Systematic Evaluation of Transferrin-Modified Porous Silicon

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Biological and Medical Applications of Materials and Interfaces

Systematic Evaluation of Transferrin-Modified Porous Silicon Nanoparticles for Targeted-Delivery of Doxorubicin to Glioblastoma Meihua Luo, Guido Lewik, Julian Charles Ratcliffe, Chung Hang Jonathan Choi, Ermei Mäkilä, Wing Yin Tong, and Nicolas H. Voelcker ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10787 • Publication Date (Web): 21 Aug 2019 Downloaded from pubs.acs.org on August 22, 2019

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ACS Applied Materials & Interfaces

Systematic Evaluation of Transferrin-Modified Porous Silicon Nanoparticles for Targeted-Delivery of Doxorubicin to Glioblastoma Meihua Luoa,b, Guido Lewika,c, Julian Charles Ratcliffed, Chung Hang Jonathan Choib, Ermei Mäkiläe, Wing Yin Tonga,d*, Nicolas H. Voelckera,b,d,f,g* aMonash

Institute of Pharmaceutics Science, Monash University, Parkville Campus, 381 Royal Parade,

Parkville VIC 3052, Australia bDepartment

of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, New Territories,

Hong Kong cFaculty

of Medicine, Ruhr-University Bochum, Bochum 44801, Germany

dCommonwealth

Scientific and Industrial Research Organization (CSIRO), Clayton, Victoria 3168,

Australia eIndustrial

Physics Laboratory, Department of Physics and Astronomy, University of Turku, Turku 20014,

Finland fMelbourne

Centre for Nanofabrication, Victorian Node of the Australian National Fabrication Facility, 151

Wellington Road, Clayton VIC 3168, Australia gMaterials

Science and Engineering, Monash University, 14 Alliance Lane, Clayton Victoria 3800, Australia

* Correspondence: [email protected], [email protected]

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Abstract There is a dire need to develop more effective therapeutics to combat brain cancer such as glioblastoma multiforme (GBM). An ideal treatment is expected to targeted deliver chemotherapeutics to glioma cells across the blood-brain barrier (BBB). The overexpression of transferrin receptor (TfR) on the BBB and the GBM cell surfaces but not on the surrounding cells renders TfR a promising target. While porous silicon nanoparticles (pSiNPs) have been intensely studied as a delivery vehicle due to their high biocompatibility, degradability, and drug loading capacity, the potential to target deliver drug with transferrin (Tf) functionalized pSiNPs remains unaddressed. Here, we developed and systematically evaluated Tffunctionalized pSiNPs (Tf@pSiNPs) as a glioma-targeted drug delivery system. These nanoparticles showed excellent colloidal stability and had a low toxicity profile. As compared to non-targeted pSiNPs, Tf@pSiNPs were selective to BBB-forming cells and GBM cells, and were efficiently internalized through clathrin receptor-mediated endocytosis. The anticancer drug doxorubicin (Dox) was effectively loaded (8.8 weight%) and released from Tf@pSiNPs in a pH-responsive manner over 24 hours. Furthermore, the results demonstrate that Dox delivered by Tf@pSiNPs induced significantly enhanced cytotoxicity to GBM cells across an in vitro BBB monolayer compared to free Dox. Overall, Tf@pSiNPs offer a potential toolbox for enabling targeted therapy to treat GBM. Key words: transferrin; porous silicon particles; blood-brain barrier; glioblastoma; targeted delivery. 1. Introduction Glioblastoma multiforme (GBM) is the most common and one of the deadliest malignant adult brain cancers.1 The current treatments for GBM includes surgical resection followed by radiotherapy and chemotherapy.2 Despite these aggressive treatments, the survival rate of patients with GBM remains relatively low over the past decades.3 Improvement of therapies 2 ACS Paragon Plus Environment

