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Biomaterials 33 (2012) 6542e6550

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Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

Amphiphilic peptide carrier for the combined delivery of curcumin and plasmidDNA into the lungs

Ji Hwan Park a, Hyun Ah Kim a,b, Jin Hyeong Park a, Minhyung Lee a,b,*

aDepartment of Bioengineering, College of Engineering, Hanyang University, Seoul 133-791, Republic of Koreab Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, Seoul 133-791, Republic of Korea

a r t i c l e i n f o

Article history:Received 9 April 2012Accepted 21 May 2012Available online 9 June 2012

Keywords:DNAGene transferGene expressionCell viabilityInflammation

* Corresponding author. Department of BioengineeHanyang University, 17 Haendang-dong, Seongdong-gKorea. Tel.: þ82 2 2220 0484; fax: þ82 2 2220 1998.

E-mail address: minhyung@hanyang.ac.kr (M. Lee

0142-9612/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.biomaterials.2012.05.046

a b s t r a c t

In this study, the R7L10 peptide, which is composed of a 7-arginine stretch and a 10-leucine stretch, wasevaluated as a carrier for the combined delivery of curcumin and plasmid DNA (pDNA) into the lungs.Curcumin is a natural product with anti-inflammatory and anti-tumor effects. Curcumin-loaded R7L10(R7L10-curucmin) was prepared by an oil-in-water (O/W) emulsion/solvent evaporation method. In vitrotransfection showed that R7L10-curcumin had higher transfection efficiency than R7L10. AlthoughR7L10-curcumin had lower transfection efficiency than polyethylenimine (25 kDa, PEI25k) and lip-ofectamine, R7L10-curcumin had lower cytotoxicity. In gel retardation assays and heparin competitionassays, R7L10-curcumin formed a more stable complex with pDNA than R7L10. The intracellular cur-cumin delivery efficiency of R7L10-curcumin was higher than that of curcumin only. Furthermore, R7L10-curcumin more efficiently decreased TNF-a level in lipopolysaccharide (LPS)-activated Raw264.7macrophage cells than curcumin only. For in vivo evaluation, pDNA/R7L10-curcumin complexes wereadministered into mouse lungs by intratracheal instillation. The results revealed that R7L10-curcumindelivered pDNA more efficiently than R7L10, poly-L-lysine (PLL), or PEI25k. In addition, R7L10-curcumin decreased TNF-a level in lung tissues in an acute lung injury mouse model. In contrast toPEI25k, R7L10-curcumin did not show liver toxicity after intravenous injection. These results suggest thatR7L10-curcumin is a useful carrier for the combined delivery of curcumin and pDNA into the lungs.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Various kinds of gene carriers have been developed for genedelivery. Non-viral carriers such as cationic polymers and lipo-somes have been evaluated as gene carriers for therapeutic appli-cations [1e6]. Recently, it was suggested that combined therapywith both drugs and genes might be effective for the treatment ofcertain types of diseases [7,8]. Drugs elicit immediate therapeuticeffects, while genes have prolonged therapeutic effects with theadded benefit of sustained expression of therapeutic proteins.Amphiphilic polymers, which are composed of both hydrophobicand hydrophilic polymer blocks and form micelles in aqueoussolution, are an example of potential carriers for dual drug and genetherapy. Amphiphilic polymers have been evaluated as carriers ofpaclitaxel and siRNA; the hydrophobic anti-tumor drug was loaded

ring, College of Engineering,u, Seoul 133-791, Republic of

).

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in the core of the micelles, and siRNA was bound on the cationicsurfaces [8].

Another example employs the use of short amphiphilicpeptides, which are composed of short hydrophobic and hydro-philic amino acid chains [9,10]. Self assembly peptides have beendeveloped as carriers for hydrophobic drug. R3V6 and R3L6 havepreviously been reported as delivery carriers for dexamethasoneand BCNU [9,10]. In this case, hydrophobic drugs such as BCNU anddexamethasone are loaded into the cores of the amphiphilicpeptide micelles. Drug-loaded peptide micelles demonstratedhigher delivery efficiency than drug administration alone.Furthermore, these hydrophobic drug-loaded peptide micelleshave improved gene delivery efficiency to a greater extent thanmicelles without drugs [9,10]. Hydrophobic drugs may provide forthe hydrophobic core, facilitating micelle formation with the shortamphiphlic peptides. As a result, drug-loaded peptide micellesmore easily and tightly form complexes with pDNA, increasingtransfection efficiency.

Hydrophobic drug-loaded polymeric micelles have someadvantages. The amphiphilic polymers increase the solubility ofhydrophobic drugs. Furthermore, nanoparticle formation with

J.H. Park et al. / Biomaterials 33 (2012) 6542e6550 6543

pDNA and drug-loaded micelles increases the cellular uptake ofhydrophobic drugs. Nanoparticles can be taken up by cells throughendocytosis, which is an active transport process that is moreefficient than simple diffusion. Considering that most hydrophobicdrugs can enter cells by simple diffusion, nanoparticle formationwith pDNA is an important step to increase the cellular uptake ofhydrophobic drugs. In this context, pDNA is not only a therapeuticagent that expresses therapeutic proteins in cells, but is alsoa structural element for efficient delivery of hydrophobic drugs. Ina previous report of dexamethasone-conjugated polyethylenimine(PEI2k-Dexa), PEI2k-Dexa with pDNA demonstrated higher cellularentry efficiency than PEI2k-Dexa alone, suggesting the effect ofpDNA as a structural element [11].

