BASIC SCIENCE

Nanomedicine: Nanotechnology, Biology, and Medicine8 (2012) 167–175

Research Article

Linear PEI nanoparticles: efficient pDNA/siRNA carriersin vitro and in vivo

Ritu Goyal, MPhila, Sushil K. Tripathi, MSca, Shilpa Tyagi, MScb, Anurag Sharma, MScb,K. Ravi Ram, PhDb, Debapratim K. Chowdhuri, PhDb, Yogeshwar Shukla, PhDb,

P. Kumar, PhDa, Kailash C. Gupta, PhDa,b,⁎aCSIR-Institute of Genomics and Integrative Biology, Delhi University, Delhi, India

bCSIR-Indian Institute of Toxicology Research, Lucknow, India

Received 16 October 2010; accepted 2 June 2011

nanomedjournal.com

Abstract

Linear polyethylenimine (lPEI, 25 kDa) nanoparticles' (LPN) series was synthesized by varying percentage of cross-linking with1,4-butanediol diglycidyl ether (BDE) and their size, surface charge, morphology, pDNA protection/release, cytotoxicity and transfectionefficiency were evaluated. Synthesized nanoparticles (NPs) were spherical in shape (size: ∼109 – 235 nm; zeta potential: +38 to +16 mV).These NPs showed increased buffering capacity with increasing percent cross-linking and also exhibited excellent transfection efficiency (i.e.,∼1.3 – 14.7 folds in case of LPN-5) in comparison with lPEI and the commercial transfection agents used in this study. LPN-5 based GFP-specific siRNA delivery resulted in ∼86% suppression of targeted gene expression. These particles were relatively nontoxic in vitro (in celllines) and in vivo (inDrosophila). In vivo gene expression studies using LPN-5 in Balb/c mice through intravenous injection showedmaximumexpression of the reporter gene in the spleen. These results together demonstrate the potential of these particles as efficient transfection reagents.

From the Clinical Editor: The authors demonstrate a novel method of synthesizing linear PEI nanoparticles to utilize these astransfection agents.© 2012 Elsevier Inc. All rights reserved.

Key words: Linear polyethylenimine; 1,4-butanediol diglycidyl ether; In vivo gene expression; Drosophila; In vivo cytotoxicity

For the successful gene therapy, the selected delivery systemmust efficiently deliver the appropriate therapeutic material intothe target tissue or cells with the least toxicity and withoutinducing an immune response. Cationic polymers have shownpromise as efficient gene-delivery vehicles,1-5 and among them,polyethylenimine (PEI) is one of the most widely studied.6

Branched polyethylenimine (bPEI) has primary, secondary andtertiary amines in the ratio of 1:2:1 and is available in molecularweights ranging from 0.8 – 800 kDa with various degrees ofbranching.7,8 In particular, bPEI (25 kDa, the gold standard) ishighly effective in transfection efficiency owing to its highbuffering capacity (due to the presence of secondary and tertiaryamines).9a,9b,10,11 Although bPEI condenses DNA very effec-tively and forms polyplexes, it is cytotoxic due to the primary

No conflict of interest was reported by the authors of this article.Financial support from the CSIR (NWP-035) and UGC-SRF (10-2(05)

2006(i)-E.U.II) to RG is gratefully acknowledged.⁎Corresponding author: Indian Institute of Toxicology Research,

Mahatma Gandhi Marg, Lucknow-226001, India.E-mail address: kcgupta@iitr.res.in (K.C. Gupta).

1549-9634/$ – see front matter © 2012 Elsevier Inc. All rights reserved.doi:10.1016/j.nano.2011.06.001

Please cite this article as: R., Goyal, et al, Linear PEI nanoparticles: efficient pDNdoi:10.1016/j.nano.2011.06.001

amines, which limits its use for in vivo applications.12 On thecontrary, linear polyethylenimine (lPEI, 25 kDa) is relativelynontoxic but is less water soluble and exhibits poor transfectionefficiency.13 The modifications of lPEI for improved transfectionefficiency involve tedious and multistep synthesis.14-19 Recentmodifications on lPEI (25 kDa) suggested that the chargeassociated cytotoxicity can be addressed by conjugation withcyclodextrin.20 However, this conjugation resulted in decreasedbuffering capacity and polyplex formation, thereby significantlyreducing transfection efficiency in vitro.20 Shuai et alconjugated α-cyclodextrin onto the linear polymeric chain oflPEI, which improved the solubility of the copolymer andimproved cell viability.21 Yamashita et al22 have suggested thatα-cyclodextrin threading decreases the charge density on lPEIand further, that polypseudorotaxane formation of lPEI andcyclodextrins significantly depends on the pH of the media.γ-Cyclodextrin based modification of lPEI also did notsignificantly increase the transfection efficiency of thesecopolymers in comparison with that of the parent polymer.22

