Evaluation of MUC1-Aptamer Functionalized Hybrid Nanoparticles for

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Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Evaluation of MUC1-Aptamer Functionalized Hybrid Nanoparticles for Targeted Delivery of miRNA-29b to Nonsmall Cell Lung Cancer Maryna Perepelyuk, Koita Sacko, Karthik Thangavel, and Sunday A. Shoyele* Department of Pharmaceutical Sciences, College of Pharmacy, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, United States ABSTRACT: The objective of this study was to evaluate the therapeutic efficacy and pharmacokinetic study of mucin1-aptamer functionalized miRNA-29bloaded hybrid nanoparticles (MAFMILHNs) in lung tumor-bearing SCID mice. MAFMILHNs were manufactured using an isoelectric point based nanotechnology. They were then fully characterized for particle size, loading capacity, zeta potential, and encapsulation efficiency. The ability of MAFMILHNs to downregulate oncoprotein DNMT3B both at the cellular level and in vivo was monitored using Western blot, while the effect of the downregulation of DNMT3B on tumor growth was assessed using bioluminescence. Results indicate that the presence of MUC1-aptamer conjugated to the surface of the nanoparticles enhanced the selective delivery of miRNA-29b to tumor cells and tissues. Further, the downregulation of DNMT3B by MAFMILHNs resulted in the inhibition of tumor growth in mouse models. KEYWORDS: nanomedicine, lung cancer, miRNA-29b, gene-silencing, oncoproteins, aptamer

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INTRODUCTION MicroRNAs (miRNAs) are frequently utilized in gene therapy and molecular medicine as research tools. These classes of compounds have recently evolved as promising candidates for the treatment of a wide variety of diseases, such as cancer, and other genetic disorders. Due to their short sequence, miRNA are often able to form a perfect base-pairing with target mRNA (mRNA) to mediate mRNA degradation.1 Often, one miRNA is able to lead to the blockade of numerous pathways that modulate cell proliferation, differentiation, apoptosis, and invasion.1,2 miRNA-29b targets DNA methyltranferases (DNMTs) and regulates DNA demethylation, thus leading to inhibition of DNA methylation in malignant cells.1 Specifically, downregulation of DNMT3B led to inhibition of cell proliferation and apoptosis of nonsmall cell lung cancer (NSCLC) cells.3,4 In view of this, miRNA-29b is seen as a very attractive candidate for miRNA-based therapeutics in NSCLC. miRNAs have the ability to silence critical molecular pathways and possibly enhance the sensitivity of NSCLC to conventional chemotherapeutic agents. However, translational application of this therapeutic molecule is limited by many challenges, including stimulation of immune response, off-target effects, rapid blood clearance degradation in serum, and poor cellular uptake.5 In order to fully harness this treatment modality in NSCLC, a smart nanoparticle delivery system must be developed in tandem with the development of miRNAs. Recently, our group developed a MUC1-aptamer hybrid nanoparticle bioconjugate for targeted delivery of miRNA-29b to the cytosol of A549 adenocarcinoma cells.5 The hybrid © XXXX American Chemical Society

nanoparticle technology is composed of human immunoglobulin G (human IgG) and poloxamer-188 (polyoxyethylene− polyoxypropylene block copolymer). This was designed to achieve optimal delivery of nucleic acid based therapeutics.6,7 The idea here is to deceive the body into believing the nanoparticle is part of itself because of the presence of human IgG. The outer layer of these hybrid nanoparticles is composed of poloxamer-188, which helps to prevent engulfment by macrophages during systemic circulation.8,9 These nanoparticles were functionalized with MUC1aptamer to facilitate active targeting of miRNA-29b to MUC1-expressing cancer cells while avoiding undesirable accumulation in healthy cells. The presence of MUC1, a transmembrane protein that is aberrantly overexpressed in NSCLCs,10 allows for the active targeting of payload to lung adenocarcinomas. MUC1 has been shown to be overexpressed in 80% of lung adenocarcinoma.10 Previously, we have been able to show that these hybrid nanoparticles were able to protect loaded oligonucleotide in the serum in vitro;6 render in vivo protection of oligonucleotides in the blood;11 avoid clearance by macrophages;6 and deliver miRNA-29b to the cytosol of cancer cells by escaping endocytic recycling.5,6 Further, loaded miRNA-29b efficiently downregulated DNMT3B in A549 cells, inhibits cell proliferation, and initiated apoptosis in these cells.5 Received: Revised: Accepted: Published: A

