A facile way of modifying layered double hydroxide ... - ACS Publications

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A Facile Way of Modifying Layered Double Hydroxide Nanoparticles with Targeting Ligand-Conjugated Albumin for Enhanced Delivery to Brain Tumour Cells Huali Zuo,† Weiyu Chen,† Helen M. Cooper,‡ and Zhi Ping Xu*,† †

Australian Institute for Bioengineering and Nanotechnology and ‡The Queensland Brain Institute, The University of Queensland, Brisbane, QLD 4072, Australia S Supporting Information *

ABSTRACT: Active targeting of nanoparticles (NPs) for cancer treatment has attracted increasing interest in the past decades. Various ligand modification strategies have been used to enhance the targeting of NPs to the tumor site. However, how to reproducibly fabricate diverse targeting NPs with narrowly changeable biophysiochemical properties remains as a major challenge. In this study, layered double hydroxide (LDH) NPs were modified as a target delivery system. Two brain tumor targeting ligands, i.e., angiopep-2 and rabies virus glycoprotein, were conjugated to the LDH NPs via an intermatrix protein moiety, bovine serum albumin (BSA), simultaneously endowing the LDHs with excellent colloidal stability and targeting capability. The ligands were first covalently linked with BSA through the heterobifunctional cross-linker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate. Then, the ligand-linked BSA and pristine BSA were together coated onto the surface of LDHs through electrostatic interaction, followed by cross-linking with the cross-linker glutaraldehyde to immobilize these BSAs on the LDH surface. In this way, we are able to readily prepare colloidally stabilized tumor-targeted LDH NPs. The targeting efficacy of the ligand-conjugated LDH delivery system has been evidenced in the uptake by two neutral cells (U87 and N2a) compared to unmodified LDHs. This new approach provides a promising strategy for rational design and preparation of target nanoparticles as a selective and effective therapeutic treatment for brain tumors. KEYWORDS: layered double hydroxide (LDH), target ligand, cell uptake mechanism, bovine serum albumin, brain tumor

1. INTRODUCTION Colloidal nanoparticle (NP) drug delivery systems are believed to preferentially accumulate in the tumor area due to an enhanced permeability and retention (EPR) effect.1 Rapid tumor growth is stimulated by enhanced angiogenesis, and the architecture of the vasculature is abnormal, leading to leakage of vessels and dysfunctional lymphatic drainage. This allows nanocarriers to extravasate into the tumor through the EPR effect.2,3 However, the challenge is confounded by the fact that ubiquitously intercellular distribution of nanoparticles within the tumor tissue may fail the cancer treatment due to suboptimal cellular uptake efficacy.4 Therefore, achieving efficient and specific cell uptake is a priority for optimizing treatment. Normally, cancer cells express a number of cell surface receptors, which uniquely interact with specific ligands. Thus, decorating the nanoparticles with such a specific targeting ligand would be expected to promote cell internalization and improve therapeutic efficacy. Moreover, active targeting will reduce the distribution to healthy tissues, thereby decreasing undesired systemic side effect.5 Layered double hydroxides (LDHs), also known as anionic clays or hydrotalcite-like materials, are a class of two© 2017 American Chemical Society

dimensional lamellar compounds made up of positively charged layers with an interlayer region containing anions and water molecules. LDHs have the general formula [M2+1−xM3+x(OH)2]x+(An−)x/n·mH2O, where M represents metal cations and A interlayer anions. Due to the unique structure of LDHs, the isomorphical substitution with divalent or trivalent cations is possible by incorporating various imaging molecules or magnetic elements, and these LDHs can be applied in nearly all imaging modalities.6 Moreover, many therapeutic agents and biochemical compounds, such as proteins/peptides, chemotherapeutic drugs, vitamins, and DNAs/RNAs, have been intercalated into the LDH interlayers and adsorbed onto the LDH surfaces through anion exchange, where LDH materials are used as carriers and vehicles for various biomedical applications. Interestingly, LDHs have a positive ζ-potential of 30−40 mV, which makes the LDHs suitable for adhering onto the negatively charged cell membrane and facilitating the subsequent cell uptake.7,8 In Received: May 8, 2017 Accepted: June 2, 2017 Published: June 2, 2017 20444

DOI: 10.1021/acsami.7b06421 ACS Appl. Mater. Interfaces 2017, 9, 20444−20453

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ACS Applied Materials & Interfaces

Figure 1. Strategy for constructing peptide-conjugated nanoparticles (A) and MALDI-TOF detection of BSA molecular weight after activation and peptide conjugation (B).