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against GBM is hampered by limited permeability across the blood-brain barrier (BBB) and inadequate accumulation of chemotherapeutics in brain tumor cells.4 Hence, development of targeted delivery carriers that are able to carry a sufficient amount of drug specifically to GBM cells across the BBB has the potential to increase the therapeutic efficiency of chemotherapeutics for GBM treatment. Porous silicon nanoparticles (pSiNPs) are promising drug delivery vehicles owing to their high surface area (up to 1125 m2/g) and porosity ( ≈ 50-80%) biodegradability and biocompatibility.5-6 By varying the pore dimensions, and optimizing the surface chemistry, pSiNPs provide remarkable loading capacity for a variety of small therapeutic molecules,7 such as chemotherapeutics,8 siRNA,9 oligos,10 antibodies,11 peptides,12 and contrast agents.13 Furthermore, the versatile surface chemistry of pSiNPs enables modifications with various targeting ligands on the surface of nanoparticles, hence offering controllable degradation, drug release profile, pharmacokinetics, cellular uptake, and specificity.14 However, the concept of applying pSiNPs to target deliver chemotherapeutics to brain tumors remain largely unexplored. One potential strategy is to couple pSiNPs with a ligand which can recognize specific receptors on brain tumor cells. GBM cell surface markers, such as EGFR,15 EphA,16 transferrin receptor (TfR),17 and other molecules that facilitate BBB penetration, such as cationic BSA,18 apolipoprotein E,19 have been studied to facilitate drug delivery. Among those, TfR has been identified as one of the most promising targets since it is expressed on both BBB and GBM cells. Tf, a hydrophilic transporter of iron ions in the blood, has been known to enter cells via TfR-mediated endocytosis.20 Indeed, many types of cancerous cells, including GBM, overexpress TfR, whilst TfR is less expressed on non-cancerous cells 21. Thereby, Tf has been widely explored as a targeting ligand for improving nanocarrier penetration into the BBB, such as micelles,22 liposomes,21 and polymersomes.23 Coating Tf on pSiNPs via hydrophobic interactions was first described by Reuter et al. in 2017.24 However, since the size, shape, and



stiffness of pSiNPs are largely different from other nanovectors, further application of Tfdecorated pSiNPs requires systematic characterization of their stability, cellular uptake specificity, efficiency, cellular internalization pathways, and the drug delivery capacity. In this study, we developed and systematically evaluated Tf-coupled pSiNPs as a targeted nanocarrier to deliver small molecular chemotherapeutics into the brain cancer cells. We hypothesized that these nanoparticles could selectively bind to TfR-overexpressing tumor cells, and deliver drug across the BBB to brain cancer cells. We first investigated the cellular uptake of nanoparticles to study their internalization selectivity and mechanisms in brain glioma cells and human BBB-forming cells. To determine the drug delivery efficiency, we further examined the cytotoxicity of anticancer drug-loaded nanoparticles on brain glioma cells in the absence and presence of the in vitro BBB. Overall, our data highlights the potential of pSiNPs conjugated with Tf as a new valuable approach to realize targeted delivery for GBM treatment. 2. Methods and Materials

2.1 Materials Human holo-transferrin (T4132), bovine serum albumin (514-0063), undecylenic acid (124672), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride(03459), sulfo-NHS(N-hydroxysulfosuccinimide) (56485), MES hydrate (M8250), chlorpromazine (C8238), nystatin (cat.no. N4014), nocodazole (cat.no. M1404), collagen type I solution (C3867) and all other nonmentioned chemicals were obtained from Sigma-Aldrich. Doxorubicin hydrochloride (15007) was purchased from Sapphire Bioscience. CellTiter-Glo luminescent cell viability assay (G7570) and VivoGlo Liciferrin (P1041) were purchased from Promega. Anti-transferrin receptor antibody (ab8598) was obtained from Abcam. Anti-beta actin antibody (sc-47778) was purchased from Santa Cruz Biotechnology. HRP-conjugated secondary antibody Immun-Star Goat Anti-Mouse HRP (1705047) for Western blotting experiments was purchased from Biorad. Fluorophore-conjugated secondary antibody Goat4 ACS Paragon Plus Environment

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Anti Mouse Alexa 647 (A-21237), CellLight Early Endosome-RFP (C10587) were purchased from Thermo Fisher Scientific. The 10 kDa cellulose snake skin dialysis tubing (68100) was obtained from Thermo Fisher. hCMEC/D3 cells were purchased from MilliPore (SCC066), HaCaT cells (PCS-200-201) were from ATCC, and U87 cells were a kind gift from Bakhos A. Tannous, MGH, Massachusetts. 2.2 Fabrication and functionalization of pSiNPs The pSiNPs were fabricated by using electrochemical anodization of a p-type boron doped Si wafer as previously described.25 Briefly, monocrystalline boron doped p+ Si wafers with a resistivity of 0.01-0.02 Ω ·cm were electrochemically anodized in a mixture of 38% hydrofluoric acid (HF) and ethanol at a 1:1(v/v) ratio, pulsed with alternating low and high current density etching profiles to fracture the surface of pSi layer at different intervals. Next, the etching current was increased to the electropolishing region to lift off the obtained multilayer film from the substrate. The films were dried and placed under N2 flow for 30 min at room temperature to eliminate oxygen and residual moisture. Then, at room temperature, acetylene (C2H2) flow (1 L/min) was added to the N2 flow for 15 min before increasing the temperature to 500oC for 15 min under the 1:1(v/v) N2/C2H2 flow. The obtained thermally hydrocarbonized pSi (THCpSi) films were then allowed to cool down to room temperature under N2 flow. The films were then immersed into neat undecylenic acid and placed at 120oC for 16 h. After this, the COOH-functionalized films were milled down into nanoparticles by ball milling in a 10 % (v/v) undecylenic acid-decane mixture. The obtained particles were washed with ethanol to remove the milling media. The size selection of the nanoparticles was done by centrifugation in ethanol and the final UA-functionalized particles (UnpSiNPs) were stored in ethanol at 4oC for further use.



