Intracellular Drug Delivery with Anodic Titanium Dioxide Nanotubes

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Functional Nanostructured Materials (including low-D carbon)

Intracellular Drug Delivery With Anodic Titanium Dioxide Nanotubes and Nanocylinders Morteza Hasanzadeh Kafshgari, Anca Mazare, Monica Distaso, Wolfgang Goldmann, Wolfgang Peukert, Ben Fabry, and Patrik Schmuki ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01211 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

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ACS Applied Materials & Interfaces

Intracellular Drug Delivery with Anodic Titanium Dioxide Nanotubes and Nanocylinders Morteza Hasanzadeh Kafshgari†‡#, Anca Mazare†, Monica Distaso§, Wolfgang H. Goldmann‡, Wolfgang Peukert§, Ben Fabry‡*, Patrik Schmuki†* † Department of Material Science and Engineering, University of Erlangen-Nuremberg, 91058 Erlangen, Germany ‡ Department of Physics, Biophysics Group, University of Erlangen-Nuremberg, Erlangen 91052, Germany § Institute of Particle Technology, University of Erlangen-Nuremberg, 91058 Erlangen, Germany * Prof.

Dr. Patrik Schmuki; Email: [email protected]

* Prof.

Dr. Ben Fabry; Email: [email protected]

KEYWORDS: Titanium dioxide, Nanotubes, Nanocylinders, Electrochemical anodization, Intracellular drug delivery

ACS Paragon Plus Environment

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ABSTRACT: Titanium dioxide (TiO2) holds remarkable promises for developing current theranostic strategies. Anodic TiO2 nanostructures as a porous scaffold have offered a broad range of useful theranostic properties; however, previous attempts to generate single and uniform TiO2 one-dimensional nanocarriers from anodic nanotube arrays have resulted in a broad cluster size distribution of arbitrarily broken tubes that are unsuitable for theraputic delivery systems due to poor biodistribution and the risk of introducing tissue inflammation. Here, we achieve well-separated, uniformly shaped anodic TiO2 nanotubes and nanocylinders through a timevarying electrochemical anodization protocol that leads to the generation of planar sheets of weakly connected nanotubes with a defined fracture point near the base. Subsequent sonication cleanly detaches the nanotubes from the base. Depending on the position of the fracture point, we can fabricate single anodic nanocylinders that are open on both ends, or nanotubes that are closed on one end. We go on to show that anodic nanotubes and nanocylinders are non-toxic at therapeutic concentrations. When conjugated with the anticancer drug doxorubicin using a pHresponsive linker, they are readily internalized by cells and subsequently release their drug cargo into acidic intracellular compartments. Our results demonstrate that uniformly sized anodic TiO2 nanotubes and nanocylinders are suitable for subcellular delivery of therapeutic agents in cancer therapy.


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In recent years, a broad range of nanocarriers for drug delivery systems has been introduced, with promising results for treating a wide variety of diseases.1 Nanocarriers can be targeted to specific cell surface receptors; they diffuse easily and are readily internalized by the targeted cells.2-3 Due to their extremely high surface-to-volume ratio, nanocarriers such as porous silicon and dendritic mesoporous silica can be loaded with considerable amounts of therapeutic agents.4 Nanocarriers have the ability to leave the bloodstream and to be resorbed by cancer cells when passing through the leaky capillary network that is often found around malignant solid tumors.5 For reasons that are currently not well understood, the shape of the nanocarriers is important for cellular adhesion and uptake.3 For example, in vivo studies have shown that elongated nanocarriers can accumulate at tumor sites after intravenous injection and can deliver effective amounts of therapeutic agents.4,

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Other studies demonstrated that porous silicon nanodiscs,

polyhedron-shaped diatom biosilica and carbon nanotubes used for targeted drug delivery systems show improved biodistribution, endothelial adhesion, and accumulation in the tumor microenvironment compared to spherical nanoparticles with a similar effective diameter.4,