2a (N2a) cells, respectively.17,18 As schematically shown in Figure 1A, the targeting peptide (Ang2 or RVG) is first conjugated to BSA (steps 1 and 2). Then peptide−BSA is mixed with pristine BSA, and the mixture is coated onto the LDH NP surface (step 3). Further, the coated BSA on the LDH NP surface is cross-linked (step 4), endowing LDHs with the targeting capability as well as colloidal stability and redispersity. It is known that Ang2 and RVG peptide ligands can bind to low-density lipoprotein (LRP) receptors and nicotinic acetylcholine receptors (nAchR) that are widely expressed in U87 and N2a cells, respectively. Interestingly, the receptors for Ang2 and RVG are also overexpressed in the brain endothelial cells of which the blood−brain barrier (BBB) is comprised.19,20 Thus, our LDHs conjugated with Ang2/RVG are expected to specifically target these cells and promote passage through the BBB. In addition, the anticancer drug 5fluorouracil (5-FU) was used as a model drug in this research, which has been widely used for the treatment of cancers (including brain cancer) for over 40 years through inhibiting thymidylate synthase,21 aiming to prolong the patient’s life and to reduce the disease symptoms.22 Therefore, the objectives of this research were (1) to develop a facile strategy to prepare in a controlled mannner BSA-coated LDH nanoparticles with Ang2/RVG ligand conjugated at a defined density, (2) to ascertain that target ligands on the LDH surface enhance delivery to U87 and N2a cells, and (3) to confirm that target ligand enhances the cytotoxicity of drug− LDH NPs to brain tumor cells. The current method for LDHs surface functionalization may provide a feasible way to apply LDH NP for in vivo target delivery.

addition, LDHs have good biocompatibility and a pHdependent dissolution property, which are beneficial for a smart drug delivery system.9 However, there are two challenges for in vivo applications of LDHsthe colloidal instability of LDH NPs in the biological environment (i.e., aggregation) and ready surface functionalization for active targetingwhich are not well addressed until now. The stability of LDHs in the biological environment is difficult to control. Upon exposure to biological fluid with high ionic strength, aggregation of LDHs occurs due to the dynamic physicochemical interactions between LDH surfaces and biological components containing proteins, ions, and nucleic acids.10,11 Thus, the aggregated LDHs either impede successful drug/gene delivery or result in quick clearance by the immune system. Therefore, it is crucial to develop the strategies to keep the colloidal stability of LDHs in biological media. Current surface modification of LDH NPs to improve colloidal stability and targeting ligand conjugation involves serial chemical reactions in organic solvent or electrolytic solution,12,13 which may release or deactivate the loaded drugs and genes. Various stabilizing agents, such as heparins, are employed to improve the colloidal stability of LDHs for better homogeneous distribution.14 Previously, we have developed a facile strategy to endow LDH nanoparticles with colloidal stability in serum and PBS by coating bovine serum albumin (BSA) onto the LDH surface through electrostatic interaction. As-formed BSA−LDH nanocomplexes are colloidally stable in electrolyte solutions.15 Furthermore, cross-linking of BSA with glutaraldehyde (GTA) allows the LDH−BSA nanocomplexes to be easily redispersed in electrolyte solutions after freeze-drying, in addition to enhancing their colloidal stability.16 Taking advantage of these findings, herein we propose a novel and facile strategy to simultaneously stabilize and functionalize LDH nanoparticles in order to actively target brain tumor cells. In this research, we chose angiopep-2 (Ang2) and rabies virus glycoprotein (RVG) peptide ligands as the examples, which are able to target U87 glioma cells and Neuron

2. MATERIALS AND METHODS 2.1. Materials. Complementary strands of dsDNAs were purchased from GeneWorks and annealed at 75 °C for 10 min. One strand of the duplex was covalently coupled to the Cy3 fluorophore at the 5′-end before annealing. Glutaraldehyde (GTA) solution (25%) was bought from Ajax Finechem, and sulfo-SMCC [sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, MW = 436 Da] 20445


