Porous Silicon Carrier Delivery System for Curcumin: Preparation

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Porous Silicon Carrier Delivery System for Curcumin: Preparation, Characterization and Cytotoxicity in Vitro Qingxia Lin, Wei Li, Di Liu, Mengyuan Zhao, Xuerui Zhu, Weiwei Li, Longfeng Wang, Tiesong Zheng, and Jianlin Li ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00645 • Publication Date (Web): 21 Jan 2019 Downloaded from http://pubs.acs.org on January 22, 2019

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ACS Applied Bio Materials

Porous Silicon Carrier Delivery System for Curcumin: Preparation, Characterization and Cytotoxicity in Vitro Qingxia Lin a,c, Wei Li b, c, Di Liu a,c, Mengyuan Zhaoa, Xuerui Zhua, Weiwei Lia, Longfeng Wanga, Tiesong Zheng a* and Jianlin Li a* a

Department of Food Science and Engineering, Nanjing Normal University, Nanjing 210024, China

b

Department of Electronic and Electrical Engineering, The University of Sheffield, Sheffield, S3 7HQ, United Kingdom

c These

authors contributed to this work equally and should be regarded as co-first authors

*Corresponding authors. Tel.: +86 25 83598286; fax: +86 25 83598901. E-mail addresses: [email protected], [email protected]

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Abstract A novel carrier delivery system for curcumin based on porous silicon (pSi) has been developed. The pSi film was prepared by electrochemical etching method and the microparticles of pSi were obtained by ultrasonication. The pSi film and particles of pSi were characterized by scanning electron microscopy (SEM). Sodium nitrite can induce curcumin into pSi and improve the drug loading (DL) and encapsulation efficiency (EE) of curcumin in double distilled water loading buffer solution. Curcumin on the pSi surface was confirmed by Fourier transform infrared spectroscopy (FTIR) and UV-spectroscopy. The optimal loading conditions of curcumin in pSi are investigated. The curcumin in pSi keeps more than 95% bioactivity for 3 h and good repeatability. The cumulative release ratio of curcumin from PSi can reach 35% after 10 h. The in-vitro cytotoxicity of curcumin loaded pSi was evaluated with HT-29 and NCM460 cell lines. The pSi delivery carrier can provide a controlled release and efficacy system for curcumin. KEYWORDS: Curcumin, porous silicon, encapsulation, release, cytotoxicity evaluation

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Introduction Curcumin is a hydrophobic yellow polyphenol compound extracted from Curcumin longa. A lot of previous reports have demonstrated that curcumin exhibits immense biology and pharmacological properties like antioxidant, anti-inflammatory, antimicrobial and anticarinogenic activities, liver and kidney protective, regulates various immune cells (T lymphocytes), B lymphocytes, natural killer cells, macrophages, dendritic cells and other immune cells, regulates multiple targets in vitro and in vivo1-2. Curcumin shows the multi-therapeutic activities by a variety of signaling pathways including Cyclin D1, COX-2, nuclear factor-kappa B(NF-κB) and matrix metalloproteinases (MMPs)3. It has been used as a traditional medicine, a food pigment and special flavor in many Asian counties for many years. However, clinical use of curcumin is restricted by pharmacokinetic obstacles such as poor oral bioavailability due to low water solubility, rapid metabolism, and elimination4-5. Various approaches have been attempted to improve the bioavailability of curcumin. Chemical synthetic curcuminoids is used to overcome the above limitations, though they often require many complicated synthetic steps2, 6. Various delivery vehicles including nano/microparticles, micelles and emulsion capsules for enhancing the water solubility, circulation time and the bioavailability of curcumin have been developed to replace the chemical synthetic method7. For example, liposomes8-10,