In view of this, we hypothesized that partial conversion ofsecondary amines in lPEI to tertiary amines, although preserving

A/siRNA carriers in vitro and in vivo.Nanomedicine: NBM 2012;8:167-175,

168 R. Goyal et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 167–175

the overall number of amines, would result in improvedbuffering capacity, which may, in turn, improve transfectionefficiency of the resulting nanoparticles (NPs) with cell viabilitycomparable with that of native lPEI. In addition, the projectedmodification also improves the dispersibility of the resulting NPsby inhibiting the formation of crystalline hydrates through H-bonding, which accounts for the poor solubility of native lPEI.For this purpose, we used 1,4-butanediol diglycidyl ether (BDE)that has been reported for cross-linking of polyamines andpolysaccharides.23,24 The unique advantage of using BDE as across-linking agent for linear PEI is that it would not compromiseon the number of functional amines in the cross-linked polymerbut will only convert secondary amines to tertiary amines.

We aimed to prepare a series of lPEI nanoparticles (LPNs)using BDE as a cross-linker to significantly improve thetransfection efficiency in comparison with bPEI, lPEI andother commercially available transfection reagents. As a proof ofprinciple, in vivo studies have been carried out in Balb/c mice tosee the gene-expression pattern. In this article, we show thatthese LPNs have significantly improved transfection efficiencywith relatively negligible cytotoxicity both in vitro and in vivo.

Methods

Materials

Specialized chemicals and reagents were purchased fromtheir respective suppliers. All other chemicals and reagents wereprocured from Sigma-Aldrich Chemical Co., St. Louis, Missouri.

Animals

Six to seven week-old male Balb/c mice (25 ± 3 g) (for in vivoluciferase gene expression), from the animal breeding colony ofIndian Institute of Toxicology Research (IITR), Lucknow, India,were acclimatized under standard laboratory conditions and givena commercial pellet diet (Ashirwad Industries, Chandigarh, India)and water ad libitum. Animals were housed in plastic cages on ricehusk bedding and maintained at 22 ± 2°C with 12 hours ofalternating darkness and light and 50%–60% humidity as per ruleslaid down by the Animal Welfare Committee of IITR.

Preparation of LPN

lPEI was cross-linked with BDE to prepare a series of LPN.Briefly, to an aqueous solution of preheated lPEI (100 mg,1 mg/mL) at 45°C, a solution of BDE (11.08 μl, 1 μl/mL inwater, for 5% cross-linking) was added dropwise over a periodof 30 minutes with continuous stirring. The stirring wascontinued overnight at the same temperature and then thevolume of the reaction mixture was reduced to one third of thetotal volume on a rotary evaporator. The remaining solution wassubjected to dialysis against water for 72 hours with intermittentchanges of water. Thereafter, the solution was concentrated in aspeed vac to obtain a white residue of LPN-1 (90 mg, ∼85%yield). These particles were then characterized by protonnuclear magnetic resonance spectroscopy (1H-NMR), fouriertransform infrared spectroscopy (FTIR), dynamic light scater-ring (DLS), zeta potential, atomic force microscopy (AFM), and

transmission electron microscopy (TEM). Similarly, NPs with10 (LPN-2), 15 (LPN-3), 20 (LPN-4), 30 (LPN-5), 40 (LPN-6),50 (LPN-7) and 60% (LPN-8) cross-linking were prepared andcharacterized, as above.

DNA retardation assay

An aqueous solution of lPEI (1 mg/mL) was added to 1 μl ofpDNA (0.3 μg/μl) at N/P ratios 1.5, 2.0, 3.0 and 4.0 to form lPEI/pDNA polyplexes and the final volume was made up to 20 μl withwater. Similarly, bPEI/pDNA polyplexes were prepared. For lPEINPs, the nanoplexes were prepared at N/P ratios 3.0, 4.0, 5.0 and6.0. The resulting samples were gently vortexed and incubated at25 ± 2°C for 30minutes. All the polyplexes and nanoplexes (20 μl)were mixed with 2 μl xylene cyanol (in 20% glycerol),electrophoresed (100 V, 1 hour) in 0.8% agarose gel, stainedwith ethidium bromide and visualized on a UV transilluminatorusing a Gel Documentation System (Syngene, Cambridge, UnitedKingdom). From the gel, the amount of sample required forcomplete retardation of 300 ng of pDNA was obtained.

