Acta Biomaterialia xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Potential application of functional porous TiO2 nanoparticles in light-controlled drug release and targeted drug delivery Tianyi Wang a, , Haitao Jiang b, , Long Wan b, Qinfu Zhao b, Tongying Jiang b, Bing Wang a,⇑, Siling Wang b,⇑ a b
College of Life Science and Health, Northeastern University, 89 Wenhuadong Road, Shenhe District, Shenyang, Liaoning Province 110015, People’s Republic of China Department of Pharmaceutics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang, Liaoning Province 110016, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 19 May 2014 Received in revised form 24 October 2014 Accepted 5 November 2014 Available online xxxx Keywords: Porous TiO2 Nanoparticle Surface functionalization Controllable release Targeting
a b s t r a c t Novel multifunctional porous titanium dioxide (TiO2) nanoparticles modified with polyethylenimine (PEI) were developed to explore the feasibility of exploiting the photocatalytic property of titanium dioxide to achieve ultraviolet (UV) light triggered drug release. Additionally, in order to further realize targeting delivery, folic acid, which chemically conjugated to the surface of the functionalized multifunctional porous TiO2 nanoparticles through amide linkage with free amine groups of PEI, was used as a cancer-targeting agent to effectively promote cancer-cell-specific uptake through receptor-mediated endocytosis. And a typical poorly water-soluble anti-cancer drug, paclitaxel, was encapsulated in multifunctional porous TiO2 nanoparticles. The PEI on the surface of multifunctional porous TiO2 nanoparticles could effectively block the channel to prevent premature drug release, thus providing enough circulation time to target cancer cells. Following UV light radiation, PEI molecules on the surface were cut off by the free radicals (OH and O2 ) that TiO2 produced, and then the drug loaded in the carrier was released rapidly into the cytoplasm. Importantly, the amount of drug released from multifunctional porous TiO2 nanoparticles can be regulated by the UV-light radiation time to further control the anti-cancer effect. This multifunctional porous TiO2 nanoparticle exhibits a combination of stimuli-triggered drug release and cancer cell targeting. The authors believe that the present study will provide important information for the use of porous TiO2 nanomaterials in light-controlled drug release and targeted therapy. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Chemotherapy is now an essential component of cancer therapies used to treat most cancers [1,2]. Although various strategies for drug delivery have been developed in recent years, in efforts to kill cancer cells effectively, many routinely used chemotherapeutic agents are not able to specifically target cancer cells, and their release is poorly controlled, often resulting in serious undesirable side effects [1,3,4]. Therefore, the ability to achieve specific drug accumulation at tumor sites is still a great challenge for successful cancer chemotherapy [5–7]. In order to improve chemotherapy, great efforts have been devoted to the development of tumor-targeted nanocarriers for the controlled delivery of anti-cancer drugs. To obtain a tumor-targeted nanocarrier, a promising strategy is to modify the carrier with ⇑ Corresponding authors. Tel./fax: +86 24 85820828 (B. Wang). Tel./fax: +86 24 23986348 (S. Wang). E-mail addresses: [email protected] (B. Wang), [email protected]. cn (S. Wang). These authors contributed equally to this work.
targeting ligands, such as peptides [8], antibodies [9] and small molecules (e.g. folate (FA)) [10], which can selectively recognize and bind to the surface receptors over-expressed on cancer cells and stimulate cancer-cell-specific uptake. Moreover, in order to further reduce systemic toxicity and undesired side effects, keeping the loaded drugs sealed during circulation in the bloodstream and rapidly releasing them on reaching and accumulating in tumor tissues is also very important [11,12]. Therefore, an ideal anti-cancer drug delivery system should be a combination of cancer cell targeting and stimuli-triggered drug release. Porous titanium dioxide (TiO2) offers the potential to achieve this challenge, owing to its interesting properties, such as a high photocatalytic activity and a functional surface. TiO2, a typical n-type semiconductor material, has been widely used in many fields, such as photocatalysis [13], energy storage and conversion [14], sunscreening [15] and sensor research [16], and especially in medical fields, as anti-cancer agents [17], implants [18] and substrates for stem cell expansion, owing to its cheapness, chemical stability, innocuity and excellent biocompatibility [19]. Among these, much attention has been drawn in recent years towards the photocatalytic degradation of organic compounds
http://dx.doi.org/10.1016/j.actbio.2014.11.010 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang T et al. Potential application of functional porous TiO2 nanoparticles in light-controlled drug release and targeted drug delivery. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.010