October 16, 2017 February 7, 2018 February 12, 2018 February 12, 2018 DOI: 10.1021/acs.molpharmaceut.7b00900 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Figure 1. Preparation and characterization of MAFMILHNs. Schematic showing the procedure involved in the production of MAFMILHNs. Nanoparticles were prepared using an isoelectric point-based technology. SEM micrograph shows the MAFMILHNs were spherical in shape. SEM scale bar = 200 nm.

1% antibiotics. A549-luciferase was obtained from PerkinElmer (Waltham, MA). All cells were cultured in a humidified air atmosphere with 5% carbon dioxide. Animals. SCID beige mice, female, 8 weeks old, were obtained from Taconic Co. (Hudson, NY). These mice weighed approximately 25 g. They were maintained in Thomas Jefferson University’s AAALAC-accredited facility. The animal protocol for this study was approved by Thomas Jefferson’s Institutional Animal Care and Use Committee. Preparation and Characterization of MAFMILHN. MAFMILHNs were prepared according to our previously reported protocol.5 Briefly, 10 mg of human IgG and 0.73 mg of MiRIDIAN mimic miR-29b were dissolved in 0.01 N HCl. Complete dissolution of all the components was ensured by using a magnetic stirrer. NaOH (0.01 N) was then slowly added to the mixture, while the pH was closely monitored with a pH meter until nanoparticles were spontaneously formed. Nanoparticles were allowed to continuously mix on a magnetic stirrer for approximately 10 min. Nanoparticles were separated from the rest of the solution by centrifuging at 2000 rpm for 5 min. Unencapsulated miRNA-29b was determined in the supernatant using ion-pair HPLC.6,7 Nanoparticles were then rinsed three times with double distilled deionized water. To coat the nanoparticles with poloxamer-188, they were suspended in 0.2% v/v poloxamer-188. Nanoparticles were then lyophilized for 48 h. MUC1-aptamer was conjugated to hybrid nanoparticles by using our previously reported protocol.5 Schematic in Figure 1 shows the sequence involved in the preparation of

The objective of this present work is to extend our study to preclinical models and evaluate the ability of MUC1-aptamer functionalized miRNA-29b-loaded hybrid nanoparticles (MAFMILHNs) to selectively deliver miRNA-29b to lung tumor while limiting accumulation in healthy tissues. Further, we aim to evaluate the ability of MAFMILHNs to downregulate DNMT3B and inhibit lung cancer growth in vivo. We hypothesize that the very limited expression of MUC1 in healthy tissues will enable selective accumulation of miRNA29b loaded hybrid nanoparticles in NSCLC while avoiding healthy tissues. We further hypothesize that delivery of MAFMILHN will lead to in vivo downregulation of DNMT3B, consequently leading to regression of NSCLC in orthotopic mouse models.

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MATERIAL AND METHODS Material. Human IgG was purchased from Equitech-Bio Inc. (Kerrville, TX, USA). MUC1-aptamer was designed and purchased from GE Healthcare Bio-Sciences Corp. (Piscataway, NJ). The sequence of the aptamer is (5′-GCA GTT GAT CCT TTG GAT ACC CTG G-3′). Poloxamer-188, RNase-free water, and fetal bovine serum (FBS) were supplied by Thermo Fisher Scientific (Waltham, MA, USA). Both miRNA-29b and control miRNA were purchased from GE Healthcare BioSciences Corp. Pierce RIPA lysis buffer was purchased from Thermo Fisher Scientific. All primary and secondary antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Cell Culture. A549 cells were purchased from American Type Culture Collection (Rockville, MD). These cells were maintained in F12K medium supplemented with 10% FBS and B