In a previous study with 1,3-bis(2-chloroethyl)-1-nitrosourea(BCNU)-loaded R3L6 micelles, BCNU delivery efficiency wasenhanced by loading the drug into the cores of the micelles [9]. Inaddition, pDNA delivery efficiency was enhanced by the drug-loaded peptide micelles [9]. As a result of this observation, RLpeptidemicelles were synthesized and evaluated as a carrier for thecombined delivery of curcumin (diferuloylmethane) and pDNA intothe lungs in the present study.

Curcumin, which originates from Curcuma longa (turmeric), hasbeen reported to have many therapeutic effects [12e17]. Due to itsanti-inflammatory effects, curcumin has been evaluated as a ther-apeutic agent in various inflammation related diseases [15],including rheumatoid arthritis, osteoarthritis, cancer, and acutelung injury [15,18,19]. Curcumin suppresses the activation of NF-kB.NF-kB is a ubiquitous transcription factor, which is involved in theexpression of various pro-inflammatory cytokines [20]. Numerouspreclinical and clinical studies involving curcumin treatment inthese diseases are currently in progress, and positive results havebeen obtained.

In this study, RL peptides of various sizes were evaluated ascarriers of pDNA. Then, curcumin was delivered to the lung in anacute lung injury animal model using R7L10 as a carrier. At thesame time, curcumin-loaded R7L10 (R7L10-curcumin) was evalu-ated as a gene carrier into the lung.

2. Materials and methods

2.1. Synthesis of R3L6, R5L8, and R7L10 peptides and preparation of curcumin-loaded R7L10

R3L6, R5L8, and R7L10 peptides were synthesized by solid phase peptidesynthesis (Peptron, Daejeon, Korea). The synthesized peptides were purified by C18reverse phase HPLC (Capcell pak C18 column, Shiseido, Tokyo, Japan) (Fig. S1). Theflow rate was 1.0 ml/min with the gradient of 0e20% mobile phase B in 2 min,20e50% in 10 min, and 50e80% in 2 min (Mobile phase A: 0.1% trifluoroacetic acid(TFA) in water, B: 0.1% TFA in acetonitrile). The purity of the peptide was more than98%. The counterions for the peptides were TFA. The mass of the purified peptidewas confirmed by Liquid chromatographyemass spectrometry (LC/MS) (Agilent,Santa Clara, CA) (Fig. S1). The RL peptides were dissolved in distilled water ata concentration of 5 mg/ml and were stored at �70 �C until use. Curcumin-loadedR7L10 (R7L10-curcumin) was prepared at R7L10/curcumin weight ratios of 1:0.2,0.4, and 0.6 (molar ratios of 1:1.2, 2.4, and 3.6). The structure of curcumin is pre-sented in Fig. S2. Curcumin was loaded into the R7L10 peptide micelles using an oil-in-water (O/W) emulsion/solvent evaporation method as described previously [10].The R7L10 and curcumin were dissolved in distilled water and methanol, respec-tively. The two solutions were mixed slowly and dispersed in distilled water viasonication for 90 s R7L10-curcumin was lyophilized using a freeze-dryer. Thelyophilized R7L10-curcumin was readily resuspended in phosphate buffered saline(PBS).

2.2. Preparation of pDNA

pb-Luc and pb-HO-1 were used as pDNAs in this study. pb-Luc was previouslyconstructed by insertion of the luciferase cDNA isolated from pGL3-promoter(Promega, Madison, WI) at the sites of HinDIII and XbaI of pb [21]. The hemeoxygenase-1 (HO-1) cDNA was isolated from pSV-HO-1 by digestion with HinDIIIand XbaI. The HO-1 cDNA was inserted into pb at the sites of HinDIII and XbaI. Theconstruction of pb-HO-1 was confirmed by directed sequencing. The pDNAs were

transformed and amplified into the JM109 E.coli strain. The pDNAs were purifiedusing the Qiagen Maxi plasmid preparation kit (Qiagen, Valencia, CA). Theconcentration and purity of pDNAwere measured by ultraviolet (UV) absorbance at260 and 280 nm, respectively.

2.3. Gel retardation assay

Gel retardation assays were performed to confirm complex formation betweencarriers and pDNA. A fixed amount of pDNA was mixed with increasing amounts ofR7L10, R7L10-curcumin, and poly-L-lysine (PLL). The mixtures were incubated atroom temperature for 30 min for complex formation and then analyzed on a 1%agarose gel by electrophoresis. The position of pDNA in the gel was visualized ona UV illuminator.

2.4. Heparin competition assay

pDNA/carrier complexes were prepared at their optimum ratios for the highesttransfection. pDNA/R7L10 and pDNA/R7L10-curcumin complexes were prepared ata weight ratio of 1:30 (pDNA:carrier), and pDNA/PLL complexes were prepared ata weight ratio of 1:2 [22]. Increasing amounts of heparin were added to thecomplexes, which were then incubated at room temperature for 30 min andanalyzed on a 1% agarose gel by electrophoresis. The position of pDNA in the gel wasvisualized on a UV illuminator.

