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

Abstract

To evaluate the potential of cell penetrating peptide (CPP)-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-Ca²⁺). This simple non-viral formulation approach showed high levels of gene expression in vitro without any cytotoxicity. In mouse studies, a single intratracheal (IT) aerosol spray or two intravenous (IV) injections of the dTAT, apoptosis-inducer gene, angiotensin II type 2 receptor (AT2R), and Ca²⁺ complexes (dTAT-pAT2R–Ca²⁺) significantly attenuated the acutely growing mouse Lewis lung carcinoma (LLC) allografts in mouse lungs. Furthermore, single IT (P=0.054) and the combination of IT and IV (P<0.05) administrations of dTAT-pAT2R-Ca²⁺ markedly attenuated slowly growing and relatively large sized H358 human bronchioloalveolar carcinoma xenografts in mouse lungs. These results indicate that the dTAT-pDNA-Ca²⁺ effectively delivered the gene to cancer cells by either IT or IV administration although the local pulmonary delivery of the dTAT-pAT2R-Ca²⁺ showed more effective growth inhibition of orthotopic lung cancer grafts. Thus, the present study offers preclinical proof of concept that a dTAT-based non-viral gene delivery method via IT administration may be an effective lung cancer gene therapy.

Keywords: dTAT peptide, Nanoparticles, Non-viral gene delivery, Lung cancer, Angiotensin II type 2 receptor, AT2R, Apoptosis

INTRODUCTION

Although lung cancer prognosis has improved due to advances in diagnosis and early surveillance, more than half (57%) of the patients are diagnosed at an advanced stage and the one- and five-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 administration or inhalation, biological 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 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 non-viral 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 non-viral 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 are the most commonly employed non-viral gene methods ^9–16. Non-viral vectors often suffer from a lower transfection efficiency compared to viral vectors ², yet numerous strategies have been put forward to advance non-viral gene delivery.

Cell-penetrating peptides (CPPs) appear to be a particularly promising component of non-viral gene therapies. CPPs consist of 30 or fewer 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 non-covalent coupling (electrostatic interactions) of CPPs with pDNA or siRNA ^20–23. When utilizing non-covalent 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-Ca²⁺ complex) was optimized using three different human cell lines: (1) A549 (a lung cancer cell), (2) HeLa (a cervical cancer cell), (3) Hek-293 (a virus-immortalized kidney cell) and one mouse cell line, LLC (a lung cancer cell) using a luciferase-reported plasmid DNA (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 plasmid DNA (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-Ca²⁺ complexes were administered intravenously or via intratracheal 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

Plasmid DNA (pDNA) encoding firefly luciferase (pGL3, 4818 bp) was obtained from Promega (Madison, WI). Plasmid DNA (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; Mw = 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) and HeLa cell line (human cervix adenocarcinoma) and H358 cell line (human bronchioloalveolar carcinoma) were obtained from American Type Culture Collection (ATCC; Rockville, MD). HEK-293 (human embryonic kidney) cell line was a gift from Dr. Nikki Cheng (University of Kansas Medical Center, KS). Calcium chloride dihydrate (CaCl₂·2H₂O) was purchased from Fisher Scientific (Pittsburgh, PA). Albumin from mouse serum (MSA) and glucose were obtained from Sigma-Aldrich.