cyclodextrin11,

chitosan12-13,

micelles14-15,

hydrogel16-17

and

degradable

microspheres17-18 have been used to establish the delivery carriers to increase the bioavailability of curcumin. Hollow, or porous nanomaterials have been drawn more and more attentions as drug delivery vehicles because of their big surface areas, good biocompatibility, sustained release and adjustable surface chemistry19-22. Among these porous delivery vehicles, porous silicon (pSi) carrier has unique properties because it contains intrinsic photoluminescence, is nontoxic and degradable to nontoxic byproducts and can self-report image19, 22-2324-26. pSi has been used as delivery carriers for oral insulin27, daunorubicin28, ciprofloxacin26, DNA29-30, RNA25, nutritional food additive31-32 and other hydrophobic drug33-34. Due to its photoelectron characteristics, pSi has been also used to real-time monitor the release properties of drug delivery22, 26, 35-36. In this work, we developed a new curcumin delivery system with pSi and investigated the load/release property and cytotoxicity of pSi-curcumin in vitro. The new delivery system for 3



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curcumin has no need for a complex modification process and loads the curcumin by physical adsorption on pSi surface. The work would provide a promising platform for the development of controllable and highly biocompatible delivery system and a new path for application of curcumin. 2. Experimental 2.1. Materials Silicon wafers (0.0008-0.0012 Ω-cm resistivity, polished on the (100) face, B-doped) were obtained from Siltronix Co. (Archamps, France). 48% Hydrofluoric acid was bought from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Absolute ethanol, sodium hydroxide, hydrochloric acid, dialysis bag (for 8000-12000 dalton), sodium chloride, sodium bicarbonate and hydrogen peroxide were obtained from Nanjing Chemistry Reagents Co., Ltd. (Nanjing, China). Curcumin (≥98%, HPLC grade), sodium nitrite, sodium dodecyl sulfate (SDS), dimethylsulfoxide (DMSO) and ethylene diamine tetraacetic acid (EDTA) were purchased from National standard material center (Beijing, China). Thiazolyl blue (MTT), Dulbecco's modified eagle medium (DMEM), phosphate buffer solution (PBS), gibco hyclone, and pancreatin were purchased from Nanjing keygen biotech co., Ltd. (Nanjing, China). HT-29 cell and NCM460 were obtained from Shanghai institute of biochemistry and cell biology (Shanghai, China). 2.2. Preparation and characteristics of pSi and thermally oxidized pSi pSi and thermally oxidized pSi were prepared according to the reference37. Briefly, silicon wafers were etched by a Teflon etch cell and a platinum counter-electrode. 3 cm×3 cm wafer was etched with 1:1(v/v0) mixture aqueous 48% HF and absolute ethanol at a constant current density of 58.5 mA/cm2 for 600 s. The pSi film was removed from the Si substrate by a current density of 0.65 mA/cm2 for 200 s in 3.3% aqueous hydrofluoric acid in ethanol. The pSi film was fractured into microparticles by ultrasonication (300 w) for 2 h. Thermally oxidized pSi was obtained from tube furnace at 500 °C for 2 h. The images of scanning electron microscopy (SEM) of pSi film and the pSi particles were taken with a FEI XL30 microscope equipped with a field emission gun and through-the-lens detector at an accelerating voltage of 5 kV. 2.3. Preparation of curcumin-load pSi particles 100 mg of curcumin was dissolved in 100 mL of absolute ethanol solution. 1 mL,1 mg/mL of curcumin solution and 1 mL, 25mM of NaNO2 solution were added into 10 mg fresh or thermally oxidized pSi and then mixed for 2 min. The sample was homogenized by ultrasonication (300 w) 4