2.5. DNase I protection assay

pDNA/R7L10 and pDNA/R7L10-curcumin complexes were prepared with 10 mgof pDNA at aweight ratio of 1:30 in 500 ml of PBS. Naked pDNAwas used as a control.After complex formation, DNase I (10 unit, Promega, Madison, WI) was added to thecomplex solutions. The reaction mixtures were incubated for the indicated timeperiod at 37 �C. One hundred microliters of the samples were collected at 30 or60 min after incubation and mixed with 100 ml of 2 � stop solution (80 mM EDTAand 2% SDS) to dissociate pDNA from the carriers. The DNAwas analyzed by agarosegel electrophoresis.

2.6. Scanning electron microscopy (SEM) image

A suspension of the complexes in deionized water was mounted on aluminumholders at room temperature, dried overnight, and then coated with platinum undera vacuum. The morphology and size of the complexes were then investigated byscanning electron microscopy (S-4800 UHR FE-SEM, Hitachi, Japan).

2.7. Cell culture and transfection assay

Human embryonic kidney 293 (HEK293) cells were maintained in Dulbecco’sModified Eagle’s Medium (DMEM, Welgene, Seoul, Korea) containing 1% penicillinand 10% fetal bovine serum (FBS) at 37 �C in a 5% CO2 atmosphere. HEK293 cellswere seeded at a density of 1 � 105 cells/well in 12-well plates 24 h before thetransfection.

For comparison of the transfection efficiencies of R3L6, R5L8, and R7L10, pDNA/R3L6, pDNA/R5L8, and pDNA/R7L10 complexes were prepared at a 1:30 weight ratiobased on previous reports [9,23]. The N/P (nitrogen of peptide/Phosphate of pDNA)ratios of pDNA/R3L6, pDNA/R5L8, and pDNA/R7L10 complexes were 1:33, 1:34, and1:34, respectively. For optimization, pDNA/R7L10-curcumin complexes wereprepared at various weight ratios. For comparison of R7L10-curcumin with othercarriers, pDNA/R7L10 and pDNA/R7L10-curcumin complexes were prepared ata 1:30 weight ratio. pDNA/PLL and pDNA/polyethylenimine (25 kDa, PEI25k)complexes were prepared at 1:2 and 1:1 weight ratios, respectively, based onprevious reports [22,24,25]. pDNA/lipofectamine complex was prepared at a 1:2.5w/v ratio, based on the manufacturer’s manual (Qiagen, Valencia, CA). Beforetransfection, the cell culture medium was replaced with serum-free DMEM. Then,the transfection mixtures were added to the cells. The cells were incubated for 4 h at37 �C in a 5% CO2 atmosphere. After the medium was replaced with DMEM con-taining 1% penicillin and 10% FBS, the cells were incubated for an additional 24 h at37 �C in a 5% CO2 atmosphere. After incubation, cells were harvested for a luciferaseassay.

2.8. Luciferase assay

After transfection and incubation, cells were washed twice with 0.5 ml of PBS,and 120 ml of reporter lysis buffer (Promega, Madison, WI) was added to the cells.The cell extracts were then harvested, transferred to microcentrifuge tubes, andcentrifuged at 13,000 rpm for 3 min to remove cell debris. Luciferase activities of thesamples were measured at room temperature using a 96-well plate luminometer(Berthold Detection System GmbH, Pforzheim, Germany). Protein concentrations inthe extracts were measured using a bicinchoninic acid (BCA) assay kit (Pierce, Iselin,NJ). The final luciferase activities were calculated as relative light units (RLU) per mgprotein.

Fig. 1. Gel retardation assay A fixed amount of pDNA was mixed with increasingamounts of R3L6, R5L8, and R7L10. The mixtures were analyzed by agarose gelelectrophoresis.

J.H. Park et al. / Biomaterials 33 (2012) 6542e65506544

2.9. Cytotoxicity assay

Cytotoxicity was measured by MTT [3-(4,5-dimethylthiazol)-2,5-dephenyl-2H-tetrazoliumbromide] assay. The complexeswere added toHEK293 cells and incubatedfor 4 h at 37 �C in serum-freemedium. After the transfectionmixtures were removed,500 ml of fresh DMEM containing 10% FBS was added to each well. The cells wereincubated for an additional 20 h at 37 �C. At the end of the transfection experiment,40 ml of 5mg/mlMTT reagent in PBSwas added. The plateswere then incubated for anadditional 4 h at 37 �C. Formazan crystals were formed by reduction of MTT in livingcells.MediumcontainingMTTsolutionwas removed, and750ml ofDMSOwasadded todissolve the formazan crystals formed by live cells. Absorbance was measured at570 nm. Cell viability (%) was calculated according to the following equation:

Cell viability (%) ¼ (OD570(sample)/OD570(control)) � 100.

2.10. Curcumin intracellular delivery assay

HEK293 cells were seeded at a density of 1 �105 cells/well in 12-well plates andincubated for 24 h at 37 �C before transfection. pDNA/R7L10, pDNA/R7L10-curcumin,and pDNA/PLL complexeswere added toHEK293 cells and incubated for 4 h at 37 �C inserum-free medium. The same amount of free curcumin as pDNA/R7L10-curcumincomplex was added to some of the cells as a control. After the media was removed,500 ml of fresh DMEM containing 10% FBS was added to each well. The cells wereincubated for an additional 20 h at 37 �C. After treatment, the cells werewashed twicewith PBS, and120ml of reporter lysis buffer (Promega,Madison,WI)was added to eachwell. The cell extracts were harvested and transferred to microcentrifuge tubes. Theextracts were centrifuged at 13,000 rpm for 3 min to remove cell debris. Absorbanceand fluorescence of the samples were measured at room temperature using a 96-wellplate and SpectraMax (Molecular Devices, Sunnyvale, CA). Absorbance read at 450 nmand fluorescence was measured at excitation 485 nm and emission 538 nm.