DOI: 10.1021/acs.molpharmaceut.7b00900 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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extract protein from tissues. The concentration of protein was determined with Bradford Assay. Samples were run on NuPAGE 4−12% Bis-Tris gels (Life Technologies, Carlsbad, CA) according to the manufacturer’s instruction. Blocking was carried out according to manufacturer’s instructions (Invitrogen, Carlsbad, CA) for 1 h at room temperature and probed with rabbit antihuman DNMT3B monoclonal antibody (1:500) and mouse antihuman β-tubulin antibody (1:5000) overnight at 4 °C. The membranes were washed 5 min, thrice in wash buffer (Invitrogen, Carlsbad, CA). Goat antimouse antibodies, conjugated with horseradish peroxidase obtained from Molecular Probes (Eugene, OR), was used as a secondary antibody. Membranes were processed in a dark room on a table top processor SRX-101A (Konica Minolta, Japan). Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Analysis. TUNEL analysis was performed with TACS 2TdT-Fluor in Situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD) according to manufacturer’s instruction. Lung tissue section slides prepared by the pathology core of Thomas Jefferson University were deparaffinized in xylene and 100%, 95%, and 70% ethanol, followed by two changes of PBS. Samples were then digested with 50 μL of Cytonin solution for 30 min and washed with water and TdT Labeling Buffer. The slides were then covered with 50 μL of labeling reaction mix, which was then incubated for 60 min at 37 °C in a humidity chamber. Positive control was generated for comparison by incubating the slide with TACS-Nuclease (generating DNA breaks in every cell) at room temperature for 40 min, which was then followed by the labeling step. Stop Buffer was then used to halt the labeling reaction, and samples were washed in PBS and covered with 50 μL of StrepFluorescein Solution for 20 min. Slides were then washed with PBS, mounted, and viewed under fluorescence microscope at 495 nm. Cell Death Detection ELISA. Apoptosis was determined by using cell death detection ELISA (Roche, Mannheim, Germany). Tissue lysates were prepared based on a modified protocol by Leist et al.15 Two percent homogenate of lung tissue was prepared in incubation buffer. This was then centrifuged for 10 min at 15 000g. Supernatant was then collected and kept at −80 °C until ready to use. Plastic wells were coated with coating solution overnight in the refrigerator. Incubation buffer was then added to the wells after decanting the coating solution. The incubation buffer was left for 30 min before being decanted and washed three times with washing solution. Tissue homogenate (100 μL) was added in the wells and incubated for 90 min at 20 °C accompanied by mild shaking. After washing the wells three times, ABTS substrate was added to the wells for 30 min until the color developed. Absorption values were obtained in a microplate spectrophotometer (BioTek, Winooski, VT) with Gen5 1.10 software.

