. Tetraethoxysilane (TEOS) (Sinopharm Chemical Reagent Co., Ltd, SCRC) and hexadecyl trimethyl ammonium Bromide (CTAB) (Shanghai Lingfeng Chemical Reagent Co., Ltd.), and



aminopropyltriethoxysilane (APTES) (Aladdin Chemistry Co., Ltd.) were used without purification. Ethylene glycol, trisodium citrate (Na3Cit), and FeCl3·6H2O were obtained by SCRC. Synthesis of Janus H-A-HMS nanomotors. The schematic illustration of the synthesis process of Janus H-A-HMS nanomotors was shown as Figure 1.

Figure 1. Schematic illustration of the synthesis process of Janus H-A-HMS nanomotors.

(1) Preparation of Fe3O4 nanoparticles with the size of 200 nm: 3.6 g of NaAc and 0.4 g of Na3Cit were added into 80 mL of ethylene glycol solution containing FeCl3·6H2O (2.7 g), which was stirred for 1 h and then transferred to autoclave for 20 h at 200℃. The product was collected, washed with ethanol, and dried. (2) Preparation of HMS nanoparticles: 50 mg of Fe3O4 with 0.858 mL of hydrazine hydrate was added into 30 mL of distilled water (DW) under ultrasonic condition for 30 min. 70 mL of DW was added subsequently under intensely stirring under room temperture, and then 32 µL of TEOS was added slowly into the resultant mixture, which was allowed to heat at 90℃ for 2 h. The solid product of the reaction was


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collected by magnets and the liquid was removed. Further, 13.6 mg of solid product was re-dispersed into 70 mL of DW, and then 0.15 g of CTAB was added into the dispersing liquid as template. The obtained mixture was heated subsequently to 90℃ under intensely stirring, and 0.2 mL of TEOS was slowly injected and the resultant mixture was continuous agitated for 2 h at 90℃. After that, the solid product of the reaction was collected by magnets and the liquid was removed. The obtained product was washed with ethanol, water and dried. The dried product was calcined under air atmosphere for 5 h at 550℃ to remove CTAB. The obtained product was named as MMS. Then, HMS was obtained by washing MMS with hydrochloric acid solution (10 mol L-1) for 24 h to remove the core of Fe3O4. (3) Preparation of A-HMS nanoparticles: The modification of APTES on the samples (MMS and HMS) was conducted according to the following procedure. 20 mg of sample was dispersed in 10 mL of ethonal solution containing 15.7 µL of APTES and stirred at room temperature for 30 min (N2 atmersphere). Then 7.2 mg of water was added to the solution with further stirring for 30 min. The obtained products were then separated, washed and dried, which named as A-MMS and A-HMS, respectively. (4) Preparation of H-A-HMS nanoparticles and Drug loading and release process: 20 mg of A-HMS sample was dispersed into 5 mL of heparin PBS solution (4 mg mL-1) for 24 h. Then the heparin-loaded A-HMS (H-A-HMS) nanoparticles were obtained. Similarly, the heparin-loaded A-MMS (H-A-MMS) nanoparticles were also obtained by the same method.



As for the release process of heparin, 20 mg of H-A-HMS was redispersed into 5 mL of PBS solution, and incubated at 37℃. During the first 24 hours of incubation, the released heparin solution was collected at appropriate time intervals (4 h, 12 h and 24 h). During the next 15 days, the released heparin solution was collected every day. After 15 days, the released heparin solution was collected every two days. Other samples include heparin loaded MMS (H-MMS), heparin loaded A-MMS (H-A-MMS) and heparin loaded HMS (H-HMS) were prepared and used as reference materials. The toluidine blue method was adopted to measure the load amount and controlled release of heparin.7,47 (5) Preparation of Janus H-A-HMS nanomotors: The ethanol aqueous suspension of H-A-HMS was dropped on slide glass. After evaporation of ethanol, platinum was deposited on the sample surface by sputtering (JEOL JFC-1600 Auto Fine Coater, JEOL, Japan). The Janus H-A-HMS nanomotors were then obtained. The “Janus” indicated that half of the surface of the H-A-HMS nanomotors was decorated by platinum, while the other half was not. This modification strategy of “Janus” can provide asymmetric surface modification of nanomotor, which is possible for asymmetric bubbles production, thus promoting the movement of nanomotor. Characterizations of MMS and HMS samples. Brunauer, Emmett, and Teller (BET) surface area/pore size characteristics of MMS and HMS were measured on ASAP2050 system (Micromeritics Instrumentcorp, USA). JEM-2100 electron microscope (200 kV, Jeol Ltd., Japan) was used to obtain transmission electron microscopy (TEM) images. The scanning electron microscopy (SEM) images were


