Combined Local Pulmonary and Systemic Delivery of AT2R Gene by Modified TAT Peptide Nanoparticles Attenuates Both Murine and Human Lung Carcinoma Xenografts in Mice

Journal of Pharmaceutical Sciences xxx (2016) 1-10

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Pharmaceutical Nanotechnology

Combined Local Pulmonary and Systemic Delivery of AT2R Gene by Modified TAT Peptide Nanoparticles Attenuates Both Murine and Human Lung Carcinoma Xenografts in Mice Susumu Ishiguro 1, Nabil A. Alhakamy 2, Deepthi Uppalapati 1, Jennifer Delzeit 1, Cory J. Berkland 2, 3, Masaaki Tamura 1, * 1 2 3

Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506 Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047 Department of Chemical and Petroleum Engineering, University of Kansas, Lawrence, Kansas 66047

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 May 2016 Revised 29 July 2016 Accepted 25 August 2016

To evaluate the potential of cell-penetrating peptide-based delivery of apoptosis-inducer gene in cancer therapy, a modified HIV-1 TAT peptide (dimerized TAT peptide, dTAT) was studied. The dTAT and plasmid DNA (pDNA) complexes (dTAT-pDNA) were condensed using calcium chloride (dTAT-pDNA-Ca2þ). This simple nonviral formulation approach showed high levels of gene expression in vitro without any cytotoxicity. In mouse studies, a single intratracheal (IT) aerosol spray or 2 intravenous (IV) injections of the dTAT, apoptosis-inducer gene, angiotensin II type 2 receptor (AT2R), and Ca2þ complexes (dTATpAT2R-Ca2þ) significantly attenuated the acutely growing mouse Lewis lung carcinoma allografts in mouse lungs. Furthermore, single IT (p ¼ 0.054) and the combination of IT and IV (p < 0.05) administrations of dTAT-pAT2R-Ca2þ markedly attenuated slowly growing and relatively large-sized H358 human bronchioloalveolar carcinoma xenografts in mouse lungs. These results indicate that the dTATpDNA-Ca2þ effectively delivered the gene to cancer cells by either IT or IV administration although the local pulmonary delivery of the dTAT-pAT2R-Ca2þ showed more effective growth inhibition of orthotopic lung cancer grafts. Thus, the present study offers preclinical proof of concept that a dTAT-based nonviral gene delivery method via IT administration may be an effective lung cancer gene therapy. © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Keywords: dTAT peptide nanoparticles nonviral gene delivery lung cancer angiotensin II type 2 receptor apoptosis

Introduction Although lung cancer prognosis has improved because of advances in diagnosis and early surveillance, more than half (57%) of the patients are diagnosed at an advanced stage and the 1- and 5-year survival rate is 26% and 4%, respectively.1 Gene therapy has become a promising approach for the treatment of numerous diseases including cancers that are considered incurable.2 Although the lungs may be accessed via intravenous (IV) administration or inhalation, biologic obstacles continue to slow the advance of lung cancer therapies.3 The sensitivity to enzymatic degradation and the poor permeability of nucleic acids considerably complicate the

The authors Susumu Ishiguro and Nabil A. Alhakamy contributed equally to this work. * Correspondence to: Masaaki Tamura (Telephone: 785-582-4825; Fax: 785-5825777). E-mail address: [email protected] (M. Tamura).

development of most gene therapy strategies. Thus, a successful gene therapy strategy largely depends on the design of efficient and safe vectors.4-8 A great deal of effort has been devoted to developing successful viral and nonviral gene delivery systems capable of improving upon a variety of limitations, including in vivo instability, low gene transfection efficiency, and toxicity.9 Viral gene therapy has dominated clinical applications, but nonviral gene therapy has been given significant attention as a gene therapy method because of the low cost, ease of synthesis, and potential for lower immunogenicity in comparison to viral methods. Plasmid DNA (pDNA) or small interfering RNA (siRNA) combined with cationic lipids (lipoplexes) or polymers (polyplexes) to form complexes is the most commonly employed nonviral gene method.9-16 Nonviral vectors often suffer from a lower transfection efficiency compared to viral vectors,2 yet numerous strategies have been put forward to advance nonviral gene delivery. Cell-penetrating peptides (CPPs) appear to be a particularly promising component of nonviral gene therapies. CPPs consist

http://dx.doi.org/10.1016/j.xphs.2016.08.023 0022-3549/© 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