2.11. Tumor necrosis factor-a (TNF-a) enzyme-linked immunosorbent assay (ELISA)

The Raw264.7 macrophage cells were seeded onto 24-well plates at a density of1 � 105 cells/well. The cells were activated by the addition of 20 ng/well lipopoly-saccharide (LPS) 2 h before the transfection. The pDNA/carrier complexes wereadded to the cells, and the cells were incubated for 4 h. After media change withfresh DMEM containing 5% FBS, the cells were incubated for an additional 20 h ina humidified 5% CO2 incubator. The culture media was harvested, and the tumornecrosis factor-a (TNF-a) levels were analyzed by ELISA according to the manufac-turer’s manual (eBioscience, San Diego, CA).

2.12. Acute lung injury mouse model

Mice were raised at an animal facility under special pathogen-free conditionswith a 12 h light/dark cycle and free access to food and water. Male BALB/c mice(20 � 1.5g) were anesthetized, orally intubated with a microsprayer aerosolizer (IA-1C; Penn-Century, Philadelphia, PA), and challenged with an intratracheal instilla-tion of 20 mg of LPS dissolved in 50 ml of saline.

2.13. Intratracheal injection of pDNA/carrier complex, luciferase assayandTNF-a ELISA

For evaluation of gene delivery efficiency, pSV-HO-1/carrier complexes wereadministered into the mice lungs by intratracheal instillation. pDNA/R7L10, pDNA/R7L10-curcumin, pDNA/PLL, and pDNA/PEI25k complexes were prepared ata volume of 100 ml in saline. pDNA/R7L10 and pDNA/R7L10-curcumin complexeswere prepared at a 1:30 weight ratio (pDNA:carrier), and pDNA/PLL and pDNA/PEI25k complexes were prepared at 1:2 and 1:1 weight ratios, respectively. Theamount of pDNA was fixed at 10 mg. Saline was administrated as a control. Twenty-four hrs after the administration of pDNA/carrier complexes, the animals wereanesthetized and sacrificed. Lungs were removed, frozen in liquid nitrogen, andstored at �80 �C until use. Frozen lung samples were homogenized using reporterlysis buffer in a homogenizer. The crude homogenates were centrifuged at 4 �C for10 min at 12,000 rpm, and the supernatants were used for HO-1 ELISA (R&DSystems, Minneapolis, MN).

For the TNF-a assay, pDNA/R7L10 and pDNA/R7L10-curcumin complexes wereprepared at a 1:30 weight ratio. The complexes were injected with the intratrachealinstillation at 2 h after the LPS challenge. The same amount of curcumin as pDNA/R7L10-curcumin complex was injected into some of the cells as a control. Naïvecontrol mice (without LPS) were injected with saline (total volume 100 ml). MouseTNF-a levels in the lung tissues were measured by an ELISA kit according to themanufacturer’s instructions (eBioscience, San Diego, CA).

2.14. Liver toxicity test

Ten or 50 mg of R7L10, R7L10-curcumin, and PEI were injected into the miceintravenously through tail veins.After 24 h, serumwas harvested from the mice, and

alanine transaminase (ALT) and aspartate transaminase (AST) levels were measuredby assay kits.

2.15. Statistical analysis

Statistical analysis was measured by analysis of variance (ANOVA) followed bythe NewmaneKeuls test. All dsata are presented as the average� standard error, andP values less than 0.05 were considered statistically significant.

3. Results

3.1. Comparison of R3L6, R5L8, and R7L10

In a previous report, the transfection efficiency of R3L6 wasevaluated by an in vitro transfection assay, showing that R3L6 hadhigher transfection efficiency than PLL [9]. In the present study, thesize of R3L6 was expanded by adding additional arginine andleucine residues at the amino- and carboxy-termini, producingR5L8 and R7L10. To confirm complex formation between thepeptides and pDNA, gel retardation assays were performed withpb-Luc. A fixed amount of pb-Luc was mixed with increasingamounts of R3L6, R5L8, and R7L10. The results showed that pDNAwas completely retarded by the peptides at a 1:2 weight ratio(Fig. 1). The transfection efficiencies of the peptides were evaluatedby in vitro transfection assays in HEK293 cells. R7L10 had a highertransfection efficiency than R3L6 or R5L8 (Fig. 2). However, thetransfection efficiencies of R3L6 and R5L8 were not differentsignificantly from each other. Therefore, R7L10 was used for all ofthe following experiments.