2
T. Wang et al. / Acta Biomaterialia xxx (2014) xxx–xxx
[20–23]. On ultraviolet (UV) light excitation, the energy absorbed from UV becomes higher than the band gap of TiO2, and the valence electrons will be excited to the conduction band, creating an electron (e ) and hole (h+) pair and further generating active free radicals (OH and O2 ) which effectively decompose the organic compounds on the surface of the TiO2 particle [24–26], providing a theoretical foundation for light-triggered drug release. It is worth mentioning that the TiO2 itself was reported to have a good anticancer effect, owing to the production of active free radicals under UV irradiation [17]. Therefore, the present authors have innovatively prepared hybrid polyethylenimine (PEI)-modified porous TiO2 nanoparticles (MTNP). PEI (a hydrophilic dendritic macromolecule) coated on the surface of MTNP could block the release of drugs as a result of the formation of a hydrophilic layer. Following UV radiation, the PEI molecules could be destroyed by free radicals (OH and O2 ) [23,24], and then allow drug release. In addition, FA was covalently linked to the PEI-MTNP. Since folate receptor (FR) is overexpressed on cancer cells, the conjugation (FA with FR) effectively facilitates cellular uptake of the FA functionalized carrier [1,10,29]. Herein, MTNP was developed using non-ionic surfactant F127 as a structure-directing agent. To achieve the targeting and lightcontrolled release goals, the targeting group (FA) was covalently linked to the bridge molecule (PEI) [30], and then the surface of the MTNP was modified by functional groups (FA-PEI) through the electrostatic adsorption effect. Finally, the anti-cancer drug paclitaxel (PTX) was loaded into the pores of the functionalized carrier by a solvent deposition method [27,28]. The loaded anticancer drug was not released during circulation in the bloodstream and normal tissues because of blocking by PEI. Multifunctional MTNP could be efficiently trapped by cancer cells through receptor-mediated endocytosis. And then under UV excitation, the PEI coated on the surface of the carrier could be destroyed, and the anti-cancer drug was rapidly released from the pores. Meanwhile, the TiO2 carrier itself could also have an anti-cancer effect. The potential of the multifunctional MTNP in tumor targeting and controllable drug release is systematically investigated in the present research. 2. Materials and methods 2.1. Materials Titanium (IV) isopropoxide (TIP) (95%) was obtained from Alfa Aesar (Lancs, UK). Pluronic block co-polymer F127 was a gift from BASF (Ludwigshafen, Germany). Thiazolyl blue tetrazolium bromide (MTT), Hoechst 33258, streptomycin and penicillin were purchased from Sigma–Aldrich (Santa Clara, USA). Fluorescein isothiocyanate isomer (FITC), PEI and FA were purchased from Aladdin (Shanghai, PRC). KB (human nasopharyngeal carcinoma cell) cell and A549 (human lung carcinoma cell line) cell were purchased from Jia He Biotechnology Co., Ltd. (Shanghai, PRC). PTX was kindly donated by Funing Pharmaceutical Co., Ltd (Shenyang, PRC). The water used for the experiment was Milli-Q (18.2 MO). Absolute ethanol (>99.7%) was purchased from Tianjin Bodi Chemical Holding Co., Ltd (Tianjin, PRC). All other solvents were chromatographic grade. 2.2. Fabrication of TiO2 nanoparticles The TiO2 nanoparticles were synthesized via a sol–gel route, using F127 as a structure-directing agent and TIP as a TiO2 source. In detail, 16 g F127 was completely dissolved in 400 ml ethanol, and then 2.4 ml Milli-Q was added to this solution with stirring. Then 7.4 ml TIP was added to the solution at ambient temperature
under vigorous stirring. When the clear solution turned into a milky white suspension, the solution was stirred continuously for 50 min, and then the solution was kept at room temperature without stirring to stand for 12 h. Finally, 2 g precursor sample was dispersed in 200 ml absolute ethanol solution, and then the solution was stirred for 2 h at 70 °C. After stirring, the white samples were collected by centrifugation. The samples were extracted three times in this way and then dried at 40 °C overnight. In order to completely remove the surfactant (F127), the sample was extracted again three times in the same way. Finally, the resulting product was dried at 200 °C for a day to remove absolute ethanol completely. The samples obtained were named MTNP. 2.3. Functional modification of MTNP Before modification of the MTNP, FA was conjugated to PEI at first. The method was as reported by Guo and co-workers [30]: briefly, 1 g FA was dissolved in 50 ml dimethylsufoxide (DMSO) and then 1.1 M excess of N-hydroxysuccinimide and N, N’-dicyclohexylcarbodiimide was added to this solution with stirring. Afterwards, the solution was stirred for 12 h at room temperature. The solution was filtrated to remove the insoluble byproduct, dicyclohexylurea. And then, PEI was dissolved in carboxy-activated FA DMSO solution at a ratio of 1:5 (molar ratio). After stirring for 12 h, the product (FA-PEI) was passed through a Sephadex G-100 column (Pharmacia Biotech) for purification. MTNP, 