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obtained from a Hitachi S4800 FE-SEM system (Hitachi Ltd., Japan). The accelerating voltage is 10 kV and the beam current is 10 mA. Cytotoxicity and blood compatibility test of MMS and HMS samples. (1) MTT assay: The viability of human umbilical vein endothelial cells (HUVECs) was detected by MTT method.48 0.1 mL of the medium containing 5×103 cells was added in 96-well plate, which was incubated in a CO2 incubator with 5% CO2 injection (37℃, 24 h). Then 0.1 mL 1 mg mL-1 of the samples were added to each well. 0.1 mL of untreated cells in the medium was used as a control. After 24 h incubation, 0.02 mL of 5 mg mL−1 MTT solution was injected into each well and dark incubated for 3 h. Then, 0.05 mL of DMSO was injected into each well. After 5 min, the absorbance at 570 nm for individual solution was tested. The viability of HUVECs = (ODexperimental / ODcontrol) × 100%, and the HUVECs viability of the control sample was 100%. (2) Percent Hemolysis and morphological changes of red blood cells (RBCs): The samples were firstly dispersed in normal saline (10 mL). After being incubated for 30 min at 37 ℃, 0.2 mL of diluted fresh rabbit blood (normal saline as diluent (volume ratio of 4/5)) was added, which were incubated at 37℃ for further 1 h, and then centrifuged with the speed of 2500 rpm for 5 min. Then the UV absorption of the supernatant solution was measured at 541 nm by using UV-visible spectrophotometer (Agilent 8453, Agilent Technologies, USA). The percentage hemolysis was calculated according to the following formula: Hemolysis (%) = (Asample - Anegative) *100%/(Apositive – Anegative). RBCs were obtained by centrifuging whole rabbit blood at 1500 rpm for 10 min,



which was re-dispersed in 0.9% saline solution. The samples were added into test tubes and formed 4 mL cell suspension (2%, v/v). The morphology of RBCs were observed and captured with an Olympus BX41microscope plus an Olympus E-620 camera. Saline and DW were denoted as negative and positive controls. (3) APTT/TT/PT assay: The coagulation time assays of the samples include activated partial thromboplastin time, prothrombin time and thrombin time (APTT, PT and TT) were tested.11 The fresh anticoagulanted whole rabbit blood sample was centrifuged with the speed of 2500 rpm for 15 min, and then the upper liquid (plasma) was obtained. Then the samples were dispersed in PBS solution (pH=7.4), and cultured with blood plasma for 1 h at 37℃. PBS solution was used as the negative control. The assay was conducted on a Semi automated Coagulometer (RT-2204C, Rayto, USA). Here, all of the coagulation time assays were repeated for three times. (4) Complement activation and platelet activation test: C3a enzyme immunoassay kit (BD Biosciences, USA) include the cleavage of complement component C3 and C3a des-Arg was utilized to detect the formation of activation peptides, which can characterize the complement activation that result from the samples.23 Whole blood was centrifuged with the speed of 3000 rpm for 15 min, and the poor platelet plasma was obtained. Then the samples we prepared were incubated with the poor platelet plasma at 37℃ for 1 h. The test procedure was conducted according to the protocol of C3a enzyme immunoassay kit for three times. Motion study of Janus H-A-HMS nanomotors. The motion of nanomotors were conducted in solution with different concentrations of H2O2 and surfactant (0.3 wt%


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of sodium dodecyl sulfate (SDS)). Optical microscope images and videos were obtained using Olympus microscope fitted with a camera (Olympus Group, Japan).