3.2. Transfection efficiency of curcumin-loaded R7L10

Curcumin-loaded R7L10 (R7L10-curcumin) was prepared at1:0.2, 1:0.4, and 1:0.6 R7L10:curcumin weight ratios by an oil-in-water (O/W) emulsion/solvent evaporation method. R7L10-curcumin/pDNA complexes were prepared at various weightratios (pDNA:R7L10-curcumin). The transfection assays were

Fig. 2. Transfection efficiencies of R3L6, R5L8, and R7L10 pDNA/R3L6, pDNA/R5L8, andpDNA/R7L10 complexes were prepared at weight ratios of 1:30. pDNA/PLL complexwas prepared at a 1:1 weight ratio. The complexes were transfected into HEK293 cells.After 24 h, transfection efficiencies were measured by luciferase assays. Luciferaseactivities are presented as mean � standard error of quadruplicated experiments.

J.H. Park et al. / Biomaterials 33 (2012) 6542e6550 6545

performed in HEK293 cells. Luciferase activities were measured24 h after the transfection. For R7L10-curcumin prepared at ratiosof 1:0.2 and 1:0.4 (R7L10-curcumin-0.2 and R7L10-curcumin-0.4),R7L10-curcumin had the highest transfection efficiency in HEK293cells at a 1:40 weight (Fig. 3). For R7L10-curcumin prepared ata R7L10:curcumin ratio of 1:0.6 (R7L10-curcumin-0.6), R7L10-curcumin had a tendency to saturate the transfection efficiency ata 1:30 weight ratio (Fig. 3). Considering that higher curcumincontent may be more efficient in terms of curcumin delivery,R7L10-curcumin-0.6 was used for all of the following experiments.The transfection efficiencies at pDNA:R7L10-curcumin weightratios of 1:30 and 1:40 were similar to each other and a 1:30 weightratio was used for all of the following experiments.

The transfection efficiency of R7L10-curcumin was comparedwith those of R7L10, PLL, lipofectamine, and PEI25k. R7L10-curcumin had higher transfection efficiency than R7L10 or PLL

Fig. 3. Transfection efficiency of R7L10-curcumin R7L10-curcumin was prepared atweight ratios of 1:0.2, 1:0.4 and 1:0.6 (R7L10:curcumin). pDNA/R7L10-curcumincomplexes were prepared at various weight ratios. The complexes were transfectedinto HEK293 cells. After 24 h, transfection efficiencies were measured by luciferaseassays. Luciferase activities are presented as mean � standard error of quadruplicatedexperiments.

(Fig. 4) but lower transfection efficiency than lipofectamine orPEI25k. Notably, PEI25k had much higher transfection efficiencythan R7L10-curcumine, with about a 105 times higher luciferaseactivity (Fig. 4).

3.3. Cytotoxicity of R7L10-curcumin

The cytotoxicity of R7L10-curcumin was measured by MTTassay. pDNA/R7L10, pDNA/R7L10-curcumin, pDNA/PLL, pDNA/lip-ofectamine and pDNA/PEI25k complexes were prepared andtransfected into HEK293 cells. R7L10 had about 90% cell viability,and R7L10-curcumin had about 83% cell viability (Fig. 5). PLL, lip-ofectamine and PEI25k had lower cell viability than R7L10-curcumin, suggesting that R7L10-curcumin was less toxic toHEK293 cells than PLL, lipofectamine or PEI25k (Fig. 5).

3.4. Physical characterization of pDNA/R7L10-curcumin complex

pDNA/R7L10-curcumin complex was prepared at a 1:30 weightratio, according to the previous transfection assays (Fig. 3). Gelretardation assays were performed to confirm complex formationbetween R7L10-curcumin and pb-Luc. A fixed amount of pDNAwasmixed with increasing amounts of R7L10-curcumin. As a control,pDNA/R7L10 complexes were prepared at various weight ratios. Gelretardation assays showed that R7L10 retarded pDNA at a 1:1.6weight ratio (pDNA:carrier) (Fig. 6), confirming the previous resultsshown in Fig. 1. On the contrary, R7L10-curcumin completelyretarded pDNA at a 1:1 weight ratio (Fig. 6). Compared with R7L10,a smaller amount of R7L10-curcumin was required for completeretardation of pDNA. In the R7L10-curcumin solution, curcuminmay have provided the hydrophobic core and facilitated themicelleformation of R7L10. R7L10-curcumin in aqueous solutionmaymoreeasily form complexes with pDNA than R7L10.

Heparin competition assayswere also performed to evaluate thestability of the pDNA/R7L10-curcumin complex. pDNA/R7L10 andpDNA/R7L10-curcumin complexes were prepared at a 1:30 weightratio (pDNA:carrier), and the pDNA/PLL complex was prepared ata 1:2 weight ratio. Increasing amounts of heparinwere added to thecomplex. pDNA began to be released from pDNA/PLL and pDNA/R7L10 complexes with 2.5 mg and 7.5 mg of heparin, respectively(Fig. 7). However, pDNA/R7L10-curcumin complex did not releasepDNA evenwith 15 mg of heparin (Fig. 7). This result does not meanthat R7L10-curcumin formed a more stable complex with pDNAthan PLL, since a 15-fold excess of R7L10-curcumin was used forcomplex formation with pDNA as compared to PLL. If a pDNA/PLLcomplex is prepared at a 1:30 weight ratio, PLL will form a morestable complex. However, in the case of R7L10, pDNA/R7L10complex was prepared at the same weight ratio as pDNA/R7L10-curcumin complex. Therefore, this result suggests that R7L10-curcumin formed a more stable complex with pDNA than R7L10.Higher stability of R7L10-curcumin may increase the transfectionefficiency, compared with R7L10.