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This apparent benefit for non-spherically shaped particles suggests that biocompatible TiO2 in an elongated geometry may be an effective nanocarrier.8-9 Elongated TiO2 geometries such as tubes, whiskers, and wires can be produced by selfassembly, thermal, hydrothermal, or template-assisted approaches as well as by self-organizing electrochemical techniques.10-11 In particular, electrochemical anodization has been widely applied to obtain layers of self-organized TiO2 nanotubular arrays on Ti substrates.12 Such arrayed layers are easy to process and can be functionalized by a variety of techniques. Although nanotube layers can be detached from the solid TiO2 substrate,12-13 it remains challenging to detach single nanotubes from the layer and to form a suspension of single and uniformly shaped


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TiO2 nanotubes (TiO2NTs) or nanocylinders (TiO2NCs). Sonication that is frequently used for lift-off or crush-off of the oxidized tubes from the metallic substrate generally leads to a dispersion of highly irregular clusters and bundles of connected structures including broken fragments of nanotubes (Figure S1, Supporting Information).10,

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The resulting suspension is

characterized by a broad cluster size distribution of arbitrarily broken nanocarriers, which renders the particles unsuitable for drug delivery systems due to poor biodistribution and the risk of introducing tissue inflammation.4 Previous studies have demonstrated that an intravenous injection of different sizes of nanoparticles including TiO2, gold and silver nanoparticles can trigger different cellular responses and cytotoxicities due to non-specific accumulation, cellular uptake, and internalization of small particles or fragments into subcellular compartment such as nucleus or mitochondria.14-16 Here, we describe a strategy to obtain well-separated single nanotubes and nanocylinders based on an electrochemical anodization and dispersion mechanism and demonstrate their potential application in nanomedicine. For this, we conjugate the nanotubes and nanocylinders with the anticancer drug doxorubicin (DOX) using a pH-responsive linker, and evaluate the cytotoxicity and cellular uptake in cancer cells. To obtain elongated nanotubes of defined size and shape, we control the multistep anodization procedure to introduce a pre-defined fracture point between the nanostructures and the solid substrate (Figure 1). Subsequent sonication generates single nanoparticles that break away at the fracture points, which prevents the formation of particle bundles or sheets. Moreover, using two different anodization techniques, we can control the position of the fracture point to either generate nanocylinders (NCs) that are open on both ends (TiO2NCs), or nanotubes (NTs) that are closed at the bottom end (TiO2NTs) (Section S1 and S2, Supporting Information). The


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anodization electrolyte used here consisted of glycerol/water (60/40 v/v) and ammonium fluoride, which was previously shown to result in well-defined intertubule separation and open tube ends. The anodization process included three voltage steps: 35 V for 240 min, 5 V for 10 min, and 35 V for 60 min. The short (10 min) anodization step at a low voltage results in a fragile layer that acts as a fracture point.12 The formation of the fragile layer in the second step allows for defined detachment of separate NTs or NCs in a mild sonication treatment and thus to obtain single nanocarriers after lift-off.

Figure 1. Schematics of the formation of anodic (a) TiO2NTs and (b) TiO2NCs. Different potentials and times were applied for each voltage cycle (1st cycle: 35 V and 240 min, 2nd cycle: 5 V and 10 min, and 3rd cycle: 35 V and 60 min) in the presence of an electrolyte composed of ammonium fluoride (0.27 M) mixed in glycerol/water (60/40 v/v) at room temperature. The


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triple-cycle electrochemical anodization process created a suitable distance among tubes (a-II) and cylinders (b-II) in arrays and a fragile layer between the first and third layer. A sonication process separated anodic TiO2NTs (a-III) and TiO2NCs (b-III). Representative SEM images of anodic tubular (a) and cylindrical (b) arrays before (IV) and after (V) sonication.