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for 0.5 h and loaded at 25 °C for 3 h. The sample was centrifuged at 1530 g for 3 min. The supernatant was collected and centrifuged at 10699 g for 10 min. The supernatant was collected in a 10 mL of brown glass flask volumetric. The curcumin-load pSi particles were obtained after pSi particles from the precipitate of double centrifugations were respectively washed with 1 mL of absolute ethanol and 10% ethanol. The control sample was prepared according to above method except for adding pSi particles. 2.4. Curcumin Ultra Violet (UV) analysis Then the supernatant above-mentioned was diluted in constant volume. The solution was filtered with a 0.45 μm filter membrane. The supernatant was collected and measured the concentration of unloaded curcumin with UV spectrophotometer (UV 6100A, Shanghai Yuanxi instrument co., Ltd, China) at 428 nm. The curcumin concentration was determined using an established standard curve of curcumin (Figure S1). 2.5. Fourier transform-infrared spectroscopy (FTIR) Chemical characteristics of the materials were examined using FTIR (Nicolet Nexus670, Thermo-Scientific) using KBr disks in the range of 400-4000cm-1 with a resolution of 0.125cm-1. 2.6. Drug loading (DL), encapsulation efficiency (EE) and loss ratios (LR) of curcumin. DL and EE are calculated according to before and after 1mL, 1 mg/mL of curcumin solution is added in 10 mg fresh or thermally oxidized pSi (see in 2.3 section), which is as follows: DL = (Total amount of added curcumin - amount of free curcumin)/(pSi+Drug)×100%

(1)

EE (%) = [1-(amount of free curcumin)/(total amount of added curcumin)]×100%

(2)

LR(%)=(Total amount of added curcumin - amount of curcumin in control sample) /(Drug)×100% (3) 2.7. Release in vitro 1 mL, 1 mg/mL of curcumin solution was placed in dialysis bag. The dialysis bag packaged with aluminum foil was incubated in 9 mL of phosphate buffered saline (pH7.4 PBS, pre-warmed to 37 °C) at 37 °C with gentle shaking 8 g. At specific time points, 1 mL of the release medium sample was taken and replaced with 1 mL of fresh release medium. The absorbance value of sample was detected by UV spectroscopy. 10 mg of curcumin-pSi and 1 mL of PBS was placed in dialysis bag. The dialysis bag packaged with aluminum foil was placed 24 mL PBS at 37 °C with gentle shaking 8 g. At specific time points, 5 mL of the external solution from dialysis bag was 5



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taken and replaced with 5 mL of fresh release medium and the absorbance value of the sample was detected by UV spectroscopy. 2.8. The stability measurement The stability measurement of curcumin and curcumin-pSi was performed according to the reference38. The optimal formula curcumin-pSi and free curcumin samples were stored at room temperature for 6 h. The storage stability of free curcumin and curcumin-pSi in normal saline solution was evaluated by monitoring the absorbance of curcumin at 428 nm at 20 °C in the specific times. The sample was centrifuged at 8000 g for 10 min, then the precipitate was collected and 2 mL of absolute ethanol was added in the sample. The sample was centrifuged at 8000 g for 10 min, then the supernatants were filtered with a 0.45 μm filter membrane. The absorbance values of filtrates were measured according to above UV method. 2.9. Cytotoxicity study in vitro The cytotoxicity assay of the materials was evaluated by MTT method. HT-29 and NCM460 Cells were plated at a density of 1×105 cells per well in 96-well plates and grown for 24 h in DMEM medium with 10% fetal bovine serum,100 units/mL penicillin and 80 units /mL streptomycin at 37°C in a CO2 incubator (95% relative humidity,5% CO2). Then, cells were exposed to a series of curcumin, pSi, and curcumin-pSi at different concentrations for 48h, respectively. After the incubation period, the cells were treated with 5 mg/mL of MTT solution for 4 h, then the solution was removed and 200 μL of DMSO was added into each well. The 96microplate packaged with aluminum foil and vibrated for 15min. Absorbance intensity of sample was detected by a microplate reader at 570 nm. The viability rates =

(

) × 100%

A1 ― A0 A0

A1 and A0 is absorbance value of sample and control, respectively. 3. Results and discussion 3.1. Synthesis and characterization of pSi

6


(3)

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Scheme1. The process of preparation of curcumin-pSi and release of curcumin. The process of preparation of curcumin-pSi and release of curcumin is shown in Scheme 1. pSi film was prepared by electrochemical etching method 37and striped off from silicon substrate by low electric current density. Microparticles of pSi was obtained by ultrasonication. Curcumin was introduced into the surfaces of pSi by sodium nitrite. Curcumin in pSi is released by the degradation of silicon surface in medium and diffusion.