The stability of pDNA in the presence of DNase I was measuredby a DNase I protection assay. pDNA/R7L10 and pDNA/R7L10-curcumin complexes were prepared at weight ratios of 1:30 andincubated with DNase I for the indicated time periods. Naked pDNAwas used as a control. The results indicate that naked pDNA wascompletely degraded by DNase I after 60 min of DNase I treatment(Fig. 8). However, R7L10 and R7L10-curcumin can protect pDNA forat least 60 min (Fig. 8). Therefore, R7L10-curcumin can protectpDNA from intracellular or extracellular nucleases.

The morphology of pDNA/R7L10-curcumin complex wasverified by SEM pDNA/R7L10 complex as a control. R7L10 andR7L10-curcumin formed spherical shaped complexes with pDNA(Fig. 9).

Fig. 4. Comparison of R7L10-curcumin with other carriers in terms of transfection efficiency pDNA/R7L10, pDNA/R7L10-curcumin, pDNA/PLL, pDNA/lipofectamine, and pDNA/PEI25k complexes were prepared as described in Materials and Methods. The complexes were transfected into HEK293 cells. After 24 h, transfection efficiencies were measured byluciferase assays. Luciferase activities are presented as mean � standard error of quadruplicated experiments.*P < 0.01 as compared with curcumin, R7L10, PLL, and lipofectamine.**P < 0.001 as compared with PEI25k.

J.H. Park et al. / Biomaterials 33 (2012) 6542e65506546

3.5. Intracellular curcumin delivery by R7L10-curcumin

To evaluate the curcumin delivery efficiency of R7L10-curcumin,HEK293 cells were treated with pDNA/R7L10-curcumin complex.pDNA/R7L10 and pDNA/PLL without curcumin complexes weretransfected into cells as negative controls. In addition, cells weretreated with curcumin only. Intracellular curcumin could be easilymeasured by UV absorbance and fluorescence. After the treatment,UV absorbance was measured with cell extracts. The results

Fig. 5. Cytotoxicity of R7L10-curcumin pDNA/R7L10, pDNA/R7L10-curcumin, pDNA/PLL, pDNA/lipofectamine, and pDNA/PEI25k complexes were prepared as described inMaterials and Methods. The complexes were transfected into HEK293 cells. After 24 h,the cytotoxicities of the complexes were measured by MTT assay. Cell viabilities arepresented as mean � standard error of quadruplicated experiments.*P < 0.05 ascompared with control, PLL, lipofectamine, and PEI25k.

showed that pDNA/R7L10-curcumin complex more efficientlydelivered curcumin into the cells than curcumin only (Fig. 10A).pDNA/R7L10 and pDNA/PLL complexes showed only a backgroundlevel of UV absorbance. These results were also confirmed byfluorescence. pDNA/R7L10-curcumin complex showed higherfluorescence than curcumin only (Fig. 10B).

Raw264.7 macrophage cells secreted increased levels of TNF-a when they were activated by LPS (Fig. 11). To evaluate the bio-logical effects of curcumin, TNF-a levels were measured aftertreatment with pDNA/R7L10-curcumin complex and curcuminonly. pDNA/R7L10-curcumin complex decreased TNF-a levels moreefficiently than curcumin only (Fig. 11). However, pDNA/R7L10complex did not show this effect, suggesting that pDNA/R7L10-curcumin complex is an efficient carrier of curcumin and that thecurcumin was released from the complex in the cells.

Fig. 6. Gel retardation assay with R7L10-curcumin A fixed amount of pDNAwas mixedwith increasing amounts of R7L10 and R7L10-curcumin. The mixtures were analyzedby agarose gel electrophoresis.

Fig. 7. Heparin competition assay pDNA/R7L10, pDNA/R7L10-curcumin and pDNA/PLL complexes were prepared as described in Materials and Methods. Increasing amounts ofheparin were added to the samples to facilitate dissociation of the pDNA/carrier complexes, after which the samples were analyzed using a 1% agarose gel.

J.H. Park et al. / Biomaterials 33 (2012) 6542e6550 6547

3.6. In vivo pDNA delivery efficiency of R7L10-curcumin afterintratracheal injection

pb-HO-1 was delivered into mice lungs by intratracheal instil-lation. pDNA/R7L10 and pDNA/R7L10-curcumin complexes wereprepared at weight ratios of 1:30, and pDNA/PLL and pDNA/PEI25kcomplexes were prepared at 1:2 and 1:1 weight ratios, respectively.At 24 h after the instillation, lungs were harvested, and tissueproteins were extracted. HO-1 expression was measured by ELISA.The results showed that R7L10-curcumin caused the highest HO-1gene expression in the mouse lungs (Fig. 12). R7L10 also led tohigher in vivo HO-1 gene expression than PLL or PEI25k (Fig. 12).

Fig. 8. DNase I protection assay naked pDNA, pDNA/R7L10, and pDNA/R7L10-curcumincomplexes were incubated with DNase I for the times indicated in Materials andMethods. The samples were mixed with 2 � stop buffer and analyzed using a 1%agarose gel.

Fig. 9. Morphology of pDNA/R7L10 and pDNA/R7L10-curcumin complexes pDNA/R7L10 and pDNA/R7L10-curcumin complexes were prepared at their optimal ratios.The morphology of the complexes was observed by scanning electron microscopy.

