AT2R Gene Delivered by Condensed Polylysine Complexes ...€¦ · 28.12.2015 · Resource Center...

Models and Technologies

AT2R Gene Delivered by Condensed PolylysineComplexes Attenuates Lewis Lung Carcinomaafter Intravenous Injection or Intratracheal SprayNabil A. Alhakamy1, Susumu Ishiguro2, Deepthi Uppalapati2, Cory J. Berkland1,3, andMasaaki Tamura2

Abstract

Transfection efficiency and toxicity concerns remain a challengefor gene therapy. Cell-penetrating peptides (CPP) have beenbroadly investigated to improve the transfection of genetic mate-rial (e.g., pDNAand siRNA).Here, a synthetic CPP (polylysine, K9peptide) was complexed with angiotensin II type 2 receptor(AT2R) plasmid DNA (pAT2R) and complexes were condensedusing calcium chloride. The resulting complexes were small(�150 nm) and showed high levels of gene expression in vitroand in vivo. This simple nonviral formulation approach showednegligible cytotoxicity in four different human cell lines (cervix,breast, kidney, and lung cell lines) and onemouse cell line (a lungcancer cell line). In addition, this K9-pDNA-Ca2þ complex dem-

onstrated cancer-targeted gene delivery when administered viaintravenous injection or intratracheal spray. The transfectionefficiency was evaluated in Lewis lung carcinoma (LLC) cell linescultured in vitro and in orthotopic cancer grafts in syngeneic mice.Immunohistochemical analysis confirmed that the complex effec-tively delivered pAT2R to the cancer cells, where it was expressedmainly in cancer cells along with bronchial epithelial cells. Asingle administration of these complexes markedly attenuatedlung cancer growth, offering preclinical proof-of-concept for anovel nonviral gene delivery method exhibiting effective lungtumor gene therapy via either intravenous or intratracheal admin-istration. Mol Cancer Ther; 15(1); 1–10. �2015 AACR.

IntroductionLung cancer is the third most prevalent form of cancer behind

prostate andbreast cancer; however, treatment options are lagging(1, 2). Even though progress has beenmade in early detection andprevention, mortality, and morbidity associated with lung canceris still unacceptably high (3). In 2015, the American CancerSociety reported >430,000 people in the United States are livingwith lung cancer yielding the largest annual financial burden ofany cancer type (1). Although incremental improvements havebeenmade in the palliative care, currently available therapies havehadminimal impact in reducingdeaths (3). In particular, theneedfor completely new therapeutics to treat the most aggressive lungcancers is especially great.

Gene therapy is experiencing a renaissance in the United Statesand around the globe. The EuropeanUnionhas recently approvedthe first gene therapy drug, Glybera, for the treatment of lipopro-tein lipase seficiency (LPLD). Promising late-stage clinical trialssuggest thefirst gene therapymaybe approved in theUnited Statesin 2016. Most gene therapy clinical trials (64.4%) have aimed attreating cancer, and yet cancer gene therapies have been slow toadvance (4, 5). Theoretical, and practical limitations still exist forgene therapy application to lung cancer. Biologic barriers andtoxicity continue to confound lung cancer treatment, even thoughthe lungs may be accessed via inhalation or intravenousadministration.

Gene vectors (viral and non-viral vectors) have steadilyimproved over the past 20 years. Viral vectors (e.g., adenoviral)are highly efficient, and facilitate strong transgene expression indifferent tumor tissues (6, 7). While these vectors are the mosteffective vectors applied in 70% of clinical trials, they continue tosuffer from safety concerns (e.g., immunogenicity and pathoge-nicity; refs. 8–11), rapid clearance from circulation, and produc-tion problems (11–14). For these reasons, nonviral vectors couldbe more promising gene carriers due to easy synthesis, low cost,and decreased immunogenicity compared with viral vectors(10, 14–18). These attributes suggest nonviral vectors could offera safer approach for repeated dosing regimens when treatingprimary as well as recurrent cancers.

Nucleic acids complexed with cationic polymers (polyplexes)or cationic lipids (lipoplexes) are the most commonly usednonviral synthetic gene carriers (16, 19–21). Cationic polymersinteract with cell membrane or extracellular components (e.g.,glycosaminoglycans) via the positive charge of the amino acidresidues (e.g., lysine and arginine; ref. 22). The molecular weight

1DepartmentofPharmaceuticalChemistry,UniversityofKansas, Lawr-ence, Kansas. 2Department of Anatomy and Physiology, College ofVeterinary Medicine, Kansas State University, Manhattan, Kansas.3Department of Chemical and Petroleum Engineering, University ofKansas, Lawrence, Kansas.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

N.A. AlhakamyandS. Ishiguro contributed equally and are co-first authors of thisarticle.

Corresponding Authors: Cory J. Berkland, University of Kansas, 2030 BeckerDrive, Lawrence, KS 66047. Phone: 785-864-1455; Fax: 785-864-1454; E-mail:[email protected]; and Masaaki Tamura, Kansas State University, 210 Coles Hall,Manhattan, KS 66506. Phone: 785-582-4825; Fax: 785-582-5777; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0448

�2015 American Association for Cancer Research.