■ RESULTS AND DISCUSSION

Characterizations of A-HMS. As shown in Figure 2a-2d, both of the TEM images of MMS before and after APTES modified showed obvious core-shell structure, which the Fe3O4 core size was about 200 nm and the mesoporous silica shell thickness was about 50 nm.And after removing the Fe3O4 core, there were not obvious change for the sizes and morphologies of HMS before and after APTES modified. However, it was distinct that the hollow structure of HMS and A-HMS can be observed. Figure 2e and 2f showed the SEM images of A-MMS and hollow structure A-HMS which were consistent with the TEM results.

Figure 2. TEM images of (a) MMS, (b) A-MMS, (c) HMS, and (d) A-HMS. The scale bar was 200 nm. SEM images of (e) A-MMS and (f) A-HMS. The scale bar was 1 µm.



Page 12 of 27

Figure 3. (a) Nitrogen (N2) adsorption-desorption isotherms of A-MMS and A-HMS, (b) Mesopore size distribution of A-MMS and A-HMS.

It can be seen from the nitrogen adsorption-desorption isotherms of samples at -196℃, they were all typical IV type isotherms (Figure 3a), indicating that A-MMS and A-HMS samples all have typical mesoporous structure with the most probable pore sizes were about 2.8 nm and 2.7 nm, respectively (Figure 3b). The specific surface area and pore volume of A-MMS and A-HMS samples were summarized as Table 1. It showed that the specific surface of the samples increases from 207 to 820 m2g-1 after removal of Fe3O4 core, which can provide greater drug loading space. However, the removal of Fe3O4 core did not affect the mesoporous size of the shell.

Table 1. Physiochemical properties of the samples. SBET

Vp

D

(m2g-1)

(m3g-1)

(nm)

Samples

A-HMS

820

0.77

3.7

A-MMS

207

0.31

3.7

Drug Release Performance of H-A-HMS. From the heparin release curves of the H-A-MMS and H-A-HMS samples (Figure 4), the sustained heparin release time of


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H-A-MMS was about 5 days (Figure 4a). Compared to H-A-MMS, the H-A-HMS sample with hollow structure showed higher drug release quantity due to its high adsorption capacity (Figure 4b), and the release time can last for 25 days, indicating that the hollow structure was of great significance to the continuous release of drugs.

Figure 4. (a) Release profiles of heparin on different samples, and (b) Adsorption amount of heparin by different samples.

Cytotoxicity Assay of H-A-HMS. The evaluation of cytotoxicity is very important for biomedical materials. The cytotoxicity assay of different samples were tested by MTT, which the survival rate data were obtained after different samples cultured with cells for 24 h (Figure 5). Results showed that, compared to the MMS (89%) and A-MMS (91%) samples, the HMS (92%), A-HMS (HMS 97%) and H-A-HMS (98%) samples had lower cell toxicity, which indicating that the hollow structure and the load of heparin were beneficial to reduce the cytotoxicity of the material.



100

Cell viability (%)

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

80 60 40 20 0 Control

MMS A-MMS

HMS

A-HMS H-A-HMS

Figure 5. MTT assay of HUVECs incubated with different samples for 24 h. Hemolysis and Related Red Blood Cell Morphology Test of H-A-HMS. The hemolysis test and red blood cell morphology can be used to evaluate the biocompatibility of biomaterials after contact with red blood cells (Figure 6) . Results showd that all of samples had low hemolytic rate (

Preparation and Properties of Janus Heparin-Loaded Ammoniated

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