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2.2.3. Preparation of Ang2/RVG−LDH NPs. Two milliliters of 4 mg/mL LDH suspension was added into 2 mL of 10 mg/mL BSA mixed with BSA−Ang2/RVG at the molar ratio of 19:1 drop by drop with vigorous stirring for 30 min to ensure saturated adsorption. Then the adsorbed BSA was cross-linked by glutaraldehyde overnight as reported previously.15,16 The resulting Ang2/RVG-conjugated LDH nanoparticles were named Ang2/RVG−NPs (Table 2). Nanoparticles were subjected to centrifugation and washed with Milli-Q water to remove the excess BSA and BSA−SMCC−Ang2/RVG and then dispersed in PBS or serum with ultrasound treatment. Similarly, 5-FU/LDH and LDH nanoparticles were coated with BSA plus BSA−Ang2/RVG at the molar ratio of 19:1, followed by association with dsDNA−Cy3 at the LDH:dsDNA−Cy3 mass ratio of 40:1, and then cross-linked,15,16 which were designated as 5-FU/ Ang2−NPs, 5-FU/RVG−NPs, dsDNA−Cy3/Ang2−NPs, and dsDNA−Cy3/RVG−NPs, respectively, in comparison with nontargeting BSA−coated nanoparticles, NPs, 5-FU/NPs, and dsDNA− Cy3/NPs (Table 2). 2.3. Characterizations. The hydrodynamic size and the surface charge (ζ-potential) of LDH NPs, Ang2−NPs, and RVG−NPs were characterized in a Nano Zetasizer instrument utilizing dynamic light scattering (DLS). To determine the ligand number in each BSA molecule, matrixassisted laser desorption ionization time-of-flight (MALDI-TOF) spectra were recorded to quantify the molecular weight of BSA before and after BSA activation and peptide conjugation. MALDI-TOF mass spectra were obtained using a Bruker Autoflex III Smartbeam TOF/ TOF 200. All spectra were recorded in linear mode. First a matrix thin layer on a ground steel target using matrix solution I (Sinapinic Acid saturated in EtOH) was prepared. Equal volumes (2 μL each) of sample solution and matrix solution II (Sinapinic Acid saturated in TA30) were premixed and 0.5 μL of this mixture was applied on top of the matrix thin layer prepared. The number-average molecular weight (MW) was calculated from the MALDI-TOF spectra using Data Explorer software (Applied Biosystems, Framingham, MA). Ellman’s reagent, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), was used to quantify the number of thiol groups in each BSA before and after BSA conjugation with peptides through the change of the DTNB concentration. During the test, 125 μL of tested sample was mixed with 50 μL of Ellman’s reagent solution and 2.625 mL of reaction buffer (0.1 M sodium phosphate, pH 8.0, containing 1 mM EDTA) and left to react for 15 min at room temperature. As a blank, 125 μL of reaction buffer was added for comparison. The absorbance at 412 nm was then measured. 2.4. Cellular Uptake Assay. U87 cells were cultured in RPMI medium, supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The neura 2a (N2a) cells were cultured in DMEM medium, supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were cultured at 37 °C with 5% CO2 under fully humidified conditions. All cell experiments were performed in the logarithmic phase of growth.

and other chemicals were from Sigma-Aldrich if not noted specifically. Water used in experiments was deionized Milli-Q water. Angiopep-2 (Ang2: TFFYGGSRGKRNNFKTEEYC) and rabies virus glycoprotein (RVG: YTIWMPENPRPGTPCDIFTNSRGKRASNGC) peptides were purchased from Biomatik. Amicon ultra-0.5 centrifugal filter units were purchased from Millipore Co. All the other solvents were of analytical or chromatographic grade. 2.2. Synthesis of Peptide-Conjugated LDH NPs. 2.2.1. Synthesis of LDH and 5-FU/LDH. Mg2Al−Cl−LDH (pristine LDH) was prepared using a coprecipitation−hydrothermal treatment method, as reported previously.23,24 In brief, LDHs were synthesized by mixing 40 mL of NaOH solution (0.15 M) with 10 mL of salt solution containing MgCl2 (3.0 mmol) and AlCl3 (1.0 mmol) under vigorous stirring. The resultant precipitate was washed and then hydrothermally treated in an autoclave (stainless steel with a Teflon lining) at 100 °C for 16 h, giving an LDH suspension with the mass concentration of 4.0 mg/mL. Loading of 5-FU into the LDH was conducted via ion exchange prior to hydrothermal treatment, resulting in 5-FU/Mg2Al−LDH hybrid (5-FU/LDH). In this process, the resultant precipitate was collected and resuspended in 40 mL of basic solution containing 0.3 mmol of 5-FU (neutralized with dilute NaOH solution, with pH 8−9) with stirring for 1 h. After washing twice, the suspension was treated at 100 °C for 16 h. After hydrothermal treatment, a transparent, homogeneous 5-FU/LDH suspension was obtained.25,26 The loading amount of 5-FU drug was determined using UV−vis at 265 nm. 2.2.2. BSA Activation and Conjugation with Peptides. BSA solution (10 mg/mL) in conjugation buffer (phosphate-buffered saline: 100 mM sodium phosphate, 150 mM sodium chloride, 3 mM EDTA, pH 7.2) was prepared and added to a solution containing a 10fold molar excess of sulfo-SMCC cross-linker over the amount of BSA protein to link sufficient maleimide groups (step 1, Figure 1). Then sulfhydryl-containing Ang2/RVG peptides were conjugated with activated BSA. The reaction of maleimide groups and thiol groups proceeded rapidly and selectively under mild coupling conditions (pH 6.5−7.5) to yield a stable, covalently linked Ang2/RVG−SMCC−BSA conjugate (step 2, Figure 1). The resulting complex was purified by Millipore ultrafiltration tube with a molecular weight cutoff (MWCO) of 30 kDa to remove the free excess peptide and salts (Table 1).

Table 1. Quantification of BSA after Activation and Peptide Conjugation

BSA BSA−SMCC BSA−SMCC− Ang2 BSA−SMCC− RVG a

MW ± SD

no. of sulfoSMCCsa

no. of peptidesa

66 593 ± 80 70 182 ± 74 86 351 ± 361

8.2 8.2

6.7

91 073 ± 856

8.2

6.2

Sulfo-SMCC MW: 436. Ang2 MW: 2405. RVG MW: 3370.