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on July 29, 2021. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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and charge of polycations play important roles in complexingnucleic acids for delivering geneticmaterials (18).Highmolecularweight polycations often condense the genetic material [e.g.,plasmid DNA (pDNA)] into small and stable complexes. On theother hand, lowmolecularweight polycations oftenproduce largeand unstable complexes (18, 23, 24). Unfortunately, low molec-ular weight polycations have historically exhibited poor transfec-tion efficiencywhile highmolecularweight polycations havebeenplagued with cytotoxicity (18).

Polylysine was one of the first polycations used for genedelivery (25). Polylysine–pDNA complexes have traditionallyrequired polylysine chains with more than 20 residues to effi-ciently complex DNA, but yielding modest transfection efficiencyand concerns about cytotoxicity (14). Many attempts were madeto increase transfection efficiency or reduce cytotoxicity by eitherchemically modifying polylysine (e.g., PEGylation) or by addingexcipients (26). Most research efforts focused on improvingtolerability of high molecular weight polylysine (10, 27, 28), butwe show that short polylysines with much lower cytotoxicity canindeed condense pDNA into small complexes when calcium isadded. Such particles offer an interesting opportunity for repeatdosing to treat lung cancer if efficient transfection can be balancedwith low cytotoxicity.

Here, a nine amino acid polylysine (K9) was complexed withpDNA and condensed with calcium chloride. This simple formu-lation (the K9-pDNA-Ca2þ complex) was explored using fourdifferent human cell lines: (i) A549 (a lung cancer cell), (ii) HeLa(a cervix cancer cell), (iii)MDA-MB-231 (a breast cancer cell), (iv)HEK-293 (a virus-immortalized kidney cell) and one mouse cellline, LLC (a lung cancer cell) using a luciferase reported plasmidDNA (pLUC) to assess transfection efficiency. Angiotensin II type2 receptor (AT2R) is known to stimulate apoptosis and inhibit cellproliferation in different cell lines such as endothelial cells,cardiomyocytes, neuronal cells, prostate cancer cells, and lungcancer cells (5, 16), therefore, AT2R plasmid DNA (pAT2R) wasdelivered to LLC tumor-bearingmice. These K9-pAT2R complexeswere administered via intravenous injection and/or via intratra-cheal spray to determine lung cancer attenuation in LLC tumor-bearing mice.

Materials and MethodsMaterials

pDNA encoding firefly luciferase (pLUC, pGL3) was obtainedfrom Promega. Plasmid DNA (pDNA) encoding human AT2R(pAT2R, pcDNA3.1þ) was obtained from the UMR cDNAResource Center (University of Missouri, Rolla, MO). K9 peptide(KKKKKKKKK; Mw ¼ 1170.65 Da; Purity > 95%) was purchasedfrom Biomatik Corporation. Branched polyethyleneimine (PEI,25 kDa), mouse serum albumin (MSA) and glucose were fromSigma-Aldrich. Calcium chloride dihydrate (CaCl2�2H2O) wasobtained from Fisher Scientific. A549 (CCL-185), Lewis lungcarcinoma (LLC; CRL-1642), and HeLa (CCL-2) cell line wereobtained from ATCC. MDA-MB-231 and HEK-293 cell linewere gifts from Dr. Nikki Cheng (University of Kansas MedicalCenter, KA).

Preparation of the K9–pDNA–Ca2þ complexFor the in vitro studies, the K9–pLUC–Ca2þ complex solution

was prepared by adding 15 mL K9 peptide solution [polymernitrogen to pLUC phosphate (N/P) ratio 10] to 10 mL pDNA

[0.1mg/mL in 1� Tris-acetate-EDTA (TAE) buffer], followed by fastpipetting for 20 seconds. Then, 15 mL calcium chloride solution(e.g., 19, 114, and 228 mmol/L) was added and mixed by fastpipetting. The K9–pDNA–Ca2þ complex solution was incubatedat 4�C for 20 to 25 minutes and used for each experiment. Forthe intravenous administration of the K9–pAT2R–Ca2þ complex,160 mL complex solution was mixed with 40 mL 1%mouse serumalbumin (MSA; the final volume of the complex is 200 mL, 4 mgpAT2R, and 53 mg K9 peptide). For the intratracheal administra-tion, 40 mL the K9-pAT2R-Ca2þ complex solution wasmixedwith10 mL 10% glucose for the osmolality adjustment (the finalvolume of the complex is 50 ml, 1 mg pAT2R, and 13 mg K9peptide).

Preparation of the PEI–pLUC complexThe PEI–pLUC complex solutionwas prepared by adding 15mL

PEI solution (N/P ratio 10) to 10 mL pLUC (0.1 mg/mL) followedby fast pipetting for 20 seconds. The PEI–pLUC complex solutionwas incubated at 4�C for 20 to 25 minutes and used for eachexperiment. The complex solution was prepared immediatelybefore each experiment and used as a control for all in vitro study.

Agarose gel electrophoresisThe K9–pLUC–Ca2þ complex solution was mixed with 4 mL

TAE buffer. Then, 4 mL SYBRGreen 1 wasmixed with the complexsolution, followed by incubation at 4�C for 20 to 25 minutes.After adding 7 mL of 6�DNA Loading Dye, the mixture solutionswere loaded onto a 1% agarose gel and electrophoresed for 30minutes at 110 V.