Figure 1. The characterization of pSi. A, The images of SEM for the top surface of pSi film; B, The images of SEM for the cross-section of pSi film; C, The image of pSi particles; D, The appearance digital photography of pSi particles. The images of SEM of pSi film and pSi particles were shown in Figure 1. The results indicate 7



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that the pore diameter size of the pSi film is about 20-50 nm (Figure1A) and the thickness is about 7.5 μm (Figure 1B). The most of diameter size of pSi particles is less than 50 μm after the pSi film is striped off from silicon substrate and broken by ultrasonication (Figure 1C). The appearance color of the pSi particles is brown (Figure 1D). 3.2. The influence of the loading formulas on the curcumin loading properties. The surface chemical characteristics of pSi play a large role in the loading properties of molecule in pSi39-41. Here, we investigated the fresh pSi, thermally oxidized pSi and sodium nitrite oxidized pSi influenced on the curcumin loading properties. Then, the different loading buffer solutions including double distilled water and absolute ethanol also have been studied. The results were shown in the Figure 1. The photographs of the different formula samples show that the appear colors of samples are different in the different formulas and the third sample keeps the color of fresh curcumin (Figure 2A). The formula of sodium nitrite oxidized pSi and double distilled water loading buffer solution shows the maximum values of EE, DL and LR among them (Figure 2B). While, the formula of thermally oxidized pSi and double distilled water loading buffer solution displays the minimum values of EE and DL among them. Sodium nitrite is a stronger oxidant and used as a food preservative, which can suppress oxidation by acting as a trap for free radicals. Sodium nitrite has been demonstrated that it can oxidize the pSi, induce trapping of drug in pSi and significantly increase drug loading efficiency39. Compared with fresh pSi, the thermally oxidized pSi has less pore volume because of swelling of the pore walls as oxygen is incorporated into the silicon skeleton, which result in the minimum values of EE, DL and LR among them. Without sodium nitrite oxidization, the EE and DL of fresh pSi display significantly less than that of sodium nitrite oxidized pSi. In the sodium nitrite loading buffer solution, pH value is 5, which is suitable for the stability of curcumin in solution. Due to the more solubility of curcumin in ethanol, double distilled water as loading buffer solution displays the higher EE and DL values for curcumin.

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B

Fresh pSi +NaNO2+

DL EE LR

double distilled water

80

Percentage(%)

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


Fresh pSi+ double distilled water

60

40

20

Fresh pSi+ethanol

NaNO2 +double distilled water

0

Thermally oxidized pSi

Fresh pSi

Fresh pSi

Fresh pSi

Figure2. The influence of the different loading formulas on the curcumin loading properties. A, Photographs of samples. Left to right: 1 mL of curcumin (in ethanol) loading with 10 mg of fresh PSi in 1 mL of double distilled water, 1 mL of curcumin loading with 10 mg of thermally oxidized pSi in 1 mL of double distilled water and 1mL, 25 mM of NaNO2 solution, 1 mL of curcumin loading with 10 mg of fresh PSi in 1 mL of double distilled water and 1 mL, 25 mM of NaNO2 solution, 1 mL of curcumin loading with 10 mg of fresh pSi in 1 mL of absolute ethanol, 1 mL of curcumin in 1 mL of double distilled water and 1 mL of curcumin in PBS. B, The influence of the different formulas on the curcumin loading properties. Left to right: thermally oxidized pSi was used as carrier and NaNO2+double distilled water as buffer solution; fresh etching pSi carrier was used as carrier and NaNO2+double distilled water, double distilled water and ethanol were respectively used as buffer solution in other formulas. 3.3. The influence of the loading temperature and time, etching current and time of pSi, stirring methods on the loading properties of curcumin To obtain the optimal conditions of loading properties of curcumin in pSi, loading temperature and time, etching current and time of pSi, stirring methods have been orderly investigated. The influence of loading temperature on the loading properties of curcumin in pSi is shown in Figure 3A. As the increase of loading temperature of curcumin from 25 °C to 65 °C, the EE and LR values of curcumin significantly increase, but the DL values (4.6%) of curcumin reach to the highest at 9



