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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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 02 Jun 2017 Downloaded from http://pubs.acs.org on June 3, 2017

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ACS Applied Materials & Interfaces

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

Australian Institute for Bioengineering and Nanotechnology, The University of Queensland,

Brisbane, QLD 4072, Australia. b

The Queensland Brain Institute, The University of Queensland, Queensland 4072, Australia

*To whom correspondence should be addressed. Tel: 61-7-33463809. Fax: 61-7-33463973. Email: [email protected] (Prof Zhi Ping Xu)

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ABSTRACT Active targeting of nanoparticles (NPs) for the 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 tumour 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 tumour targeting ligands, i.e. Ang2 and RVG, were conjugated to the LDHs via an inter-matrix 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 crosslinker sulfo-SMCC. Then, the ligand-linked BSA and pristine BSA were together coated onto the surface of LDHs through electrostatic interaction, followed by crosslinking with crosslinker glutaraldehyde to immobilise these BSAs on the LDH surface. In this way, we are able to readily prepare colloidally-stabilised tumour-targeted LDH NPs. The targeting efficacy of 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 tumours.


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Keywords: Layered double hydroxide (LDH); target ligand; cell uptake mechanism; bovine serum albumin, brain tumour. 1. INTRODUCTION Colloidal nanoparticle (NP) drug delivery systems are believed to preferentially accumulate in the tumour area due to enhanced permeability and retention (EPR) effect.1 Rapid tumour 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 tumour through the EPR effect.2-3 However, the challenge is confounded by the fact that ubiquitously intercellular distribution of nanoparticles within the tumour tissue may fail the cancer treatment due to suboptimal cellular uptake efficacy.4 Therefore, achieving efficient and specific cell uptake is a priority for optimising 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 internalisation and improve therapeutic efficacy. Moreover, active targeting will reduce the distribution to healthy tissue, 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 dimensional lamellar compounds made up of positively charged layers with an interlayer region containing anions and water molecules. LDHs have a 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,



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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 zeta 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 addition, LDHs have good biocompatibility and pH-dependent dissolution property, which are beneficial for a smart drug delivery system.9 However, there are two challenges for LDHs’ in vivo applications: the colloidal instability of LDH NPs in the biological environment (i.e. aggregation) and surface functionalisation for active targeting, which are not well addressed until now. The stability of LDHs in biological environment is difficult to control. Upon exposure into the 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 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 stabilising agent 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 the 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, crosslinking of BSA by glutaraldehyde (GTA) allows the LDH-BSA nanocomplexes to be easily


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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 stabilise and functionalise LDH nanoparticles in order to actively target brain tumour 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 2a (N2a) cells, respectively.17-18 As schematically shown in Figure 1A, the targeting peptide (Ang2 or RVG) is firstly conjugated to BSA (Step 1 and 2). Then peptide-BSA is mixed with pristine BSA, and the mixture 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 5-fluorouracil (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 prolonging patient’s life and reducing the disease symptoms.22 Therefore, the objectives of this research were to: (1) develop a facile strategy to controlprepare BSA-coated LDH nanoparticles with Ang2/RVG ligand conjugated at a defined density, (2) ascertain that target ligands on the LDH surface enhance delivery to U87 and N2a cells, and (3) confirm that target ligand enhances the cytotoxicity of drug-LDH NPs to brain



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tumour cells. The current method for LDHs surface functionalisation may provide a feasible way to apply LDH NP for in vivo target delivery.

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) and other chemicals were from Sigma-Aldrich if not illustrated specifically. Water used in experiments was deionized Milli-Q water. Angiopep-2 (Ang2: TFFYGGSRGKRNNFKTEEYC) and Rabies virus glycoprotein (RVG: YTIWMPENPRPGTPCDIFTNSRGKRASNGC) peptide were purchased from Biomatik. Amicon ultra-0.5 centrifugal filter units were purchased from Millipore Company. All the other solvents were of analytical or chromatographic grade. 2.2. Synthesis of Peptide-Conjugated LDH NPs. Synthesis of LDH and 5-FU/LDH. Mg2Al-Cl-LDH (pristine LDH) was prepared using a co-precipitation-hydrothermal treatment method, as reported previously.23-24 In brief, LDHs were synthesised by mixing 40 ml NaOH solution (0.15 M) with 10 ml 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 oC 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 6 ACS Paragon Plus Environment