Buffering capacity

A suspension of LPN-2 (6 mg/30 mL) in 0.1N NaCl wasadjusted to pH 10 with 0.1N NaOH and then the pH was broughtto 3.0 with 50 μl aliquots of 0.1 N HCl; pH values were recordedafter each addition. The slope of the line in the plot for pH andthe amount of HC1 consumed indicates the intrinsic bufferingcapacity of the system.25 Similarly, the projected assay wasrepeated with LPN-5 and 8.

DNA release assay

LPEI was complexed with pDNA (300 ng) at the N/P ratio,where it exhibited the highest transfection efficiency, andincubated for 30 minutes, as described above. Subsequently, anaqueous solution of heparin was added, varying from 0.1–2 U.The samples were then incubated for 20 minutes and electro-phoresed (100 V, 1 hour) in 0.8% agarose, stained with ethidiumbromide and imaged on a UV transilluminator using a GelDocumentation system. The amount of DNA released fromcomplexes after heparin treatment was estimated by densitom-etry. Likewise, the assay was repeated with bPEI and LPNs.

In vitro transfection

The transfection experiments were carried out on HEK293,HepG2 and HeLa cells for all the LPNs, lPEI, bPEI andcommercial transfection reagents in absence and presence ofserum. The study was performed at various N/P ratios rangingfrom 4 to 30 for LPNs, lPEI and bPEI. N/P ratio is indicative ofthe amount of cationic polymer (containing positively chargednitrogens responsible for binding with negatively chargedphosphates of DNA) used for attaining a maximum transfectionefficiency. In case of commercial transfection reagents, theoptimum ratios suggested by the respective manufacturer wereused due to non-availability of concentration and molecularweight of these standards. The quantification of GFP expressionwas carried out using Nano Drop 3300 spectrofluorometer.

The knockdown of two genes, namely, GFP and PLK 1, wasobserved using LPN-5 in the HEK293 and A431 cells,

169R. Goyal et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 167–175

respectively, and the comparisons were made with commerciallyavailable transfection reagents (please see SupplementaryMaterial which can be found in the online version of this article).

Percentage of EGFP-expressing cells was determined byFluorescence Activated Cell Sorting (FACS) analysis (please seeSupplementary Material). Approximately, 5,000 cells wereanalyzed to generate data for statistical purposes. All experi-ments were performed at least in triplicate.

Toxicity evaluation: In vitro and in vivo

The cytotoxicity of lPEI and bPEI/pDNA polyplexes, LPN/pDNA nanoplexes as well as Superfect,™ Fugene,™GenePORTER 2™ and Lipofectamine™/pDNA complexeswas evaluated in vitro on HEK293, HepG2 and HeLa cells byMTT colorimetric assay. In vivo toxicity evaluation of theLPN-5 and LPN-5/DNA nanoplex was carried out in Droso-phila melanogaster (Oregon-R+), by analyzing the stress geneexpression. Each group contained 30 larvae/replicates and theexposures/analyses were carried out in triplicate. Thedifferences between control and treated from each groupwere analyzed for statistical significance using t-test. Inanother set of experiments, after a 36 hours' exposure, larvaeof both control and treated groups were kept on normal foodand observed for emergence of adults (please see Supplemen-tary Material).

Intracellular trafficking

LPN-5 and pDNA were labeled with TRITC and YOYO-1iodide, respectively, and the intracellular trafficking of thefluorescently tagged LPN-5/pDNA nanoplex was monitoredusing confocal laser scanning microscopy (CLSM) (please seeSupplementary Material). All confocal images were capturedusing a Zeiss LSM 510 inverted LSC microscope.

DNase I protection assay

To assess the ability of the NPs (LPN-5) to protect thecondensed pDNA from nucleases, a DNase I protection assaywas performed. Native pDNA and LPN-5/pDNA nanoplex(at N/P ratio 10) were incubated at 37°C for 0.25, 0.5, 1 and 2hours with DNase I (l μl, 1 unit/μl in a buffer containing100 mM Tris, 25 mM MgCl2 and 5 mM CaCl2). Treatment withPBS alone served as control. After incubation, a solution ofEDTA (5 μl of 100 mM) was added and the mixture incubatedat 75°C for 10 minutes to inactivate DNase I. The mixture wasfurther incubated for 2 hours (25 ± 2°C) with heparin (10 μl,5 mg/mL) to release bound DNA from the cationic polymer.Subsequently, samples were electrophoresed and imaged, asdescribed above (DNA release assay). The amount of pDNAreleased from complexes after heparin treatment was estimatedby densitometry.