2.3 Preparation of covalently conjugated Tf nanoparticles (Tf@pSiNPs) A two-step EDC/NHS reaction was employed to conjugate Tf to UnpSiNPs. Briefly, 5 mg of UnpSiNPs dispersed in ethanol were centrifuged (20,000 rcf, 15 min) and resuspended into 0.1 M MES buffer (pH=6). EDC and sulfo-NHS were directly added to UnpSiNPs with final concentration of 2.6 mM and 5 mM, respectively. The reaction components were mixed well and allowed to react at room temperature (RT) for 15 min. After the NHS ester activation, MES buffer was replaced by PBS buffer (pH =7.4). Then the suspension was added dropwise to Tf solution in PBS buffer (Tf concentration 10 mg/mL). The mixture was agitated for 2 h at RT. 50 mM Tris was added to quench the reaction for 15 min. Tf@pSiNPs were washed with PBS three times to remove the free Tf by centrifugation and stored at 4oC for further use. The supernatant after the centrifugation was collected for Tf quantitation. 2.4 Fourier transform infrared spectroscopy (IR) The chemical bonding formed between Tf and pSiNPs were evaluated via attenuated total reflectance infrared (ATR-IR) spectroscopy (PerkinElmer, USA). Samples were first rinsed free of salt with MilliQ, and resuspended into EtOH. The suspension was then deposited and dried onto the diamond crystal. The ATR spectra were then acquired over 24 scans with a frequency range of 650 to 4000 cm-1. 2.5 Size and zeta potential To study the hydrodynamic size and zeta-potential of nanoparticles, the particles were first washed and resuspended into PBS buffer and sonicated for 2 min. The particles were then analyzed using a Zetasizer Nano ZS (Malvern, UK). The analysis was carried out at the scattering angle θ=173˚ at a temperature of 25oC. Each measurement was an average of thirteen repetitions and repeated three times.


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2.6 Transmission electron microscopy (TEM) The dimensions and architecture of pSiNPs with or without functionalization were studied using a Tecnai T12 Transmission Electron Microscope (FEI, Eindhoven, The Netherlands) equipped with a tungsten filament operating at 120 kV. Images were recorded using an FEI Eagle 4k × 4k CCD camera. Briefly, NPs dispersed in ethanol at 1 mg/mL were dropped onto 300-mesh copper grids (ProSciTech, Qld, Australia) coated with thin carbon film made using an HHV BT150 (Ezzi Vision, Australia). Excess fluid was drawn off. Next, the sample was allowed to dry at RT before visualization under TEM. Particle size was measured by counting 50 measurement of randomly selected particles using ImageJ. The pore diameter was determined by measuring wall-to-wall distance of individual pores. For cryo-TEM experiments, the functionalized pSiNPs were diluted in PBS. The samples were pipetted onto 300-mesh copper grids coated with lacey carbon film (ProSciTech, Qld, Australia) then blotted with Whatman 541 filter paper and plunged into liquid ethane using a lab-built plunge freezer and examined using a Gatan 626 cryoholder (Gatan, Pleasanton, CA, USA). At all times low dose procedures were followed, using an electron dose of 8-10 electrons/Å2. Images were recorded using a Megaview III CCD camera and AnalySIS camera control software (Olympus). 2.7 Cell culture In this study we used U87 cells of human origin that stably express cytoplasmic mCherry, a kind gift from Bakhos A.Tannous, MGH, Massachusetts, USA. It is a well-recognized model cell line for GBM. This cell line was used because mCherry expressing GBM cells allow live cell tracking of cell body, and thus enable time-resolved study of cell-nanoparticle colocalization. U87 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, 11995073) supplemented with 10% fetal bovine serum (FBS, Life Technologies, 10437028) and 1% antibiotic-antimycotic (Life Technologies, 15240062) at 37oC and 5% CO2 in a



