Receptor-Mediated Enhanced Cellular Delivery of Nanoparticles

Dec 1, 2016 - Antibodies and peptides are often used to home nanoparticles (NPs) to specific cells. Here in this work, we have used recombinant recept...
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Receptor-Mediated Enhanced Cellular Delivery of Nanoparticles Using Recombinant Receptor-Binding Domain of Diphtheria Toxin Mahesh Agarwal,† Amaresh Kumar Sahoo,‡,§ and Biplab Bose*,†,‡ †

Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, India Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, India

‡

S Supporting Information *

ABSTRACT: Antibodies and peptides are often used to home nanoparticles (NPs) to specific cells. Here in this work, we have used recombinant receptor-binding domain of diphtheria toxin (RDT) as a homing molecule for NPs. Diphtheria toxin binds to heparin binding EGF-like growth factor (HB-EGF) through its receptor-binding domain. HB-EGF is often overexpressed as cell surface molecule in various types of cancer. We have prepared monodispersed, spherical PLGA NPs and coated these NPs with RDT. These NPs are characterized by FESEM and FT-IR spectroscopy. Using flow cytometry and fluorescence spectroscopy, we show that coating with RDT increases cellular uptake of PLGA NPs. We further show that RDT-coated nanoparticles are internalized through clathrin-dependent receptor-mediated endocytosis that can be reduced by specific inhibitor. These RDT-coated nanoparticles (RDT-NP) were further used for preferential delivery of Irinotecan, a chemotherapeutic agent, to cells overexpressing HB-EGF. We show that receptor-mediated enhanced uptake of RDT-NPs increases the potency of irinotecan in these cells. KEYWORDS: diphtheria toxin, nanoparticles, endocytosis, HB-EGF, targeted delivery

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glioblastoma, ovarian cancer, and hepatocellular carcinoma.6−9 Expression of HB-EGF has significant correlation with disease progression, development of drug resistance, and clinical outcome in cancer.10−12 HB-EGF is initially expressed as a membrane-anchored protein. Subsequently, it is cleaved and released by metalloproteases.6 Being a membrane anchored protein, HB-EGF is a good target for cell-specific drug delivery. Several research groups have developed antibodies against HB-EGF and have used those antibodies for targeted delivery.13,14 Cell surface HB-EGF is the receptor for diphtheria toxin (DT). DT belongs to the group of AB toxins and has three independent domains: N-terminal catalytic domain (Cdomain), C-terminal receptor-binding domain (R-domain), and T-domain.15 The C-domain is responsible for the toxicity of DT. DT binds to cell surface HB-EGF through its R-domain and enters the cell by receptor-mediated endocytosis.15 Earlier, we cloned, expressed, and purified the R-domain of diphtheria toxin (RDT) using an E. coli based expression system.16 Molecular weight of RDT is approximately 20 kDa, and it is nontoxic.16 This protein retains the affinity for HBEGF and binds to HB-EGF expressed on cell surface.16 In the present work, we have used this protein as homing agent for

INTRODUCTION Drug formulations using nanoparticles are gaining popularity. FDA has already approved several nanoparticle-based formulations.1,2 The physical dimension of nanoparticles gives rise to several advantageous properties to nanoformulations. These include enhanced tissue penetration and ability to cross blood− brain barrier.3,4 The microvasculatures around solid tumors are leaky, and nanoparticles can get through those into the tissue. Therefore, nanoparticles in systemic circulation preferentially accumulate in and around tumors. This leads to passive targeting of nanoparticles to tumor microenvironment.4 However, such passive targeting is still nonspecific. Cellular specificity can be achieved by tagging nanoparticles to a homing molecule.5 A homing molecule recognizes a surface molecule on target cells and helps in preferential accumulation of the nanoparticles around the target cells. Some homing molecule may also trigger receptor-mediated endocytosis of the nanoparticles by the target cells. As a whole, a homing molecule increases effective delivery of drug to target cells.5 Antibodies, peptides, and small molecules are often used as homing molecules.5 They recognize and bind to cell surface molecules present differentially on targeted cells. In this work, we have used a protein derived from a bacterial toxin as a homing molecule. This protein targets heparin binding EGFlike growth factor (HB-EGF). HB-EGF is a growth factor and is involved in various processes linked to development and progression of cancer.6 It is overexpressed in various types of cancers including © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