Size and zeta potentialThe particle size [effective diameter (nm)] of the K9–pLUC

complex with or without calcium chloride was determined bydynamic light scattering (Brookhaven Instruments). The zetapotentials of the complexes weremeasured by Zeta PALS dynamiclight scattering (Brookhaven Instrument). Particle size was mea-sured after dispersing the complexes into nuclease-free water(NFW) or serum-free media (SFM). Zeta potential was measuredafter dispersing the complexes into 1mmol/L potassium chloridesolution.

Cell cultureA549 cell line were grown in F-12K Nutrient Mixture media

(Mediatech, Inc.) supplemented with 10% (v/v) FBS (FBS;Hyclone) and 1% (v/v) penicillin/streptomycin (MB Biomedical,LLC). HeLa, MDA-MB-231, LLC, and HEK-293 cell lines weregrown in DMEM(Invitrogen/Life Technologies) supplementedwith 10% FBS and 1% penicillin/streptomycin. These cell lineswere incubated at 37�C in 5% CO2 humidified the air. Cell linewas authenticated by short tandem repeat (STR) DNA profiling.The cells were maintained in low passage (<15) for this study.

Transfection efficiency of the K9–pDNA–Ca2þ complexes tocultured cells

A549, HeLa, MDA-MB-231, LLC, and HEK-293 cell (80,000cells/well) were cultured in 96-well plates for 24 hours prior to thetransfection. The cells were washed once with SFM and 100 mLtransfection solution (a mixture of 20 mL of the K9–pLUC–Ca2þ

complex and 80 mL of SFM, 0.5 mg pLUC/well) was added to eachwell. After 5 hours incubation, the transfection solution wasreplaced with 100 mL fresh growth medium. After 48-hour

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incubation, total cellular protein was collected by using BCAProtein Assay Reagent (Thermo Fisher Scientific Inc.). The effi-ciency of the gene transfection by the complexes was determinedby Luciferase Reporter Assay using Luciferase Assay SystemFreezerPack (Promega). The Luciferase expression was measured by amicroplate reader (SpectraMax; Molecular Devices Corp). Thetransfection efficiency was expressed as relative light units (RLU)per milligram (mg) of cellular protein.

Cytotoxicity of K9 peptide, PEI, and calcium chloride in vitroCytotoxicity of K9 peptide, PEI, and calcium chloride was

determined using a CellTiter 96 AQueous Non-Radioactive CellProliferation Assay (MTS) obtained from Promega. A549, HeLa,MDA-MB-231, LLC, and HEK-293 cells were cultured in a 96-wellplate in growth media as described previously. Cells were treatedwith the samples (0–5 mg/mL as indicated in the Fig. 4) forapproximately 24 hours. After 24 hours of incubation, the mediawere replaced with a mixture of 100 mL of fresh serum mediumand 20 mL ofMTS. Then, the plate was incubated for 3 hours in theincubator. To determine cell viability, the absorbance of eachwellwasmeasured by amicroplate reader (SpectraMax) at 490nmandnormalized to untreated control cells.

Assessment of DNA accessibility to the K9–pLUC complex bySYBR Green assay

The degree of pLUC accessibility in the K9- or PEI–pLUCcomplex was assessed by the double stranded DNA bindingreagent SYBR Green. Briefly, 10 mL pLUC (0.1 mg pDNA/mL)was mixed with 15 mL of K9 peptide (N/P ratio 10), then 15 mLdeionized water or 38 mmol/L calcium chloride solution wasadded. After a 30-minute incubation at room temperature,120 mL PBS, and 160 mL 10� SYBR Green solutions (Invitrogen)were added. And then 100 mL sample was added to one well ofa 96-well cell culture plate. The fluorescence was measured usinga fluorescence plate reader (SpectraMax M5; excitation, 250 nm;emission, 520 nm).

AnimalsSix-week-old wild-type male C57BL/6 mice were obtained

from Charles River Laboratories International, Inc. All mice werehoused in a clean facility and held for 10 days to acclimatize. Allanimal experiments were carried out under strict adherence to theInstitutional AnimalCare andUseCommittee (IACUC)protocols

and the Institutional Biosafety Committee set by Kansas StateUniversity (Manhattan, KS).

Lung cancer graft in syngeneic mouse and treatment with theK9–pAT2R–Ca2þ complex

Seven-week-old C57BL/6 mice were intravenously injectedwith 1.2 � 106 LLC cells suspended in 200 ml PBS via the tailvein using a 1mL syringewith a 27Gneedle. TheK9–pAT2R–Ca2þ

complex solution was prepared immediately before injection asdescribed above. For the intravenous administration of theK9–pAT2R–Ca2þ complex, 160 mL complex solution was mixedwith 40 mL 1% MSA. For the intratracheal administration, 40 mLthe K9–pAT2R–Ca2þ complex solution was mixed with 10 mL10% glucose for the osmolality adjustment. After 7 days of LLCinjection, the mice were injected with 200 and 50 mL of thecomplexes with each composition via intravenous and intratra-cheal, respectively. PBS and K9-Ca2þ solution without pAT2Rwere used as control. In addition, K9–pLUC–Ca2þ complexprepared as described above was also injected into LLC tumor-bearing mice via intravenous and intratracheal as a nonspecificgene control. Mice were sacrificed by cervical dislocation underdeep anesthesia at 14 days after the complex treatment. The lungswere dissected and tumor burden was analyzed.