humidified incubator. All experiments were conducted by using cells that were passaged at least twice after thawing from the frozen stock. The immortalized human brain endothelial cell line (hCMEC/D3) was obtained from MilliPore (SCC066). The tight and polarized monolayers formed by hCMEC/D3 have low paracellular flux and expression of transporters, closely mimicking the in vivo condition. Thus, they are commonly used for in vitro BBB model.26 hCMEC/D3 was cultured in EBM-2 medium (Lonza, CC-3156) supplemented with 5% FBS, bFGF, hydrocortisone, ascorbic acid, CD-pipid, penicillin-streptomycin and HEPES. Cells were maintained at 37oC in 5% CO2. Only cells between Passage 10 and Passage 20 were used in this study to maintain the barrier phenotype of the microvascular cells. The immortalized human keratinocyte (HaCaT) was used as a control cell type. It is documented that this cell line has low expression of TfR.27 HaCaT cells were cultured under the same condition as described for U87 cells. All experiments were conducted on cells that were passaged at least twice after thawing from the frozen stock. 2.8 Cellular biocompatibility The biocompatibility of blank Tf@pSiNPs (without drug payload) was determined using the Glo-luminescence cell viability assay (Promega, G7570) to measure ATP content in cells. Briefly, three cell lines were seeded onto 96-well white plates at a density of 5,000 cells per well and maintained in medium for 1 or 2 days until confluence. Subsequently, the cells were treated with different concentrations of blank Tf@pSiNPs. Cells without any treatment were used as control and each group was triplicated. After incubating for 24 h, the cell viability was then determined using a Glo-luminescence cell viability assay. In particular, 100 µL of the solution was added into 100 µL of medium in each well and the plates were gently shaken at room temperature for 2 min. The samples were then read under a microplate reader to measure


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the luminescence intensity. The data was expressed as mean and standard deviation of 3 replicates. 2.9 Immunofluorescence imaging of TfR Cells were seeded onto a coverslip in 6-well plate at the density of 20,000 cells per cm2. After 24 h of culture, cells were rinsed with warm PBS, fixed with freshly made 4% paraformaldehyde (PFA) for 10 min, and permeabilized with 0.1% Triton-100 for 5 min. The cells were stained with phalloidin Atto 565 (Sigma Ardrich, 94072) with an excitation of 563 nm, and an emission of 592 nm. After 3 times of washing, the cells were incubated with blocking buffer (5% goat serum in PBS) for 30 min, followed by incubation with primary antibody against TfR diluted 1:50 in blocking buffer overnight at 4oC in a humidified chamber. After washing, cells were incubated with F(ab’)2-Goat anti-Mouse IgG (H+L) Cross-Adsorbed secondary antibody (Alexa Fluor 488) (Thermo Fisher Scientific, A-11017) diluted 1:250 in blocking buffer for 1 h in dark. The fluorophore of this secondary antibody was excited at 494 nm, and the emission was collected at 517 nm, Nucleus were then counter stained with DAPI, which was excited with ultraviolet light (345 nm). Coverslips were mounted onto glass slides and visualized using laser scanning confocal microscope (Leica SP8). 2.10 Western blot analysis of TfR expression To study the relative protein expression of TfR in hCMEC/D3, U87-mCherry and HaCaT, protein lysates were analyzed by western blotting. The cells were washed in warm PBS and directly lysed in a sodium dodecyl sulphate (SDS) lysis buffer (2% SDS, 50 mM Tris pH 6.8) supplemented with protease inhibitor cocktail (Sigma, P8340). The protein concentration was quantified by the EZQ protein quantitation kit (Invitrogen, R3320). The proteins were then mixed with NovexTM BoltTM LDS Sample loading buffer (Thermo Fisher Scientific, B0007) with β-mercaptoethanol, and boiled at 95°C for 10 min. To analyze TfR, proteins were electrophoresed in gradient 4-12% bis-acrylamide gels (NuPAGETM), and transferred onto 9 ACS Paragon Plus Environment


nitrocellulose membranes overnight. Membranes were blocked with 5% (w/v) goat serum in PBST (0.05% Tween-20 in PBS) for 60 min, followed by washing with PBST and incubation with anti-TfR primary antibody (1:50) in blocking buffer overnight with rocking at 4°C. After PBST washing, the blot was incubated with HRP conjugated secondary antibodies at 1:30,000 dilution in blocking buffer for 60 min. After washing, membranes were developed with SuperSignalTM West Pico PLUS Chemiluminescent substrate (Thermo Fisher Scientific, 34577) and imaged by using a Chemi-Doc with a cool CCD camera (Bio-rad ChemiDoc MP). Expression was measured by densitometry analysis of the bands using Fiji ImageJ, deducting the background intensity, and normalized to the integrated density of the β-actin housekeeping protein band. 2.11 Evaluating cell selectivity To visualize the specificity of Tf@pSiNPs to different cell types, Tf@pSiNPs were first labelled with Cy5 fluorescent dye (Lumiprobe, 13020). This NHS-Cy5 was expected to conjugate to the free amines of lysine available on Tf already attached to pSiNPs. U87mCherry, hCMECD3 and HaCaT cells were seeded in a 96 well black microplate at a density of 5,000 cells per well. After cultured until confluence, these cells were exposed to Cy5Tf@pSiNPs at a concentration of 100 𝜇g/mL. After 3 h, Cy5-Tf@pSiNPs that were not associated with the cells were washed off by warm PBS, and the fluorescence intensity was measured by using a fluorescence microplate reader (PerkinElmer EnSight Multimode). The cells were then lysed and protein content measured as mentioned above. To normalize the effect resulting from the variations of cell number between wells, normalized Cy5 intensity which equals to the measured intensity divided by protein amount was reported. 2.12 Visualization of internalization dynamics under confocal microscopy To visualize the internalization of pSiNPs by cells, nanoparticles (Tf@pSiNPs and BSA@pSiNPs) were again labelled with Cy5 fluorescent dye. The conjugation efficiency of 10 ACS Paragon Plus Environment