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50 °C (Figure 3A). The highest LR value (56.2%) of curcumin may result from the degradation of curcumin at 65 °C. Therefore, 50 °C is selected as the optimal temperature for the following experiments. The influence of loading time of curcumin in pSi on the DL, EE and LR values of curcumin is shown in Figure 3B. As the increase of loading time of curcumin, the DL (5.1%) and EE (66.4%) values of curcumin reach to the highest at 16 h. The LR value of curcumin also increases as loading time increase and reaches the highest value (15.6%) at 21 h. Loading time at 16 h is used as the optimal time for next experiments. The influence of the different etching currents on the loading properties of curcumin is shown in Figure 3C. As the increase of etching currents, the pore sizes and morphologies of pSi change (Figure S2). The DL and EE values of curcumin reach to the highest (6.6% and 74.9%, respectively) at 360 mA of etching current and the LR values of curcumin almost do not change during the ranges of etching currents. Generally, the porosity of pSi is increased as increase of etching currents. However, the etching current density is too large, the wafer might be electropolished. In addition, too much pore size of pSi will reduce the EE value of curcumin. Therefore, the optimal etching currents is used at 360 mA as the condition of following experiments. As the increase of etching time, the EE and DL values of curcumin increase and reach to the plateau (68.8% and 5.6%, respectively), while the LR values reduce (Figure 3D). The porosity of pSi will also increase as the increase of etching time, which result in the increase of the EE and DL values of curcumin. The optimal etching time is used at 800 s as the condition of the next experiments. The different stirring methods including water bath and magnetic stirring at 25°C and 50°C water bath on the curcumin loading properties have been investigated. The results are shown in the Figure 3E. The higher temperature of water bath remarkably increases the EE, DL and LR values of curcumin in pSi. This is ascribed to rapid molecular motion at higher temperature. The stirring has not increased the EE and DL values of curcumin in pSi at the same temperature. This may be attributed to that curcumin molecules in the pores of pSi move out during stirring. The maximum EE (80.0%) and DL (6.2%) values can be obtained at water bath at 50°C. Therefore, the optimal stirring method is water bath at 50°C for loading curcumin in pSi.

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B

LR EE DL

Percentage(%)

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C

LR EE DL

3

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90

Percentage(%)

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LR EE DL

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270 360 450 Etching current(mA)

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Figure 3. The influence of the loading temperature (A) and time (B), etching current(C) and time(D) of pSi, stirring methods (E) of curcumin on the loading properties of curcumin. 3.4. FTIR The FTIR spectra of pSi, pSi-curcumin and curcumin are shown in Figure 4. Absorption peak positions at 2090 cm-1 and 1110 cm-1 assign to Si-O-Si stretching vibration of silanol group. Absorption peak at 3508 cm-1 come from OH bending vibration. The absorption peak positions at 1424, 1508,1602 and 1628 cm-1 are ascribed to C=O stretching and C=C stretching vibrations, 11



respectively, which come from curcumin. These characteristics of FTIR of pSi-curcumin indicate that curcumin is physically adsorbed on the surface of pSi.

Absorbance intensity(a.u)

PSi PSi-Cur Cur

100

1424

2090

50

3508

1628 1602 1508 1110

0 500

1000

1500

2000

2500 Wavenumber(cm-1)

3000

3500

Figure 4. The FTIR spectra of pSi, pSi-curcumin and curcumin. 3.5. The stability and recovery ratios of the curcumin in pSi. PSi-curcumin Free curcumin

100

Curcumin (%)

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

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80

60

40

0

2

Time (h)