Immunohistochemical analysis for AT2R, cell proliferation,and apoptosis in LLC allografts

Fixed lung tissues were sectioned at 4 mm and stained withhematoxylin and eosin (H&E) staining for histologic examina-tion. To analyzeAT2R expression and cell proliferation byKi-67 inLLC tumors, sections were deparaffinized and heat-induced epi-tope unmasking was performed in the citrate buffer followedby incubation with 3% H2O2/methanol for 3 minutes to blockendogenous peroxidase activity. Sections were incubated withpolyclonal anti-AT2R (1:100 dilution, for 18 hours at 4�C,Abcam) and polyclonal anti-Ki-67 (1:500 dilution, for 18hours at 4�C, Abcam) antibodies. After the incubation withprimary antibodies, sections were induced into reaction with abiotin-conjugated anti-rabbit IgG antibody (Vector Laboratories)at a 1:100 dilution for 1 hour at 37�C, followed by reaction withthe avidin–biotin–peroxidase complex reagent (Vector Laborato-ries) for 40minutes at 37�C. Reactions were developed with 3, 30-diaminobenzidine tetrahydrochloride (Sigma) and counter-stained lightly with Mayer hematoxylin. The cell proliferation

Figure 1.Agraose gel electrophoresis study of K9–pDNA–Ca2þ complexes at N/P ratios of 5, 10, or 30 with or without 38 mmol/L calcium chloride (A), K9–pDNAcomplexes without calcium chloride at N/P ratios of 4, 3, 2, 1, 0.5, or 0.1 (B), and pLUC/calcium chloride complexes with 0, 19, 38, or 114 mmol/L of calciumchloride (C). InA,A–C refer toN/P5,N/P 10, andN/P30 in the presenceof 38mmol/L calciumchloride, respectively,whereas a, b, and c refer toN/P5,N/P 10, andN/P30 in the absence of calcium chloride, respectively. M, size marker.

pAT2R Gene Therapy Inhibits Lung Cancer via IV/IT Injection

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index was assessed as the fold change of average percentage ofpositive cells in three randomly selected fields in the tumornodules.

Apoptotic cells in LLC tumors were detected by a TUNEL assayby using the DeadEnd Colorimetric TUNEL System (Promega),according to the manufacturer's instructions with a slight mod-ification (29). The apoptotic indexwas assessed as the fold changeof average number of positive cells in three randomly selectedfields in the tumor nodules.

Statistical analysisData were analyzed by using GraphPad software. All values

were expressed as the mean � SEM. All experiments were con-ducted with multiple sample determinations. A statistical evalu-ation comparing the significance of the difference in gene expres-sion (RLUs/mg protein) between the means of two datasets wasperformed using a t test. One-way ANOVA, Tukey post test wasused to analyze the differences whenmore than two datasets werecompared.

ResultsFormation of the K9–pLUC–Ca2þ complex

The K9–pDNA–Ca2þ and PEI–pDNA complexes were preparedbymixing pLUCor pAT2Rwith K9peptide at variousN/P ratios asdescribed in the Materials and Methods section. To demonstratecomplex formation, agarose gel electrophoresis assay was per-formedusing 1%agarose gel, and electrophoresed for 30minutes.Uncomplexed pLUC (naked pLUC) was used as a control. The K9peptide and pLUC mixture showed ability to form complexes ofpLUC and K9 peptide regardless of the presence of 38 mmol/Lcalcium chloride at N/P ratios 5, 10, and 30. As the net charges ofthese complexes are positive, the complexes stayed in the loadingwells without migrating into the gel and no bands were observedin the electrophoresis (Fig. 1A). Although lower N/P ratios (1–4)also showed no bands in the absence of calcium chloride, the N/Pratios lower than 0.5 showed bands (Fig. 1B). In addition, mixingwith calcium chloride and pLUC did not form stable complexes(Fig. 1C).

Physical characterization of the K9–pLUC–Ca2þ and thePEI–pLUC complexes

The effect of calcium chloride concentration on the surfacecharge and particle size of the K9-pLUC complexwas investigated.As shown in Fig. 2A, addition of calcium chloride of 37.7 and113 mmol/L (final concentration) significantly decreased theparticle size of the K9–pLUC–Ca2þ complex, with relativelynarrow polydispersity (�0.1), in both NFW and in SFM. The zetapotential of the K9–pLUC–Ca2þ and PEI–pLUC complexesincreased significantly with increases in the concentration ofcalcium chloride (Fig. 2B).

SYBR Green assay provides a nondestructive and simplemethod to examine pDNA accessibility within complexes. Asshown in Fig. 2C, pLUC was accessible in the K9–pLUC com-plex, whereas pLUC accessibility in the PEI–pLUC complex waslow as compared with pLUC alone. Although pLUC accessibil-ity in the K9–pLUC complex was significantly decreased in thepresence of 38 mmol/L calcium chloride, DNA accessibility waslower in the PEI–pLUC complex. These results suggest that theaddition of calcium chloride significantly decreased the size ofthe K9–pLUC complex, but the pLUC in the complex was moreaccessible than in the PEI–pLUC complex.