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Cy5 is determined by free amines available on Tf and BSA conjugated to pSiNP. We have measured and compared the fluorescence yield per mg of the two pSiNPs and found there was no significant between them (Figure S1). To demonstrate the internalization of Tf@pSiNPs, U87-mCherry, hCMECD3 and HaCaT cells were seeded onto 6-well plates with glass coverslips at a density of 20,000 cells/cm2. After 24 h, cells were exposed to Cy5-labelled Tf@pSiNPs, which had an excitation of 633 nm and an emission of 647 nm, at a concentration of 100 𝜇g/mL for 3 h. The cells were then washed with PBS and fix with 4% PFA at room temperature for 10 min. Subsequently, the cells were washed with PBS for 3 times and stained with Hoechst (2 𝜇g/mL) at 37oC for 45 min. Then, the cells were washed with PBS 3 times and stained with Vybrant CM-Dil dye (Invitrogen, V22888) with an excitation of 553 nm and an emission of 570 nm at a concentration of 10 𝜇g/mL at 37oC for 4.5 min, and then incubated at 4oC for 15 min. The cells were washed with PBS at 37oC thrice. After washing, the cells were fixed again with 4% PFA at room temperature for 10 min and at 37˚C for 5 min. At last, the cells were mounted onto microscope slides and visualized using a laser scanning confocal microscope (Leica SP8). Time-lapse confocal microscopy was used to visualize the dynamics of cellular uptake. U87-mCherry and hCMEC/D3 were seeded onto glass-bottom 24-well plates at a density of 20,000 cells per cm2. After 24 h, Cy5-labelled Tf@pSiNPs were added into the culture, fluorescence images of the co-localization of nanoparticles and cells were taken every 10 min by a confocal microscope (Nikon Ti eclipse) fitted with cell culture chamber that maintains CO2 at 5% and temperature at 37oC. The cell body of hCMEC/D3 was tracked by live cell CellTracker Green CMFDA (ThermoFisher, C7025) pre-labelled before each experiment, which had an excitation of 492 nm and an emission of 517 nm. The cell body of U87 was traced by mCherry stably expressed in the cytosol, which had an excitation of 587 nm and an emission of 610 nm. Snapshots at 1, 2, 3, and 4 h post-exposure were reported. 11 ACS Paragon Plus Environment


2.13 Visualization of internalization under TEM U87 and hCMEC/D3 cells were seeded onto 6-well culture plate at a density of 25,000 cells per cm2 and cultured in a 37oC, 5% CO2 humidified atmosphere for 24 h prior to incubation with NPs. After 24 h, the medium was removed and cells were exposed to culture medium containing 0.1 mg/mL of Tf@pSiNPs at 37oC, 5 % CO2 as specific above. Controls (untreated cells) were generated by incubating U87 and hCMEC/D3 cells in medium without NPs for an identical time period. Following incubation 24 h, the cells were washed with PBS to remove the NPs that were not internalized. Then the cells were harvested by trypsin and centrifuged for 3 min at 180 RCF. The resultant cell pellets were fixed by glutaraldehyde (3% in Sorenson’s phosphate buffer, pH 7.2-7.4)(ProSciTech, Thuringowa, QLD) at 4oC overnight, washed in Sorenson’s buffer, post-fixed in 1% osmium tetroxide for 1 h. Pellets were washed in buffer and then gradually dehydrated in increasing ethanol gradients, transferred to propylene oxide, infiltrated and embedded in Epson-Araldite (ProSciTech, Thuringowa, QLD) and polymerized at 55oC for 48 h. Ultrathin sections were collected on 200-mesh copper grids and stained with 8% methanolic uranyl acetate, followed by Reynolds’ lead citrate, each for 5 min using conventional methodology. Samples were viewed by using TEM (FEI, Eindhoven, The Netherlands) at an accelerating voltage of 120 kV. 2.14 Mechanism of cellular uptake To investigate the mechanisms involved in Tf@pSiNPs uptake in U87 and hCMEC/D3 cells, pharmacological blockers of different endocytic pathways and were used. Cells were first seeded onto coverslips in a 6 well plate at a density of 20,000 cells per cm2. After 24 h, they were pretreated with pharmacological blockers chlorpromazine (5 𝜇g/mL, Sigma, C8138), nystatin (50 𝜇g/mL, Sigma, N4014) and nocodazole (10 𝜇M, Sigma, M1404) for 30 min.28 The blockers were then washed off, and cells were exposed to fresh media containing either Cy5Tf@pSiNPs or Cy5-BSA@pSiNPs, and exposed for 1 h. The cells were then washed with PBS, 12 ACS Paragon Plus Environment