The morphology of TiO2NTs and TiO2NCs with a closed and open bottom, respectively, is shown in scanning electron microscopy (SEM) images (Figure 1). The entrance diameters of tubes and cylinders (top view) are in the range of 150 – 200 nm (Figure S10, Supporting Information). Around 75% of the nanotubes (based on the SEM evaluation, Figure S13, Supporting Information) and almost all nanocylinders are individually separated from each other after sonication and detachment from the layer. The mean lengths, evaluated by SEM, are 973.6±86 nm (TiO2NTs) and 724.7±13 nm (TiO2NCs). The colloidal stability of the suspension was assessed by measuring the -potential at different pH values. With the ammonium fluoride/glycerol electrolyte used during the fabrication step we obtained a tube-tube termination that leads to a point of zero charge (PZC) of TiO2 nanocarriers at around pH 4 (Figure S14, Supporting Information), and much lower than the PZC of pH 6.6 expected for TiO2 anatase.17-18 The absolute maxima of the measured -potentials (+45 mV at pH < 3 and -30 mV at pH > 7) also highlight the stability effect of the surface charge under the experimental conditions. To the best of our knowledge, this is the first report describing the production of a dispersion of single and uniformly shaped TiO2 nanotubes and nanocylinders, whereas previous efforts mainly generated a suspension of clusters and bundles of broken fragments of anodic nanotubes.13


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Figure 2. (a) Schematics depicting the conjugation of pH-cleavable DOX on the surface of TiO2NTs or TiO2NCs. (b) XPS N1s spectra of (I) CDI, (II) tert-Butyl carbazate, (III) deprotected BOC-carbazate, and (IV) pH-cleavable DOX-conjugated TiO2NT arrays. (c) Representative release of pH-cleavable DOX from TiO2NTs and TiO2NCs. Release medium: PBS, pH 2 (), 5 (), and 7.4 (); T=37 °C.

To functionalize the nanocarriers, we modified anatase TiO2NT arrays (see X-ray diffraction spectra, Section S3, Supporting Information) with linkers and drugs prior to separation by sonication (Figure 2a). First, a monolayer of carbonyldiimidazole (CDI) was used as a linker to bind tert-Butyl carbazate (BOC-hydrazide). The BOC-hydrazide was then removed using trifluoroacetic acid to form a pH-responsive hydrazone linker. After deprotection of the BOCgroup, the positively charged anticancer drug DOX was subsequently conjugated. The pHresponsive hydrazone is a cleavable linker and releases the DOX at low pH (< 5) found in cell


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lysosomes and endosomes containing the cell-internalized nanocarriers.19 The intracellular drug delivery as opposed to conventional chemotherapy is important for exerting therapeutic actions inside specific organelles to improve the therapeutic efficiency and reduce side effects.2-3 After functionalization with DOX, the ζ-potentials increased to 15 mV for nanotubes and to 31 mV for nanocylinders, suggesting improved stability of the dispersion compared to nonfunctionalized nanoparticles (Figure S15, Supporting Information).17-18 The increased ζ-potential can be attributed to the primary amine functional group of the conjugated DOX molecules with a positive charge at pH 7.4. X-ray photoelectron spectroscopy (XPS) characterization (N1s spectra) was performed prior to sonication to track the various stages leading to the DOX conjugation (Figure 2b, the spectrum of the unconjugated TiO2NT array is included in Figure S17, Supporting Information). The N1s signals confirm the attachment of carbonyldiimidazole on the surface, the signals at ~398.9, ~400.3, and ~401.3 eV (Figure 2b-I) are assigned to free amine groups and the two nitrogen atoms in the imidazole rings.20-22 After tert-butyl carbazate coupling to CDI, the presence of the bound BOC-hydrazide was confirmed by the N1s peaks at ~399.5, ~400.6, and ~401.4 eV (Figure 2b-II), corresponding to amides, NH–C=O, and positively charged nitrogen, respectively.23-24 After BOC de-protection (Figure 2b-III), similar N1s peaks at ~399.6, ~400.5, and ~401.4 eV were observed, with a higher fraction of free amide. At the final step, DOX molecules were conjugated to the de-protected tert-butyl carbazate (Figure 2b-IV). N1s peaks were found at ~400 (amides or amines), ~401 (quaternary or graphitic nitrogen), and ~402.3 eV (–NH3+ ),23, 25-26 with a significant increase in the signal of the free amino group located at the side group of DOX, demonstrating the successful conjugation of DOX on the surface of the nanocarriers.