MAFMILHN. The appearance of MAFMILHNs was captured using SEM. Minimum Tolerated Dose Determination (MTD). The minimum tolerated dose to be used for preclinical work was determined by administering different doses to 12 female SCID beige mice, divided into four groups of three animals/group. Different doses, 0.6, 0.9, 1.5, and 2.5 mg/kg, of free miRMA29b were administered to these animals by intraperitoneal administration. These animals were monitored for a week for changes in their feeding, weight, and mortality. Mice treated with doses up to 1.5 mg/kg showed no change in weight and were generally healthy, and none died. However, two of the three mice treated with 2.5 mg/kg died after 4 days after a significant weight loss. Animal Treatment and Blood Sampling for Pharmacokinetic and Biodistribution Studies. Female SCID beige mice were injected with 5 × 106 A549-luciferase cells suspended in sterile PBS through tail vein intravenous injection to create metastatic models of NSCLC. Xenogen IVIS bioluminescence imaging system was used to monitor tumor growth by administering 100 μL of 30 mg/mL Xenolight Rediject D-luciferein intraperitoneally into the mice approximately 10 min before imaging. Once tumors were established, tumor-bearing mice were divided into three groups of three animals each. The first group was treated with MAFMILHNs containing an equivalent miRNA-29b dose of 1.5 mg/kg twice weekly. The second group was treated with MUC1-aptamer functionalized control miRNA-loaded hybrid nanoparticles (NC-nano) containing an equivalent control miRNA dose of 1.5 mg/kg twice weekly by intraperitoneal injection. The third group was treated with PBS twice weekly also, by peritoneal injection. Nanoparticles were dispersed in sterile PBS. All mice were treated for a total of 4 weeks. Tumor progression was evaluated using bioluminescence. For biodistribution and pharmacokinetic study, tumor bearing SCID beige mice were divided into two groups of three animals each. The first group was treated with a single dose of MAFMILHN loaded with an equivalent dose of 1.5 mg/kg miRNA-29b. The second group was treated with a single dose of nonfunctionalized miRNA-29b loaded hybrid nanoparticles loaded with an equivalent dose of 1.5 mg/kg. Blood samples were collected from each group over a 48 h period by retroorbital puncture into EDTA tubes. Animals were also euthanized 48 h after dose administration. The following organs were collected for analysis: lungs, heart, kidneys, and liver. miRNA-29b was isolated from tissues using Clarity OTX kit (Phenomenenex, CA) with Thermo Scientific HyperSep Vacuum Manifold according to manufacturer’s instruction. Samples were then concentrated to 100 μL with speed vacuum before ion-pair HPLC analysis.12−14 Pharmacokinetic (PK) Data Analysis. PK parameters were estimated by WinNonlin software version 6.0 (Pharsight, Mountain View, CA), using noncompartment analysis of the composite data. Hyperspectral Microscopy. Cells and lung tissue section slides were prepared for hyperspectral microscopy. Images of nanoparticles in cells and tissues were obtained by the CytoViva hyperspectral imaging system (Auburn, AL). Western-Blot Analysis. Lung tissues of treated mice were sampled for Western blot. These samples were kept at −80 °C until use. Pierce RIPA buffer (Thermo Scientific, Rockford, IL) containing Pierce Protease Inhibitor Mini Tablets was used to

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RESULTS Characterization of MAFMILHNs. MAFMILHNs prepared were examined for morphology using SEM. These nanoparticles were spherical in shape and well dispersed. Encapsulation efficiency (EE) and loading capacity (LC) were determined to be 98.8 ± 0.4% and 8.6 ± 0.1%, respectively. Prior to conjugation of MUC1-aptamer, the nonfunctionalized hybrid nanoparticles had a particle size of 236 nm and a polydispersity index (PDI) of 0.242. MAFMILHNs had a particle size of 595 nm and a PDI of 0.554. Zeta potential of C

DOI: 10.1021/acs.molpharmaceut.7b00900 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 2. Hyperspectral imaging of nanoparticle internalization. (A) MUC1-aptamer functionalized miR-29b-loaded hybridnanoparticles, (B) nonfunctionalized miR-29b-loaded hybrid nanoparticles, (C) untreated A549 cells, (D) untreated MRC-5 cells, (E) MUC1-aptamer miR-29b-loaded hybrid nanoparticles-treated A549 cells, (F) nonfunctionallized miR-29b-loaded hybrid nanoparticled-treated A549 cells, (G) MUC1-aptamer miR29b-loaded hybrid nanoparticles-treated MRC-5 cells, and (H) nonfunctionallized miR-29b-loaded hybrid nanoparticled-treated MRC-5 cells. (I) Flow cytometry measurement of nanoparticle uptake.