80 mg, was suspended in FA-PEI phosphate (PBS) buffer (pH 7.4), and then the solution was stirred for 5 h at room temperature. These modified particles (FA-PEI-MTNP) were then collected by centrifugation (5000 rpm, 5 min) and were dried at room temperature. The FA-PEI-functionalized particles were further labeled with FITC (fluorescein isothiocyanate) by immersing 50 mg TiO2 particles in carbonate buffer (pH 8.2) and mixing with 500 ll FITC-ethanol solution (1 mg ml 1), followed by stirring this solution for 40 min [30]. After this, the FITC-FA-PEI-functionalized particles were collected by centrifugation (5000 rpm, 5 min). Electrokinetic titrations were measured using a Malvern Zetasizer-3000. The sample solutions were adjusted to different pH values with 0.1 M NaOH and HCl. 2.4. Characterization Field-emission scanning electron microscopy (SEM), using a SUPRA 35 instrument (ZEISS, GER, operated at 15 kV) and transmission electron microscopy (TEM) (TECNAI G2 20, FEI, USA) were used to examine the morphology of the MTNP obtained. A surface area and pore size analyzer (V-sorb 2800P, Gold App Instruments, CN) was used to measure the pore characteristics of the samples. Samples were degassed at 150 °C for 16 h prior to analysis. The Brunauer–Emmett–Teller (BET) method was used to determine the surface area of the samples. The Barrett–Joyner–Halenda (BJH) method was used to determine the pore size distribution. X-ray diffractometry (XRD; PW3040/60 PANALYII CALB.V NED) was used to determine the crystals of MTNP over the angle range from 5° to 80° (2h) with a scan rate of 6° min 1 and Cu K-Alpha1 KAlpha2 radiation (k = 1.54 Å, 30 mA, 30 kV). 2.5. Drug loading and release PTX was loaded into the pores of the MTNP by a solvent deposition method [27,28], involving a combination of soaking equilibrium and solvent evaporation. The details of the drug loading are described in the Supporting Information. PTX released from FA-PEI-PTX-MTNP was determined by the dialysis method. A certain amount of sample was added to 2 ml PBS (pH 7.4) with 0.1% Tween 80, and then this solution was placed in a dialysis
Please cite this article in press as: Wang T et al. Potential application of functional porous TiO2 nanoparticles in light-controlled drug release and targeted drug delivery. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.010
T. Wang et al. / Acta Biomaterialia xxx (2014) xxx–xxx
bag (MWCO 8000, Fisher Scientific). The dialysis bag was immersed in 40 ml release medium, which was maintained at 37 °C and stirred at a speed of 100 rpm. At pre-determined time intervals, samples (0.5 ml) were withdrawn and were centrifuged at 10,000 rpm for 15 min, and then replaced by 0.5 ml fresh release medium. The concentration of PTX was determined by high-performance liquid chromatography (HPLC), and the mobile phase consisted of water and acetonitrile (50:50 v/v) at a flow rate of 1.0 ml min 1. The oven temperature was set at 40 °C, the column was DiamohsilTMC18 (250 4.6 mm) and the detection wavelength was 227 nm.
2.6. Degree of drug loading The actual drug loading of drug-loaded MTNP was also determined by HPLC. In detail, the drug-loaded samples (5 mg) were transferred to 20 ml dichloromethane with stirring for 8 h. After centrifugation, the supernatant was diluted with methanol in a ratio of 1:100, and then the dilute solution was examined by HPLC at 227 nm. Drug loading = (weight of drug in MTNP/weight of MTNP) 100.
2.10. Assessment of cytotoxicity The cell line was seeded into 96-well plates at a density of 5 103 cells per well for 24 h. The medium was then removed, and 100 ll of culture medium containing different concentrations of samples were added to 96-well plates and incubated for selected times. Then, 10 ll MTT (5 mg ml 1) solution was added to the wells and incubated for 24 h. After incubation, the solution containing MTT was removed, and 100 ll DMSO was added to each well, followed by stirring for 10 min. Finally, the absorbance was recorded at 492 nm (n = 6), and the cell survival was determined from the equation: cell survival (%) = absorbance of sample/absorbance of control 100%. 2.11. Statistical analysis Average and standard deviations were calculated from three repeats on each sample, except for all data in Figs. 4, 9 and 10, where the measurements were repeated six times. Error bars on the graphs represent one standard deviation in both positive and negative orientations.
2.7. Confocal laser scanning microscopy (CLSM)
3. Results and discussion
A549 and KB cells were seeded in 24-well plates for 48 h. The A549 and KB cells were washed three times with PBS (pH 7.4), and then incubated with FITC-FA-PEI-MTNP or FITC-PEI-MTNP (20 lg ml 1) in the culture medium at 37 °C for 5 h. The cells were washed three times with PBS and then fixed in 4% formaldehyde PBS solution at 37 °C for 20 min. Finally, the cells were washed three times with PBS and then incubated with 0.1% Triton X-100 PBS solution including 1% bovine serum albumin at 37 °C for 4 min. The cells were then stained with Hoechst 33258 (200 ll, 1 lg ml 1) at 37 °C for 20 min. After staining, the cells were fixed and examined by CLSM.