Fig. 10. Intracellular delivery efficiency of curcumin by R7L10-curcumin pDNA/R7L10,pDNA/R7L10-curcumin and pDNA/PLL complexes were prepared at their optimal ratiosand added to HEK293 cells. Curcumin only was also used as a control. After 4 h, theintracellular uptake of curcumin was measured by UV absorbance (A) and fluorescence(B). Data are presented as mean � standard error of the quadruplicated experiments.*,***P < 0.01 compared with control, R7L10, curcumin, and PLL. **,****P < 0.01compared with control, R7L10 and PLL.

Fig. 11. TNF-a ELISA after pDNA/R7L10-curcumin complex treatment Raw264.7 cellswere activated by LPS treatment. pDNA/R7L10 and pDNA/R7L10-curcumin complexeswere prepared at weight ratios of 1:30. The complexes were added to the Raw264.7cells. Curcumin only was also used as a control. At 24 h after treatment, TNF-a levelswere measured by ELISA. Data are presented as mean � standard error of thequadruplicated experiments. *P < 0.01 compared with control, LPS control, R7L10 andcurcumin, **P < 0.05 compared with control, LPS control, and R7L10.

Fig. 12. Gene delivery efficiency of R7L10-curcumin in the mouse lung pb-HO-1/R7L10and pb-HO-1/R7L10-curcumin complexes were prepared at weight ratios of 1:30. pb-HO-1/PLL and pb-HO-1/PEI25k complexes were prepared at 1:2 and 1:1 weight ratios,respectively. The complexes were administrated into the mice lungs by intratrachealinstillation. Twenty four hours after the administration, the lung tissues were har-vested, and the HO-1 expression levels were measured by ELISA. The data are pre-sented as mean � standard error (n ¼ 8). *P < 0.05 compared with control, PLL andPEI25k but no statistical significance compared with R7L10. **P < 0.05 compared withcontrol, PLL, and PEI25k.

J.H. Park et al. / Biomaterials 33 (2012) 6542e65506548

3.7. Lung TNF-a level in acute lung injury mice

To evaluate the in vivo delivery efficiency of curcumin by R7L10-curcumin, pDNA/R7L10, pDNA/R7L10-curcumin and curcumin onlywere administered into the lungs of acute lung injury mice. Theacute lung injury mice were produced by intratracheal instillationof LPS. The anti-inflammatory effect of the delivered curcumin wasmeasured by the TNF-a ELISA assay in the lung tissues. The resultsshowed that curcumin only reduced TNF-a level slightly comparedwith the LPS control group (Fig. 13). pDNA/R7L10-curcumincomplex further decreased TNF-a level compared with curcuminonly (Fig. 13). pDNA/R7L10 complex did not decrease TNF-a level.These results suggest that R7L10-curcumin is more efficient thancurcumin only in terms of curcumin delivery.

3.8. Liver toxicity of R7L10-curcumin

To evaluate the liver toxicity, R7L10-curcuminwas intravenouslyinjected into the mice through tail veins. R7L10 and PEI25k wereinjected as controls. After 24 h, serumwas harvested from themice,and the ALT and AST activities were measured. Fifty microgram of

Fig. 13. TNF-a level in the acute lung injury mice models after curcumin deliverypDNA/R7L10 and pDNA/R7L10-curcumin complexes were prepared at weight ratios of1:30. Curcumin only was used as a positive control. Twenty four hours after intra-tracheal instillation of the complexes into the acute lung injury mice models, the lungtissues were harvested, and the TNF-a levels were measured by ELISA. Data are pre-sented as mean � standard error (n ¼ 6). *P < 0.05 compared with control, LPS control,R7L10 and curcumin.

Fig. 14. Liver toxicity of R7L10-curcumin R7L10, R7L10-curcumin, and PEI25k wereinjected intravenously into the mice through tail veins. Twenty four hours after theinjection, sera from the mice was harvested and subjected to ALT and AST analysis.Data are presented as mean � standard error of quadruplicated experiments. *P < 0.01compared with all other samples.

J.H. Park et al. / Biomaterials 33 (2012) 6542e6550 6549

PEI25k significantly increased serum ALTand AST levels, suggestingextensive liver toxicity of PEI25k (Fig. 14). However, 10 mg of R7L10and R7L10-curcumin did not increase serum ALT or AST level(Fig. 14). Fifty micrograms of R7L10-curcumin increased the ALTlevel slightly compared with the control. However, it did notincrease the AST level. Therefore, R7L10 and R7L10-curcumin hadlower liver toxicities than PEI25k.

4. Discussion

Curcumin has various therapeutic effects such as anti-tumorand anti-inflammatory effects [15e17,19] and has been investi-gated as a therapeutic agent for the treatment of acute lung injury[18]. In addition, gene delivery to the lung is also emerging asa therapeutic option [26e30]. A few anti-inflammatory genes andanti-apoptotic genes have been suggested as therapeutic agents forthe treatment of acute lung injury [27,29e31]. Together, these factssuggest that the combined delivery of curcumin and therapeuticgenes may be useful in the treatment of acute lung injury. In thisresearch, R7L10 was evaluated for the combined delivery of cur-cumin and pDNA.