Table 2. Average Particle Size and Zeta Potential of LDH and the Conjugates abbreviation LDH BSA−LDH BSA−LDH−GTA BSA−LDH−Ang2−GTA BSA−LDH−RVG−GTA 5-FU/LDHa 5-FU/BSA−LDH 5-FU/BSA−LDH−GTA 5-FU/BSA−LDH−Ang2−GTA 5-FU/BSA−LDH−RVG−GTA

NPs Ang2−NPs RVG−NPs 5-FU/NPs 5-FU/NPs 5-FU/Ang2−NPs 5-FU/RVG−NPs

Z-average particle size (d, nm)/PDI

ζ-potential (mV)

103.3/0.179 170.5/0.198 170.4/0.197 169.9/0.201 170.3/0.185 90.8/0.185 165.3/0.196 167.3/0.189 165.9/0.210 168.9/0.195

30.5 −22.9 −25.5 −18.4 −16.0 28.9 −22.1 −26.3 −17.9 −15.3

a

5-FU/LDH hybrids had 14.6 wt % of 5-FU, determined by the UV−vis absorbance at 265 nm. 5-FU/LDH NPs were also modified into 5-FU/ Ang2−NPs and 5-FU/RVG−NPs. 20446


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ACS Applied Materials & Interfaces U87 and N2a cells were seeded at a density of 1× 105 cells/well in 12-well plates (Corning Coaster) and incubated for 24 h. Then U87 and N2a cells were incubated with dsDNA−Cy3-labeled Ang2/RVG− NPs at the dsDNA−Cy3 concentration range of 20−120 nM for 2 h in 37 °C. Cells treated without any nanoparticles were used as a control. In order to confirm that the prepared LDH NPs were able to effectively transport nucleic acids into cells, the Cy3-labeled mimic dsDNA was used to replace siRNA due to high stability and low cost. Time-dependent cellular uptake was also tested by incubating cells for 0.5, 1, and 2 h at the concentration of 60 and 120 nM dsDNA−Cy3, respectively. After treatment, the culture medium was removed and the cells were washed three times with ice-cold PBS (pH 7.4) and harvested. The cellular uptake was quantified by flow cytometry analysis using a FL-2 log filter for collection of fluorescence intensity. 2.5. Cellular Uptake Mechanism of Ang2/RVG−NPs. U87 and N2a cells were seeded at a density of 1× 105 cells/well in 12-well plates (Corning Coaster) and incubated for 24 h. After checking the confluency and morphology, chlorpromazine hydrochloride (CPZ, 10 μg/mL), sucrose (0.45 M), nystatin (25 μg/mL), and Ang2/RVG (200 μg/mL) were added into each well and incubated for 20 min. Then the culture medium was withdrawn, and a medium containing dsDNA−Cy3-labeled Ang2/RVG−NPs was added. After 2 h incubation, the medium was discarded, and the cells were washed three times with ice-cold PBS. The cellular uptake was measured by flow cytometry analysis using FL-2 log filter for collection of fluorescence intensity. 2.6. Intracellular Tracking of LDH NPs and Ang2/RVG−LDH NPs. U87 and N2a cells were seeded onto coverslips in 6-well plates. After 24 h, cells were incubated in the medium with dsDNA−Cy3labeled Ang2/RVG−NPs and NPs for 4 h at 37 °C. Cells treated without any nanoparticles were used as a control. Cells were washed twice with PBS, fixed, and moved to the glass slides containing DAPI mounting medium. Confocal images were obtained using a Zeiss LSM 710 confocal microscope equipped with an inverted microscope. For U87 and N2a cells, using Z-stacks of typically 0.3 μm, 30−40 slices were imaged, each slice being the average of four laser scans. An Axio Imager Azure microscope was also used to take images. 2.7. In Vitro Cytotoxicity and Antiproliferative Activity against U87 and N2a Cells. U87 and N2a cells were seeded in a 96-well at a density of 3000 cells/well in growth medium (RPMI and DMEM medium respectively, containing 10% fetal calf serum, 100 U/ mL penicillin, and 100 μg/mL streptomycin) and incubated at 37 °C in a humidified atmosphere with 5% CO2. Twenty-four hours after seeding, the cells were treated with growth medium containing LDH NPs at varied concentrations, and their cytotoxicity was assessed by the MTT (Sigma) assay. Absorbance was measured at 490 nm on a SpectraMax M5 microplate reader, and cell viability was calculated as (absorbance in the treatment well)/(absorbance in the control well) × 100%. To assess proliferative capacity, U87 and N2a cells were seeded into 96-well plates at a density of 3000 cells/well and cultured at 37 °C for 24 h, incubated with 5-FU/NPs and 5-FU/Ang2−NPs or 5-FU/ RVG−NPs at the 5-FU concentration of 0−20 μg/mL for 72 h, and the culture medium was used as a control. The MTT (Sigma) assay was conducted to evaluate the cell viability. Concentrations of 5-FU showing 50% reduction in cell viability (i.e., IC50) were calculated. 2.8. Hemolysis Assay. All studies were in accordance with guidelines of the Animal Ethics Committee of The University of Queensland (UQ), and the Australian Code for the Care and Use of Animals for Scientific Purposes. Fresh ethylenediaminetetraacetic acid (EDTA)-stabilized blood samples were collected from C57BL/6 male mice in the AIBN animal facility, University of Queensland Biological Resources. Whole blood was diluted in PBS (1 mL blood in 10 mL PBS), and the packed red blood cells (RBCs) were isolated via centrifugation at 1600 rpm for 5 min and further washed more than five times with sterile isotonic PBS (until no red color was seen in supernatant). Then, 200 μL of packed RBCs were diluted into 4 mL of PBS. The diluted RBC suspension (0.2 mL) was then mixed with NPs, Ang2−NPs, or RVG−NPs PBS solution (0.8 mL) at various concentrations. PBS and water (0.8 mL) were used instead of LDH