nonfunctionalized hybrid nanoparticles was −2.1. This figure increased to +4.1 following the conjugation of MUC1-aptamer to the surface of the hybrid nanoparticles to convert them to MAFMILHNs. To monitor the physical stability of MAFMILHN in PBS, nanoparticles were dispersed in PBS, and the size and PDI were measured. The particle size in PBS was 499.1 ± 0.3 with a PDI of 0.233 ± 0.02. Intracellular Uptake of MAFMILHNs. Cellular uptake was monitored with hyperspectral microscopy (Cytoviva Inc. Auburn, AL).16,17 Figure 2, shows micrographs captured by hyperspectral microscopy. Figures 2A and 2B, demonstrates the spherical morphology of both MAFMILHN and nonfunctionalized miRNA-29b-loaded hybrid nanoparticles. This is consistent with the image of the morphology obtained by SEM in Figure 1. Figure 2C and 2D show the untreated A549 and MRC5 cells respectively, showing lack of presence of nanoparticles in the cells. However, Figure 2E demonstrates the presence of numerous MAFMILHN in A549 cells following 2htreatment. This is in contrast to A549 cells in Figure 2F treated with nonfunctionalized hybrid nanoparticles, showing limited amount of internalized nanoparticles. This demonstrates the influence of MUC1-aptamer in the uptake of these nanoparticles. Figures 2G and 2H, demonstrates that both MAFMILHN and nonfunctionalized hybrid nanoparticles were not taken up significantly by MRC5 due to the limited expression of MUC1 in this cell. Nanoparticle uptake by A549 and MRC5 was quantitatively analyzed using flow cytometer. Figure 2I, demonstrates that MAFMILHN were efficiently internalized by A549 cells in comparison to MRC5. Further, nonfunctionalized nanoparticles were not efficiently internalized by both A549 and MRC5 cells.

Tissue Distribution and Pharmacokinetic Study. Following the harvest of essential organs from euthanized lung-tumor bearing SCID beige mice, the deposition of miRNA-29b in the heart, tumor-bearing lungs, kidneys, and liver was quantified. Figure 3A,B demonstrates the importance of MUC1-aptamer in ensuring that miRNA-29b loaded in MAFMILHN was preferentially delivered to tumor bearing lungs while limiting accumulation in healthy organs such as kidney, liver, and heart. In Figure 3A, delivery of miRNA-29b to tumor-bearing lung was significantly (P ≤ 0.001) greater than other organs. In the absence of MUC1-aptamer, as demonstrated in Figure 3B, nonselective delivery of miRNA-29b was observed by nonfunctionalized hybrid nanoparticles with more miRNA-29b detected in the liver and kidney than in the lungs. The delivery of miRNA-29b to tumor-bearing lung by MAFMILHNs was significantly (P ≤ 0.001) greater than that by nonfunctionalized miRNA-29b loaded hybrid nanoparticles. This demonstrates the importance of MUC1-aptamer in targeted delivery of miRNA-29b to lung tumor with minimal delivery to other organs. To visualize the presence of nanoparticles in the lungs, tissue sections of lungs obtained from the two experimental groups were imaged using hyperspectral microscopy. Figure 3C shows high deposition of MAFMILHNs in lung tumor of mice treated with these nanoparticles. In contrast, lung tumor of mice treated with nonfunctionalized miRNA-29b loaded hybrid nanoparticles show limited amount of nanoparticles (Figure 3D), further demonstrating the selective and specific delivery of miRNA-29b using MUC1-aptamer functionalized hybrid nanoparticles. Figure 4 shows the plasma concentration−time curves of miRNA-29b. The maximum concentration (Cmax) of miRNAD

DOI: 10.1021/acs.molpharmaceut.7b00900 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 3. (A) Tissue distribution of MAFMILHNs in the lung of tumor bearing mice. (B) Comparison of MAFMILHNs and nonfunctionalized miR-29b-loaded hybrid nanoparticles. ***P ≤ 0.001. (C) Hyperspectral imaging of MAFMILH. (D) Hyperspectral imaging of Nonfunctionalized miR-29b-loaded hybrid nanoparticles Scale bar = 10 μm.