Preparation of the dTAT-pDNA-Ca²⁺ Nanoparticles

For the in vitro studies, the dTAT-pDNA nanoparticles were prepared by adding 15 µL of dTAT solution (N/P 10, different polymer nitrogen to pDNA phosphate (N/P) ratios) to 10 µL (0.1 µg/µL) of pDNA [Tris-acetate-EDTA (TAE) Buffer (1 x) was used as a solution for DNA storage], followed by fast pipetting for 20 seconds. At that point, 15 µL of identified molarity (e.g., 50, 300, and 600 mM) CaCl₂ was added and mixed by fast pipetting. The total volume was 40 µL. After preparing the nanoparticles, they were stored at 4°C for 20–25 minutes. For the mouse studies with intravenous (IV) administration, the dTAT-pDNA nanoparticles were prepared by adding 60 µL (0.88 µg/µL) of dTAT solution to 40 µL (0.1 µg/µL) of pDNA (pAT2R or pLUC) (TAE Buffer (1 x) was used as a solution for DNA storage), followed by fast pipetting for 20 seconds. At that point, 60 µL of identified molarity (100 mM) CaCl₂ was added and mixed by fast pipetting. Then, 40 µL of mouse serum albumin (MSA, 1%) was added to the solution. The total volume was 200 µL. After preparing the nanoparticles, they were stored at 4°C for 20–25 minutes. For the mouse studies with intratracheal (IT) administration, the dTAT-pDNA nanoparticles were prepared by adding 15 µL (0.88 µg/µL) of dTAT solution to 10 µL (0.1 µg/µL) of pDNA (pAT2R or pLUC) (TAE Buffer (1 x) was used as a solution for DNA storage), followed by fast pipetting for 20 seconds. At that point, 15 µL of identified molarity (100 mM) CaCl₂ was added and mixed by fast pipetting. Then, 10 µL of glucose (10%) was added to the solution. The total volume was 50 µL. After preparing the nanoparticles, they were stored at 4°C for 20–25 minutes.

Preparation of the PEI-pDNA Nanoparticles

The PEI-pDNA nanoparticles were prepared by adding 15 µL of PEI solution (N/P 10) to 10 µL (0.1 µg/µL) of pDNA followed by fast pipetting for 20 seconds. After preparing the nanoparticles, they were stored at 4°C for 20–25 minutes. The nanoparticles were prepared immediately before each experiment.

Agarose Gel Electrophoresis

The nanoparticles were prepared as defined previously and subsequently, 4 µL of TAE buffer was added to the nanoparticles. Then, 4 µL of SYBR Green 1 was mixed with the nanoparticles. Afterward, the mixture was stored at 4°C for 20–25 minutes. After storage, 7 µL of 6X DNA Loading Dye was added. A one 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 minutes 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 (FBS) at 37°C in 5% CO₂ humidified air.

Transfection Studies

A549, HeLa, LLC, and HEK-293 cell lines were cultured in 96-well plates for 24 hours before transfection. The concentration of the cells in every well was approximately 100,000 cells/ml. The wells were washed once with SFM and afterwards a 100 µL sample (which consisted of 20 µL of nanoparticle and 80 µL of SFM) was added to each well. Subsequently, a 96-well plate was incubated for 5 hours in an incubator. After 5 hours incubation, the sample was replaced with 100 µL of fresh serum medium and then incubated again for approximately 48 hours. 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 (RLU) per milligram (mg) of cellular protein and PEI-pDNA was used as a control. BCA 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 BCA were measured by a microplate reader (SpectraMax; Molecular Devices LLC., Sunnyvale, CA). HEK-293 and LLC cells are semi-adherent, 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 µL of gelatin solution (2%) to each well followed by incubation for at least one hour 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 minutes before use.

Cytotoxicity assay of dTAT, PEI, CaCl₂, and dTAT-pDNA-Ca²⁺ Nanoparticles

Cytotoxicity of dTAT, PEI, CaCl₂, and dTAT-pLUC-Ca²⁺ Nanoparticles at N/P 10 with 38mM CaCl₂ were determined using a CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) obtained from Promega. A549, HeLa, HEK-293, and LLC cells were cultured in a 96-well plate as described previously. After 24 hours of incubation, the media were replaced with a sample consisting of 100 µL of fresh serum medium and 20 µL of MTS. Then, the plate was incubated for 3 hours. To determine cell viability, the absorbance of each well was measured by a microplate reader at 490 nm and normalized to untreated control cells.