May 30, 2016 October 23, 2016 December 1, 2016 December 1, 2016 DOI: 10.1021/acs.molpharmaceut.6b00480 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

centrifugation. The supernatant was discarded. NPs were resuspended and incubated in blocking buffer (2% BSA in PBS), for 2 h at room temperature. Subsequently, NPs were separated by centrifugation, resuspended, and washed with PBS. These NPs were resuspended in 100 μL of 2% BSA-PBS with mouse anti-His antibody (1:1000 dilution, Calbiochem) and incubated for 1 h at room temperature. NPs were collected by centrifugation, washed with PBS, and resuspended in 100 μL of HRP-conjugated antimouse antibody (1:1000 dilution, Sigma-Aldrich) in 2% BSA-PBS. After 1 h of incubation at room temperature, NPs were harvested by centrifugation, washed by PBS, and resuspended in 400 μL of OPD substrate. After few minutes of incubation at room temperature, 100 μL of sample was transferred to wells of ELISA plate. Reaction was stopped by adding 50 μL of 8 N H2SO4, and absorbance was measured at 492 nm. Stability of RDT, adsorbed on NPs, was also determined by ELISA. For this, RDT-coated NPs were resuspended in serumfree media and incubated for different durations at 37 °C. Subsequently, RDT bound to NPs was detected by ELISA as described above. Amount of protein adsorbed on NPs was also determined by Bradford assay20 as used by Kockbek et al.18 Coated or uncoated NPS were resuspended in 200 μL of PBS. Two hundred microliters of Bradford reagent (Sigma) was added to each sample and incubated for few minutes at room temperature. Subsequently, absorbance was measured at 595 nm. Solutions of BSA with known concentrations were used to generate a standard curve, and the amount of protein attached to NPs was estimated using this standard curve. Cellular Uptake Assay of NPs. Uptake of various NPs by U-87 MG and RAW 264.7 cells was measured by flow cytometry and fluorescence spectroscopy. Cells in 6-well tissue culture plates were treated with rhodamine-123-loaded NPs, RDT-NPs and BSA-NPs for 2 h at 37 °C in serum-free media. Subsequently, cells were washed thrice thoroughly by PBS and trypsinized. Thorough wash and trypsinization remove NPs bound to cell surface. Trypsinized cells were harvested by centrifugation and resuspended in serum free media. Fluorescence intensity of rhodamine 123 in these cells was measured by flow cytometry in FL-1 channel (FACSCalibur, BD, USA). Similarly, for fluorescence spectroscopy based assay of cellular uptake, cells treated with rhodamine-123-loaded NPs were washed thrice, thoroughly, by PBS and then trypsinized. Trypsinized cells were harvested by centrifugation, resuspended in PBS, and lysed by sonication. Cell lysates were further diluted in PBS, and fluorescence intensity of rhodamine 123 was measured by Fluoromax-4 (Horiba scientific) (λEx = 505 nm, λEm = 528 nm; path length =1 cm; excitation and emission slit widths were 1 and 5 nm, respectively). For competitive assay, cells were pretreated with excess RDT for 30 min and then incubated with various types of NPs in the presence of excess RDT. Percentage uptake of NPs was calculated by comparing fluorescence of rhodamine 123 in cell lysate with total fluorescence of NPs used. In Vitro Drug Release Assay. The Irinotecan-loaded NPs, BSA-NPs, and RDT-NPs were dispersed in 1 mL of PBS (pH 7.4) and incubated at 37 °C with gentle shaking. At different time points, Irinotecan-loaded NPs were harvested by centrifugation at 10,000 rpm for 5 min at 25 °C. Release of irinotecan was measured by measuring the absorbance of the supernatant at 255 nm. A standard curve was made by

drug-loaded polymeric nanoparticles. We have coated these nanoparticles with RDT and have shown that these coated nanoparticles are preferentially internalized by cells expressing human HB-EGF.