Table 1. Comparison of the Pharmacokinetics of miRNA29b Loaded in MAFMILHN and Nonfunctionalized Hybrid Nanoparticles in Orthotopic Models of NSCLC parameters peak plasma concentration, Cmax (ng/mL) time to peak plasma concentration, Tmax (h) area under the plasma concentration− time curve from time zero to time of last measurable concentration, AUClast (h·ng/mL) apparent clearance, CL/F (mL/min/kg) elimination rate constant, λz (1/h) plasma terminal half-life, T1/2 (h) mean residence time, MRT (h) apparent volume of distribution, VZ/F (L/kg)

Figure 4. miRNA-27b plasma concentration over time. Tumor bearing mice were given a single dose of 1.5 mg/kg of miRNA-29b in MAFMILHNs or in nonfunctionalized miRNA-29b-loaded hybrid nanoparticles. n = 3.

29b delivered MAFMILHNs was 15289.5 ng/mL, slightly lesser than 18289.5 ng/mL observed for the nonfunctionalized miRNA-29b-loaded hybrid nanoparticles. These maximum concentrations were observed at 60 min (Tmax) for both nanoparticles. Table 1 demonstrates the similarities in the pharmacokinetics of both MAFMILHN and nonfunctionalized miRNA-29bloaded hybrid nanoparticles. Both MAFMILHN and nonfunctionalized miRNA-29b-loaded hybrid nanoparticles produced a half-life of 13.3 and 14.6 h, respectively. Further, both

MAFMILHN

nonfunctionalized hybrid nanoparticles

15289.5

18289.5

1.0

1.0

443991.8

540341.8

5.04 × 10−8 0.05208 13.3 16.4 5.8 × 10−5

4.04 × 10−8 0.04763 14.6 16.7 5.1 × 10−5

formulations produced similar mean residence time (MRT) and apparent clearance. No significant difference (P ≥ 0.001) was found in all the PK parameters between MAFMILHNs and nonfunctionalized miRNA-29b-loaded hybrid nanoparticles Downregulation of DNMT3B by MAFMILHN. The ability of MAFMILHNs to downregulate the oncoprotein DNMT3B was elucidated both at the cellular and tissues levels. Figure 5A demonstrates that MAFMILHN effectively downregulated DNMT3B in A549 cells, in a superior manner when E

DOI: 10.1021/acs.molpharmaceut.7b00900 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 5. Downregulation of target oncoprotein DNMT3B. (A) Western blot analysis of DNMT3B downregulation in A549 cells. (B) In vivo evaluation of DNMT3B downregulation in tumor bearing lungs of SCID mice. miR29b-lipo represents lipofectamine transfected miRNA-29b.

Figure 6. Apoptosis in treated tumor-bearing lungs. (A) Evaluation of apoptosis using TUNEL. The tumor-bearing lung of mice treated with MAFMILHN showed higher level of apoptosis, while tumor-bearing lungs of mice treated with both negative control (NC)-loaded hybrid nanoparticles and PBS did not show any apoptosis. (B) Evaluation of apoptosis using cell death detection ELISA. ***p ≤ 0.001, n = 3. The result obtained with TUNEL analysis was replicated with cell death detection ELISA. O.D. stands for optical density.

compared to MUC1-aptamer functionalized negative control miRNA-loaded hybrid nanoparticles (NC-nano) and lipofectamine-miRNA-29b (miRNA-29b lipo). To confirm that DNMT3B could be downregulated in vivo, lungs harvested from SCID mice treated with MAFMILHNs, NC-nano, and phosphate buffered saline (PBS) were analyzed for DNMT3B levels using Western blot. Figure 5B demonstrates that DNMT3B was effectively downregulated in tumor-bearing lung of SCID mice treated with MAFMILHN. In contrast, both NC-nano and PBS were not effective in downregulating DNMT3B in treated mice. Initiation of Apoptosis in Lung Tumor. The ability of MAFMILHNs to induce apoptosis in lung tumor was evaluated using both TUNEL assay and cell death-detection ELISA. The lungs of tumor-bearing mice treated with MAFMILHNs demonstrated a high level of apoptosis as measured by TUNEL in Figure 6A. Conversely, mice treated with NCnano and PBS demonstrated very a low level of apoptosis. Results generated from TUNEL assay were validated with cell