3.1. Fabrication of TiO2 nanoparticles
2.8. Flow cytometric analysis of particle uptake A549 and KB cells were seeded in 12-well plates at a density of 1 104 and cultured in culture medium at 37 °C for 3 days. The cells were then treated with FITC-FA-PEI-MTNP or FITC-PEI-MTNP (with different concentrations) in culture medium at 37 °C for 5 h. The cells were then washed three times with PBS and examined by flow cytometry.
2.9. In vivo distribution of FA-FITC-PEI-MTNP nanoparticles In order to study the tumor targeting effect of FA-PEI-MTNP, the in vivo distribution of FITC-FA-PEI-MTNP in Kunming mice bearing hepatic H22 tumors was observed by a Carestream Molecular Imaging In-Vico Ms. FX PRO system and Carestream Molecular Imaging software. The murine hepatoma H22 cells at a density of 5 106 cells per mouse were injected subcutaneously into the right flank of the Kunming mice (n = 3). The tumor volume was monitored closely and calculated as V (mm3) = (W2 L)/2, where L and W are the longest and shortest axes of the tumor. When the tumors in the mice reached 300 mm3, the mice were injected in the tail vein with FITC-FA-PEI- MTNP (as the experimental group) and FITC-PEI- MTNP (as the control group), respectively (at a concentration of 1.0 mg ml 1, 200 ll). At the pre-determined time intervals, tumors and tissues were excised from the sacrificed mice, and imaged using the optional wavelength (kem, 535 nm; kex, 470 nm).
3
The morphology of MTNP was observed by SEM and TEM. Fig. 1a and b shows that the average diameter of the prepared MTNP is 150 nm, with pores of 2–3 nm. After modification of MTNP by FA-PEI, the shape and diameter of the FA-PEI-MTNP show no significant change (Fig. 1c), while the porous channel becomes blurry after functionalization, owing to the presence of a macromolecule (FA-PEI) layer on the surface of the MTNP. (Fig. 1d). The pore characteristics of the MTNP and FA-PEI-MTNP were further investigated by surface analyzer. As shown in Table 1, the pore size, specific surface area and total pore volume of MTNP were 2–3 nm, 555 m2 g 1 and 0.294 cm3 g 1, respectively. Compared with MTNP, the pore size of FA-PEI-MTNP was still 2–3 nm, while the specific surface area (519 m2 g 1) and total pore volume (0.257 cm3 g 1) showed a slight decrease, indicating that the pore structure can still keep its morphology after functionalization, and only a small part of the pores are blocked by the functional group FA-PEI. PEI with molecular weight up to 25,000 was too large to enter the pores 2–3 nm in diameter. In addition, although the portion of the pores was possibly covered by FA-PEI, the small molecule can still enter nearly all the pores of the carrier, owing to the mutually connected channels (Fig. 1b). Therefore, after functionalization, the pore volume and the specific surface area do not change dramatically. 3.2. Functional modification of MTNP The process of FA-PEI modification is illustrated in Fig. 2a and b, and was further confirmed by zeta-potential measurements. As expected (Fig. 2d), the zeta potential curve of FA-PEI-functionalized MTNP (FA-PEI-MTNP) shifted significantly to the right in comparison with that of MTNP and the isoelectric point (IEP) increased from 5.2 for MTNP to 8.1 for FA-PEI-MTNP, demonstrating that PEI with a lot of positive charge (amino group) has wrapped onto the surface of MTNP [29]. Under physiological conditions of pH 7.4, positively charged FA-PEI was absorbed onto the negatively charged surface of MTNP through electrostatic interaction, as shown in Fig. 2d. Therefore, the desorption of FA-PEI reduces the zeta potential of the precipitate. As shown in Fig. S1 (Supporting Information), the zeta potential of the samples remains almost
Please cite this article in press as: Wang T et al. Potential application of functional porous TiO2 nanoparticles in light-controlled drug release and targeted drug delivery. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.010
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Fig. 1. SEM images of (a) MTNP, (c) FA-PEI-MTNP and (e) FA-PEI-PTX-MTNP, and TEM images of (b) MTNP, (d) FA-PEI-MTNP and (f) FA-PEI-PTX-MTNP.