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MATERIALS AND METHODS Human glioma cell line U-87 MG and mouse macrophage cell line RAW 264.7 were procured from National Centre for Cell Science (NCCS), Pune. These cells were maintained in DMEM (HiMedia) supplemented with 10% fetal bovine serum (Gibco) and antibiotics (Anti-anti, Gibco). The cells were maintained at humidified 5% CO2 incubator at 37 °C. Recombinant RDT was expressed in E. coli and purified as described earlier.16 BSA was procured from Sigma-Aldrich. Synthesis and Characterization of Nanoparticles. The poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) were synthesized by emulsification solvent evaporation method as described by Chaubey et al.17 Five milligrams of PLGA (50:50, MW 7000−17000, Sigma-Aldrich) was dissolved in 1 mL of chloroform (Merck). Further, this PLGA solution was added dropwise in a solution of poly(vinyl alcohol) (PVA, 0.5 mg/mL in water, Sigma-Aldrich), under continuous sonication for 2 min at room temperature. Whenever required, rhodamine 123 (Sigma-Aldrich) was dissolved in the PVA solution (final concentration 2.5 μg/mL) to synthesize rhodamine-loaded NPs. For drug-loaded nanoparticles, irinotecan in water was mixed with PLGA solution and sonicated well to create a wellformed emulsion. This was subsequently added dropwise to PVA solution. NPs were washed thrice with water and dried in vacuum to remove excess solvent. Subsequently, NPs were resuspended in water/serum free media for further uses. NPs were coated with RDT or BSA by surface adsorption.18 This was achieved by incubating 10 μg of RDT or BSA in PBS with 250 μg of NPs for 2 h at 37 °C. Subsequently NPs were centrifuged at 8000 rpm for 5 min at room temperature. The supernatant was removed, and pellet was resuspended in water/ serum-free media. Size and morphology of PLGA NPs and RDT-coated NPs (RDT-NPs) were analyzed by field emission scanning electron microscope (FESEM, Zeiss, model: Sigma). NPs or RDT-NPs was drop-casted on aluminum foil wrapped coverslip. Samples were dried for overnight at room temperature. Before visualization, samples were coated with gold layer and imaged at 3 kV. FESEM images were analyzed by ImageJ19 to calculate size of nanoparticles. Size distribution of nanoparticles was also measured by dynamic light scattering method (DLS, Malvern Zetasizer Nano ZS, USA). For this, NPs were resuspended in water. Zeta potentials of NPs were measured in the same instrument. FTIR spectroscopy was used to confirm coating of NPs by RDT. NPs and RDT-NPs were mixed, separately, with IR grade, potassium bromide (Sigma) in the ratio of 1:100 and corresponding pellets were prepared by applying 5.5 t of pressure with a hydraulic press. The infrared absorption spectra were collected in an inert atmosphere over a wavenumber range of 4000−450 cm−1 in (PerkinElmer; Spectrum Two, FT-IR instrument). Recombinant RDT has a His-tag. Adsorption of RDT on NPs was checked by ELISA using anti-His antibody. NPs were incubated with different amounts of RDT in PBS for 2 h at 37 °C. Subsequently, NPs were separated by centrifugation and resuspended in 1 mL of PBS. One hundred microliters of resuspended NPs was taken in 1.5 mL tubes and separated by B

DOI: 10.1021/acs.molpharmaceut.6b00480 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics measuring absorbance of irinotecan solutions of different concentrations. Percentage release of the drug, from NPs, was calculated from the absorbance data using this standard curve. In Vitro Cell Viability Assay. Cells in 96-well tissue culture plates were treated, for specific duration, with NPs of various types as mentioned in the results section. Subsequently, viability of these cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.21 Percentage cell viability was calculated relative to cells treated with equivalent amount of serum free media. Data Analysis. SigmaPlot was used to create graphs and for all statistical analysis. Wherever required, means of multiple data points, from repeated experiments, are shown. Error bars represent standard deviation in data. One-way ANOVA with multiple comparisons was used.