Animals

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 (IACUC) and carried out under strict adherence to the IACUC 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, seven week old C57BL/6 mice were intravenously injected with 1.2×10⁶ LLC cells/200µl PBS via the tail vein using a 1 mL syringe with a 27G needle. For xenogeneic lung cancer mouse model, C.B-17 SCID mice were intravenously injected twice with 1.0×10⁶ and 1.2×10⁶ H358 cells suspension in 200µl PBS with an 8 week interval. The dTAT-pDNA-Ca²⁺ complex solution was prepared immediately before injection as described above. For the IV administration of the dTAT-pDNA-Ca²⁺ complexes, 160 µL complex solution was mixed with 40 µL 1% mouse serum albumin (MSA). For the IT administration, 40 µL the dTAT-pDNA-Ca²⁺ complex solution was mixed with 10 µL 10% glucose for the osmolality adjustment. In the allograft study, seven days after the LLC cell inoculation, the mice were treated with either IV, IT, or both IV and IT. In the xenograft study, seven 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-Ca²⁺ 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 µm 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 heat-induced epitope unmasking was performed in the citrate buffer followed by incubation with 3% H₂O₂/methanol for 3 minutes to block endogenous peroxidase activity. Sections were incubated with polyclonal anti-AT2R antibodies (1:200 dilution, for 18 hours at 4°C, Santa Cruz Biotechnology, Inc., Dallas, TX). After the incubation with primary antibodies, sections were incubated with a biotin-conjugated anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA) at a 1:100 dilution for 1 hour at 37°C, followed by a reaction with the avidin-biotin-peroxidase complex reagent (Vector Laboratories) for 40 minutes 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 by using GraphPad software. All values are expressed as the mean ± standard deviation of the mean. All experiments were conducted with multiple sample determinations. A statistical evaluation comparing the significance of the difference in gene expression (RLUs/mg protein) between the means of two data sets was performed using t-test. One-way ANOVA, Tukey post test was used to analyze the differences when more than two data sets were compared.

RESULTS

Formation and physical characterization of the dTAT-pDNA-Ca²⁺ nanoparticles

The dTAT-pDNA-Ca²⁺ 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 minutes. 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 38mM 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 lower than 0.5 showed bands (Fig. 1B). Furthermore, mixing with calcium chloride and pLUC did not form stable complexes (data not shown).

Figure 1

A–B, 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, N/P 10, and N/P 30 in the presence of 38 mM calcium chloride, respectively, whereas a, b, and c refer to N/P 5, N/P 10, and N/P 30 in the absence of calcium chloride, respectively. M, 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 DLS in nuclease free water (NFW), or serum-free F-12 media (SFM). D, Zeta potentials of the dTAT-pLUC and PEI-pLUC complexes at an N/P 10 were determined by Zeta PALS dynamic light scattering in the presence of various concentrations of calcium chloride (0, 38, and 114 mM). Results are presented as mean ± SD (n = 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 Fig. 1C, addition of calcium chloride of 38 and 114 mmol/L (final concentration) significantly decreased the particle size of the dTAT-pLUC-Ca²⁺ complexes, with relatively narrow polydispersity (0.1), in both NFW (475 and 446 nm at 38 and 114 mM CaCl₂, respectively) and in SFM (382 and 321 nm at 38 and 114 mM CaCl₂, respectively). The zeta potential of both the dTAT-pLUC-Ca²⁺ 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-Ca²⁺ nanoparticle caused efficient gene transfection with low cytotoxicity in vitro

The in vitro transfection efficiency of the dTAT-pLUC-Ca²⁺ nanoparticles was studied using the three different human cancer cell lines including lung cancer (A549), cervix cancer (HeLa), kidney cancer (HEK-293) and one mouse lung cancer cell line (LLC). Luciferase gene expression was evaluated 48 hours after the transfection using the dTAT-pLUC-Ca²⁺ nanoparticles at N/P 5, 10, 20, and 30, and at various calcium chloride concentrations during the complex formulation (Fig. 2A). The dTAT-pLUC-Ca²⁺ nanoparticles had a high level of gene expression at higher calcium chloride concentration from 38–114 mM in all four 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–E). In all cell lines examined, the transfection efficiency of the dTAT-pLUC-Ca²⁺ was significantly higher than those by PEI-pLUC.