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After sonication for 45 min, fluorospectrometry was used to determine the amount of conjugated DOX, yielding 83 g/mg for DOX-TiO2NTs and 46 g/mg for DOX-TiO2NCs. We next examined the pH-responsive release of DOX from TiO2NTs and TiO2NCs (Figure 2c and d) at different pH values (2, 5, and 7.4) over 36 h at 37°C. The release kinetics clearly proved a pH-responsive DOX release from the nanocarriers, characterized by a faster release at pH 2 and 5, whilst the release of DOX was suppressed at pH 7.4. Moreover, we found a fast release of DOX within the first hour, followed by a sustained release behavior over 36 h. By comparison, the release rate of loaded DOX (non-conjugated) from tubular nanocarriers was slightly pHdependent but not similar (Section S4, Supporting Information). The internalization of DOX-TiO2NTs and DOX-TiO2NCs into cervical cancer cells (HeLa) was evaluated using laser-scanning confocal microscopy (Figure 3 and Section S5, Supporting Information). HeLa cells were washed and fixed one hour after incubation with DOX-conjugated nanocarriers. We found that 100% of the cells either bind or internalize the particles, as shown by the multiple punctate red dots from the intrinsic fluorescence of particle-bound DOX (Figure 3c,d). SEM images confirm the particle binding and internalization, where we find particles bound to the cell surface and also particles that are in the process of being internalized (Figure 3h,i). A cross-view of Z-stack images acquired using confocal microscopy confirms that a substantial fraction of the particles is internalized by the cell after 1 h of incubation (Figure S21, Supporting Information). After the incubation of particles for 24 h, the punctuated red dots have disappeared and the DOX is spread throughout the cytoplasm (Figure 3e,f and Section S5, Supporting Information), suggesting that all bound particles have been internalized, trapped in endosomes with low pH, and released their cargo. To verify that particle uptake is an intrinsic feature of the nanoparticles and is not facilitated by the pH-responsive linker or the loading with


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DOX, we also incubated the cells with fluorescein isothiocyanate modified TiO2NTs (FITCTiO2NTs) for one hour and found a similar cellular uptake behavior (Figure S22c-II, Supporting Information). FITC-conjugated nanoparticles were also imaged after 24 h of incubation. The green punctuated spots caused by FITC persisted over time, and no spreading of the fluorescent signal was observed, confirming that the intracellular release of DOX was not unspecific but due to the pH-responsive linker (Figure S22c-III, Supporting Information).

Figure 3. Uptake of DOX-TiO2NTs and DOX-TiO2NCs by HeLa cells. a) Schematic of the intracellular

pH-cleavable

DOX

release

from

DOX-TiO2NTs

and

DOX-TiO2NCs.


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Representative laser-scanning confocal microscopy images of (b) control HeLa cells without the nanocarriers DOX-TiO2NTs (c, red punctate) and DOX-TiO2NCs (d, red punctate) in HeLa cells after one hour of the incubation. pH-cleavable release of DOX into the intracellular compartments after 24 h of incubation of DOX-TiO2NTs (e, reddish color), and DOX-TiO2NCs (f, reddish color). Cells were stained by fluorescein diacetate (green). Representative SEM images show the cells after the treatment without (g) nanocarriers and with (h) TiO2NTs and (i) TiO2NCs. Insets show the magnified images of internalizing TiO2NTs and TiO2NCs. Scale bars of the inset frames are 500 nm.