nanoparticle solution as negative and positive control, respectively. The mixture was gently shaken and incubated at room temperature for 2 h, followed by centrifugation at 1600 rpm for 5 min. The supernatant absorbance at 541 nm (Ab) was measured by a multifunctional microplate reader (infinite M200, Tecan). The percentage of RBC hemolysis was calculated using the following formula:

hemolysis percentage =

(Abssample − Absnegative control ) (Abs positive control − Absnegative control )

× 100%

2.9. Statistics. The data are presented as the mean ± SD (t test), and one-way or two-way ANOVA with Bonferroni’s posthoc test was used to assess statistical significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

3. RESULTS AND DISCUSSION 3.1. Targeting Peptide Conjugation to BSA. As described in Figure 1A, the first reaction step in our strategy was to conjugate a heterobifunctional cross-linker sulfo-SMCC to activate BSA, where the amine group of BSA reacted with Nhydroxysuccinimide (NHS) ester to form the amide bond (step 1). In step 2, the thiol group of the targeting peptide was reacted with the maleimide group in activated BSA, thus conjugating the targeting peptide with BSA (BSA−Ang2 or BSA−RVG).27,28 MALDI-TOF is a powerful analytical technique for the analysis of biomolecules (such as DNA, proteins, peptides, and sugars) and large organic molecules (such as polymers, dendrimers, and other macromolecules)29−31 by providing accurate quantification of molecular weight.32 As shown in Figure 1B and listed in Table 1, the pristine BSA had a molecular weight of 66 593 Da, similar to the reported value.33 After activation by the linker sulfo-SMCC, the obtained complex BSA−SMCC had a molecular weight of 70 182 Da, which indicates that approximately 8.2 sulfo-SMCC were conjugated to each BSA molecule. The peptide-conjugated BSA, BSA−Ang2 and BSA−RVG, had a molecular weight of 86 351 and 91 073 Da, respectively. Calculation indicates that there were around 6.7 Ang2 and 6.2 RVG peptides conjugated to each BSA molecule. In addition, the Ellman’s method was also used to semiquantitatively determine whether the targeting peptide was successfully conjugated to activated BSA.34 As shown in Table S1 of the Supporting Information (SI), the absorbance value at 412 nm decreased after the reaction, indicating that the sulfydryl groups in the peptides reacted with the maleimide groups of the activated BSA. Thus, both peptides were successfully conjugated to BSA. 3.2. Preparation and Characterization of LigandConjugated LDH Nanoparticles. Following our previously published method, the mixture of pristine BSA and BSA−Ang2/RVG was prepared at a molar ratio of 19:1 (BSA:BSA−Ang2/RVG = 19:1) and then coated onto the surface of LDHs through electrostatic interaction. Subsequent cross-linking by GTA (steps 3 and 4, Figure 1) led to ligandconjugated LDH−BSA nanocomplexes with both colloidal stability and targeting capability.15,16,35,36 Similarly, Ang2 or RVG peptides were conjugated to the LDH surface through coating with BSA molecules. The nanocomplexes BSA−Ang2/ RVG−LDH (i.e., Ang2−NPs or RVG−NPs; Table 2) were used to test the efficacy of brain tumor cell targeting. Table 2 shows the average particle size and the ζ-potential of these LDH nanocarriers. The particle size was increased from 20447