Table 1 Characterization of properties of carriers before and after drug loading.
a b c
Sample
SBETa (m2 g
MTNP FA-PEI-MTNP FA-PEI-PTX-MTNP
555 519 4.74
1
)
Vtb (cm3 g 0.294 0.257 0.059
1
)
WBJHc (nm)
Loading content (%, HPLC)
2–3 2–3
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Potential application of functional porous TiO2 nanoparticles in light-controlled drug release and targeted drug delivery Tianyi Wang a, , Haitao Jiang b, , Long Wan b, Qinfu Zhao b, Tongying Jiang b, Bing Wang a,⇑, Siling Wang b,⇑ a b
College of Life Science and Health, Northeastern University, 89 Wenhuadong Road, Shenhe District, Shenyang, Liaoning Province 110015, People’s Republic of China Department of Pharmaceutics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang, Liaoning Province 110016, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 19 May 2014 Received in revised form 24 October 2014 Accepted 5 November 2014 Available online xxxx Keywords: Porous TiO2 Nanoparticle Surface functionalization Controllable release Targeting
a b s t r a c t Novel multifunctional porous titanium dioxide (TiO2) nanoparticles modified with polyethylenimine (PEI) were developed to explore the feasibility of exploiting the photocatalytic property of titanium dioxide to achieve ultraviolet (UV) light triggered drug release. Additionally, in order to further realize targeting delivery, folic acid, which chemically conjugated to the surface of the functionalized multifunctional porous TiO2 nanoparticles through amide linkage with free amine groups of PEI, was used as a cancer-targeting agent to effectively promote cancer-cell-specific uptake through receptor-mediated endocytosis. And a typical poorly water-soluble anti-cancer drug, paclitaxel, was encapsulated in multifunctional porous TiO2 nanoparticles. The PEI on the surface of multifunctional porous TiO2 nanoparticles could effectively block the channel to prevent premature drug release, thus providing enough circulation time to target cancer cells. Following UV light radiation, PEI molecules on the surface were cut off by the free radicals (OH and O2 ) that TiO2 produced, and then the drug loaded in the carrier was released rapidly into the cytoplasm. Importantly, the amount of drug released from multifunctional porous TiO2 nanoparticles can be regulated by the UV-light radiation time to further control the anti-cancer effect. This multifunctional porous TiO2 nanoparticle exhibits a combination of stimuli-triggered drug release and cancer cell targeting. The authors believe that the present study will provide important information for the use of porous TiO2 nanomaterials in light-controlled drug release and targeted therapy. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Chemotherapy is now an essential component of cancer therapies used to treat most cancers [1,2]. Although various strategies for drug delivery have been developed in recent years, in efforts to kill cancer cells effectively, many routinely used chemotherapeutic agents are not able to specifically target cancer cells, and their release is poorly controlled, often resulting in serious undesirable side effects [1,3,4]. Therefore, the ability to achieve specific drug accumulation at tumor sites is still a great challenge for successful cancer chemotherapy [5–7]. In order to improve chemotherapy, great efforts have been devoted to the development of tumor-targeted nanocarriers for the controlled delivery of anti-cancer drugs. To obtain a tumor-targeted nanocarrier, a promising strategy is to modify the carrier with ⇑ Corresponding authors. Tel./fax: +86 24 85820828 (B. Wang). Tel./fax: +86 24 23986348 (S. Wang). E-mail addresses: [email protected] (B. Wang), [email protected]. cn (S. Wang). These authors contributed equally to this work.
targeting ligands, such as peptides [8], antibodies [9] and small molecules (e.g. folate (FA)) [10], which can selectively recognize and bind to the surface receptors over-expressed on cancer cells and stimulate cancer-cell-specific uptake. Moreover, in order to further reduce systemic toxicity and undesired side effects, keeping the loaded drugs sealed during circulation in the bloodstream and rapidly releasing them on reaching and accumulating in tumor tissues is also very important [11,12]. Therefore, an ideal anti-cancer drug delivery system should be a combination of cancer cell targeting and stimuli-triggered drug release. Porous titanium dioxide (TiO2) offers the potential to achieve this challenge, owing to its interesting properties, such as a high photocatalytic activity and a functional surface. TiO2, a typical n-type semiconductor material, has been widely used in many fields, such as photocatalysis [13], energy storage and conversion [14], sunscreening [15] and sensor research [16], and especially in medical fields, as anti-cancer agents [17], implants [18] and substrates for stem cell expansion, owing to its cheapness, chemical stability, innocuity and excellent biocompatibility [19]. Among these, much attention has been drawn in recent years towards the photocatalytic degradation of organic compounds