The pH-responsive drug release was furthermore confirmed by employing a washing step (described in Section S1-6, Supporting Information). HeLa cells were incubated at different concentrations of nanocarriers (DOX-conjugated and DOX-loaded) for 30 min, followed by replacing the culture medium to remove all unattached nanocarriers. After 72 h of incubation, the percentage of surviving HeLa cells was approximately 2-fold lower when then cells were treated with pH-cleavable DOX-TiO2NTs and DOX-TiO2NCs as compared to treatment with DOXloaded TiO2 nanocarriers (Figure 4).


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Figure 4. Cell viability of HeLa cells treated for 72 h with native, DOX-conjugated and DOXloaded (a) anodic TiO2NTs and (b) TiO2NCs after 72 h with and without a washing step. Washing step: HeLa cells were incubated at different concentrations of the nanocarriers for 30 min, the cell culture medium was subsequently replaced to remove unbound nanocarriers. Control experiments were performed with and without performing the washing step. Conditions: T=37 °C ± 0.2 and 5% CO2 (n = 3; mean ± standard deviation).

To test that the cytotoxicity from DOX-TiO2NTs and DOX-TiO2NCs is attributed to the intracellular release DOX, and not through non-specific particle toxicity, HeLa cells were incubated with the uncoated nanocarriers for 72 h, after which 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyltetrazolium bromide (MTT) assays were employed to evaluate the cytotoxicity (Figure 4). The results indicate that anodic TiO2NTs and TiO2NCs are biocompatible at a low concentration (0.1 mg/mL); however, at higher concentrations (1 mg/mL), a significant cytotoxicity was induced after 72 h of incubation. Moreover, TiO2NCs showed slightly higher cytotoxicity compared to TiO2NTs at all concentrations (Section S5-2, Supporting Information).


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We point out that recent and previous studies showed a significant cytotoxicity induced by clusters and bundles of broken fragments of TiO2 nanotubes (Section S5-3, Supporting Information).13 By contrast, single intact TiO2NTs and TiO2NCs roughly show lower cytotoxicity compared to multiwall carbon nanotubes.27-29 In summary, our data demonstrate that single and uniformly shaped pH-responsive anodic TiO2 nanotubes and nanocylinders show low cytotoxicity and are highly efficient vehicles for the intracellular delivery of therapeutic agents in cancer therapy.

ASSOCIATED CONTENT Supporting Information: The Supporting Information is available free of charge on the ACS Publications website at DOI: ... Section S1: Materials and methods, Section S2: Tubular and cylindrical one-dimensional TiO2 nanostructures, Section S3: XRD and XPS analysis, Section S4: Non-conjugated DOX release from TiO2 nanocarriers, and Section S5: Cellular uptake, cell viability and drug delivery in vitro (PDF). AUTHOR INFORMATION Corresponding Author Prof. Dr. Patrik Schmuki; Email: [email protected], Phone: +49 9131 852 7575 and Prof. Dr. Ben Fabry; Email: [email protected], Phone: +49 9131 852 5610. Present Addresses # Department of Engineering Physics, Polytechnique Montreál, Montreál, Quebec, Canada, H3C3A7.