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ACS Applied Materials & Interfaces the original 103 nm to approximately 170 nm, whereas the ζpotential changed from 30.5 to −22.9 mV after BSA coating, which is in consistence with our previous study.15,16 After GTA cross-linking, the size remained the same, while the ζ-potential was slightly more negative due to the reaction of amino groups during cross-linking. Clearly, the particle size distribution fell within a moderately narrow range in the cases, with the polydispersity index (PDI) being close to 0.20. Conjugating Ang2 or RVG peptide to BSA−LDH did not affect the average size and the size distribution. Meanwhile, the ζ-potential slightly changed from −22.9 to −18.4 mV (Ang2 peptide) and −16.0 mV (RVG peptide) after conjugation, probably because the side chain of both peptides carries two net positive charges (two negative charges and four positive charges). Altogether, these data suggest that BSA−Ang2 or BSA−RVG was successfully coated onto LDH NPs.37 Above we have developed a new and facile method to synthesize colloidally stabilized tumor-targeted LDH systems. The preconjugation of BSA with the desired ligand offers a simple and amenable procedure and avoids the complicated postparticle modification often applied to LDHs. This significantly differs from previous methods that involve serial chemical reactions in organic solvents.12,38 Thus, BSA coating onto LDH does not only retain the colloidal stability but also provides a platform for ready conjugation of the other modalities, to make the colloidally stable LDH a targeting delivery nanoplatform.12,15,16 Compared with pristine LDHs, the average size of 5-FUincorporated LDHs (5-FU/LDH) was decreased to 90.8 nm (Table 2), which can be attributed to the inhibition of incorporation of the anionic organic drug 5-FU to the hydroxide layer lateral growth, as previously suggested.39 The 5-FU concentration was determined to be 0.58 mg/mL (5-FU) in 4.0 mg/mL (LDH) suspension, where the 5-FU loading in LDHs was 14.6 wt %, as further confirmed by element analysis (Table S2, SI). As shown in Figure S2A (SI), the XRD pattern of LDH is typical of the layered structure, characteristic with diffractions from planes (003) and (006), identical to the previous report.40 In comparison, the (003) and (006) reflections of 5-FU/LDH became weaker and broader, suggesting the reduced crystallinity of the LDH phase due to intercalation of 5-FU. However, 5-FU intercalation did not lead to a new phase (with the interlayer d-spacing of 0.83 nm),26 so 5-FU molecules are possibly irregularly intercalated as well as adsorbed on the surface. The FTIR spectrum of LDHs (Figure S2B, SI) shows typical peaks, as previously reported,41 while the additional peaks (1672 and 1540 cm−1) in 5-FU/LDH indicate the successful loading of 5-FU by LDHs.26 Note that coating 5-FU/LDH with BSA or BSA−Ang2/RVG and subsequent cross-linking led to a size distribution, average particle size, and ζ-potential similar to those of corresponding LDH samples (Table 2). 3.3. Improved Uptake of Ang2/RVG−NPs by U87 and N2a Cells. In this study, Cy3-labeled dsDNA−LDH NPs were used to determine concentration- and time-dependent cellular uptake kinetics. Ang2 and RVG peptides were conjugated to Cy3−dsDNA−LDH NPs for targeting U87 and N2a cells. As shown in Figure 2A,C, the U87 cell uptake of Ang2−NPs increased with the dose and incubation time, compared to ligand-unconjugated NPs. In particular, the U87 cell uptake efficiency of Ang2−NPs was significantly higher at the dsDNA−Cy3 concentrations of 40 and 60 nM with the incubation time of 2 h (Figure 2A). Similarly, the N2a cellular

Figure 2. Cellular uptake of (A) Ang2−NPs/NPs by U87 cells and (B) RVG−NPs/NPs by N2a cells at the dsDNA−Cy3 concentration range of 20−120 nM for 2 h. Cellular uptake of (C, E) Ang2/RVG− NPs/NPs (60 nM dsDNA−Cy3) by U87 cells and (D, F) Ang2/ RVG−NPs/NPs (120 nM dsDNA−Cy3) by N2a cells for 0.5−2 h.

uptake of Cy3-labeled RVG−NPs also demonstrated a timeand concentration-dependent mode (Figure 2B,2D). In particular, N2a cellular uptake of Cy3-labeled RVG−NPs was significantly higher than that of Cy3-labeled NPs at the dsDNA−Cy3 concentrations of 60 and 120 nM, increasing by 41% and 36%, respectively, after 2 h incubation (Figure 2B). To further confirm whether the enhanced uptake was induced by ligand−receptor-mediated endocytosis rather than the change in surface properties, we incubated U87 cells with RVG−LDHs and N2a cells with Ang2−LDHs. Figure 2E,F shows that there was no enhancement of uptake. Thus, we conclude that there is no effect of changes of surface properties, as U87 cells do not have the receptors for RVG and N2a cells lack the receptors for Ang2. These results are in agreement with previous reports that target ligands (Ang2 and RVG) facilitate NP receptor-mediated uptake by the target cells.42 In a similar study using an Ang2conjugated poly(ethylene glycol)-co-poly(caprolactone) (Ang2−PEG−PCL) drug delivery system for U87 cells, the cellular uptake of rhodamine isothiocyanate (RBITC)-labeled Ang2−PEG−PCLs exhibited a time-dependent mode and was significantly higher than RBITC-labeled PEG−PCLs when the incubation time was from 30 to 120 min.43 Therefore, conjugation with Ang2 or RVG endows LDH NPs with enhanced cellular uptake efficiency. 3.4. Intracellular Localization of NPs and Ang2/RVG− NPs and Uptake Mechanism. The intracellular localization of Cy3-labeled Ang2/RVG−NPs and NPs in U87 and N2a cells was evaluated by confocal microscopy (Figure 3A,B) and 20448