death-ELISA data presented in Figure 6B. Similar to the TUNEL results, cell death ELISA showed that apoptosis in the lungs of mice treated with MAFMILHNs was significantly higher than in the lungs of mice treated with NC-nano and PBS. Antitumor Effect of MAFMILHN in Mice Models. Tumor suppression capability of MAFMILHNs was evaluated using IVIS bioluminescence imaging system to monitor tumor burden in tumor-bearing SCID mice over a four-week period. Figure 7 demonstrates the ability of MAFMILHN to suppress tumor growth when compared to different controls. In Figure 7A, the intensity of bioluminescence was quantified and plotted against time. While the tumor in mice treated with NC-nano and PBS continued to grow over the study period, tumor in mice treated with MAFMILHN decreased in intensity over the same period. Representative bioluminescence images are shown in Figure 7B. These images are constituent with the graphical presentation in Figure 7A. F

DOI: 10.1021/acs.molpharmaceut.7b00900 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 7. In vivo antitumor effect in SCID beige mice monitored using IVIS bioluminescence imaging system. (A) Graph of tumor burden over four week period. Bioluminescence intensity was plotted against time. MAFMILHN was able to inhibit tumor growth in treated animals in comparison to controls. (B) Representative bioluminescence images of tumor-bearing mice over 4-week treatment period.

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DISCUSSION

could lead to agglomeration of particles, hence the increase of particle size. This is further reflected in the PDI increase from 0.243 to 0.554 following the aptamer conjugation procedure. Intracellular delivery of the nanoparticles in both A549 cells and lung tumor tissues was probed using hyperspectral microscopy. Hyperspectral microscopy combines hyperspectral imaging (HSI) with advanced optics, which allows it to focus on specialized dark-field reflectance systems.16,17 HSI has been used as a tool for detecting nanoparticle state in biological systems.16,17 Hyperspectral microscopy data in Figure 2 confirms the selective delivery of MAFMILHN to mucin expressing cells in comparison to cells with limited mucin-1 expression. Reverse transcription PCR was previously used to compare the expression of mucin-1 in lung adenocarcinoma cells, A549, and normal healthy lung cells, MRC-5.5 As reported, A549 cells showed very high level of expression of mucin-1 as compared to MRC-5, which showed very limited level of mucin-1. Hyperspectral microscopy showed that MAFMILHNs were preferentially delivered to A549 cells when compared to MRC-5. This correlates nicely with the comparative level of mucin-1 in both cells. Further, hybrid

miRNAs are potential therapeutic agents for NSCLC due to their ability to downregulate different pathways that drive cancer.2 They participate as backups of transcriptional control in signaling networks.18 miRNA-29b has been shown to target DNMTs, which subsequently leads to the inhibition of global DNA methylation in malignant cells.19 Previously, our research group was able to show that by incorporating miRNA-29b in our MAFMILHNs, oncogenes DNMT3B and MCL-1 can be effectively downregulated in A549 cells, subsequently leading to apoptosis and antiproliferative effect.5 The objective of this present work was to extend our study to animal models and evaluate the ability of MAFMILHNs to selectively deliver miRNA-29b to lung tumor while limiting accumulation in healthy tissues. We also carried out PK studies to determine some critical PK parameters for MAFMILHNs. Conjugation of MUC1-aptamer to the hybrid nanoparticles increased the particle size from 236 to 595 nm. This increase could be due to the presence of the aptamer in the nanoparticles coupled with extra steps involved in the conjugated process, especially the use of lyophilization, which G

DOI: 10.1021/acs.molpharmaceut.7b00900 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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CONCLUSION On the basis of all the generated data, we conclude that MUC1aptamer functionalized hybrid nanoparticles can potentially be used as a platform-targeted nanoparticle delivery system for efficient delivery of miRNAs, especially miRNA-29b to nonsmall cell lung cancer for the downregulation of target oncogene.