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5-FU (neutralised with dilute NaOH solution, with pH 8–9) under 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 5FU drug was determined using UV-Vis at 265 nm. 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 10-fold molar excess of sulfo-SMCC crosslinker over the amount of BSA protein to link sufficient maleimide groups (Step 1, Figure 1). And 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. Preparation of Ang2/RVG-LDH NPs. Two millilitres 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 crosslinked by glutaraldehyde overnight as reported previously.15-16 The resulted Ang2/RVG-conjugated LDH nanoparticles were named as Ang2/RVG-NPs (Table 1 and 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 similarly, 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-



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FU/Ang2-NPs,

5-FU/RVG-NPs,

dsDNA-Cy3/Ang2-NPs

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and

dsDNA-Cy3/RVG-NPs,

respectively, in comparison with non-targeting BSA-coated nanoparticles, NPs, 5-FU/NPs and dsDNA-Cy3/NPs (Table 1 and 2). 2.3. Characterisations. The hydrodynamic size and the surface charge (zeta potential) of LDH NPs, Ang2-NPs and RVG-NPs were characterised in a Nano Zetasizer instrument utilizing dynamic light scattering (DLS). To determine the ligand number in each BSA molecule, matrix assisted laser desorption ionisation - 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 volume (2 µl each) of sample solution and matrix solution II (Sinapinic Acid saturated in TA30) was pre-mixed 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’-dithio-bis-(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 ul tested sample was mixed with 50 µl of Ellman’s reagent solution and 2.625 ml of reaction buffer (0.1M 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


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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. 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 labelled Ang2/RVG-NPs at the dsDNA-Cy3 concentration range of 20-120 nM for 2 h at 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 labelled mimic dsDNA replaced 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 of 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 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), 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 labelled Ang2/RVG-NPs were 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 cells and N2a cells were seeded onto coverslips in 6-well plates. After 24 h, cells were incubated in the



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medium with dsDNA-Cy3-labeled 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, Z-stacks of typically 0.3 µm, 30-40 slices were imaged, each slice being the average of four laser scans. Microscopes Axio Imager Azure was also used to take images. 2.7. In Vitro Cytotoxicity and Anti-proliferative 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% foetal 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 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℃ for 24 h, incubated with 5-FU/NPs and 5FU/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 control. 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. Haemolysis Assay. All studies were in accordance with guidelines of the Animal Ethics Committee of The University of Queensland (UQ), and Australian Code for the Care and Use of Animals for Scientific Purposes. Fresh ethylenediamine tetraacetic acid (EDTA)stabilised blood samples were collected from C57BL/6 male mice in the AIBN animal facility,


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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 RBCs 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 nanoparticles 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: =

(Abs − Abs !" #$ %$ ) × 100 % (Abs$! !" #$ %$ − Abs !" #$ %$ )

2.9. Statistics. The data are presented as the mean ± SD. T-test, one-way or two-way ANOVA with Bonferroni’s post-hoc 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 crosslinker sulfo-SMCC to activate BSA where the amine group of BSA reacts with N-hydroxysuccinimide (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



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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 66593 D, similar to the reported value.33 After activation by the linker sulfo-SMCC, the obtained complex BSA-SMCC had a molecular weight of 70182 D which indicates approximately 8.2 sulfo-SMCC were conjugated to each BSA molecule. The peptideconjugated BSA, BSA-Ang2 and BSA-RVG, had a molecular weight of 86351 D and 91073 D, respectively. Calculation indicates that there were around 6.7 Ang2 and 6.2 RVG peptides conjugated to each BSA molecule.


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Figure 1. The strategy for constructing peptide-conjugated nanoparticles (A) and MALDITOF detection of BSA molecular weight after activation and peptide conjugation (B).

Table 1. Quantification of BSA after activation and peptide conjugation.

MW ± SD

Sulfo-SMCC*

Peptide*

number

number

BSA

66593 ± 80

-

-

BSA-SMCC

70182 ± 74

8.2

-

BSA-SMCC-Ang2

86351 ± 361

8.2

6.7

BSA-SMCC-RVG

91073 ± 856

8.2

6.2

* Sulfo-SMCC MW: 436; Ang2 MW: 2405; RVG MW: 3370. In addition, the Ellman’s method was also used to semi-quantitatively determine whether the targeting peptide was successfully conjugated to activated BSA.34 As shown in Table S1, the absorbance value at 412 nm decreased after the reaction, indicating that the sulfydrylgroups in the peptides reacted with maleimide groups of the activated BSA. Thus both peptides were successfully conjugated to BSA.