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for clathrin-mediated [chlorpromazine (CPZ) and sucrose] or caveolin-mediated [nystatin (Nys)] endocytosis were selected. As shown in Figure 4A, both CPZ and sucrose were found to significantly inhibit the U87 cellular uptake of Ang2−NPs, decreasing by 53% and 62%, respectively. Incubation of U87 cells with Nys also significantly reduced the cellular uptake of Ang2−NPs, but to a smaller extent. These data suggest that clathrin-mediated endocytosis is the main contributor to internalization of Ang2−NPs by U87 cells, together with some contribution from caveolae-mediated endocytosis, as well as nonreceptor-mediated endocytosis. Similarly, Figure 4B shows that incubation of N2a cells with CPZ and sucrose effectively reduced the cellular uptake of RVG−NPs, indicating the involvement of clathrin-mediated endocytosis in the cellular uptake of RVG−NPs. However, nystatin was not found to significantly affect the cellular uptake of RVG−NPs by N2a cells. Thus, clathrin-mediated endocytosis may contribute to the majority of internalization of RVG− NPs by N2a cells. Our observations are similar to the reports that cellular uptake by both U87 and N2a cells is mostly dependent on clathrin-mediated endocytosis.45,18 LDH NPs are taken up by various mammalian and neural cells, mainly via clathrindependent endocytosis.8,40,46 The advantages of clathrindependent endocytosis over caveolin-mediated endocytosis using LDH as the vehicle include efficient cellular uptake and drug release in the cytoplasm (Figure 3) of tumor cells, leading to efficient proliferation inhibition.41,47,48 Therefore, the current ligand-modified strategy makes LDHs promising vehicles for cancer therapy. Furthermore, the effect of the free target ligands in inhibiting active endocytosis was further investigated. Preincubation of U87 or N2a cells with Ang2 or RVG peptide significantly reduced cellular uptake of Ang2−LDHs or RVG−LDHs, because the receptors were competitively prebound with free ligands. If this portion of the cellular uptake after preincubation with the free ligands is regarded as the nontargeting uptake, the peptide − receptor-mediated targeting uptake is probably 30%−40% of the total whole cellular uptake, which is comparable with previous reports43,49 and further supports the substantial contribution of cellular uptake by ligand− receptor-mediated endocytosis. 3.5. Enhanced Apoptosis of U87 and N2a Cells. Cytotoxicity and blood biocompatibility of various LDH NPs were further evaluated by MTT and hemolysis assays, respectively. In general, in all treatment groups (LDH concentrations of 10−500 μg/mL), the viability of U87 and N2a cells was greater than 80% after exposure to LDH formulations for 3 days (Figure 5A−D), demonstrating that the modified LDH NPs have good biocompatibility with U87 and N2a cells. Compared with the cytotoxicity reported previously for pristine LDHs, where cell viability was over 90% within 3 days incubation at the concentration range of 10−500 μg/ mL,50 the LDHs NPs used in this study were slightly toxic to U87 and N2a cells. The increased cytotoxicity is possibly due to the unreacted aldehyde groups of GTA, which is known to induce some toxicity.51 Overall, Ang2/RVG−NPs were demonstrated to have low cytotoxicity. The hemolysis and its percentage for groups BSA−LDHs, NPs, Ang2−NPs, and RVG−NPs are presented in Figure 5E,F, with distilled water and PBS as positive and negative control. As shown in Figure 5F, LDH NPs exhibited no hemolytic toxicity at concentrations up to 400 μg/mL, indicating that all LDH

Figure 3. Intracellular localization of Ang2−NPs/NPs within U87 cells incubated for 4 h (A) and RVG−NPs/NPs within N2a cells incubated for 4 h (B). The nuclei were stained with DAPI. The center merged confocal ortho-images of Z-stacks were X−Y view, the images above and right were X−Z and Y−Z views (cross section at the red and green lines).

fluorescence microscope (Figure S3A,B, SI). Both confocal and fluorescence images show that U87/N2a cells treated with Cy3labeled Ang2/RVG−NPs overall exhibited higher fluorescence intensity in comparison with nontargeting Cy3-labeled NPs, while cells without treatment show no red fluorescence within the cells. In particular, confocal ortho-images of Z-stacks illustrated that these NPs were mainly present in cytoplasm. The images indicate that Ang2/RVG-peptide-modified LDH NPs could efficiently deliver genes and facilitated the internalization process, leading to enhanced accumulation throughout the cytoplasm of U87 and N2a cells, with the localization seeming to be very similar for both target and nontarget LDH nanoparticles, mainly in the perinuclear cytoplasm, which is consistent with previous reports.44 In order to understand the internalization mechanism of Ang2/RVG−NPs, the effects of endocytosis inhibitors on cellular uptake kinetics were evaluated quantitatively. Inhibitors 20449


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Figure 4. Cellular uptake of NPs and Ang2/RVG−NPs by U87 cells (A) and N2a cells (B) in the presence of specific inhibitors. Cells were pretreated with various inhibitors for 20 min, followed by treating with Ang2/RVG−NPs for 2 h. Asterisks stand for the difference between individual groups and the Ang2−NPs group, respectively.