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followed by fixation, and counter stained for nucleus and cell membrane with the abovementioned method. Immediately samples were imaged under laser scanning confocal microscopy as described above. The cellular uptake of NPs was assessed by quantifying and comparing the Cy5 fluorescence intensity in the cytoplasm of the cells for each group. Quantitation was averaged from random measurements of at least 100 cells in 5 samples for each cell line. 2.15 Colocalization of Tf@pSiNPs and early endosome To determine the colocalization of Tf@pSiNps with early endosomes, U87-mCherry cells were transfected with CellLight Early Endosome-RFP (BacMam 2.0, C10587, ThermoFisher), which had an excitation of 555 nm and an emission 584 nm, to visualize the early endosomes. Specifically, U87-mCherry cells were seeded onto glass-bottom 96-well plates at a density of 5,000 cells per well. After 24 h incubation, cells were washed with warm PBS thrice and replaced with fresh medium. Cells were then transfected with Rab5a-RFP by adding 10 µL of CellLight early endosome-RFP reagents to the culture medium, achieving the ratio of 40 particles per cell (PPC). After 24 h transfection, cells were washed with warm PBS thrice and replaced with medium with Cy5-labeled Tf@pSiNPs (0.1 mg/mL). U87 cells were exposed to nanoparticles for 1 h and then the particle-containing medium was then removed, followed by PBS washing three times. Fresh, particle-free medium was added to the cells. Cells were observed by using the confocal microscopy (Nikon Ti eclipse) and images were captured at the time point of 1 h. 2.16 Drug loading and release We use doxorubicin (Dox) as a model drug to evaluate the passive loading and release capacity of Tf@pSiNPs. Tf@pSiNPs were first dispersed into PBS buffer (1 mg/mL) at a pH value of 7.4, and then Dox stock solution (10mg/mL in MilliQ H2O) was added dropwise to a final concentration of 200 µg/mL. The mixture was allowed to stir for 24 h at 0 oC. After drug 13 ACS Paragon Plus Environment


loading, the particles were collected by centrifugation and the supernatant which contained free Dox was collected for further quantification. The loading amount of Dox in the NPs were determined by using the following equations: (1)

Loading amount of Dox = Initial amount of Dox used – Amount of Dox in

supernatant. (2)

Loading amount of Dox

Loading efficiency = Initial amount of Dox used × 100%

In vitro release of Dox from Tf@pSiNPs was conducted using a dialysis method in PBS buffer. The pH value of solutions was adjusted by NaOH or HCl to 7.4 and 5.0, respectively. 2 mg of Dox-loaded Tf@pSiNPs were resuspended into 1 mL of corresponding buffer, and the solution was transferred to a dialysis bag (cellulose membrane, 10 kDa MWCO). The sealed dialysis bag was immersed into 8 mL PBS buffer (pH=5 or pH=7.4). The release tests were performed at 37oC with gentle agitation. At each predetermined time point, the dialysis bag was removed to new fresh buffer, and the amount of Dox released in the original buffer was quantified. 2.17 HPLC analysis of Dox The amount of Dox was quantified using high performance liquid chromatography (HPLC, Shimadzu Nexera, Japan) and a C18 column (50 × 2 mm2, 5 µm, Phenomenex, USA). For the HPLC detection, the mobile phase consisted of H2O with 0.1% formic acid and acetonitrile (ACN), under a gradient system. The initial ratio of H2O with 0.1% formic acid and ACN was 90:10 (v/v), which was changed to 50:50 (v/v) during 4 min, and then changed back to 80:20 (v/v) for the next 1 min. The flow rate was 0.2 mL/min and the injection volume of the sample was 1 µL. The column temperature was set to room temperature and the detection wavelength was 480 nm. The area under the Dox peak was used to quantify the concentration.