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of 30 amino acids (cationic or amphiphilic in nature), which can mediate transport across the cell membrane.17-19 The charge, size, and molecular weight of CPPs have a significant role in condensing and delivering genetic material to the target cells.17 Two main approaches have been investigated: covalent coupling (chemical linkage) and noncovalent coupling (electrostatic interactions) of CPPs with pDNA or siRNA.20-23 When using noncovalent coupling, strong binding between CPPs and pDNA is essential to stabilize the resulting complexes and to achieve high levels of gene expression; however, binding between CPPs and pDNA must not be too strong, to facilitate the release of the cargo after cellular uptake. One particular CPP of interest is TAT derived from HIV (RKKRRQRRR), which has been demonstrated to translocate across cell membranes.17,20,24,25 Here, a longer “double” TAT (dTAT, RKKRRQRRRHRRKKR) was investigated as a potential carrier for pDNA. When used alone, the transfection efficiency of the dTAT-pDNA complexes is quite low; however, calcium has been shown to regulate the delicate balance of binding strength within polyplexes.9,17,20 The addition of calcium chloride (CaCl2) to the dTAT-pDNA complexes directly affects particle size and gene expression.9 In the present study, a simple formulation (dTAT-pDNA-Ca2þ complex) was optimized using 3 different human cell lines: (1) A549 (a lung cancer cell), (2) HeLa (a cervical cancer cell), (3) HEK293 (a virus-immortalized kidney cell) and one mouse cell line, Lewis lung carcinoma (LLC, a lung cancer cell) using a luciferasereported pDNA (pLUC) to evaluate transfection efficiency. Next, the formulation was tested in vivo (LLC tumor-bearing mice) using apoptosis-inducer gene pDNA: angiotensin II type 2 receptor pDNA (pAT2R). AT2R is recognized to inhibit cell proliferation and stimulate apoptosis in various cells (e.g., neuronal, endothelial, prostate, and lung cancer cells).3,25,26 The dTAT-pAT2R-Ca2þ complexes were administered intravenously or via intratracheal (IT) aerosol spray to determine lung cancer attenuation in acutely growing murine lung carcinoma (LLC) and slowly growing human bronchioloalveolar carcinoma (H358) graft-bearing mice. Materials and Methods Materials pDNA encoding firefly luciferase (pGL3, 4818 bp) was obtained from Promega (Madison, WI). pDNA encoding human AT2R (agtr2 pcDNA3.1þ) was obtained from the UMR cDNA Resource Center (University of Missouri, Rolla, MO). The pDNA purity level was determined by UV spectroscopy and agarose gel electrophoresis. dTAT (RKKRRQRRRHRRKKR; molecular weight ¼ 2201.7 Da) peptide was purchased from Biomatik USA, LLC (Wilmington, DE). Branched polyethylenimine (PEI, 25 kDa) was obtained from Sigma-Aldrich (Milwaukee, WI). A549 cell line (human lung carcinoma), LLC (mouse Lewis lung carcinoma), HeLa cell line (human cervix adenocarcinoma), and H358 cell line (human bronchioloalveolar carcinoma) were obtained from American Type Culture Collection (Rockville, MD). HEK-293 (human embryonic kidney) cell line was a gift from Dr. Nikki Cheng (University of Kansas Medical Center, Lawrence, KS). Calcium chloride dihydrate (CaCl2$2H2O) was purchased from Fisher Scientific (Pittsburgh, PA). Mouse serum albumin (MSA) and glucose were obtained from Sigma-Aldrich. Preparation of the dTAT-pDNA-Ca2þ Nanoparticles For the in vitro studies, the dTAT-pDNA nanoparticles were prepared by adding 15 mL of dTAT solution (N/P 10, different

polymer nitrogen to pDNA phosphate [N/P] ratios) to 10 mL (0.1 mg/mL) of pDNA (Tris-acetate-EDTA [TAE] buffer [1x] was used as a solution for DNA storage), followed by fast pipetting for 20 s. At that point, 15 mL of identified molarity (e.g., 50, 300, and 600 mM) CaCl2 was added and mixed by fast pipetting. The total volume was 40 mL. After preparing the nanoparticles, they were stored at 4 C for 20-25 min. For the mouse studies with IV administration, the dTAT-pDNA nanoparticles were prepared by adding 60 mL (0.88 mg/mL) of dTAT solution to 40 mL (0.1 mg/mL) of pDNA (pAT2R or pLUC; TAE buffer [1x] was used as a solution for DNA storage), followed by fast pipetting for 20 s. At that point, 60 mL of identified molarity (100 mM) CaCl2 was added and mixed by fast pipetting. Then, 40 mL of MSA (1%) was added to the solution. The total volume was 200 mL. After preparing the nanoparticles, they were stored at 4 C for 20-25 min. For the mouse studies with IT administration, the dTAT-pDNA nanoparticles were prepared by adding 15 mL (0.88 mg/mL) of dTAT solution to 10 mL (0.1 mg/mL) of pDNA (pAT2R or pLUC; TAE buffer [1x] was used as a solution for DNA storage), followed by fast pipetting for 20 s. At that point, 15 mL of identified molarity (100 mM) CaCl2 was added and mixed by fast pipetting. Then, 10 mL of glucose (10%) was added to the solution. The total volume was 50 mL. After preparing the nanoparticles, they were stored at 4 C for 20-25 min. Preparation of the PEI-pDNA Nanoparticles The PEI-pDNA nanoparticles were prepared by adding 15 mL of PEI solution (N/P 10) to 10 mL (0.1 mg/mL) of pDNA followed by fast pipetting for 20 s. After preparing the nanoparticles, they were stored at 4 C for 20-25 min. The nanoparticles were prepared immediately before each experiment. Agarose Gel Electrophoresis The nanoparticles were prepared as defined previously and subsequently: 4 mL of TAE buffer was added to the nanoparticles. Then, 4 mL of SYBR® Green 1 was mixed with the nanoparticles. Afterward, the mixture was stored at 4 C for 20-25 min. After storage, 7 mL of 6X DNA Loading Dye was added. A 1-kb DNA ladder was used as a reference marker. The mixture of the solutions was loaded onto a 1% agarose gel and electrophoresed for 30 min at 110 V. Size and Zeta Potential The particle size (effective diameter [nm]) of the dTAT-pDNA nanoparticle with or without calcium chloride was determined by dynamic light scattering (Brookhaven Instruments, Holtsville, NY). The zeta potentials of the nanoparticles were measured by Zeta PALS dynamic light scattering (Brookhaven Instruments). All samples intended for particle size measurements were prepared using PBS, nuclease-free water (NFW), and serum-Free media (SFM). All samples intended for zeta potential measurements were prepared using KCL (1 mM). Cell Culture A549, HeLa, LLC, HEK-293, and H358 cell lines were grown in F-12K Nutrient Mixture media (Kaighn's modified with L-glutamine; Mediatech, Inc., Manassas, VA) for A549; Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, Grand Island, NY) for HeLa, LLC, and HEK-293; and RPMI 1640 medium (Mediatech, Inc.) with 1% (v/v) penicillin/streptomycin and 10% (v/v) fetal bovine serum at 37 C in 5% CO2 humidified air.