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RESULTS Characterization of RDT-Coated PLGA Nanoparticles. We have used PLGA, an FDA approved biodegradable polyester polymer,22 to prepare nanoparticles (NPs) by emulsification solvent evaporation technique.17 Another biodegradable polymer PVA was used as a nonionic emulsifying agent to synthesize these NPs. Nanoparticles were loaded with rhodamine 123, for tracking. Subsequently, nanoparticles were coated by RDT. The size and morphology of NPs were analyzed by FESEM. It was observed that NPs were monodisperse and spherical in shape having average diameter of 33.10 ± 7.29 nm (Figure 1a,b). RDT-coated NPs were also monodisprese but slightly bigger (54.42 ± 5.14 nm) probably due to protein coating (Figure 1a,b). Size distributions of these NPs were also measured by DLS (Figure 1c). Average size of NPs estimated by DLS (154.9 and 157.5 nm for NPs and RDT-NPs, respectively) was higher than the size estimated from FESEM data. Such size difference would arise as layer of hydration around NPs affects DLS measurement. Such layer of hydration is absent in samples of FESEM. Additionally, PLGA nanoparticles would dehydrate and may shrink during sample preparation for FESEM. Cellular uptake of NPs depends upon size of NPs.23,24 Coated and uncoated PLGA NPs, prepared in this work, are in the optimal size range for endocytosis.24−26 We have also measured zeta potentials of uncoated NPs and RDT-coated NPs. These were −0.127 and +0.717 mV for uncoated NPs and RDT-coated NPs, respectively. Therefore, these NPs are close to neutral. The surface charge of NPs may play a role in the cellular uptake of NPs. Positively charged NPs are considered to have higher cellular uptake due to interaction with negatively charged cell membrane.24 However, strong surface charge of NPs may also cause cytotoxicity.27 However, positive surface charge is not crucial when specific receptormediated endocytosis is used for cellular uptake of NPs. In this case, the interactions between the homing molecules on NPs with the receptor on cell surface would play the major role. Coating of NPs with RDT was confirmed by FT-IR spectroscopy (Figure 2). It has been reported28 that PLGA shows prominent peak of carbonyl-stretching in the range of 1690−1760 cm−1. In addition to that, PLGA shows strong C− O stretching band near 1100 cm−1. In our analysis for PLGA NPs, the peak of CO stretching bond appears at 1755.05 cm−1 and CC stretching bond at 1638.85 cm−1 (Figure 2a). Characteristic peaks for amid bonds are visible in the FT-IR spectrum of RDT-NP (Figure 2b). A peak at 1647.00 cm−1 is due to amide I having dominant CO stretching bond.29,30 A

Figure 1. Physical characterization of uncoated and RDT-coated PLGA NPs. (a) FESEM images of NPs and RDT-coated NPs. (b) Histogram showing size distribution of NPs and RDT NPs as observed in FESEM images. (c) Histogram showing size distribution of NPs and RDT-NPs as measured by DLS.