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2.18 Establishment of the in vitro BBB model To assess the potential of Tf@pSiNPs penetrating BBB, we set up a conventional Transwell BBB model by using immortalized hCMEC/D3 cells. The in vitro BBB formed by hCMEC/D3 cells expresses all signature tight junction proteins and gives a transepithelial electrical resistance (TEER) reading of 30-40 Ω·cm2, a value well accepted for in vitro BBB model.29 Briefly, hCMEC/D3 were seeded onto the Transwell inserts (3 𝜇m, polyester membrane, Sigma, CLS3472), which were pre-coated with type I collagen, at a density of 50,000 cells per cm2. The upper and lower chamber were filled with medium. After cultured for 4 days, the TEER of the hCMEC/D3 monolayer was measured every 1-2 days using Millicell ERS-2 voltammeter EVOM2 and a STX02 chopstick-style electrode (Millipore). Every time after TEER measurement, the media was refreshed in both upper and lower chambers. To calculate TEER (Ω・cm2), electrical resistance across collagen I-coated insert without cells (Rblank) was subtracted from the resistance readings obtained on inserts with cells (Rcells), and this value was multiplied by the surface area (A) of the insert (0.33 cm2). The following is the equation used to calculate the TEER value: (3)

TEER=(Rcells-Rblank) x A (Ω・cm2)

2.19 In vitro cytotoxicity of drug-loaded Tf@pSiNPs To evaluate whether the optimized properties of Dox loaded Tf@pSiNPs (Dox-Tf@pSiNPs) enhance the killing of GBM cells, we studied the cytotoxicity of Dox-Tf@pSiNPs firstly in the absence of BBB. The cytotoxicity of Dox-Tf@pSiNPs, empty Tf@pSiNPs and free dox on U87 cells were determined using vivoGlo luciferin cell viability assay (Promega, G7570). Briefly, U87 cells were seeded onto 24-well white microplates at a density of 25,000 cells per cm2. After 24 h, the cells were treated with Dox-Tf@pSiNPs (5 µg/mL), Tf@pSiNPs (56.81



µg/mL) and free dox (1.25 µg/mL) for 1 h. The concentration of free dox was equivalent to the concentration of Dox released from Tf@pSiNPs in 1 h, which was pre-determined by drug loading and release test. Cells without any treatment were used as control. After exposure, the cells were washed with PBS and replaced with fresh growth medium and incubated for another 48 h. The cell viability was then determined by adding 30 µL of the vivoGlo luciferin stock solution into 600 µL of medium in each well (final luciferin concentration was 150 µg/mL). The luminescence intensity of each well was then measured by using a microplate reader (PerkinElmer EnSight Multimode). The average intensity of 3 replicates and error equals to 1 standard deviation were reported. After confirming the U87 killing capacity of Dox-Tf@pSiNPs in the absence of BBB, we studied the cytotoxicity of Dox-Tf@pSiNPs across the BBB on U87 cells using the above described in vitro BBB model. U87 cells were seeded at a density of 25,000 cells per cm2. After 24 h, the Transwell inserts covered with hCMEC/D3 cell monolayers with TEER measured between 30 and 40 Ω·cm2 were placed on the wells cultured with U87. Subsequently, DoxTf@pSiNPs, Tf@pSiNPs and free Dox were added to the upper inserts at the aforementioned concentration and incubated for 1 h. Cells without any treatment were used as control. After 1 h, the inserts were removed to discard non-transcytosed particles or drug. U87 were further incubated for another 48 h. The cell viability was then determined as described above. The average intensity of 3 replicates and error equals to 1 standard deviation (SD) were reported. 2.20 Statistical Analysis All experiments were at least triplicated. Statistical analysis was performed using GraphPad Prism7 (San Diego, USA). Statistically significance was determined by using Student’s t test (between two groups) or one-way ANOVA test (three or more groups). The hypothesis was accepted at a 95% significant level (p < .05). Results are reported as mean ± SD of N=3.


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3. Results and Discussion Despite medical breakthroughs, the survival rate of patients with GBM has not improved over the past decades.3 Tf has been intensely studied as a targeting ligand to target small molecular drugs and polymeric nanovectors to brain tumors. Whilst pSiNP is a better alternative which is biocompatible, degradable, and is capable to deliver relatively higher amount of drug, their promise in target-delivering drug into GBM remains largely unexplored. To our best knowledge, the potential of Tf-conjugated pSiNPs in treating brain cancers has never been explored. Reuter et al. targeted human breast cancer cells using a composite of pSiNPs and Tf-hydrophobin fusion protein, and demonstrated that the presence of the fusion protein led to enhanced cellular uptake of nanoparticles in breast cancer cells.24 In their study, the hydrophobic domain of the protein was physically adsorbed on the hydrophobic pSiNPs surface. However, in another study conducted by Pitek et al. physical adsorption of Tf onto nanoparticles was shown to cause a complex interaction pattern with serum proteins and a high degree of opsonization problems.30 Therefore, we decided to explore covalently conjugated Tf@pSiNPs and investigated their potential application in GBM treatment. To the best of our knowledge, it was the first time that Tf was covalently conjugated to pSiNPs and applied to brain cancer treatment. 3.1 Surface characterizations of Tf-coated pSiNPs The synthesis scheme of Tf-conjugated pSiNPs is shown in Figure 1A. pSiNPs with carboxyl termination (UnpSiNPs), which was formed by hydrosilylation of pSiNPs in undecylenic acid, was confirmed by the appearance of C=O peaks at 1716 cm-1 from the carboxyl group on the surface in FTIR spectra (Figure 1B). Subsequently, carboxylated pSiNPs were covalently coupled with transferrin (Tf) via EDC/NHS chemistry reaction, resulting in the formation of stable conjugation between Tf and pSiNPs, a particle system termed Tf@pSiNPs. In the IR spectrum of Tf@pSiNPs, we observed the appearance of characteristic 17 ACS Paragon Plus Environment


peaks at 1657 and 1544 cm-1 for amide I and amide II vibrations, respectively, and the disappearance of peaks corresponding to carboxyl groups. These results confirmed that Tf was successfully conjugated to UnpSiNPs for Tf@pSiNPs group.