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Figure 2.Evaluation of the particle sizes (effective diameters), zeta potentials, andSYBR Green assay of the K9–pLUC and PEI–pLUC complexes with orwithout calcium chloride. A, the particle size of the K9–pLUC complexes(N/P ratio ¼ 10) were determined by DLS in the presence of variousconcentrations of calcium chloride (0, 38, and 114 mmol/L) in NFW or SFM.B, zeta potentials of the K9–pLUC and PEI–pLUC complexes (N/Pratio ¼ 10) were determined by Zeta PALS dynamic light scattering in thepresence of various concentrations of calcium chloride (0, 38, and 114mmol/L). C, effect of calcium chloride concentration at 0 and 38 mmol/Lon pLUC accessibility in K9–pLUC and PEI–pLUC complexes at an N/Pratio of 10 was evaluated using the SYBR Green assay. The pLUC alonein the solution was used as a control. Results are presented as mean � SD(n ¼ 3). ��, P < 0.0001, one-way ANOVA, Tukey post test comparisonto pDNA.

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The K9–pLUC–Ca2þ complex caused efficient gene transfectionwith low cytotoxicity in vitro

The in vitro transfection efficiency of the K9–pLUC–Ca2þ andthe PEI–pLUC complexes was studied using the four differenthuman cell lines and the mouse cell line. Luciferase gene expres-sion was evaluated 48 hours after the transfection using the K9–pLUC–Ca2þ and the PEI–pLUC complexes at N/P ratios 5, 10, 20,and 30, and at various calcium chloride concentrations during thecomplex formulation (Fig. 3A). The K9–pLUC–Ca2þ complexeshad a high level of gene expression at a calcium chloride con-centration of 38 mmol/L (N/P ratio 10) in A549 cells. Luciferasegene expression was decreased when calcium concentration washigher than 114 mmol/L in all N/P ratios tested. This result wasfurther confirmed in other cell lines: A549 cells (Fig. 3B), HeLacells (Fig. 3C), MDA-MB-231 cells (Fig. 3D), HEK-293 cells (Fig.3E), and LLC cells (Fig. 3F). In all cells lines examined, thetransfection efficiency of the K9–pLUC–Ca2þ with 38 mmol/Lcalcium chloride was significantly higher than those with PEI–pLUC and the K9–pLUC–Ca2þ with 114 mmol/L calcium chlo-ride (Fig. 3). Significance of including K9 peptide in pDNAtransfectionwas further examinedby comparing pLUCexpressionefficiency in the presence or absence of the peptide in various celllines (Supplementary Fig. S1). The transfection efficiency of thepLUC–Ca2þ complex (without K9 peptide) was significantlylower than that of the K9–pLUC–Ca2þ complex at the samecalcium chloride concentration.

High transfection efficiency and low cytotoxicity are vitalattributes for nonviral gene vectors. To examine whether K9,PEI, and calcium chloride affected the viability of live cells, amembrane translocalization signal (MTS) cytotoxicity assay wasconducted using five different cell lines. The five cell lines wereindividually incubated with up to 5 mg/mL K9, PEI, or calciumchloride for 24 hours and then MTS assay was performed(Fig. 4). The K9 peptide showed no cytotoxicity to 2.5 mg/mL.Only HEK-293 cell viability was decreased at 1 mg/mL levels ofK9 peptide. Calcium chloride also did not show strong cyto-toxicity until 1 mg/mL. However, PEI induced significant cyto-toxicity even at 10 mg/mL in the four cell lines. Although HEK-293 cells were resistant to PEI-induced cytotoxicity, their cellviability was gradually decreased at higher concentrations. ThisMTS assay strongly suggested that the K9–pDNA–Ca2þ complexis a low cytotoxic pDNA transfection vector.

Treatment with the K9–pAT2R–Ca2þ complex via intravenousinjection or intratracheal spray caused significant growthattenuation of lung tumors

The effect of the K9–pAT2R–Ca2þ complex delivering theendogenous apoptosis-inducer gene AT2R was examined usingorthotopic LLC allografts in syngeneic C57BL/6mice. To evaluatethe effect of the complex on the lung tumor growth, LLC cells (1.2� 106) were injected via the tail vein. Seven days after cancer cellinjection, the mice were treated with a single dose of the K9–pAT2R–Ca2þ complexes containing 4 mg pAT2R intravenously or1 mg pAT2R intratracheally. The tumor growth attenuating theeffect of these complexes was observed both macroscopically(Fig. 5A and C) and microscopically (Fig. 5B). Macroscopically,a large number and large size of tumor nodules were detected inPBS- or K9-treatedmouse lungs. Average lungweights (mg) of theK9–pAT2R–Ca2þ intratracheal (190.6 � 48.3) and the K9–pAT2R–Ca2þ intravenous (201.6 � 67.0) treated groups weresignificantly smaller than that of the control PBS group (325.7 �

69.4, P < 0.05, Fig. 5C and Supplementary Table S1). Histologicexamination of tumors in H&E-stained lung sections also dis-played only a small number and small size of LLC tumor nodulesin mouse lungs treated with the K9–pAT2R–Ca2þ complexes(Fig. 5B). Average numbers of tumor nodules in the lungs in theintratracheal (6.0� 4.2) and the intravenously (4.6� 3.4) groupswere significantly smaller than that of the control PBS group (17.8� 6.0, P < 0.01, Fig. 5D; Supplementary Table S2). On the otherhand, treatment with K9–pLUC–Ca2þ complexes did not showany effect on average lung weight (246.8 � 70.3 in PBS, 213.9 �52.3 in K9–pLUC–Ca2þ intravenous, and 267.3 � 6.9 in K9–pLUC–Ca2þ intratracheal) and average numbers of tumornodules (20.4 � 4.0, 20.2 � 11.9, and 25.7 � 1.5 in the samegroup order as the lung weights described above) in LLC tumor-bearing mouse lung (Supplementary Fig. S2). Both local (i.e.,pulmonary) and systemic treatment with the K9–pAT2R–Ca2þ

complexes were equally effective in attenuating the growth of LLClung tumor grafts in immunocompetent mice.

Immunohistochemical analysis of AT2R expression, cellproliferation, and apoptosis in LLC grafts

The expression of AT2R gene in the lung was determinedimmunohistochemically (Fig. 6). As shown in Fig. 6A, AT2Rexpression was upregulated in the tumor cells in the K9–pAT2R–Ca2þ intravenous and intratracheal spray groups but notin other groups. These observations suggested that the K9–pAT2R–Ca2þ administration via intravenous and intratrachealsignificantly attenuated the lung tumor growth by expressingAT2R in the tumor cells. Immunohistochemical analysis of cellproliferation in tumors using anti-Ki-67 antibody did not showany difference (Fig. 6B). Although average number of apoptoticcells detected by TUNEL assay tended to increase in the K9–pAT2R–Ca2þ treatment groups (Fig. 6C), there was no statisticalsignificance between groups due to large variations within thesamples. As the analysis was carried out 14 days after treatmentwith the K9–pAT2R–Ca2þ complex, these resultsmay suggest thatthe treatment with the K9–pAT2R–Ca2þ complex attenuatedtumor growth during an early stage of LLC tumorigenesis. Nev-ertheless, these results support that both intravenous and intra-tracheal administration of the K9–pAT2R–Ca2þ offer effectivemodalities for lung cancer targeted nonviral gene therapy.

DiscussionDevelopment in the field of gene therapy is presently hindered

by the lack of robust gene delivery methods. Synthetic, nonviralgene vectors such as polycationic peptides (e.g., polylysine) arepromising vectors (10, 17, 18). The toxicity of these CPPs may beminimized or eliminated by reducing their molecular size. How-ever, the level of transfection efficiency mediated by smallerpolypeptides is typically low compared with larger polypeptides(17). CPPs have been used to deliver various anticancer agents(e.g., smallmolecules, proteins, andnucleic acids) into cells in vivoandhave been observed to be effective in inhibiting tumor growthin preclinical tumor models (17, 30, 31). The main goals of thiswork were to examine the transfection efficiency of the K9–pDNA–Ca2þ complex and to determine whether pDNA (pAT2R)can be distributed to lung cancer cells, resulting in therapeuticallyeffective gene expression.

The in vitro transfection efficiency of the K9–pDNA–Ca2þ

complex was evaluated using luciferase pDNA (pLUC) in the






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Figure 3.The transfection efficiencies of the K9–pLUC complex. A, the transfection efficiencies in various concentrations of calcium chloride (0, 19, 38, 114, and 228 mmol/L)in A549 cells at N/P ratios of 5, 10, 20, and 30. The transfection efficiencies of the K9–pLUC complexes with different concentrations of added calciumchloride (0, 38, and 114mmol/L) at anN/P ratio 10 inA549 cells (B), HeLa cells (C),MDA-MB-231 cells (D), HEK-293 cells (E), and LLCcells (F). RLUs, relative light units.Results are presented as mean � SD (n ¼ 4). �� , P < 0.0001; �� , P < 0.001; one-way ANOVA; Tukey post test.

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four different human cell lines (kidney, cervix, breast, and lung)and one mouse cancer cell line (lung). Over expression of theAT2R gene in lung cancer cells (32), or other type of CPP–pDNA–Ca2þ complex-based intraperitoneal delivery of the AT2R genewas known to reduce lung cancer cell growth (16). Intravenousinjection or intratracheal spray of the K9–pAT2R–Ca2þ complexesyielded robust gene expression, primarily in lung cancer cells, andsignificantly attenuated cancer growth. Therefore, this study pre-sents an effective in vitro and in vivo gene delivery system using alow molecular weight cationic CPP (K9 peptide) for lung cancertherapy.