peak at 1546.18 cm−1 is due to amide II bond having N−H bend and C−H stretch.30 We have estimated the amount of RDT bound to NPs by Bradford assay. It was observed that 12.6 ng of RDT was absorbed per microgram of NPs. Binding of RDT to NP was further confirmed by ELISA. The recombinant RDT used in this work has His-tag. PLGA NPs were incubated with different amount of RDT for 2 h, and bound RDT was detected by mouse anti-His antibody, followed by antimouse-HRP conjugate. As shown in Figure 2c, adsorption of RDT increased with concentration of RDT used. The dose-dependent binding of RDT to NPs has a hyperbolic behavior similar to Langmuir adsorption model.31 We used this ELISA based method to investigate the stability of coating by RDT on NPs. Nanoparticles coated with RDT were incubated for different durations in serum-free media at 37 °C. Subsequently, RDT bound to NPs was detected by ELISA. As shown in Figure 2d, coating of RDT on NPs is stable even after 24 h of incubation in serum-free media. Receptor-Mediated Endocytosis of RDT-NPs. We have performed in vitro assays to study cellular uptake of RDT-NPs. Human glioma cell line U-87 MG expresses HB-EGF on cell surface.16 Mouse cells also express HB-EGF. However, due to sequence variation, DT does not bind to mouse HB-EGF and mouse cells are not sensitive to DT.32,33 Therefore, we have used a mouse cell line, RAW 264.7, as a negative control in our experiments. C

DOI: 10.1021/acs.molpharmaceut.6b00480 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 2. Adsorption of RDT on PLGA NPs. Panels (a) and (b) show FT-IR spectra of NPs and RDT-coated NPs, respectively. (c) Concentrationdependent adsorption of RDT on NPs was measured by ELISA. Filled circles are experimental data points. The data was fitted to a rectangular hyperbola (solid line). (d) NPs coated with RDT were incubated in serum-free media for different durations, and bound RDT was detected by ELISA. Filled circles are experimental data points. The data was fitted to an exponential decay curve (solid line).

NPs loaded with rhodamine 123 were used for measuring endocytosis of NPs. Rhodamine 123 may leach out of the PLGA NPs on prolonged storage in aqueous environment. That will affect the endocytosis assays. To estimate the extent of leaching, we incubated rhodamine 123-loaded NPs in serumfree culture media, at 37 °C, for different durations, with mild shaking. Subsequently, NPs were separated by centrifugation and resuspended in PBS. Fluorescent intensity of these NPs was measured by spectrofluorometry. We observed that with time fluorescence intensity of these NPs decreased (Figure S1). However, these NPs retained measurably high fluorescence intensity even after 24 h of incubation. For our endocytosis experiments, dye-loaded NPs were never kept more than 4−5 h in aqueous environment and retained very high fluorescence. Therefore, rhodamine 123loaded PLGA NPs are stable enough for experiments to measure endocytosis either by flow cytometry or fluorescence spectroscopy. We treated both U-87 MG and RAW 264.7 cells with different NPs for 2 h at 37 °C and measured extent of endocytosis by flow cytometry. Rhodamine 123-loaded NPs, uncoated and coated with RDT, were used for these experiments. We have also prepared BSA-coated NPs and used those as control in our experiments. BSA does not have any specific receptor on U-87 MG and RAW 264.7 cells. Both coated and uncoated NPs are internalized by U-87 MG cells (Figure 3). However, the extent of endocytosis of RDTNPs was much higher than uncoated and BSA-coated NPs (Figure 3). However, both RDT and BSA-NPs have similar uptake in RAW 264.7 cells (Figure 4). These observations indicate that probably RDT-NPs are preferentially internalized by U-87 MG cells through HB-EGF-mediated endocytosis.