Figure 5. Cell viability of U87 (A and B) and N2a (C and D) treated with NPs (A and C), Ang2−NPs (B), and RVG−NPs (D) at 0−500 μg/mL. Experiments were carried out in duplicate and repeated three times. The data presented were the mean ± SD. Photographs (E) and hemolysis assay (F) of RBCs treated with functionalized LDH NPs at 0−800 μg/mL.

NPs are biocompatible with blood.52 In comparison with BSA− LDHs (11%), the other groups showed a slightly higher hemolytic activity at 800 μg/mL (14−15%). The higher hemolysis may be ascribed to aldehyde groups on the LDH surface and the higher negative charge.16 The hemolysis activity

of these LDH NPs was also confirmed by photoimage (Figure 5E). There was no hemoglobin released from damaged cells, and all groups exhibited almost colorless supernatants at all concentrations except for the positive control group. 20450


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Figure 6. Antiproliferation effect of different formulations at 5-FU concentrations from 0 to 20 μg/mL against U87 (A) and N2a (B) cells; n = 3. “*” and “#” stand for the difference between the 5-FU group and 5-FU/NPs or 5-FU/Ang2−NPs, respectively.

and subsequent ligand−receptor-mediated endocytosis. The targeting capacity has been clearly demonstrated by delivering 5-FU to inhibit the growth of U87 and N2a cells. Thus, the current research suggests that the functional BSA can be used to uniformly prepare large-scale target LDH NPs, and the current technology may pave the way for in vivo application of LDHs, particularly for brain tumor therapy.

Figure 6 shows the antiproliferative effect of 5-FU-loaded LDH NPs on U87 and N2a cells. The cell viability was decreased with the 5-FU dose for both U87 and N2a cells in free 5-FU or upon association with targeting/nontargeting LDH nanoparticles. In general, 5-FU/NPs were more efficient than free 5-FU in inhibiting cell growth (Figure 6 and Table 3),

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Table 3. Cytotoxic Activity (IC50) of Various 5-FU Compounds against U87 and N2a Cells

S Supporting Information *

cytotoxicity assay (IC50, μg/mL, mean ± SD)

free 5-FU 5-FU/NPs 5-FU/Ang2−NPs 5-FU/RVG−NPs

U87

N2a

>20 2.86 ± 0.3522 0.917 ± 0.0575

∼20 12.35 ± 2.054 7.25 ± 1.216

ASSOCIATED CONTENT

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b06421. Ellman’s method test of peptide conjugation; element composition of LDH and 5-FU/LDH samples; particle size distribution of LDH, NPs, Ang2−NPs, and RVG− NPs in water; XRD and FT-IR of LDH nanoparticles and 5-FU/LDH nanocomplex; fluorescence images of Ang2− NPs/NPs incubated with U87 cells and RVG−NPs/NPs incubated with N2a cells (PDF)

p (vs 5-FU/ NPs)

0.0007 0.0208

demonstrating that LDH NPs facilitate the cellular uptake of drugs. Moreover, 5-FU/Ang2/RVG−NPs showed no significant inhibition of cell growth compared with nontargeted 5FU/NPs at the same 5-FU concentrations. However, as listed in Table 3, the IC50 of 5-FU/NPs for U87 cells was 2.86 μg/ mL, which was significantly reduced to 0.917 μg/mL for 5-FU/ Ang2−NPs. Similarly, the IC50 for N2a cells was also significantly reduced from 12.35 μg/mL for 5-FU/NPs to 7.25 μg/mL for 5-FU/RVG−NPs. The IC50 value was reduced by 1.7−3.0-fold when 5-FU/LDH NPs were conjugated with the target ligand (Ang2 or RVG), demonstrating the contribution of target delivery to the anticancer therapy. Note that the amount of LDH NPs in all NP formulations was around 137 μg/mL at 20 μg/mL 5-FU, which does not cause any toxicity to cancer cells (Figure 5). Thus, the cell death in this study is induced solely by the 5-FU and is directly related to the 5-FU amount taken up by the cells.16 These observations are also consistent with previous reports that 5-FU inhibits the proliferation of U87 and N2a cells,53,54 which is enhanced using LDH NPs.55 This current research has clearly demonstrated that conjugation with a targeting ligand is able to further enhance the antiproliferation of brain cancer cells.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 61-7-33463809. Fax: 61-7-33463973. E-mail: gordonxu@ uq.edu.au. ORCID

Zhi Ping Xu: 0000-0001-6070-5035 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support of Australian Research Council (ARC) Future Fellowship (FT120100813). The Australian Government Research Training Program Scholarship (RTP) for financial support is also acknowledged. We are grateful for use of the facilities and for the scientific and technical assistance of the Australian National Fabrication Facility (ANFF) and the Australian Microscopy and Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland.

4. CONCLUSIONS In conclusion, our current strategy provides a facile way to conjugate targeting peptide ligands to the LDH surface with excellent colloidal stability and targeting capability. The Ang2/ RVG-conjugated LDH NPs enhanced cellular uptake by the target brain tumor cells, which is attributed to ligand targeting 20451


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