nanoparticles that were not conjugated with MUC1-aptamer were not efficiently delivered to either A549 cells or MRC-5. The effect of MUC1-aptamer on the selective delivery of MAFMILHNs to tumor tissues in mouse models was compared to nonfunctionalized hybrid nanoparticles. MAFMILHNs were found to favorably deliver miRNA-29b to tumor-bearing lung tissues in contrast to heart liver and kidney in Figure 3A. However, the nonfunctionalized hybrid nanoparticles were not able to achieve this in Figure 3B. Further, hyperspectral microscopy images in Figure 3C,D were able to confirm that MAFMILHN were indeed accumulated in tumor bearing lung tissues, while such could not be said for nonfunctionalized hybrid nanoparticles. The liver and kidneys also had minor amounts of miRNA-29b delivered to them by MAFMILHN. The liver, among other organs, such as spleen, belongs to the reticuloendothelial endothelial system organs.20 This is probably because liver, being a highly perfused organ, enables rapid distribution of nanoparticles to this organ, whether the nanoparticle is targeted or not.20,21 In addition, large fenestrations of the microvessels of liver often permits entry of particles up to 200 nm.22 The kidney also demonstrated a limited amount of miRNA-29b from MAFMILHN. This is probably because it is the main organ for elimination. Further, the kidney is a highly perfused organ. Although, a limited amount of miRNA-29b was delivered to tumor-bearing lungs by nonfunctionalized hybrid nanoparticles regardless of these nanoparticles not being functionalized by MUC1-aptamer. Enhanced permeation and retention effect could be responsible for the accumulation of nonfunctionalized hybrid nanoparticles in the tumor-bearing lungs.20 The pharmacokinetic parameters of both MAFMILHNs and nonfunctionalized hybrid nanoparticles were very similar suggesting that, although the conjugation of MUC1-aptamer to the nanoparticles in MAFMILHN enhanced the discriminatory delivery of miRNA-29b to lung tumor tissues, it did not influence the pharmacokinetics to any significant extent when compared to the nonfunctionalized hybrid nanoparticles. This is expected since the presence of the nanoparticles in both formulations protected the loaded miRNA-29b from marauding endonuclease enzymes and phagocytes, hence helping to enhance the circulation time of miRNA-29b in the blood. Western blot was used to monitor DNMT3B in both in vitro and in vivo models. As demonstrated in Figures 5A, DNMT3B was downregulated in A549 cells following treatment with MAFMILHNs. miRNA-29b delivered with lipofectamine as well as MUC1-aptamer functionalized control miRNA-29bloaded hybrid nanoparticles were not as effective. This result consisted in SCID mice (in vivo), as demonstrated by the result in Figure 5B in which MAFMILHN was able to downregulate the expression of DMNT3B. DNMT3B is a member of the DNA methyltrasferase family that accounts for the inactivation of tumor-suppressor genes in many cancer cells.5,23 However, miRNA-29b directly targets DNMT3B in cancer cells, which subsequently leads to the suppression of tumor growth.5,6,23 Hence, we hypothesized that the downregulation of DNMT3B would lead to the induction of apoptosis in tumor tissues, hence leading to the inhibition of tumor growth in SCID mouse models. Figures 6 and 7 strongly confirm our hypothesis as apoptosis was observed in treated mice, which consequently led to the inhibition of tumor growth in the animals.

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AUTHOR INFORMATION

Corresponding Author

*Address: Department of Pharmaceutical Sciences, College of Pharmacy, Thomas Jefferson University, Room 908, 901 Walnut Street, Philadelphia, Pennsylvania 19107, United States. Tel: 215-503-3407. Fax: 215-503-9052. E-mail:sunday. shoyele@jefferson.edu. ORCID

Sunday A. Shoyele: 0000-0002-7856-3538 Notes

The authors declare the following competing financial interest(s): S.S. is the Chief Scientific Officer for Atocan LLC, licensee of the nanoparticle technology used in this study.

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ACKNOWLEDGMENTS This project was supported by the QED grant number: S1402 awarded to S.A.S. by the Science Center, Philadelphia, and Thomas Jefferson University.

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DOI: 10.1021/acs.molpharmaceut.7b00900 Mol. Pharmaceutics XXXX, XXX, XXX−XXX