The formation of complexes between K9 and pDNA wasobserved in both the K9–pLUC–Ca2þ and K9–pLUC complexesas observed via agarose gel electrophoresis (Fig. 1) when theN/P ratio is higher than 0.5. By itself, calcium chloride showednegligible ability to complex naked pLUC even at a highconcentration (114 mmol/L; Fig. 1C). However, the K9–pLUCcomplex without calcium chloride exhibited very low geneexpression (Fig. 3A), and the size of this complex was inap-propriately large (500–1,500 nm) for gene delivery (Fig. 2A).The addition of calcium chloride in the K9–pLUC complexsignificantly decreased the complex size in water and culturemedium (Fig. 2A) and correspondingly increased gene trans-fection (Fig. 3A). Therefore, calcium chloride acted as aneffective condensing agent to decrease the particle size of the

K9–pLUC complex and enhance transfection efficiency. Theseobservations are in good agreement with our previous study inwhich calcium chloride also decreased particle sizes of CPP–pLUC complexes with other types of CPP (33). In this regard, itis of interest to note that the PEI–pLUC complex did not show adecrease in particle sizes as calcium chloride increased (17).Calcium ion–dependent increase of the total positive charge ofthe K9–pLUC–Ca2þ complex may also play an important roleenhancing transfection efficiency by the stronger interactionwith the negatively charged plasma membrane (34). In addi-tion, accessibility of SYBR green to the pLUC in the K9–pLUCcomplex was significantly decreased when calcium chloride(38 mmol/L) was added to the complex solution (Fig. 2C),suggesting the K9–pLUC complex was effectively condensed,and calcium chloride played an essential role in condensationof the complex. The reduction in the particle size of the K9–pDNA–Ca2þ complexes likely led to an increase in transfectionefficiency.

The effect of calcium chloride on the transfection efficiency ofthe K9–pDNA complex was first assessed using A549 cells. Thebest transfection efficiency was achieved at 38 to 76 mmol/Lcalcium chloride (Fig. 3A). Interestingly, no significant level ofgene expression was detected without calcium chloride. As calci-um chloride appeared to be an essential component in thecondensation of the K9–pLUC complex, yielding small

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complexes with optimal pLUC expression, the transfection effi-ciency of the K9–pLUC–Ca2þ and PEI–pLUC complexes (N/Pratio 10) was assessed across five cell lines using three differentcalcium chloride concentrations (0, 38, and 114 mmol/L, Fig. 3).This experiment confirmed that pLUC transfection by the K9–pLUC–Ca2þ complex was highest at 38mmol/L calcium chloride,and the LUC expression was significantly better than that ofPEI–pLUC complex (25 kDa PEI; ref. 35).

In another study, the importance of the K9 peptide in geneexpression was evaluated by comparing pLUC–Ca2þcomplex(without K9 peptide) and the K9–pLUC–Ca2þ complex in thesame calcium concentration. The result in Supplementary Fig. S1clearly shows that pLUC expression resulting from the pLUC–Ca2þ complex (without K9 peptide) was significantly lower thanthat of the K9–pLUC–Ca2þ complex suggesting that K9 in thecomplex is indeed important to achieve the high gene expressionfrom the K9–pLUC–Ca2þ complex. The PEI–pDNA complex hadhigh transfection efficiency in the absence of calcium chloride(17, 18, 33).

A successful gene vector should be able to deliver geneticmaterial to the target cells without influencing the viability ofthe host cells. The present study clearly indicated negligiblecytotoxicity in vitro up to 2.5 mg/mL for K9 peptide and 1 mg/mL for calcium chloride after 24 hours for all five cell lines,whereas PEI exhibited strong cytotoxicity at low micromolarconcentrations in all cell lines except for HEK-293 cells (Fig.4). In addition, the low toxicity of the K9–pAT2R–Ca2þ com-plexeswas also observed in themouse study after intravenous andintratracheal applications, in which all mice receiving K9 alone orthe K9–pAT2R–Ca2þ complex survived during the experimentalperiod and did not show acute inflammatory reaction or any

histologically detectable abnormality. Therefore, data stronglysuggested that the K9–pAT2R–Ca2þ complex represents a safeand efficient gene transfection vector.

The robust gene expression in various cell lines with negligiblecytotoxicity compelled in vivo gene transfection studies. An endog-enous apoptosis inducer gene, AT2R, was delivered using K9–pAT2R–Ca2þ complexes administered to LLC tumor-bearingmice. As shown in Fig. 5, the treatment with the K9–pAT2R–Ca2þ

complex via either intratracheal or intravenous administrationssignificantly attenuated tumor growth macroscopically andmicroscopically, while the treatment with the nonspecific controlgene complex (K9–pLUC–Ca2þ) did not show any effect ontumor growth attenuation effect (Supplementary Fig. S2). Immu-nohistochemical analysis of AT2R protein expression in the lungindicated that the expression of this gene was detected mainly inlung cancer cells, but not in normal lung tissues, which suggeststhat the K9–pAT2R–Ca2þ complex preferentially delivered thetherapeutic gene into cancer cells. It has been known that anincreased expression of anionic molecules in the membrane ofcancer cells, such as: the phosphatidylserine, O-glycosylatedmucins, sialylated gangliosides, and heparin sulfate, resulted inan increased net negative charge compared to the nonmalignantcell membrane (36). These electric characteristics of the cancercell's surfaces support that polycationic peptide-based nanopar-ticles are suited for cancer-targeted gene therapy. The observedincrease of apoptotic cells in the K9–pAT2R–Ca2þ complex trea-ted groups (Fig. 6) suggested AT2R expression in the tumorsinduced apoptosis in cancer cells. These results are consistentwith earlier studies confirming that AT2R expression attenuatesgrowth of various cancer cells including lung cancer cells(16, 32, 37–39). Therefore, AT2R gene expression is a potential

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treatment scheme for primary as well asmetastatic lung cancers. Asingle intrvenous bolus injection or intratracheal spray of K9alone did not attenuate cancer growth significantly as comparedwith the PBS controls (Fig. 5). Dosing K9 alone or K9–pLUCneither exhibit any side effects such as abnormal clinical orhistologic signs in the native tissue of the lung nor alter the tumorgrowth. These results suggest that K9 peptide is a useful and safevector for cancer gene therapy.