3.2. Preparation and Characterisation of Ligand-Conjugated LDH Nanoparticles. Following our previously published method, the mixture of BSA-Ang2/RVG and BSA 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 crosslinking by GTA (Step 3



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and 4, Figure 1) led to ligand conjugated 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 BSAAng2/RVG-LDH (i.e. Ang2-NPs or RVG-NPs; Table 2) were used to test efficacy of brain tumour cell targeting. Table 2 shows the average particle size and the zeta potential of these LDH nano-carriers. The particle size was increased from the original 103 nm to approximately 170 nm, whereas the zeta potential changed from 30.5 to -22.9 mV after BSA coating, which is in consistence with our previous study.15-16 After GTA crosslinking, the size remained the same while the zeta potential was slightly more negative due to the reaction of amino groups during crosslinking. Clearly, the particle size distribution fell within a moderately narrow range in the three cases, with the polydispersity index (PDI) close to 0.20. Conjugating Ang2 or RVG peptide to BSA-LDH did not affect the average size and the size distribution. Meanwhile, the zeta potential slightly changed from -22.9 mV to -18.4 mV (Ang2 peptide) and -16.0 mV (RVG peptide) after conjugation, probably because the side chain of both peptides carries 2 net positive charges (2 negative charges and 4 positive charges). Altogether, these data suggest that BSA-Ang2 or BSA-RVG were successfully coated onto LDH NPs.37 Table 2. Average particle size and zeta potential of LDH and the conjugates.

Abbreviation

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

Zeta potential (mV)

LDH

--

103.3/0.179

30.5

BSA-LDH

--

170.5/0.198

-22.9

BSA-LDH-GTA

NPs

170.4/0.197

-25.5

BSA-LDH-Ang2-

Ang2-NPs

169.9/0.201

-18.4 14

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GTA BSA-LDH-RVGGTA

RVG-NPs

170.3/0.185

-16.0

5-FU/LDH*

5-FU/NPs

90.8/0.185

28.9

5-FU/BSA-LDH

--

165.3/0.196

-22.1

5-FU/BSA-LDHGTA

5-FU/NPs

167.3/0.189

-26.3

5-FU/BSA-LDHAng2-GTA

5-FU/Ang2-NPs

165.9/0.210

-17.9

5-FU/BSA-LDHRVG-GTA

5-FU/RVG-NPs

168.9/0.195

-15.3

* 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.

Above we have developed a new and facile method to synthesise colloidally stabilised tumour-targeted LDH systems. The pre-conjugation of BSA with the desired ligand offers a simple and amenable procedure, and avoids the complicated post-particle 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-FU incorporated LDHs (5-FU/LDH) was decreased to 90.8 nm (Table 2), which can be attributed to the inhibition of incorporation of 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) 15 ACS Paragon Plus Environment


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suspension where the 5-FU loading in LDHs was 14.6 wt%, as further confirmed by element analysis (Table S2). As shown in Figure S2A, 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 5FU. 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. FTIR spectrum of LDHs (Figure S2B) shows typical peaks as previously reported,41 while the additional peaks (1672 cm-1 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 crosslinking led to the similar size distribution, the average particle size and the zeta potential to corresponding LDH samples (Table 2).

3.3. Improved Uptake of Ang2/ RVG-Nps By U87 and N2a Cells. In this study, Cy3labeled 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 and 2C, 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 dsDNACy3 concentration of 40 and 60 nM with the incubation time of 2 h (Figure 2A). Similarly, the N2a cellular uptake of Cy3-labeled RVG-NPs also demonstrated a time- and concentrationdependent mode (Figure 2B and 2D). In particular, N2a cellular uptake of Cy3-labeled RVGNPs was significantly higher than Cy3-labeled NPs at the dsDNA-cy3 concentration of 60 and 120 nM, increasing by 41% and 36%, respectively, after 2 h incubation (Figure 2B).


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Figure 2. (A) and (B) Cellular uptake of Ang2-NPs/NPs by U87 cells and RVG-NPs/NPs by N2a cells at the dsDNA-Cy3 concentration range of 20-120 nM for 2 h. (C, E) and (D, F) Cellular uptake of Ang2/RVG-NPs/NPs (60 nM dsDNA-Cy3) by U87 cells and Ang2/RVGNPs/NPs (120 nM dsDNA-Cy3) by N2a cells for 0.5-2 h. 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 RVGLDHs and N2a cells with Ang2-LDHs. Figure 2E and 2F show that there was no enhancement of uptake. Thus we conclude that there is no effect of charge 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 Ang2-



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conjugated poly(ethylene glycol)-co-poly(caprolactone) (Ang2-PEG-PCLs) drug delivery system for U87 cells, the cellular uptake of rhodamine isothiocyanate (RBITC)-labelled Ang2PEG-PCLs exhibited a time-dependent mode and was significantly higher than RBITClabelled 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 Localisation of NPs and Ang2/RVG-NPs and Uptake Mechanism. The intracellular localisation of Cy3-labelled Ang2/RVG-NPs and NPs in U87 and N2a cells was evaluated by confocal microscopy (Figure 3A and 3B) and fluorescence microscope (Figure S3A and S3B). Both confocal and fluorescence images show that U87/N2a cells treated with Cy3-labelled Ang2/RVG-NPs overall exhibited higher fluorescence intensity in comparison with non-targeting 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 internalisation process, leading to enhanced accumulation throughout the cytoplasm of U87 and N2a cells, with the localisation seems to be very similar for both target and non-target LDH nanoparticles, mainly in the perinuclear cytoplasm, which is in consistence with previous reports.44


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Figure 3. Intracellular localisation 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.