Subsequently, we have performed endocytosis assay in presence and absence of excess free RDT. In these experiments, extent of endocytosis was estimated by measuring fluorescence intensity of rhodamine 123 in cell lysates using fluorescence spectrophotometer. We observed that the fluorescence intensity of rhodamine 123 was higher in case of RDT-NPs treated U-87 MG cells as compared to BSA-NPs treated cells (Figure 5a). This implies that the extent of endocytosis of RDT-NPs in U-87 MG cells was much higher than BSA-NPs. However, such difference was not observed when the same experiment was performed with prior incubation of cells with excess, free RDT (Figure 5a). This proved that RDT-NPs are internalized by receptor-mediated endocytosis and pretreatment with excess RDT is reducing this by saturating the receptor, HB-EGF. Further, we have performed the endocytosis experiment in the presence of different physical and chemical inhibitors of endocytosis. Low temperature reduces endocytosis.34 We have treated U-87 MG cells with rhodamine-loaded RDT-NP and BSA-NP at 37 °C and at 4 °C. It was observed that extent of cellular uptake is reduced by several fold for both BSA-and RDT-NPs at 4 °C (Figure 5b). We have treated U-87 MG cells, with RDT-NPs and BSANPs, in the presence of chlorpromazine and filipin. Chlorpromazine is an inhibitor of clathrin-dependent, receptor-mediated endocytosis.35 It inhibits receptor-mediated endocytosis of DT.36 In our experiment, chlorpromazine inhibited cellular uptake of RDT-NPs (Figure 5b). However, chlorpromazine did not affect uptake of BSA-NPs as it does not involve any receptor-mediated endocytosis. Filipin disturb caveolae-dependent endocytosis, but does not disturb receptor-mediated endocytosis of DT.36 In our experiment, filipin D

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Figure 4. RDT-NPs and BSA-NPs have similar uptake in RAW 264.7 cells. Uptake of BSA-coated and RDT-coated NPs was measured by flow cytometry. These NPs are loaded with rhodamine 123. (a) Histogram of rhodamine 123 intensity in different treatment groups in a representative experiment. (b) Median fluorescence intensity of rhodamine 123 in cells treated with different NPs as calculated from three independent experiments. UN: untreated U-87 MG cells.

Figure 3. Cellular uptake of RDT-NPs is more in U-87 MG cells. Uptake of uncoated, BSA-coated and RDT-coated NPs was measured by flow cytometry. These NPs are loaded with rhodamine 123. (a) Histogram of rhodamine 123 intensity in different treatment groups in a representative experiment. (b) Median fluorescence intensity of rhodamine 123 in cells treated with different NPs as calculated from three independent experiments. UN: untreated U-87 MG cells. Oneway ANOVA with pairwise comparison: *do not have any significant difference (p > 0.05).

indicate that receptor-mediated enhanced uptake of RDT-NPs increases the potency of irinotecan in U-87 MG cells.

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DISCUSSION In this work, we have used recombinant receptor-binding domain of diphtheria toxin (RDT) to achieve specific and enhanced cellular uptake of nanoparticles. We have coated PLGA NPs with RDT and have shown that these coated NPs are internalized by cells expressing human HB-EGF through receptor-mediated endocytosis. Such receptor-mediated endocytosis provides cellular specificity and increases effective uptake of the NPs. Targeted delivery of NPs is usually achieved using antibodies and peptides. Here, we have used a protein derived from a toxin to achieve the same. Diphtheria toxin binds to cell-surface HBEGF and is internalized through receptor-mediated endocytosis. HB-EGF is overexpressed in several types of cancer, and it can be targeted for cell specific delivery of a therapeutic cargo. CRM197, a mutated diphtheria toxin, has been utilized for targeting cells expressing HB-EGF both in vitro and in vivo.38−41 CRM197 does not bind to mouse HB-EGF.42 However, Tosi et al.43 have shown that CRM197-conjugated PLGA NPs crosses blood−brain barrier in a mouse model and have proposed that such uptake may be due to CRM197-induced upregulation of Caveolin-1-mediated transport. CRM197 is full-length diphtheria toxin, with all three domains, but having mutation in the catalytic domain. Though, generally considered as a nontoxic mutant, there are reports that CRM197 has residual toxicity.44,45 Earlier, we have cloned and expressed the receptor-binding domain of diphtheria toxin (RDT).16 This recombinant protein