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. RLUs refer to relative light units. Results are presented as mean ± SD (n = 4). *, P < 0.05, **, P < 0.001, ***, P < 0.0001 were evaluated by one-way ANOVA followed by Tukey post hoc analysis as indicated in the figure panels. F, the transfection efficiency of dTAT-pLUC-Ca²⁺ (shaded bars) and pLUC-Ca²⁺ (white bars) nanoparticles were determined at an N/P 10 in the presence of 38 mM CaCl₂ in A549, HeLa, HEK-293, and LLC cells. RLUs refer to relative light units. Results are presented as mean ± SD (n = 4). *, P < 0.05, **, P < 0.01, ***, P < 0.0001 (t-test).

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-Ca²⁺ complexes (without the dTAT peptide) were significantly lower than those of the dTAT-pLUC-Ca²⁺ 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 membrane translocalization signal (MTS) cytotoxicity assay was carried out using four different cell lines. The four cell lines were individually incubated with up to 5 mg/ml dTAT, PEI, or calcium chloride for 24 hours 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 µg/ml). Calcium chloride also did not show strong cytotoxicity until 1 mg/ml level. However, PEI induced significant cytotoxicity even at 1 µg/ml in three 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-Ca²⁺ complexes on four different cell lines was evaluated using firefly luciferase pDNA (pLUC). As shown in figure 4, the dTAT-pLUC-Ca²⁺ nanoparticles at N/P 10 with 38 mM calcium chloride did not show any cytotoxicity in all four different cell lines. These MTS assays strongly suggest that the dTAT-pDNA-Ca²⁺ complexes are a low cytotoxic pDNA transfection vector.

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 % of culture medium control. Results are presented as mean ± SD (n = 3).

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 % of untreated cells. Results are presented as mean ± SD (n = 3).

Treatment with apoptosis inducer gene by the dTAT-pDNA-Ca²⁺ 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-Ca²⁺ complexes containing 4 µg of pAT2R intravenously (IV) twice with a 3 day interval or a single dose of 1 µg pAT2R intratracheally (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 B) 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-Ca²⁺ IT (140.0±14.6) and the dTAT-pAT2R-Ca²⁺ IV (174.7±42.5) treated groups were significantly smaller than that of the control PBS group (325.7±69.4, P<0.05, Fig. 5B). Histological 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-Ca²⁺ complexes (Fig. 5C). The average number of tumor nodules in the lungs in the dTAT-pAT2R-Ca²⁺ IT (0.8±1.2) and the dTAT-pAT2R-Ca²⁺ IV (3.6±2.1) treated groups were significantly smaller than that of the control PBS group (17.8±6.0, P<0.01, Fig. 5C). Although treatment with the dTAT-Ca²⁺ complexes in IT and IV significantly decreased average lung weight (217.1±54.0 in dTAT-Ca²⁺ IT, and 191.6±45.2 in dTAT-Ca²⁺ IV), the average number of tumor nodules in dTAT-Ca²⁺ IT (3.5±3.8) was insignificant as compared with 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-Ca²⁺ complexes) by injecting it via IV or IT spray into LLC tumor bearing mouse. However, as have reported previously by our group ^3,25, this 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-Ca²⁺ IV and IT spray groups but not in other control groups. These results indicate that both pulmonary and systemic treatments with the dTAT-pAT2R-Ca²⁺ complexes were equally effective in attenuating the growth of LLC lung tumor grafts grown in immunocompetent mice.

Figure 5

Intravenous (IV) or intratracheal (IT) administration of apoptosis-inducer gene, AT2R, by dTAT-pDNA-Ca²⁺ nanoparticles inhibited LLC lung tumor growth in syngeneic mice. A, comparison of average lung weights was carried out when mice were sacrificed 14 days after the treatments with PBS IT, dTAT-Ca²⁺ (IT or IV), or dTAT-pAT2R-Ca²⁺ (IT or IV),. B, 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 intratracheal (dTAT alone IT), dTAT alone intravenous (dTAT alone IV), dTAT-pAT2R-Ca²⁺ intratracheal (dTAT-AT2R IT), or dTAT-pAT2R-Ca²⁺ intravenous (dTAT-AT2R IV) -treated mice. Microscopic images of immunohistochemistry for AT2R expression (Original magnification at 200x). 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 = 5). *, P < 0.05 as compared with the PBS treated group (t-test).