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Transfection Studies A549, HeLa, LLC, and HEK-293 cell lines were cultured in 96-well plates for 24 h before transfection. The concentration of the cells in every well was approximately 100,000 cells/mL. The wells were washed once with SFM, and afterward, a 100 mL sample (which consisted of 20 mL of nanoparticle and 80 mL of SFM) was added to each well. Subsequently, a 96-well plate was incubated for 5 h in an incubator. After 5-h incubation, the sample was replaced with 100 mL of fresh serum medium and then incubated again for approximately 48 h. To determine the gene expression of the nanoparticles, the Luciferase Reporter Assay using Luciferase Assay System Freezer Pack (Promega) was conducted. The results of the transfections were expressed as relative light units per milligram of cellular protein, and PEI-pDNA was used as a control. Bicinchoninic acid Protein Assay Reagent (bicinchoninic acid) from Thermo Fisher Scientific Inc. (Waltham, MA) was used to measure total cellular protein concentration in the cell extracts. The Luciferase Assay and bicinchoninic acid assay were measured by a microplate reader (SpectraMax; Molecular Devices LLC., Sunnyvale, CA). HEK-293 and LLC cells are semiadherent and can easily lift from the growth surface during the transfection assays that required multiple washes. The gelatin solution was used to coat the 96-well plate for the culture of HEK-293 and LLC cells. The HEK-293 and LLC cells plates were prepared by adding 300 mL of gelatin solution (2%) to each well followed by incubation for at least 1 h at 37 C. The wells were washed 2 times with sterile water. After washing, the plate was left to dry in the TC hood for 30-60 min before use. Cytotoxicity Assay of dTAT, PEI, CaCl2, and dTAT-pDNA-Ca2þ Nanoparticles Cytotoxicity of dTAT, PEI, CaCl2, and dTAT-pLUC-Ca2þ nanoparticles at N/P 10 with 38 mM CaCl2 was determined using a CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (membrane translocalization signal [MTS]) obtained from Promega. A549, HeLa, HEK-293, and LLC cells were cultured in a 96-well plate as described previously. After 24 h of incubation, the media were replaced with a sample consisting of 100 mL of fresh serum medium and 20 mL of MTS. Then, the plate was incubated for 3 h. To determine cell viability, the absorbance of each well was measured by a microplate reader at 490 nm and normalized to untreated control cells.

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IV administration of the dTAT-pDNA-Ca2þ complexes, 160 mL complex solution was mixed with 40 mL 1% MSA. For the IT administration, 40 mL of the dTAT-pDNA-Ca2þ complex solution was mixed with 10 mL 10% glucose for the osmolality adjustment. In the allograft study, 7 days after the LLC cell inoculation, the mice were treated with either IV, IT, or both IV and IT. In the xenograft study, 7 days after the second H358 cell inoculation, the mice were treated with either IV, IT, or both IV and IT. The IV administration was always conducted twice with a 3-day interval. PBS and dTAT-Ca2þ solutions without pDNA were used as the control. Mice were sacrificed by cervical dislocation under anesthesia 14 days after the complex treatment. The lungs were dissected, and tumor burden was analyzed. Immunohistochemical Analysis for AT2R Expression in Lung Tumor Grafts Lung tissues fixed with 10% buffered formalin were sectioned at 4 mm and stained with hematoxylin and eosin (H&E) for histologic examination. To analyze AT2R expression in both LLC and H358 tumor grafts, sections were deparaffinized, and heatinduced epitope unmasking was performed in the citrate buffer followed by incubation with 3% H2O2/methanol for 3 min to block endogenous peroxidase activity. Sections were incubated with polyclonal anti-AT2R antibodies (1:200 dilution, for 18 h at 4 C; Santa Cruz Biotechnology, Inc., Dallas, TX). After the incubation with primary antibodies, sections were incubated with a biotinconjugated antirabbit IgG antibody (Vector Laboratories, Burlingame, CA) at a 1:100 dilution for 1 h at 37 C, followed by a reaction with the avidin-biotin-peroxidase complex reagent (Vector Laboratories) for 40 min at 37 C. Reactions were developed with 3, 30-diaminobenzidine tetrahydrochloride (Sigma), and the sections were counterstained lightly with Mayer hematoxylin. Statistical Analysis Data were analyzed using GraphPad software. All values are expressed as the mean ± SD of the mean. All experiments were conducted with multiple sample determinations. A statistical evaluation comparing the significance of the difference in gene expression (relative light units per milligram of protein) between the means of 2 data sets was performed using t-test. One-way ANOVA and Tukey post hoc test were used to analyze the differences when more than 2 data sets were compared.

Animals Results Six-week-old male C57BL/6 and C.B-17 SCID mice were obtained from Charles River Laboratories International, Inc. All mice were housed in a clean facility and held for 10 days to acclimatize. All animal experiments were approved by the Institutional Animal Care and Use Committee and carried out under strict adherence to the Institutional Animal Care and Use Committee protocols and the Institutional Biosafety Committee set by Kansas State University (Manhattan, KS). Preparation of Lung Cancer Grafts in Syngeneic and Xenogeneic Mice and Treatment With dTAT-Apoptosis-Inducer Gene Complex For syngeneic lung cancer mouse model, 7-week-old C57BL/6 mice were IV injected with 1.2  106 LLC cells/200 mL PBS via the tail vein using a 1 mL syringe with a 27-ga needle. For xenogeneic lung cancer mouse model, C.B-17 SCID mice were IV injected twice with 1.0  106 and 1.2  106 H358 cells suspension in 200 mL PBS with an 8-week interval. The dTAT-pDNA-Ca2þ complex solution was prepared immediately before injection as described previously. For the