4

6

Figure 5. The stability of curcumin in pSi. Curcumin in aqueous solution is easily to hydrolysis or degradation38. The stability of curcumin in pSi has been studied by monitoring the changes of curcumin at different times in 6 h and the result is shown in Figure 5. Almost 95% and 80% curcumin in pSi remained stable after storage for 3 h and 6 h, respectively. On the contrary, only 60% free curcumin remained stable after storage for 6 h. This indicate that pSi can significantly improve the stability of curcumin. The stable 12


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properties of curcumin in pSi are remarkedly higher than that of curcumin in soy protein carrier38. To investigate the repeatability of curcumin-pSi, the different quantities of curcumin were added in pSi according to above the optimal conditions. Then, the curcumin in pSi was extracted by ethanol and quantitative determination with UV. The result is shown in table S1. The recovery ratios of curcumin in pSi containing the different quantities are more than 90%. This indicates that the prepared method for curcumin-pSi has a good repeatability. 3.6. the curcumin release behavior in vitro The curcumin release properties in vitro have been investigated. The results are shown in Figure 6. In the SDS PBS release medium, only 11% of curcumin was released from curcumin-pSi after 12 h, whereas 40% of curcumin released into normal saline containing 20% ethanol medium (Figure 6A). The slow release of curcumin in PBS medium is consistent with the previous report in the other release system13, which is ascribed to the low solubility in the solution. As the increase of ethanol concentration, the release ratio of curcumin reaches to 93% after 4 h in normal saline containing 40% ethanol medium (Figure 6B). The release kinetics of curcumin from curcumin-pSi fit well with Higuchi equation (Q=45.53t1/2 +5.28, Q is the cumulation release ratio; t is time; R2=0.982). The results indicate that curcumin-pSi can realize sustained release and keep a long lifetime in human body. The curcumin release properties in the PBS medium solutions with the different pH values (containing 40% ethanol) are shown in Figure 6C. The cumulative release ratio reached to the maximum value after 4 h at pH8.5 PBS medium and then decreased, which may attribute to the degradation of pSi and curcumin in alkaline solution. The cumulative release ratio in pH 7.4 PBS medium is higher than that in pH 5.0 PBS medium at the same time.

Cumulative release (%)

normal saline containing 20% ethanol 0.5% SDS PBS

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pH7.4 pH5.0 pH8.5

30

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10 0

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Figure 6. The properties of curcumin in the vitro. A, The release of curcumin in the different buffer solutions; B, The cumulative release of curcumin in normal saline containing 40% ethanol. C, The cumulative release of curcumin in different pH PBS medium containing 40% ethanol. Except for pSi delivery system, degradable macro/mesoporous silica structure (DMSS) is often used as drug delivery system42. Compared with DMSS, pSi has more activity and autofluorescence property. Therefore, the drug loading in pSi carrier is more complicated than that in DMSS carrier. As for drug releasing performance in the two delivery systems, the releasing properties of drug are related to the properties of drugs and surfaces of pSi. 3.7. In vitro cytotoxicity The cytotoxicity of curcumin in pSi was evaluated by the cell viability ratios when the materials with the different concentrations were respectively exposed to HT-29 and NCM460 cell lines. As depicted in Figure 7A, curcumin shows an obvious inhibition effect on HT-29 and NCM460 cells viability at 2.5 μg/mL and 5.0 μg/mL, respectively. The result is in agreement with the bioactivity of curcumin. Both HT-29 and NCM460 cells can keep 78% cell viability ratios when the concentrations of pSi are in range of 10-125 μg/mL (Figure 7B). While cell viability ratios both HT-29 and NCM460 cells are respectively decreased to 19.12% and 60.14% at 250 μg/mL pSi. The HT-29 cancer cell displays more sensitive to pSi than NCM460 cell. The result indicates that pSi has some cytotoxicity at high concentration of pSi. The cytotoxicity of curcumin-pSi was shown in Figure 7C. The curcumin-pSi shows an additive and synergistic toxic effects on both HT29 and NCM460 cell viability. The cell viability ratios of the two kinds of cells in curcumin-pSi system are remarkably lower than that of individual curcumin and pSi system at the same concentration. The cancer HT-29 cell only keeps 6.57% cell viability at 125 μg/mL curcumin-pSi; 14