Co-treatment with the dTAT-pAT2R-Ca²⁺ 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-Ca²⁺ complexes on relatively large human lung tumor growth in the orthotopic xenograft model, H358 human bronchioloalveolar carcinoma cells were injected via the tail vein twice with eight weeks interval. Seven days after second cancer cell injection, the mice were treated with the dTAT-pAT2R-Ca²⁺ complexes containing 4 µg of pAT2R IV twice with 3 days interval, a single dose of 1 µg pAT2R IT, or combined treatment with IV and IT with the same doses described above. Among these three types of administrations, only combined treatment with IV and IT of the AT2R was effective in inhibiting tumor growth (Figs. 6A and B). Macroscopically, a large number and size of tumor nodules were detected in PBS, the dTAT alone IT or the dTAT-pAT2R-Ca²⁺ IV treated mouse lungs. Average lung weights (mg) of the dTAT-pAT2R-Ca²⁺ IT (537.0±154.0, P=0.054) and the dTAT-pAT2R-Ca²⁺ 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-Ca²⁺ IV group (729.3±360.7, Fig. 6B). Histological 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-Ca²⁺ 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-Ca²⁺ 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-Ca²⁺ IT and IV, and their combination treatment groups. These results suggest that pulmonary treatment, but not systemic treatment with the dTAT-pAT2R-Ca²⁺ complexes, is more effective in attenuating the growth of relatively large H358 human lung tumor xenografts grown in immunodeficient mice for nine weeks. However, systemic treatment with the dTAT-pAT2R-Ca²⁺ complexes appear to enhance pulmonary treatment-induced tumor growth inhibition in this apoptosis-inducer gene therapy.

Figure 6

Combination of intratracheal and intravenous delivery of the AT2R gene by dTAT-AT2-Ca²⁺ 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-Ca²⁺ IT and combination of dTAT-pAT2R-Ca²⁺ IT+IV treated groups; B and C. 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 dTAT alone IT, dTAT-pAT2R-Ca²⁺ IT, dTAT-pAT2R-Ca²⁺ IV, or dTAT-pAT2R-Ca²⁺ IT+IV-treated mice. Microscopic images of immunohistochemistry for AT2R expression (Original magnification at 200x). High AT2R expression was observed in the tumor cells from dTAT-AT2R IT, dTAT-AT2R IV or dTAT-AT2R IT+IV-treated mice. Results are presented as mean ± SD (n = 5). *, P < 0.05 as compared with the PBS treated group (t-test).