Formation and Physical Characterization of the dTAT-pDNA-Ca2þ Nanoparticles The dTAT-pDNA-Ca2þ and the PEI-pDNA nanoparticles were prepared by mixing pLUC with each polycation at various N/P ratios as described in the Materials and Methods section. To demonstrate nanoparticle formation, agarose gel electrophoresis assay was performed using 1% agarose gel and electrophoresed for 30 min. Uncomplexed pLUC (naked pLUC) was used as a control. The dTAT nanoparticles showed the ability to form stable complexes with pLUC regardless of the presence of 38 mM calcium chloride at N/P 5, 10, and 30. As the net charges of these complexes are positive, the complexes stayed in the loading wells without migrating into the gel, and no bands were observed in electrophoresis (Fig. 1a). Although lower N/P ratios (N/P 1-4) also showed no bands in the absence of calcium chloride, the N/P ratios <0.5 showed bands (Fig. 1b). Furthermore, mixing with calcium chloride and pLUC did not form stable complexes (data not shown).

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Figure 1. Complex formulation of dTAT and pLUC at N/P ratios of 5, 10, and 30 was determined by agarose gel electrophoresis in the presence or absence of 38 mM CaCl2 (a) or in the absence of calcium chloride at N/P ratios of 4, 3, 2, 1, 0.5, or 0.1 (b). In panel (a), A-C refer to N/P 5, 10, and 30 in the presence of 38 mM CaCl2, respectively, whereas a-c refer to N/P 5, 10, and 30 in the absence of calcium chloride, respectively. M ¼ size marker. (c) Evaluation of the particle sizes (diameters) at an N/P ratio 10 in the presence of calcium chloride (0, 38, and 114 mM) was determined by dynamic light scattering (DLS) in nuclease-free water (NFW) or SFM. (d) Zeta potentials of the dTAT-pLUC and PEI-pLUC complexes at an N/P 10 were determined by Zeta PALS DLS in the presence of various concentrations of calcium chloride (0, 38, and 114 mM). Results are presented as mean ± SD (n ¼ 3).

The effect of calcium chloride concentration on the particle size and the surface charge of the dTAT-pLUC complexes were also investigated. As shown in Figure 1c, addition of calcium chloride of 38 and 114 mmol/L (final concentration) significantly decreased the particle size of the dTAT-pLUC-Ca2þ complexes, with relatively narrow polydispersity (0.1), in both nuclease-free water (475 and 446 nm at 38 and 114 mM CaCl2, respectively) and SFM (382 and 321 nm at 38 and 114 mM CaCl2, respectively). The zeta potential of both the dTAT-pLUC-Ca2þ and PEI-pLUC complexes increased significantly with increases in the concentration of calcium chloride (Fig. 1d). The increases were recorded from 15.5 to 22.7 for the dTAT complexes and 13.1 to 32.2 mV for the PEI complexes. The dTAT-pLUC-Ca2þ Nanoparticle Caused Efficient Gene Transfection With Low Cytotoxicity In Vitro The in vitro transfection efficiency of the dTAT-pLUC-Ca2þ nanoparticles was studied using the 3 different human cancer cell lines including A549, HeLa, HEK-293, and LLC. Luciferase gene expression was evaluated 48 h after the transfection using the dTAT-pLUC-Ca2þ nanoparticles at N/P 5, 10, 20, and 30 and at various calcium chloride concentrations during the complex formulation (Fig. 2a). The dTAT-pLUC-Ca2þ nanoparticles had a high level of gene expression at higher calcium chloride concentration from 38 to 114 mM in all 4 N/P ratios tested in A549 cells. This result was also confirmed in other cell lines including A549 cells, HeLa cells, HEK-293 cells, and LLC cells (Figs. 2b-2e). In all cell lines

examined, the transfection efficiency of the dTAT-pLUC-Ca2þ was significantly higher than those by PEI-pLUC. The significance of the dTAT peptide inclusion in pLUC transfection was further examined by comparing pLUC expression efficiency in the presence or absence of the peptide in various cell lines including A549 cells, HeLa cells, HEK-293 cells, and LLC cells (Fig. 2f). The transfection efficiency of the pLUC-Ca2þ complexes (without the dTAT peptide) was significantly lower than that of the dTAT-pLUC-Ca2þ complexes at the same calcium chloride concentration. High transfection efficiency and low cytotoxicity are the essential components of gene transfection vector and keys to successful gene therapies. To examine whether dTAT, PEI, and calcium chloride affect the viability of live cells, a MTS cytotoxicity assay was carried out using 4 different cell lines. The 4 cell lines were individually incubated with up to 5 mg/mL dTAT, PEI, or calcium chloride for 24 h, and then, MTS assay was performed (Fig. 3). The dTAT peptide showed no cytotoxicity to 1.0 mg/mL for A549 cells and approximately 0.3 mg/mL for HeLa and LLC cells. Only HEK-293 cell viability was gradually decreased at lower concentrations of the dTAT peptide (10 mg/mL). Calcium chloride also did not show strong cytotoxicity until 1 mg/mL level. However, PEI induced significant cytotoxicity even at 1 mg/mL in 3 cell lines including A549, HeLa, and LLC cells. Although HEK-293 cells were resistant to PEI-induced cytotoxicity, their cell viability gradually decreased at higher concentrations. Furthermore, the cytotoxicity of the dTAT-pDNA-Ca2þ complexes on 4 different cell lines was evaluated using firefly