In terms of gene delivery, curcumin has synergistic effects thatincrease the transfection efficiency of R7L10. R7L10-curcumin hadhigher transfection efficiency than R7L10 alone, both in vitro andin vivo (Figs. 4 and 12). R7L10 in aqueous solution may formmicelles and behave like a larger molecular weight peptide withmore cationic charges on the surface. Hydrophobic curcumin mayfacilitate the formation of R7L10 micelles in aqueous solution byproviding a hydrophobic core. Indeed, gel retardation assaysshowed that R7L10-curcumin formed complexes with pDNA withsmaller quantities of carrier than R7L10 only (Fig. 1). Furthermore,pDNA/R7L10-curcumin complexes were more stable than pDNA/R7L10 complexes in the presence of heparin (Fig. 2). Therefore,higher complex stability may enhance the transfection efficiency ofR7L10-curcumin. In addition, R7L10 was less toxic than PEI25k.In vitro toxicity tests showed that R7L10 reduced cell viability less

than PLL, lipofectamine, or PEI25k (Fig. 5). In vivo liver toxicity testsalso showed that R7L10 did not induce significant liver toxicity aftersystemic administration (Fig. 14).

R7L10-curcumin had much lower in vitro transfection efficiencythan PEI25k. However, in vivo transfection efficiencies showed theopposite trend, with R7L10-curcumin demonstrating highertransfection efficiency than PEI25k. This kind of discrepancy hasbeen observed in other studies. In a previous report, reduciblepoly(oligo-arginine) (rPOA) was evaluated as a gene carrier in micelungs [32]. In that study, rPOA had lower in vitro transfection thanPEI25k, but rPOA had much higher in vivo transfection efficiencythan PEI25k. In another study, dexamethasone-conjugated PEI (PEI-Dexa) had higher transfection efficiency in mice lungs than PEI25k,although PEI25k had higher transfection efficiency than PEI-Dexain cultured lung epithelial cells [30]. It is not clear why this oppo-site tendency has been observed in gene delivery to the lung. It isspeculated that in vivo gene delivery into the lungs may involveconditions that are different from in vitro cultured cells and whichaffect the transfection efficiencies of the carriers, such as mucus.

In terms of curcumin delivery, R7L10 also has some advan-tages. First, curcumin is hydrophobic with low solubility inaqueous solution. Loading curcumin into the R7L10 micelle

J.H. Park et al. / Biomaterials 33 (2012) 6542e65506550

dramatically increased the solubility of curcumin. Second, thecellular uptake efficiency of curcumin was enhanced by loadingcurcumin into the cores of the R7L10 peptide micelles. Hydro-phobic drugs usually enter cells by simple diffusion. However,nanoparticles such as pDNA/carrier complexes are taken up bycells via endocytosis. Endocytosis is an energy-consumingprocess, which is more efficient than simple diffusion. There-fore, nanoparticle formation with pDNA and R7L10-curcumin mayresult in increased endocytosis and cellular uptake of curcumin.As in Figs. 10 and 11, the pDNA/R7L10-curcumin complex moreefficiently delivered curcumin into the cells than curcumin only.Further, the enhanced delivery of curcumin into the cells showedincreased anti-inflammatory effects, reducing TNF-a levels in theLPS activated Raw264.7 macrophage cells (Fig. 11). The anti-inflammatory effect of R7L10-curcumin may be increased by thecombination with an anti-inflammatory gene such as the HO-1gene. However, the combined delivery of gene and drug usuallyhas a limitation for clinical application. Curcumin may berequired repeatedly, but the therapeutic genes may be deliveredless frequently than curcumin.

Curcumin has various biological effects, mainly inflammationreduction. It has been reported that curcumin suppresses nuclearfactor-kappa B (NF-kB) by inhibiting phosphorylation of I-kappa B(IkB) [15]. The suppression of NF-kB reduces cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Clinical trials to test theeffects of curcumin on various diseases including rheumatoidarthritis, inflammatory bowel disease, chronic pancreatitis, andvarious types of cancers have been attempted [15]. Acute lung injuryhas also been suggested as a disease in which curcumin may havetherapeutic effects. In the present study, the intratracheal instillationof pDNA/R7l10-curcumin effectively reduced TNF-a level in ananimal model. This anti-inflammatory effect of R7L10-curcumin inthe lungs was stronger than that for curcumin only, confirming thein vitro results seen with Raw264.7 cells. It has been reported thatcurcumin is rapidly cleared after in vivo administration, suggestingthat complex formation of curcumin with other materials mayincrease the bio-availability of curcumin [15]. Therefore, R7L10-curcumin may increase the in vivo half-life of curcumin.

5. Conclusions

In summary, R7L10-curcumin is an efficient gene carrier fordelivery into mice lungs with higher transfection efficiency thanPEI25k. In addition, R7L10-curcumin is an efficient carrier of cur-cumin, showing higher intracellular curcumin delivery than is seenwith curcumin only. Considering the anti-inflammatory effect ofcurcumin, R7L10-curcumin may be useful as a carrier for thecombined delivery of curcumin and pDNA for the treatment ofacute lung injury.

Acknowledgments

This work was supported by a grant from the National ResearchFoundation of Korea funded by the Ministry of Education, Scienceand Technology (2011K000803, 20110026013).

Appendix A. Supplementary material

Supplementarymaterial associatedwith this article can be found,in the online version, at doi:10.1016/j.biomaterials.2012.05.046.

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