failed to reduce cellular uptake of RDT-NPs (Figure 5b). These experiments confirmed that RDT-NPs are internalized by clathrin-dependent, HB-EGF-mediated endocytosis. RDT-NPs Enhances Potency of a Drug. Irinotecan is a prodrug that gets hydrolyzed to generate SN-38.37 SN-38 acts as a chemotherapeutic agent by inhibiting topoisomerase I.37 Irinotecan loaded PLGA NPs were prepared as described in the methods section, and these NPs were coated with RDT or BSA. Such coating does not affect in vitro release of the drug from NPs and most of the drug gets released by 6 h (Supplementary Figure S2). We treated U-87 MG and RAW 264.7 cells with these NPs and effect of the drug on viability of these cells was measured by MTT assay. Without the drug, RDT-NPs and BSA-NPs do not have considerable cytotoxicity even at higher doses (Supplementary Figure S3). The dose-dependent effect of the drug in solution and drugloaded NPs, on these two cells is shown in Figure 6. As shown in Figure 6b, these nanoformulations do not change the effect of the drug in RAW 264.7 cells. Rather, at higher concentrations, irinotecan in solution has more effect than its nanoformulations. Such variation may arise due to slow release of the drug from NPs. However, RDT-coated NPs increase the potency of irinotecan for U-87 MG cells (Figure 6a). In our experiment, the IC50 of irinotecan is 18.74 μM, but when used as RDTcoated NPs, the IC50 is only 8.69 μM. BSA-coated NPs does not increase the effect of the drug and shows lesser potency as observed in the case of RAW 264.7 cells. These observations E

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Figure 5. RDT-NPs enter U-87 MG cells by receptor-mediated endocytosis. U-87 MG cells treated with different NPs in different conditions were lysed and fluorescence intensity of rhodamine 123 was measured in cell lysates to estimate extent of endocytosis. (a) Endocytosis experiment was performed with or without excess, free RDT as mentioned in methods section. (b) Experiment was performed at 4 °C or in presence Chloropromazine (CHPZ, 5 μM) and Filipin (7.5 μM). One-way ANOVA with pairwise comparison: **have significant difference (p < 0.01); *do not have significant difference (p > 0.05); #do not have significant difference p > 0.05.

Figure 6. RDT-NP increases potency of irinotecan in U-87 MG cells. Effects of irinotecan in solution or loaded in RDT-NP and BSA-NP on viability of (a) U-87 MG and (b) RAW 264.7 cells were measured by MTT assay. Cells, in serum-free media, were treated for 48 h. Data points were fitted to exponential decay equation to calculate IC50. The gray horizontal line represents 50% cell viability.

Further, we performed competitive experiment and experiment with specific inhibitors of receptor mediated endocytosis. In our earlier work,16 we have shown that RDT binds to cell surface HB-EGF. However, it was not known whether the receptor-binding domain of diphtheria toxin (RDT), alone, can be able to induce receptor-mediated endocytosis. We have performed cellular uptake assay for RDT-NPs in the presence of excess, free RDT. Excess RDT will block cell surface HB-EGF and will inhibit interaction of RDT-NPs with cell surface HB-EGF. As expected, we observed that excess, free RDT blocks internalization of RDT-NPs, but not of BSA-NPs. This confirmed that RDT-NPs are internalized through interaction with cell surface HB-EGF. Receptor-mediated endocytosis of diphtheria toxin is clathrin-dependent, and chlorpromazine blocks such clathrindependent endocytosis.36 However, filipin, an inhibitor of caveolae-dependent endocytosis, has no effect on endocytosis of diphtheria toxin.36 In our experiments, chlorpromazine reduced cellular uptake of RDT-NPs, but it did not affect uptake of BSA-NPs. Further, Filipin had no effect on uptake of RDT-NPs. This proved that, just like, full-length diphtheria toxin, RDT is also internalized through clathrin-dependent, receptor-mediated endocytosis. Receptor-mediated endocytosis allows cell specific, enhanced drug delivery, which improves the potency of a drug. We have