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Funding Sources This work was kindly supported by Alexander von Humboldt-Foundation (MHK), European Union Horizon 2020 programme Phys2BioMed (WHG) and Deutsche Forschungsgemeinschaft (BF and PS). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT MHK was supported by a Georg Forster Research Fellowship from the Alexander von Humboldt-Foundation. We thank Dr. Ning Liu and Mr. Imgon Hwang for performing SEM, Ms. Ulrike Marten-Jahns for carrying out XRD, Ms. Helga Hildebrand for the XPS measurements, Dr. M.S. Killian for helpful discussions, and Ms. Astrid Mainka for help with cell culture. REFERENCES (1) Shi, J.; Kantoff, P. W.; Wooster, R.; Farokhzad, O. C. Cancer Nanomedicine: Progress, Challenges and Opportunities. Nat. Rev. Cancer 2017, 17, 20−37. (2) Kafshgari, M. H.; Voelcker, N. H.; Harding, F. J. Applications of Zero-Valent Silicon Nanostructures in Biomedicine. Nanomedicine (Lond) 2015, 10, 2553−2571. (3) Kafshgari, M. H.; Harding, F. J.; Voelcker, N. H. Insights into Cellular Uptake of Nanoparticles. Curr. Drug Deliv. 2015, 12, 63−77. (4) Parodi, A.; Quattrocchi, N.; van de Ven, A. L.; Chiappini, C.; Evangelopoulos, M.; Martinez, J. O.; Brown, B. S.; Khaled, S. Z.; Yazdi, I. K.; Enzo, M. V.; Isenhart, L.; Ferrari, M.; Tasciotti, E. Synthetic Nanoparticles Functionalized with Biomimetic Leukocyte Membranes Possess CellLike Functions. Nat. Nanotechnol. 2013, 8, 61−68.


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Figure 1. Schematics of the formation of anodic (a) TiO2NTs and (b) TiO2NCs. Different potentials and times were applied for each voltage cycle (1st cycle: 35 V and 240 min, 2nd cycle: 5 V and 10 min, and 3rd cycle: 35 V and 60 min) in the presence of an electrolyte composed of ammonium fluoride (0.27 M) mixed in glycerol/water (60/40 v/v) at room temperature. The triple-cycle electrochemical anodization process created a suitable distance among tubes (a-II) and cylinders (b-II) in arrays and a fragile layer between the first and third layer. A sonication process separated anodic TiO2NTs (a-III) and TiO2NCs (b-III). Representative SEM images of anodic tubular (a) and cylindrical (b) arrays before (IV) and after (V) sonication. 160x117mm (300 x 300 DPI)



Figure 2. (a) Schematics depicting the conjugation of pH-cleavable DOX on the surface of TiO2NTs or TiO2NCs. (b) XPS N1s spectra of (I) CDI, (II) tert-Butyl carbazate, (III) de-protected BOC-carbazate, and (IV) pH-cleavable DOX-conjugated TiO2NT arrays. (c) Representative release of pH-cleavable DOX from TiO2NTs and TiO2NCs. Release medium: PBS, pH 2 (), 5 (), and 7.4 (); T=37 °C. 224x118mm (300 x 300 DPI)


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Figure 3. Uptake of DOX-TiO2NTs and DOX-TiO2NCs by HeLa cells. a) Schematic of the intracellular pHcleavable DOX release from DOX-TiO2NTs and DOX-TiO2NCs. Representative laser-scanning confocal microscopy images of (b) control HeLa cells without the nanocarriers DOX-TiO2NTs (c, red punctate) and DOX-TiO2NCs (d, red punctate) in HeLa cells after one hour of the incubation. pH-cleavable release of DOX into the intracellular compartments after 24 h of incubation of DOX-TiO2NTs (e, reddish color), and DOXTiO2NCs (f, reddish color). Cells were stained by fluorescein diacetate (green). Representative SEM images show the cells after the treatment without (g) nanocarriers and with (h) TiO2NTs and (i) TiO2NCs. Insets show the magnified images of internalizing TiO2NTs and TiO2NCs. Scale bars of the inset frames are 500 nm. 123x115mm (300 x 300 DPI)



Figure 4. Cell viability of HeLa cells treated for 72 h with native, DOX-conjugated and DOX-loaded (a) anodic TiO2NTs and (b) TiO2NCs after 72 h with and without a washing step. Washing step: HeLa cells were incubated at different concentrations of the nanocarriers for 30 min, the cell culture medium was subsequently replaced to remove unbound nanocarriers. Control experiments were performed with and without performing the washing step. Conditions: T=37 °C ± 0.2 and 5% CO2 (n = 3; mean ± standard deviation). 160x67mm (300 x 300 DPI)


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Intracellular Drug Delivery with Anodic Titanium Dioxide Nanotubes

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