In the current study, K9–pDNA–Ca2þ complex-based genedelivery was found to be a useful tool for an in vivo gene deliverysystem for lung cancer treatment in immunocompetentmice. As itis obvious that both cellular and humoral immune systems in thetumor microenvironment play a significant role in regulation oftumor growth (40), use of immunocompetent mice for theevaluation of the newly developed treatment regimen is appro-priate. Although our study has provided first proof-of-principledata using murine lung carcinoma cells and syngeneic immuno-competent mouse model, further evaluation of this gene therapyusing multiple types of human lung cancer cells in mouse xeno-graft models will solidify this discovery.

A recent study has evaluated the utilization of polylysinedendrimers for the delivery of Polo-like kinase 1 (PLK1)-spe-cific siRNA for the treatment of non–small cell lung cancer invitro and in vivo (41). The apparent safety and acceptabletransfection efficiency of polylysine dendrimers may supportthe calcium condensed polylysine complexes used for pDNAdelivery in the current study. The K9–pDNA–Ca2þ complex is asimple approach, thus usefulness in future translational studiesappears to be very high.

ConclusionThe addition of calcium chloride to nascent complexes of

polylysine CPP (K9 peptide) and pDNA (pLUC and pAT2R)produced small and stable complexes. These complexes exhibitedhigh gene expression in various human and mouse cancer cellscomparable with PEI–pDNA complexes. Single intravenous orintratracheal administrations of the K9–pAT2R–Ca2þ complexes

significantly attenuated the growth of LLC cell allografts inmouselungs, suggesting that K9 peptide–based gene therapy is effectiveand that the AT2R gene is potentially useful for lung cancer genetherapy. K9 peptide showed negligible cytotoxicity in cell culture,supporting the notion that this CPP is a safe delivery vehicle forgenetic materials (e.g., siRNA and pDNA). Although, furtherstudies are required to confirm the in vivo safety of the K9–pAT2R–Ca2þ complexes by formal pharmacokinetics, pharma-codynamics, and multispecies toxicity studies, these data showedthat the K9–pDNA–Ca2þ complexes could be an effective and safenonviral gene transfection tool.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: N.A. Alhakamy, S. Ishiguro, C.J. Berkland, M. TamuraDevelopment of methodology: N.A. Alhakamy, C.J. Berkland, M. TamuraAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N.A. Alhakamy, M. TamuraAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): N.A. Alhakamy, S. Ishiguro, D. Uppalapati,C.J. Berkland, M. TamuraWriting, review, and/or revision of the manuscript: N.A. Alhakamy, S. Ishi-guro, D. Uppalapati, C.J. Berkland, M. TamuraAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. TamuraStudy supervision: C.J. Berkland, M. Tamura

AcknowledgmentsThe authors acknowledge financial support from the following sources:

Savara Pharmaceuticals (to C.J. Berkland, a co-founder of Savara Pharmaceu-ticals, and M. Tamura), and Faculty of Pharmacy of King Abdulaziz University,Jeddah, Saudi Arabia (to N.A. Alhakamy). The authors also thank Macromol-ecule and Vaccine Stabilization Center (University of Kansas) for the use oflaboratory equipment.

Grant SupportThis work was supported by King Abdulaziz University, Jeddah, Saudi

Arabia (to N.A. Alhakamy), Kansas State University Johnson Cancer ResearchCenter (to M. Tamura), NIH grants U43 CA165462 (to M. Tamura), P20

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Figure 6.Immunohistochemical analysis ofAT2R expression (A), cellproliferation (B), and apoptosis (C) inLLC graft tumors in PBS, K9 aloneintratracheal, K9–pAT2R–Ca2þ

intracheal (complex IT), K9 aloneintravenous, or K9–pAT2R–Ca2þ

intravenous (complex IV)-treatedmice (n ¼ 5). A, microscopic imagesof immunohistochemistry for AT2Rexpression (original magnification at200�). AT2R expression wasobserved in the tumor cells fromcomplex IT or complex IV–treatedmice (insets). Scale bars, 100 mm (A).Cell proliferation index (B) andapoptotic index (C) were evaluatedby Ki-67 expressions and TUNELassay in the tumor cells, respectively.K9, nine amino acid polylysine.






GM103418 (M. Tamura), Kansas Bioscience Authority Collaborative CancerResearch grant (to M. Tamura), and Savara Pharmaceuticals (to C. Berklandand M. Tamura).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 29, 2015; revised October 6, 2015; accepted October 16, 2015;published OnlineFirst December 4, 2015.

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Alhakamy et al.




Published OnlineFirst December 4, 2015.Mol Cancer Ther Nabil A. Alhakamy, Susumu Ishiguro, Deepthi Uppalapati, et al. Injection or Intratracheal SprayAttenuates Lewis Lung Carcinoma after Intravenous AT2R Gene Delivered by Condensed Polylysine Complexes

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