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Figure 2. (a) The transfection efficiency of dTAT-pLUC nanoparticles was determined in the different concentrations of CaCl2 (0, 19, 38, 76, and 114 mM) in A549 cells at N/P ratios of 5, 10, 20, and 30. (b-e) The transfection efficiencies of the dTAT-pDNA nanoparticles with different concentrations of CaCl2 (0, 38, and 114 mM) at an N/P 10 in A549 cells (b), HeLa cells (c), HEK-293 cells (d), and LLC cells (e) were determined. The PEI-pDNA nanoparticle at an N/P 10 was used as a positive control. Results are presented as mean ± SD (n ¼ 4). *p < 0.05, **p < 0.001, ***p < 0.0001 were evaluated by 1-way ANOVA followed by Tukey post hoc analysis as indicated in the figure panels. (f) The transfection efficiency of dTAT-pLUC-Ca2þ (shaded bars) and pLUC-Ca2þ (white bars) nanoparticles was determined at an N/P 10 in the presence of 38 mM CaCl2 in A549, HeLa, HEK-293, and LLC cells. Results are presented as mean ± SD (n ¼ 4). *p < 0.05, **p < 0.01, ***p < 0.0001 (t-test). RLU, relative light unit.

luciferase pDNA (pLUC). As shown in Figure 4, the dTAT-pLUC-Ca2þ nanoparticles at N/P 10 with 38 mM calcium chloride did not show any cytotoxicity in all 4 different cell lines. These MTS assays strongly suggest that the dTAT-pDNA-Ca2þ complexes are a low cytotoxic pDNA transfection vector. Treatment With Apoptosis-Inducer Gene by the dTAT-pDNA-Ca2þ Complexes Via Either IV Injection or IT Aerosol Spray Caused Significant Growth Attenuation of Lung Tumors in Allograft Model in Syngeneic Mice To evaluate the effect of the endogenous apoptosis-inducer gene types and administration routes on the lung tumor growth in an acute orthotopic LLC allograft model, LLC cells (1.2  106) were injected via the tail vein. Seven days after the cancer cell inoculation, the mice were treated with the dTAT-pDNA-Ca2þ complexes containing 4 mg of pAT2R IV twice with a 3-day interval or a single dose of 1 mg pAT2R IT. The tumor growth attenuation effect of the complexes by either IV or IT administration was essentially identical in this allograft mouse model in both macroscopically (Figs. 5a and 5b) and microscopically (Fig. 5c). Macroscopically, a large number and size of tumor nodules were detected in PBS- or dTAT-treated mouse lungs. Average lung weights (mg) of the dTAT-

pAT2R-Ca2þ IT-treated (140.0 ± 14.6) and the dTAT-pAT2R-Ca2þ IV-treated (174.7 ± 42.5) groups were significantly smaller than those of the control PBS group (325.7 ± 69.4, p < 0.05, Fig. 5b). Histologic examination of tumors in H&E-stained lung sections also displayed only a small number and small size of LLC tumor nodules in mouse lungs treated with the dTAT-pAT2R-Ca2þ complexes (Fig. 5c). The average number of tumor nodules in the lungs in the dTAT-pAT2R-Ca2þ IT-treated (0.8 ± 1.2) and the dTAT-pAT2R-Ca2þ IV-treated (3.6 ± 2.1) groups was significantly smaller than that of the control PBS group (17.8 ± 6.0, p < 0.01, Fig. 5c). Although treatment with the dTAT-Ca2þ complexes in IT and IV significantly decreased average lung weight (217.1 ± 54.0 in dTAT-Ca2þ IT and 191.6 ± 45.2 in dTAT-Ca2þ IV), the average number of tumor nodules in dTAT-Ca2þ IT (3.5 ± 3.8) was insignificant compared to that in PBS IT-treated group (Fig. 5c). In addition, the effect of a control gene expression was evaluated using the firefly luciferase gene (dTAT-pLUC-Ca2þ complexes) by injecting it via IV or IT spray into LLC tumor-bearing mouse. However, as have reported previously by our group,3,25 these control gene complexes did not show any effect on the growth of LLC tumor (data not shown). The expression of AT2R gene in the lung was determined using immunohistochemical technique (Fig. 5d). High AT2R expression was detected in the tumor cells in the dTAT-pAT2R-Ca2þ IV and IT spray groups but not in

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Figure 3. Cytotoxicity profiles of dTAT, PEI, and CaCl2 in A549 cells (a), HeLa cells (b), HEK-293 cells (c), and LLC cells (d) were determined by MTS assay. Cell viability is expressed as percent of culture medium control. Results are presented as mean ± SD (n ¼ 3).

other control groups. These results indicate that both pulmonary and systemic treatments with the dTAT-pAT2R-Ca2þ complexes were equally effective in attenuating the growth of LLC lung tumor grafts grown in immunocompetent mice. Cotreatment With the dTAT-pAT2R-Ca2þ Complexes Via IV and IT Aerosol Spray Caused Significant Growth Attenuation of Lung Tumors in Orthotopic Human Bronchioloalveolar Carcinoma Xenograft Model in CB-17 SCID Mice To evaluate the effect of the dTAT-pAT2R-Ca2þ complexes on relatively large human lung tumor growth in the orthotopic

Figure 4. Cytotoxicity profiles of dTAT-pLUC nanoparticles with 38 mM CaCl2 concentration at an N/P 10 in A549, HeLa cells, HEK-293 cells, and LLC cells were determined. Cell viability is expressed as percent of untreated cells. Results are presented as mean ± SD (n ¼ 3).