Figure 1. Schematic and surface characterizations of holo-transferrin modified porous silicon nanoparticles. A) Schematic of pSiNPs fabrication and EDC/NHS chemistry. B) FTIR characterization of undecanoic acid modified, and Tf covalently conjugated pSiNPs. C) Architecture of Tf@pSiNPs imaged under cryo-TEM in PBS. (UnpSiNP was imaged under conventional TEM owing to poor colloidal stability in physiological buffer). D) Hydrodynamic size distribution, and zeta-potential of UnpSiNPs and Tf@pSiNPs, which were characterized in PBS using dynamic light scattering with zeta potential analyzer.


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3.2 Physical properties and biocompatibility of Tf-modified pSiNPs To study the size and the structure of Tf-decorated pSiNPs, cryo-transmission electron microscopy (cryo-TEM) was performed (Figure 1C). The images show that Tf@pSiNPs were dispersed in PBS with an average size of 167 ± 16 nm. Besides, the nanoparticles displayed an irregular plate shape with pore size of 16 ± 4 nm. DLS was used to further characterize the size distribution and zeta potential of UnpSiNPs and Tf@pSiNPs in PBS at pH7.4. As shown in Figure 1D, before Tf coating, UnpSiNPs displayed colloidal instability under those conditions, with the size of 1857 ± 315 nm (PDI: 0.62). By contrast, after Tf functionalization, Tf@pSiNPs showed obvious improvement of colloidal stability in PBS, with the size of 182 ± 1 nm. Besides, we observed that the polydispersity index (PDI) changed from 0.62 to less than 0.12 for Tf@pSiNPs. This indicates that Tf modification resulted in relatively narrower particle size distribution and improved dispersion of the pSiNPs. The particle size obtained from TEM image was slightly smaller than that determined by DLS measurement. This may due to the two different principles of particle size measurement. Specifically, “static” particle size is measured in TEM, while hydrodynamic size of NPs is measured in DLS.31 For zeta potential, Tf@pSiNPs remained negative after Tf modification, where the zeta potential of Tf@pSiNPs was significantly higher than unmodified UnpSiNP. This is consistent with the IR results that the carboxyl group has been consumed in forming amide links with Tf to form Tf@pSiNPs. 3.3 Analysis of TfR expression Since we hypothesized that Tf@pSiNPs were specific to brain cancer cells and microvascular cells by their overexpression in TfR, we first determined the relative TfR expression levels in U87, hCMEC/D3 and HaCaT cells by Western blot (WB) and



immunofluorescence. WB analysis revealed that the expression of TfR at 95 kDa for U87 cells was much higher than the expression levels in the other two cells (Figure 2A). However, we were unable to distinguish expression levels in hCMEC/D3 and HaCaT cells by WB. We further sought to study the TfR expression by immunofluorescence. It was obvious that TfR expression was observable for all U87 cells (Figure 2B). While strong TfR expression could only be observed in some the hCMEC/D3 cells, and the expression in HaCaT cells was barely observable. These data together with the result obtained in WB experiment, allow us to conclude that U87 had the highest level of TfR expression, followed by hCMEC/D3 and HaCaT cells (Figure 2B).

Figure 2. Transferrin receptor (TfR) expressions in cells, cell selectivity and biocompatibility of Tf@pSiNPs. A) Western blotting analysis of TfR. B) Laser scanning confocal microscopy images of immunofluorescence against TfR. (Blue: DAPI; Green: F(ab’)2-Goat anti-Mouse IgG (H+L) Cross-Adsorbed secondary antibody (Alexa Fluor 488); Red: Phalloidin Atto 565.) C) Biocompatibility of Tf@pSiNPs demonstrated by ATP viability assay on cells (U87, hCMEC/D3, and HaCaT) exposed to the particles. Error bars equal ± 1 SD, N=3. D) Association of Cy520 ACS Paragon Plus Environment

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Tf@pSiNPs with U87, hCMEC/D3, and HaCaT cells as measured by Cy5 fluorescence intensity normalized to total protein content. Error bars equal ± SD, N=3, and * indicates p

Systematic Evaluation of Transferrin-Modified Porous Silicon

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