DISCUSSION

Synthetic non-viral 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 anti-cancer 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-Ca²⁺ complexes in vitro and to determine whether the endogenous apoptosis inducer gene delivery by the dTAT-pDNA-Ca²⁺ 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 luciferase pDNA (pLUC). The complexes’ formation was observed in both the dTAT-pLUC-Ca²⁺ and the dTAT-pLUC complexes as observed via agarose gel electrophoresis (Fig. 1) when the N/P ratio is higher than 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 1,000 nm) for gene delivery (Fig. 1C). The addition of calcium chloride in the dTAT-pLUC complexes significantly decreased the complexes’ 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-Ca²⁺ complexes may also play an important role enhancing transfection efficiency by the stronger interaction with the negatively charged cell membrane ²⁹. However, although the reduction in the particle size of the dTAT-pDNA-Ca²⁺ complexes appears to increase in transfection efficiency and this dTAT vector-dependent transfection efficiency is higher than those by PEI vector in all four cell lines tested (Figs. 2B–E), the average particle size of 450 nm is larger than typical drug delivery nanoparticles ^30–32. This result suggests that the dTAT-pDNA-Ca²⁺ 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-Ca²⁺ complexes were evaluated using pLUC in the A549 cells (Fig. 2A) and subsequently in three different human cell lines (kidney, cervix and lung) and one mouse lung cancer cell line (Figs. 2B–F). As shown in these figures the best transfection efficiency was achieved at 38–114 mM calcium chloride (Figs. 2A–E). Interestingly, no significant level of gene expression was detected without calcium chloride. Since 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-Ca²⁺complexes (without dTAT peptide) and the dTAT-pLUC-Ca²⁺ complexes in the same calcium concentration. The results in Figures 2B–F clearly show that pLUC expression resulting from the pLUC-Ca²⁺complexes (without dTAT peptide) was significantly lower than that of the dTAT-pLUC-Ca²⁺ complexes suggesting that dTAT in the complexes is indeed important to achieve the high gene expression by the dTAT-pLUC-Ca²⁺ 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 sub-millimolar concentration for dTAT peptide and 1 mg/ml for calcium chloride after 24 hours in three 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 three 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-Ca²⁺ complexes did not show any cytotoxicity on cell growth in four different cell lines (Fig. 4). Furthermore, the low cytotoxicity of the dTAT-pAT2R-Ca²⁺ complexes was also observed in the mouse study after IV and IT applications, in which all mice receiving dTAT-alone or the dTAT-pDNA-Ca²⁺ 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-Ca²⁺ complexes represents a safe and efficient gene transfection vector.

The high level of gene expressions in various cell lines with negligible cytotoxicity have 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-Ca²⁺ complexes. As shown in Figure 5, the treatment with the dTAT-pAT2R-Ca²⁺ complexes via either IT or IV administration significantly attenuated tumor growth macroscopically and microscopically, while the treatment with the control dTAT-Ca²⁺ or dTAT-pLUC-Ca²⁺ 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 non-malignant 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, since AT2R over expression 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-Ca²⁺ complex-based delivery of endogenous apoptosis inducer genes such as AT2R gene 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 did not attenuate cancer growth significantly as compared with the PBS controls (Fig. 6) nor exhibited any side effects such as abnormal clinical or histological 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-pAT2R-Ca²⁺ complexes. As shown in Figure 6, the combination treatment with the dTAT-pAT2R-Ca²⁺ complexes via aerosol IT spray and IV significantly attenuated tumor growth macroscopically and microscopically, while the treatment with the control dTAT-Ca²⁺ complexes IT or the dTAT-pAT2R-Ca²⁺ complexes IV showed a negligible effect on tumor growth attenuation. Since the growth of the H358 human bronchioloalveolar carcinoma required a significantly longer time (study duration was 12 weeks and mice were treated nine weeks after the initial tumor cell inoculation) than that of the LLC tumor (study duration was 4 weeks and mice were treated one 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-Ca²⁺ 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-Ca²⁺ complex-based delivery of endogenous apoptosis inducer gene such as AT2R gene 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-Ca²⁺ 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. Since 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 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-Ca²⁺ complexes have been shown to attenuate the growth of LLC allografts and H358 human bronchioloalveolar carcinoma xenografts in mouse lungs by single IT or two IV administrations. In vitro, the dTAT peptide showed negligible cytotoxicity. These data support the notion that the dTAT CPP is effective, safe to use to deliver genetic materials, and shows that endogenous apoptosis inducer genes such as AT2R are potentially useful for lung cancer gene therapy. These data reveal that the dTAT-pDNA-Ca²⁺ complexes could be an effective and safe non-viral gene transfection tool; however, further in vivo studies are needed to confirm the safety of the dTAT-pAT2R-Ca²⁺ complexes by formal pharmacokinetics, pharmacodynamics, and multispecies toxicity studies.

Acknowledgments

This work was supported in part by Kansas State University Johnson Cancer Research Center (MT), NIH grants U43 CA165462 (MT), P20 GM103418 (MT), and Kansas Bioscience Authority Collaborative Cancer Research grant (MT). 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 (NA).

Footnotes

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The authors disclose no potential conflicts of interest.

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