xenograft model, H358 human bronchioloalveolar carcinoma cells were injected via the tail vein twice with 8-week interval. Seven days after second cancer cell injection, the mice were treated with the dTAT-pAT2R-Ca2þ complexes containing 4 mg of pAT2R IV twice with 3-day interval, a single dose of 1 mg pAT2R IT, or combined treatment with IV and IT with the same doses described previously. Among these 3 types of administrations, only combined treatment with IV and IT of the AT2R was effective in inhibiting tumor growth (Figs. 6a and 6b). Macroscopically, a large number and size of tumor nodules were detected in PBS-, the dTAT alone IT-, or the dTAT-pAT2R-Ca2þ IV-treated mouse lungs. Average lung weights (mg) of the dTAT-pAT2R-Ca2þ IT (537.0 ± 154.0, p ¼ 0.054) and the dTAT-pAT2R-Ca2þ IV and IT combination (485.1 ± 115.7, p < 0.05) treated groups were smaller than those of the control PBS (923.5 ± 224.0), the dTAT alone IT (796.1 ± 479.9), or the dTAT-pAT2R-Ca2þ IV group (729.3 ± 360.7, Fig. 6b). Histologic examination of tumors in H&E-stained lung sections also displayed a small number and small size of tumor nodules in mouse lungs treated with the IV and IT combination of the dTAT-pAT2R-Ca2þ complexes (Fig. 6c). Average numbers of tumor nodules in the lungs in the IT alone (60.3 ± 25.2) and the IT and IV combination (60.6 ± 17.8) groups tended to be smaller than those of the control PBS group (83.5 ± 23.0), the dTAT alone IT (94.4 ± 74.2), or the dTAT-pAT2R-Ca2þ IV group (88.0 ± 61.1, Fig. 6c). The immunohistochemical analysis of the AT2R expression revealed that the AT2R expression was markedly increased in the tumor cells in the dTAT-pAT2R-Ca2þ IT and IV and their combination treatment groups. These results suggest that pulmonary treatment, but not systemic treatment with the dTATpAT2R-Ca2þ complexes, is more effective in attenuating the growth of relatively large H358 human lung tumor xenografts grown in immunodeficient mice for 9 weeks. However, systemic treatment with the dTAT-pAT2R-Ca2þ complexes appears to

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Figure 5. IV or IT administration of apoptosis-inducer gene, AT2R, by dTAT-pDNA-Ca2þ nanoparticles inhibited LLC lung tumor growth in syngeneic mice. (a) Macroscopic view of the lungs in PBS-, dTAT alone IT-, dTAT alone IV-, dTAT-pAT2R-Ca2+ IT (dTAT-AT2R IT)-, or dTAT-pAT2R-Ca2+ IV (dTAT-AT2R IV)-treated mice. (b) Comparison of average lung weights was carried out when mice were sacrificed 14 days after the treatments with PBS IT, dTAT-Ca2þ (IT or IV), or dTAT-pAT2R-Ca2þ (IT or IV). (C) Tumor nodule numbers in microscopic views of the lungs in each treatment group were determined by viewing H&E-stained lung sections. (d) Immunohistochemical analysis of AT2R expression in LLC tumors in PBS-, dTAT alone IT-, dTAT alone IV-, dTAT-AT2R IT-, or dTAT-AT2R IV-treated mice. Microscopic images of immunohistochemistry for AT2R expression (original magnification at 200). High AT2R expression was observed in the tumor cells from dTAT-AT2R IT- or dTAT-AT2R IV-treated mice. Results are presented as mean ± SD (n ¼ 5). *p < 0.05 Compared to the PBS-treated group (t-test).

enhance pulmonary treatment-induced tumor growth inhibition in this apoptosis-inducer gene therapy. Discussion Synthetic nonviral gene vectors such as CPP including HIV-1 TAT peptide are potentially usable vectors in gene therapies.3,25 The major benefit of these CPP vectors is the low cytotoxicity.27 Although the level of transfection efficiency mediated by CPP vectors is typically lower than that by viral vectors,2 its safety and ability to target gene delivery are attractive properties justifying further study of the CPP vector. Indeed, CPPs have been used to deliver various anticancer agents into cancer cells in vivo and have been observed to be effective in inhibiting tumor growth in preclinical mouse models.3,25 Therefore, the primary goals of this study were to further examine the safety and transfection efficiency of the dTAT-pDNA-Ca2þ complexes in vitro and to determine whether the endogenous apoptosis-inducer gene delivery by the dTAT-pDNA-Ca2þ complexes can be resulting in therapeutically effective tumor growth suppression in lung cancer mouse models.

The formation of the complexes between dTAT and pDNA was evaluated using pLUC. The complex formation was observed in both the dTAT-pLUC-Ca2þ and the dTAT-pLUC complexes as observed via agarose gel electrophoresis (Fig. 1) when the N/P ratio is >1.0. However, the dTAT-pLUC complexes without calcium chloride exhibited very low gene expression (Fig. 2a), and the size of these complexes in serum-free culture medium was inappropriately large (average size of 1000 nm) for gene delivery (Fig. 1c). The addition of calcium chloride in the dTAT-pLUC complexes significantly decreased the complex size in water and serum-free culture medium (Fig. 1c) and correspondingly increased gene transfection (Fig. 2a). Therefore, calcium chloride acted as an effective condensing agent to decrease the particle size of the dTAT-pLUC complexes and enhance transfection efficiency. These observations are in good agreement with our previous study in which calcium chloride also decreased particle sizes of CPP-pLUC complexes with other types of CPP.3,28 Calcium ion-dependent increase of the total positive charge of the dTAT-pLUC-Ca2þ complexes may also play an important role in enhancing transfection efficiency by the stronger interaction with the negatively charged cell membrane.29 However, although the reduction in the particle size of the