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Even, HT-29 cell cannot survive and NCM460 cell only keeps11.06% cell viability at 250 μg/mL curcumin-pSi, respectively. pSi particles have phototoxicity against cancer cells when they are exposed to white light43. The photoactivity of pSi particles and bioactivity of curcumin may result in the large decrease of cell viability at the 125 μg/mL curcumin-pSi. The detail mechanism needs to be further investigated. 100

A

NCM460 HT-29

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Cell viability (%)

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1

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2.5 5 10 20 The concentration of curcumin (μg/mL)

30

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125 25 50 250 The concentration of PSi(μg/mL)

500

NCM460 HT-29

75 50 25 0

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10 25 50 125 250 The concentration of curcumin-PSi (μg/mL)

Figure 7. The cytotoxicity evaluation in vitro. A, The cytotoxicity of curcumin; B, The cytotoxicity of pSi; C, The cytotoxicity of curcumin-pSi. Cell damage is one of the vital features for apoptotic cells and cytotoxicity evaluation. The cytotoxicity evaluation of curcumin-pSi has been further investigated by observing HT-29 and NCM460 cell morphological characteristics with microscopy. The result is shown in Figure S3. The cells in the control sample show normal cell morphology (Figure S3A for HT-29, Figure S3E for NCM460), while the free curcumin at 30 μg/mL will kill HT-29 cells and cell membrane will break (Figure S3B) and can partially damage NCM460 cell (Figure S3F). When HT-29 and NCM460 cells were respectively cultivated with 125 μg/mL pSi and curcumin-pSi, both of them show partial damage and HT-29 cell is more sensitive than NCM460 cell to these drugs (Figure 15



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S3C, D, G and H). The morphological characteristics of cell treated with these materials are consistent with that of Figure 7. Although the cytotoxicity evaluation of porous silicon has been previously reported40,

44,

cytotoxicity of curcumin-pSi in vitro was first investigated in this work and their additive and synergistic toxic effects were found. In addition, we also observed that there was a high absorption value of MTT assay on the cytotoxicity assessment of pSi, which may cause high viabilities of cells and underestimate the cytotoxicity of drug-loaded pSi microparticles44. By subtracting the reference value from the cell tests, the cell viability is in the reasonable range of values in this work, which is different with the previous report44. The difference is ascribed to the two possible reasons: one is the different quantity reagents; the other is the different cell lines between the two works. The primary results of curcumin-pSi properties indicate that pSi can load, stabilize and controlled release curcumin. Cytotoxicity of curcumin-pSi for HT-29 cells and NCM460 cells in vitro displays some toxicity at125µg/mL. Although the properties of curcumin-pSi in vivo should be further researched, the work paved the base way for application of curcumin-pSi. pSi has been reported in drug delivery42 and real-time monitoring for tumor treatment23, which has a good biocompatibility, big internal surface areas, easily modified surfaces, degradable and autofluorescence. These properties may make pSi to be used as a promising platform for the development of controllable and highly biocompatible system. 4. Conclusion In summary, we have developed a new curcumin delivery system using pSi. The curcumin loading conditions have been optimized. The FITR spectra, stability, release properties and cytotoxicity in vitro of curcumin-PSi have been investigated. The pore size of pSi, surface chemical characteristics, loading time, loading buffer solution and temperature can influence the curcumin loading properties. Curcumin in pSi shows a good stability for 6 h. The release medium of curcumin plays an important role in the release ratios of curcumin in pSi. The cytotoxicity evaluation in vitro indicates the curcumin-pSi has an additive and synergistic toxic effects on both HT-29 and NCM460 cells viability and the different sensitivity to HT-29 and NCM460 cells. This work provides a new pathway for efficient application of curcumin. ASSOCIATED CONTENT 16


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Porous Silicon Carrier Delivery System for Curcumin: Preparation

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