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The centre 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 line).

In order to understand the internalisation mechanism of Ang2/RVG-NPs, the effects of endocytosis inhibitors on cellular uptake kinetics were evaluated quantitatively. Inhibitors 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 internalisation of Ang2-NPs by U87 cells, together with some contribution from caveolae-mediated endocytosis as well as non-receptor mediated endocytosis.

Figure 4. Cellular uptake of NPs and Ang2/RVG-NPs by U87 cells (A) and N2a cells (B) in presence of specific inhibitors. Cells were pretreated with various inhibitors for 20 min,


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followed by treating with Ang2/RVG-NPs for 2 h. Stars stand for the difference between individual groups and Ang2-NPs group respectively. 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 internalisation 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 clathrin-dependent endocytosis.8, 40, 46 The advantages of clathrin-dependent endocytosis over caveolin-mediated endocytosis using LDH as the vehicle include efficient cellular uptake and drug release in the cytoplasm (Figure 3) of tumour 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. Pre-incubation 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 pre-bound with free ligands. If this portion of the cellular uptake after preincubation with the free ligands is regarded as the non-targeting uptake, the peptide-receptor mediated targeting uptake is probably 30%-40% of 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 assay, respectively. In general, in all treatment groups (LDH concentrations of 10-500 µg/ml), the viability of U87



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and N2a cells was greater than 80% after exposure to LDH formulations for 3 days (Figure 5A-5D), demonstrating that the modified LDH NPs have good biocompatibility with U87 and N2a cells. Compared with pristine LDH’s cytotoxicity reported previously where cell viability were over than 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.

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 to 500 µg/ml. Experiments were carried out duplicate


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and repeated three times. The data presented were the mean ± SD. Photographs (E) and haemolysis assay (F) of RBCs treated with functionalised LDH NPs at 0 to 800 µg/ml.

The hemolysis and its percentage for groups of BSA-LDHs, NPs, Ang2-NPs and RVG-NPs are presented in Figure 5E and 5F, 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 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 microscopy (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.

Figure 6. Anti-proliferation 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 5-FU group and 5-FU/NPs or 5-FU/Ang2-NPs, respectively.



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Figure 6 shows the anti-proliferative 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 association with targeting/non-targeting LDH nanoparticles. In general, 5-FU/NPs were more efficient than free 5-FU in inhibiting cell growth (Figure 6 and Table 3), demonstrating that LDH NPs facilitate the cellular uptake of drugs. Moreover, 5-FU/Ang2/RVG-NPs showed no inhibition of cell growth compared with non-targeted 5-FU/NPs at the same 5-FU concentrations. However, as listed in Table 3, 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, 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 folds 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 directly related to the 5-FU amount taken up by the cells.16 Table 3. Cytotoxic activity (IC50) of various 5-FU compounds against U87 and N2a cells. Cytotoxicity assay (IC50, µg/ml, Mean ± SD) U87

N2a

P (vs. 5-FU/NPs)

Free 5-FU

>20

∼20

--

5-FU/NPs

2.86 ± 0.3522

12.35 ± 2.054

--

5-FU/Ang2-NPs

0.917 ± 0.0575

--

0.0007

5-FU/RVG-NPs

--

7.25 ± 1.216

0.0208


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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 anti-proliferation of brain cancer cells. 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 tumour cells, which is attributed to ligand targeting 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 tumour therapy.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. 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 Ang2NPs/NPs incubated with U87 cells and RVG-NPs/NPs incubated with N2a cells.

AUTHOR INFORMATION Corresponding Author *Zhi Ping Xu. E-mail: [email protected]. Tel: 61-7-33463809. Fax: 61-7-33463973. 25 ACS Paragon Plus Environment


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ORCID Zhi Ping Xu: 0000-0001-6070 5035 Huali Zuo: 0000-0002-7308-5434 Weiyu Chen: 0000-0003-4436-2743 Helen M. Cooper: 0000-0003-4590-9384 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.

ACKNOWLEDGEMENTS 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 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.

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