http://dx.doi.org/10.1016/j.actbio.2014.11.010 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang T et al. Potential application of functional porous TiO2 nanoparticles in light-controlled drug release and targeted drug delivery. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.010
2
T. Wang et al. / Acta Biomaterialia xxx (2014) xxx–xxx
[20–23]. On ultraviolet (UV) light excitation, the energy absorbed from UV becomes higher than the band gap of TiO2, and the valence electrons will be excited to the conduction band, creating an electron (e ) and hole (h+) pair and further generating active free radicals (OH and O2 ) which effectively decompose the organic compounds on the surface of the TiO2 particle [24–26], providing a theoretical foundation for light-triggered drug release. It is worth mentioning that the TiO2 itself was reported to have a good anticancer effect, owing to the production of active free radicals under UV irradiation [17]. Therefore, the present authors have innovatively prepared hybrid polyethylenimine (PEI)-modified porous TiO2 nanoparticles (MTNP). PEI (a hydrophilic dendritic macromolecule) coated on the surface of MTNP could block the release of drugs as a result of the formation of a hydrophilic layer. Following UV radiation, the PEI molecules could be destroyed by free radicals (OH and O2 ) [23,24], and then allow drug release. In addition, FA was covalently linked to the PEI-MTNP. Since folate receptor (FR) is overexpressed on cancer cells, the conjugation (FA with FR) effectively facilitates cellular uptake of the FA functionalized carrier [1,10,29]. Herein, MTNP was developed using non-ionic surfactant F127 as a structure-directing agent. To achieve the targeting and lightcontrolled release goals, the targeting group (FA) was covalently linked to the bridge molecule (PEI) [30], and then the surface of the MTNP was modified by functional groups (FA-PEI) through the electrostatic adsorption effect. Finally, the anti-cancer drug paclitaxel (PTX) was loaded into the pores of the functionalized carrier by a solvent deposition method [27,28]. The loaded anticancer drug was not released during circulation in the bloodstream and normal tissues because of blocking by PEI. Multifunctional MTNP could be efficiently trapped by cancer cells through receptor-mediated endocytosis. And then under UV excitation, the PEI coated on the surface of the carrier could be destroyed, and the anti-cancer drug was rapidly released from the pores. Meanwhile, the TiO2 carrier itself could also have an anti-cancer effect. The potential of the multifunctional MTNP in tumor targeting and controllable drug release is systematically investigated in the present research. 2. Materials and methods 2.1. Materials Titanium (IV) isopropoxide (TIP) (95%) was obtained from Alfa Aesar (Lancs, UK). Pluronic block co-polymer F127 was a gift from BASF (Ludwigshafen, Germany). Thiazolyl blue tetrazolium bromide (MTT), Hoechst 33258, streptomycin and penicillin were purchased from Sigma–Aldrich (Santa Clara, USA). Fluorescein isothiocyanate isomer (FITC), PEI and FA were purchased from Aladdin (Shanghai, PRC). KB (human nasopharyngeal carcinoma cell) cell and A549 (human lung carcinoma cell line) cell were purchased from Jia He Biotechnology Co., Ltd. (Shanghai, PRC). PTX was kindly donated by Funing Pharmaceutical Co., Ltd (Shenyang, PRC). The water used for the experiment was Milli-Q (18.2 MO). Absolute ethanol (>99.7%) was purchased from Tianjin Bodi Chemical Holding Co., Ltd (Tianjin, PRC). All other solvents were chromatographic grade. 2.2. Fabrication of TiO2 nanoparticles The TiO2 nanoparticles were synthesized via a sol–gel route, using F127 as a structure-directing agent and TIP as a TiO2 source. In detail, 16 g F127 was completely dissolved in 400 ml ethanol, and then 2.4 ml Milli-Q was added to this solution with stirring. Then 7.4 ml TIP was added to the solution at ambient temperature
under vigorous stirring. When the clear solution turned into a milky white suspension, the solution was stirred continuously for 50 min, and then the solution was kept at room temperature without stirring to stand for 12 h. Finally, 2 g precursor sample was dispersed in 200 ml absolute ethanol solution, and then the solution was stirred for 2 h at 70 °C. After stirring, the white samples were collected by centrifugation. The samples were extracted three times in this way and then dried at 40 °C overnight. In order to completely remove the surfactant (F127), the sample was extracted again three times in the same way. Finally, the resulting product was dried at 200 °C for a day to remove absolute ethanol completely. The samples obtained were named MTNP. 2.3. Functional modification of MTNP Before modification of the MTNP, FA was conjugated to PEI at first. The method was as reported by Guo and co-workers [30]: briefly, 1 g FA was dissolved in 50 ml dimethylsufoxide (DMSO) and then 1.1 M excess of N-hydroxysuccinimide and N, N’-dicyclohexylcarbodiimide was added to this solution with stirring. Afterwards, the solution was stirred for 12 h at room temperature. The solution was filtrated to remove the insoluble byproduct, dicyclohexylurea. And then, PEI was dissolved in carboxy-activated FA DMSO solution at a ratio of 1:5 (molar ratio). After stirring for 12 h, the product (FA-PEI) was passed