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Figure 6. Combination of IT and IV delivery of the AT2R gene by dTAT-AT2-Ca2þ nanoparticle significantly attenuated chronically grown H358 human lung bronchioloalveolar carcinoma xenografts in SCID mice. (a) Representative lung images from each group at end of the study show decreases in tumor nodules in dTAT-pAT2R-Ca2þ IT and combination of dTAT-pAT2R-Ca2þ IT þ IV treated groups. Comparison of average lung weights (b) and number of tumor nodules (c) in the lung H&E sections among groups. (d) Immunohistochemical analysis of AT2R expression in H358 xenograft tumors in PBS-, dTAT alone IT-, dTAT-pAT2R-Ca2þ IT-, dTAT-pAT2R-Ca2þ IV-, or dTAT-pAT2R-Ca2þ IT þ IV-treated mice. Microscopic images of immunohistochemistry for AT2R expression (original magnification at 200). High AT2R expression was observed in the tumor cells from dTAT-AT2R IT-, dTAT-AT2R IV-, or dTAT-AT2R IT þ IV-treated mice. Results are presented as mean ± SD (n ¼ 5). *p < 0.05 Compared to the PBS-treated group (t-test).

dTAT-pDNA-Ca2þ complexes appears to increase transfection efficiency and this dTAT vector-dependent transfection efficiency is higher than those by PEI vector in all 4 cell lines tested (Figs 2b-2e), the average particle size of 450 nm is larger than typical drug delivery nanoparticles.30-32 This result suggests that the dTAT-pDNACa2þ complexes may be effectively taken up to cancer cells by its cell membrane penetrating property and induces an efficient gene expression despite the larger particle size. The in vitro transfection efficiency of the dTAT-pDNA-Ca2þ complexes was evaluated using pLUC in the A549 cells (Fig. 2a) and subsequently in 3 different human cell lines (kidney, cervix, and lung) and one mouse lung cancer cell line (Figs. 2b-2f). As shown in these figures, the best transfection efficiency was achieved at 38-114 mM calcium chloride (Figs. 2a-2e). Interestingly, no significant level of gene expression was detected without calcium chloride. Because calcium chloride is considered to be an essential component in the condensation of the dTAT-pLUC complexes, it is proposed that the yield of small complexes with higher surface charge may result in an optimal pLUC expression. In another study, the importance of the dTAT peptide in gene expression was evaluated by comparing pLUC-Ca2þ complexes (without dTAT peptide) and the dTAT-pLUCCa2þ complexes in the same calcium concentration. The results in Figures 2b-2f clearly show that pLUC expression resulting from the pLUC-Ca2þ complexes (without dTAT peptide) was significantly lower than that of the dTAT-pLUC-Ca2þ complexes suggesting that

dTAT in the complexes is indeed important to achieve the high gene expression by the dTAT-pLUC-Ca2þ complexes. It is of interest to know that the PEI-pDNA complexes had high transfection efficiency in the absence of calcium chloride.9,17,28 A useful gene delivery vector should deliver genetic material to the target cells without influencing the viability of the host cells. The present study clearly indicated negligible cytotoxicity in vitro up to submillimolar concentration for dTAT peptide and 1 mg/mL for calcium chloride after 24 h in 3 cell lines that include A549, HeLa, and LLC cells, whereas PEI exhibited strong cytotoxicity at low micromolar concentrations in all cell lines except for HEK-293 cells (Fig. 3). Although the HEK-293 cells responded differently from the other 3 cell lines as both dTAT and PEI caused a gradual decrease in cell viability, relatively high cell viability was sustained up to millimolar concentration for both dTAT and PEI. In addition, dTAT-pLUC-Ca2þ complexes did not show any cytotoxicity on cell growth in 4 different cell lines (Fig. 4). Furthermore, the low cytotoxicity of the dTAT-pAT2R-Ca2þ complexes was also observed in the mouse study after IV and IT applications, in which all mice receiving dTAT alone or the dTATpDNA-Ca2þ complexes survived during the experimental period and did not show any acute inflammatory reaction or histologically detectable abnormality. Therefore, data strongly suggested that the dTAT-pAT2R-Ca2þ complexes represent a safe and efficient gene transfection vector.

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The high level of gene expressions in various cell lines with negligible cytotoxicity has led us to carry out in vivo gene transfection studies in multiple lung cancer mouse models. First, endogenous apoptosis-inducer gene, AT2R, was delivered to LLC tumor-bearing mice by the dTAT-pDNA-Ca2þ complexes. As shown in Figure 5, the treatment with the dTAT-pAT2R-Ca2þ complexes via either IT or IV administration significantly attenuated tumor growth macroscopically and microscopically, whereas the treatment with the control dTAT-Ca2þ or dTAT-pLUC-Ca2þ complexes IT or IV showed only a small effect on tumor growth attenuation. It has been known that an increased expression of anionic molecules in the membrane of cancer cells resulted in an increased net negative charge compared to the nonmalignant cell membrane.33 These electric characteristics of the cancer cell's surfaces support that cationic amino acid-rich dTAT peptide-based nanoparticles are suited for cancer-targeted gene therapy. Furthermore, because AT2R overexpression is shown to induce apoptosis in various cancer cells and attenuate their growth which includes lung cancer cells,3,25,34,35 it is conceivable to speculate that tumor growth attenuation was accomplished by apoptosis-inducer gene delivered by the dTAT vector. Therefore, the dTAT-pDNA-Ca2þ complexebased delivery of endogenous apoptosis-inducer genes such as AT2R is a potential treatment scheme for primary as well as metastatic lung cancers. Two times IV bolus injection or a single IT spray of dTAT alone neither attenuated cancer growth significantly compared to the PBS controls (Fig. 6) nor exhibited any side effects such as abnormal clinical or histologic signs in the normal lung epithelium. These results suggest that the dTAT peptide is a useful and safe vector for cancer gene therapy. In the second mouse study, AT2R was delivered to H358 human bronchioloalveolar carcinoma-bearing mice by the dTAT-pAT2RCa2þ complexes. As shown in Figure 6, the combination treatment with the dTAT-pAT2R-Ca2þ complexes via aerosol IT spray and IV significantly attenuated tumor growth macroscopically and microscopically, whereas the treatment with the control dTAT-Ca2þ complexes IT or the dTAT-pAT2R-Ca2þ complexes IV showed a negligible effect on tumor growth attenuation. Because the growth of the H358 human bronchioloalveolar carcinoma required a significantly longer time (study duration was 12 weeks and mice were treated 9 weeks after the initial tumor cell inoculation) than that of the LLC tumor (study duration was 4 weeks and mice were treated 1 week after the tumor cell inoculation), tumor sizes in this human xenograft mouse model were much bigger than those in the LLC tumor model. This model is better mimicking human lung cancer. Although the local pulmonary treatment is more effective than the systemic treatment in attenuating the growth of relatively large H358 human lung tumor xenografts grown, systemic treatment with the dTAT-pAT2R-Ca2þ complexes appear to enhance pulmonary treatment-induced tumor growth inhibition by the apoptosis-inducer gene therapy. Therefore, the second mouse study indicates that the dTAT-pDNA-Ca2þ complex-based delivery of endogenous apoptosis-inducer gene such as AT2R via combination delivery by both IT and IV is a potentially useful treatment scheme for primary as well as metastatic lung cancers. In the present study, the dTAT-pDNA-Ca2þ complex-based gene delivery was found to be a useful tool for an in vivo gene delivery system for lung cancer treatment in both mouse and human lung cancer mouse models in immunocompetent and immunodeficient mice, respectively. Because it is obvious that both cellular and humoral immune systems in the tumor microenvironment play a significant role in the regulation of tumor growth,36 use of a cancer model in immunocompetent mice for the evaluation of the newly developed treatment regimen is appropriate. Simultaneously, it is also important to incorporate a slow-growing human lung cancer