does not have the cytotoxic N-terminal catalytic domain of diphtheria toxin. It also does not have the translocation-domain (T-domain). We had shown that RDT binds to HB-EGF with equivalent affinity as the full-length toxin.16 Therefore, we have used this recombinant protein as the homing agent for PLGA nanoparticles. PLGA is a biodegradable polymer, and it is widely used for making nanoparticles.22 We have prepared monodisperse, spherical PLGA NPs of less than 50 nm. These PLGA NPs were loaded with a fluorescence dye for ease of tracking. We have coated these NPs with proteins, RDT or BSA. It was assumed that RDT will bind to cell surface HB-EGF and would facilitate internalization of NPs. As BSA does not have any specific receptor, BSA-coated will not have any cell-specific enhanced uptake. In fact, using flow cytometry and fluorescence spectroscopy, we observed that cellular uptake of RDT-NPs is more than uncoated NPs and BSA-NPs in U-87 MG cells. These cells express the receptor of Diphtheria toxin, human HB-EGF, on cell-surface. However, no such enhanced uptake was observed in mouse RAW 264.7 cells as diphtheria toxin does not bind to mouse HB-EGF. This showed that through RDT we can achieve both enhanced cellular uptake and cell specificity. F

DOI: 10.1021/acs.molpharmaceut.6b00480 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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loaded protein-coated NPs with irinotecan, a chemotherapeutic drug. U-87 MG and RAW 264.7 cells were treated with different doses of these drug-loaded NPs. We observed that delivery through RDT-NPs increases cytotoxicity of irinotecan, but such effect is cell specific. Similar to cellular uptake experiments, RDT-NPs decreased the IC50 of irinotecan only in U-87 MG cells that express human HB-EGF on cell surface. No such effect was observed in RAW 264.7 cells or when drugloaded BSA-NPs were used. This study showed that the receptor-binding domain of diphtheria toxin, alone, can be used to create a drug delivery system for cell-specific and enhanced delivery through receptormediated endocytosis. We have coated nanoparticles with RDT through surface adsorption. Covalent conjugation of this protein to the polymer may enhance the stability of the system and make it more suitable for long-term in vivo uses. In vivo experiments are also required for future clinical translation of RDT-mediated cell-specific drug delivery systems. Such studies are required to understand stability, biodistribution, tissue penetration, and organ/tissue specific preferential accumulation of RDT-tagged drug delivery systems in a live organism. Targeted-delivery of nanoformulations is a challenging field of study. Most of the works focus on use of antibodies or peptides for achieving cellular specificity. However, several pathogen-associated molecules, like viral coat proteins, bacterial toxins, have cellular specificity and are internalized through specific receptor-mediated endocytosis. Our work reaffirms that such protein can be manipulated to create novel drug-delivery systems. Diphtheria toxin belongs to the group of AB toxins that have separate domains for receptor binding and for toxicity. We have used only the receptor-binding domain that has no toxicity. We have proved this domain is completely independent, for receptor binding and receptor-mediated endocytosis. This domain may be further modified to reduce its size without affecting cell-specific receptor-mediated endocytosis.

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*Phone: +91-361-2582216. Fax: +91-361-2582249. E-mail: [email protected]. Present Address §

Department of Applied Sciences, Indian Institute of Information Technology Allahabad, Allahabad 211012, India.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Department of Electronics and Information Technology, Government of India, for financial support through the project No. 5(9)/ 2012-NANO (Vol. II) and DBT Programme Support facility at IIT Guwahati (project no. BT/PR13560/COE/34/44/2015, Department of Biotechnology, Government of India) for providing facilities for experiments. G

DOI: 10.1021/acs.molpharmaceut.6b00480 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

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DOI: 10.1021/acs.molpharmaceut.6b00480 Mol. Pharmaceutics XXXX, XXX, XXX−XXX