through a Sephadex G-100 column (Pharmacia Biotech) for purification. MTNP, 80 mg, was suspended in FA-PEI phosphate (PBS) buffer (pH 7.4), and then the solution was stirred for 5 h at room temperature. These modified particles (FA-PEI-MTNP) were then collected by centrifugation (5000 rpm, 5 min) and were dried at room temperature. The FA-PEI-functionalized particles were further labeled with FITC (fluorescein isothiocyanate) by immersing 50 mg TiO2 particles in carbonate buffer (pH 8.2) and mixing with 500 ll FITC-ethanol solution (1 mg ml 1), followed by stirring this solution for 40 min [30]. After this, the FITC-FA-PEI-functionalized particles were collected by centrifugation (5000 rpm, 5 min). Electrokinetic titrations were measured using a Malvern Zetasizer-3000. The sample solutions were adjusted to different pH values with 0.1 M NaOH and HCl. 2.4. Characterization Field-emission scanning electron microscopy (SEM), using a SUPRA 35 instrument (ZEISS, GER, operated at 15 kV) and transmission electron microscopy (TEM) (TECNAI G2 20, FEI, USA) were used to examine the morphology of the MTNP obtained. A surface area and pore size analyzer (V-sorb 2800P, Gold App Instruments, CN) was used to measure the pore characteristics of the samples. Samples were degassed at 150 °C for 16 h prior to analysis. The Brunauer–Emmett–Teller (BET) method was used to determine the surface area of the samples. The Barrett–Joyner–Halenda (BJH) method was used to determine the pore size distribution. X-ray diffractometry (XRD; PW3040/60 PANALYII CALB.V NED) was used to determine the crystals of MTNP over the angle range from 5° to 80° (2h) with a scan rate of 6° min 1 and Cu K-Alpha1 KAlpha2 radiation (k = 1.54 Å, 30 mA, 30 kV). 2.5. Drug loading and release PTX was loaded into the pores of the MTNP by a solvent deposition method [27,28], involving a combination of soaking equilibrium and solvent evaporation. The details of the drug loading are described in the Supporting Information. PTX released from FA-PEI-PTX-MTNP was determined by the dialysis method. A certain amount of sample was added to 2 ml PBS (pH 7.4) with 0.1% Tween 80, and then this solution was placed in a dialysis
Please cite this article in press as: Wang T et al. Potential application of functional porous TiO2 nanoparticles in light-controlled drug release and targeted drug delivery. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.010
T. Wang et al. / Acta Biomaterialia xxx (2014) xxx–xxx
bag (MWCO 8000, Fisher Scientific). The dialysis bag was immersed in 40 ml release medium, which was maintained at 37 °C and stirred at a speed of 100 rpm. At pre-determined time intervals, samples (0.5 ml) were withdrawn and were centrifuged at 10,000 rpm for 15 min, and then replaced by 0.5 ml fresh release medium. The concentration of PTX was determined by high-performance liquid chromatography (HPLC), and the mobile phase consisted of water and acetonitrile (50:50 v/v) at a flow rate of 1.0 ml min 1. The oven temperature was set at 40 °C, the column was DiamohsilTMC18 (250 4.6 mm) and the detection wavelength was 227 nm.
2.6. Degree of drug loading The actual drug loading of drug-loaded MTNP was also determined by HPLC. In detail, the drug-loaded samples (5 mg) were transferred to 20 ml dichloromethane with stirring for 8 h. After centrifugation, the supernatant was diluted with methanol in a ratio of 1:100, and then the dilute solution was examined by HPLC at 227 nm. Drug loading = (weight of drug in MTNP/weight of MTNP) 100.
2.10. Assessment of cytotoxicity The cell line was seeded into 96-well plates at a density of 5 103 cells per well for 24 h. The medium was then removed, and 100 ll of culture medium containing different concentrations of samples were added to 96-well plates and incubated for selected times. Then, 10 ll MTT (5 mg ml 1) solution was added to the wells and incubated for 24 h. After incubation, the solution containing MTT was removed, and 100 ll DMSO was added to each well, followed by stirring for 10 min. Finally, the absorbance was recorded at 492 nm (n = 6), and the cell survival was determined from the equation: cell survival (%) = absorbance of sample/absorbance of control 100%. 2.11. Statistical analysis Average and standard deviations were calculated from three repeats on each sample, except for all data in Figs. 4, 9 and 10, where the measurements were repeated six times. Error bars on the graphs represent one standard deviation in both positive and negative orientations.
2.7. Confocal laser scanning microscopy (CLSM)
3. Results and discussion
A549 and KB cells were seeded in 24-well plates for 48 h. The A549 and KB cells were washed three times with PBS (pH 7.4), and then incubated with FITC-FA-PEI-MTNP or FITC-PEI-MTNP (20 lg ml 1) in the culture medium at 37 °C for 5 h. The cells were washed three times with PBS and then fixed in 4% formaldehyde PBS solution at 37 °C for 20 min. Finally, the cells were washed three times with PBS and then incubated with 0.1% Triton X-100 PBS solution including 1% bovine serum albumin at 37 °C for 4 min. The cells were then stained with Hoechst 33258 (200 ll, 1 lg ml 1) at 37 °C for 20 min. After staining, the cells were fixed and examined by CLSM.