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model, in which cancer treatment was started several weeks after tumor growth was stated, in this kind of evaluation. However, it is obvious that further evaluation of this gene therapy using multiple types of human lung cancer cells in mouse xenograft models and naturally occurring lung cancer in domestic animals will solidify this discovery. Conclusion Dimerized TAT CPP (dTAT peptide) and pDNA (pLUC and pAT2R) can produce small and stable complexes with the addition of calcium chloride, and when compared to the PEI-pDNA complexes, the dTAT-pDNA complexes manifest higher gene expression in various human and mouse cancer cells. The dTAT-pAT2R-Ca2þ complexes have been shown to attenuate the growth of LLC allografts and H358 human bronchioloalveolar carcinoma xenografts in mouse lungs by single IT or 2 IV administrations. In vitro, the dTAT peptide showed negligible cytotoxicity. These data support the notion that the dTAT CPP is effective and safe to use to deliver genetic materials and show that endogenous apoptosis-inducer genes such as AT2R are potentially useful for lung cancer gene therapy. These data reveal that the dTAT-pDNA-Ca2þ complexes could be an effective and safe nonviral gene transfection tool; however, further in vivo studies are needed to confirm the safety of the dTAT-pAT2R-Ca2þ complexes by formal pharmacokinetics, pharmacodynamics, and multispecies toxicity studies. Acknowledgments This work was supported in part by Kansas State University Johnson Cancer Research Center (M.T.), National Institutes of Health grants U43 CA165462 (M.T.), P20 GM103418 (M.T.), and Kansas Bioscience Authority Collaborative Cancer Research grant (M.T.). This work was also supported by Savara Pharmaceuticals, Higuchi Biosciences Center and Faculty of Pharmacy of the University of Kansas, King Abdulaziz University, Jeddah, Saudi Arabia (N.A.A.). References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin. 2015;65(1):5-29. 2. Aldawsari H, Edrada-Ebel R, Blatchford DR, Tate RJ, Tetley L, Dufes C. Enhanced gene expression in tumors after intravenous administration of arginine-, lysine- and leucine-bearing polypropylenimine polyplex. Biomaterials. 2011;32(25):5889-5899. 3. Alhakamy NA, Ishiguro S, Uppalapati D, Berkland CJ, Tamura M. AT2R gene delivered by condensed polylysine complexes attenuates Lewis lung carcinoma after intravenous injection or intratracheal spray. Mol Cancer Ther. 2016;15(1): 209-218. 4. Chen S, Han K, Yang J, Lei Q, Zhuo RX, Zhang XZ. Bioreducible polypeptide containing cell-penetrating sequence for efficient gene delivery. Pharm Res. 2013;30(8):1968-1978. 5. Khondee S, Baoum A, Siahaan TJ, Berkland C. Calcium condensed LABL-TAT complexes effectively target gene delivery to ICAM-1 expressing cells. Mol Pharm. 2011;8(3):788-798. 6. Margus H, Padari K, Pooga M. Cell-penetrating peptides as versatile vehicles for oligonucleotide delivery. Mol Ther. 2012;20(3):525-533. 7. Nakamura Y, Kogure K, Futaki S, Harashima H. Octaarginine-modified multifunctional envelope-type nano device for siRNA. J Control Release. 2007;119(3): 360-367. 8. Zuhorn IS, Engberts JB, Hoekstra D. Gene delivery by cationic lipid vectors: overcoming cellular barriers. Eur Biophys J. 2007;36(4-5):349-362. 9. Baoum A, Xie SX, Fakhari A, Berkland C. “Soft” calcium crosslinks enable highly efficient gene transfection using TAT peptide. Pharm Res. 2009;26(12):26192629. 10. Davis ME. Non-viral gene delivery systems. Curr Opin Biotechnol. 2002;13(2): 128-131. 11. Felgner PL, Gadek TR, Holm M, et al. Lipofection: a highly efficient, lipidmediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987;84(21): 7413-7417.

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