3.1. Fabrication of TiO2 nanoparticles
2.8. Flow cytometric analysis of particle uptake A549 and KB cells were seeded in 12-well plates at a density of 1 104 and cultured in culture medium at 37 °C for 3 days. The cells were then treated with FITC-FA-PEI-MTNP or FITC-PEI-MTNP (with different concentrations) in culture medium at 37 °C for 5 h. The cells were then washed three times with PBS and examined by flow cytometry.
2.9. In vivo distribution of FA-FITC-PEI-MTNP nanoparticles In order to study the tumor targeting effect of FA-PEI-MTNP, the in vivo distribution of FITC-FA-PEI-MTNP in Kunming mice bearing hepatic H22 tumors was observed by a Carestream Molecular Imaging In-Vico Ms. FX PRO system and Carestream Molecular Imaging software. The murine hepatoma H22 cells at a density of 5 106 cells per mouse were injected subcutaneously into the right flank of the Kunming mice (n = 3). The tumor volume was monitored closely and calculated as V (mm3) = (W2 L)/2, where L and W are the longest and shortest axes of the tumor. When the tumors in the mice reached 300 mm3, the mice were injected in the tail vein with FITC-FA-PEI- MTNP (as the experimental group) and FITC-PEI- MTNP (as the control group), respectively (at a concentration of 1.0 mg ml 1, 200 ll). At the pre-determined time intervals, tumors and tissues were excised from the sacrificed mice, and imaged using the optional wavelength (kem, 535 nm; kex, 470 nm).
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The morphology of MTNP was observed by SEM and TEM. Fig. 1a and b shows that the average diameter of the prepared MTNP is 150 nm, with pores of 2–3 nm. After modification of MTNP by FA-PEI, the shape and diameter of the FA-PEI-MTNP show no significant change (Fig. 1c), while the porous channel becomes blurry after functionalization, owing to the presence of a macromolecule (FA-PEI) layer on the surface of the MTNP. (Fig. 1d). The pore characteristics of the MTNP and FA-PEI-MTNP were further investigated by surface analyzer. As shown in Table 1, the pore size, specific surface area and total pore volume of MTNP were 2–3 nm, 555 m2 g 1 and 0.294 cm3 g 1, respectively. Compared with MTNP, the pore size of FA-PEI-MTNP was still 2–3 nm, while the specific surface area (519 m2 g 1) and total pore volume (0.257 cm3 g 1) showed a slight decrease, indicating that the pore structure can still keep its morphology after functionalization, and only a small part of the pores are blocked by the functional group FA-PEI. PEI with molecular weight up to 25,000 was too large to enter the pores 2–3 nm in diameter. In addition, although the portion of the pores was possibly covered by FA-PEI, the small molecule can still enter nearly all the pores of the carrier, owing to the mutually connected channels (Fig. 1b). Therefore, after functionalization, the pore volume and the specific surface area do not change dramatically. 3.2. Functional modification of MTNP The process of FA-PEI modification is illustrated in Fig. 2a and b, and was further confirmed by zeta-potential measurements. As expected (Fig. 2d), the zeta potential curve of FA-PEI-functionalized MTNP (FA-PEI-MTNP) shifted significantly to the right in comparison with that of MTNP and the isoelectric point (IEP) increased from 5.2 for MTNP to 8.1 for FA-PEI-MTNP, demonstrating that PEI with a lot of positive charge (amino group) has wrapped onto the surface of MTNP [29]. Under physiological conditions of pH 7.4, positively charged FA-PEI was absorbed onto the negatively charged surface of MTNP through electrostatic interaction, as shown in Fig. 2d. Therefore, the desorption of FA-PEI reduces the zeta potential of the precipitate. As shown in Fig. S1 (Supporting Information), the zeta potential of the samples remains almost
Please cite this article in press as: Wang T et al. Potential application of functional porous TiO2 nanoparticles in light-controlled drug release and targeted drug delivery. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.010
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T. Wang et al. / Acta Biomaterialia xxx (2014) xxx–xxx
Fig. 1. SEM images of (a) MTNP, (c) FA-PEI-MTNP and (e) FA-PEI-PTX-MTNP, and TEM images of (b) MTNP, (d) FA-PEI-MTNP and (f) FA-PEI-PTX-MTNP.
Table 1 Characterization of properties of carriers before and after drug loading.
a b c
Sample
SBETa (m2 g
MTNP FA-PEI-MTNP FA-PEI-PTX-MTNP
555 519 4.74
1
)
Vtb (cm3 g 0.294 0.257 0.059
1
)
WBJHc (nm)
Loading content (%, HPLC)
2–3 2–3