Intravenous injection of LPN-5 in Balb/c mice

pGL3 (25 μg) was complexed with LPN-5, bPEI and nakedDNA at an N/P ratio of 10, 10 and 0, respectively, and the finalvolume was made to 100 μl using normal saline. The complexeswere incubated for 30 minutes at 25 ± 2°C for injection in Balb/cmice. The complexes and naked DNA were injected through tail

vein using a syringe of 40 U with needle of size 0.30 × 8 mm.The same experiment was repeated twice. The animals were keptfor 7 days on a normal diet after which they were sacrificed bycervical dislocation and all the vital organs were dissected out.The organs were washed with chilled normal saline, weighed,chopped and made 25% (w/v) homogenate in 1x cell lysis buffer(Promega Corp., Madison, Wisconsin) containing 1x proteaseinhibitor cocktail (Sigma). After 3 cycles of freezing andthawing, the homogenate was centrifuged at 10,000 rpm for 10minutes at 4°C. Next, 100 μl of the cell lysate from each samplewas assayed for luciferase activity by adding 100 μl luciferinsubstrate on the luminometer (Berthold, Herts, United King-dom). Standard curve was made using the luciferase enzymeprovided with the kit.

Statistical analysis

Statistical analysis, wherever needed,was carried out by one-wayanalysis of variance followed by Student's t-test after ascertaininghomogeneity of variance and normality of data. A value of P b 0.05was considered statistically significant. JMPversion 6.0.0, StatisticalDiscovery™ from SAS was used for analysis.

Results

Preparation and characterization of lPEI NPs

lPEI was cross-linked (5%, 10%, 15%, 20%, 30%, 40%, 50%and 60%) with BDE to obtain a series of LPN (Scheme S1). Thepercentage of cross-linking was controlled by adjusting themolar ratio of the cross-linker and lPEI, and the resulting LP NPs(LPN-1 to LPN-8) were obtained in ∼80%–85% yield. Thepercentage of cross-linking was estimated using 1H-NMR(Table 1) and the realized substitution was nearly 30% of theattempted substitution. In IR spectroscopy, the peak at 1160 cm-1

showed the C-O stretching, which further confirmed the cross-linking of lPEI with BDE.

Size, surface morphology and zeta potential measurements

The size of lPEI NPs (LPN) by DLS was found to be in therange 109 – 235 nm (Table 1) with polydispersity index of b 0.4.On increasing the percent cross-linking, a concomitant decreasein the size of NPs was observed, which decreased further oncomplexing with plasmid DNA (pDNA) (Table 1). In thepresence of 10% FBS, the pDNA complexed LPN showed anadditional decrease in the size. TEM and AFM analyses haveshown spherical shaped NPs and nanoplexes but the size of NPswas smaller than that determined by DLS (Figure S1). Zetapotential of NPs also decreased upon complexing with DNA(Table 1) but those particles still had positive values. However,in 10% serum, the zeta potential of the LP nanoplexes, lPEI andbPEI polyplexes was found to be negative.

Buffering capacity

Figure 1 shows the buffering capacity of different LPNs (2,5 and 8) and lPEI in the pH range 10 to 3. We found that theamount of 0.1N HCl required in bringing the pH from 10 to 3

Table 1Biophysical characterization of LPNs

Sample ID Average particle size in nm ± SD (PDI) Zeta potential in mV ± SD Ratio ofNP : DNA(N/P)

Attemptedsubstitution(%)

Realizedsubstitutionestimatedusing 1H-NMR (%)

NP (in H2O) DNA-loaded NP(in H2O)

DNA-loaded NP(in DMEM)

NP (in H2O) DNA-loaded NP(in H2O)

DNA-loaded NP(in DMEM)

LPN-1 235.3 ± 0.3(0.117)

212.2 ± 0.5(0.158)

157.1 ± 12.2(0.198)

38.7 ± 0.8 29.7 ± 1.3 −10.6 ± 2.2 10 5 1.79

LPN-2 215.6 ± 9.6(0.178)

188.4 ± 1.5(0.180)

125.4 ± 12.4(0.176)

35.3 ± 4.8 24.3 ± 1.8 −9.3 ± 1.6 10 10 3.40

LPN-3 198.6 ± 10.8(0.275)

186.0 ± 3.6(0.169)

101.3 ± 1.5(0.276)

24.3 ± 0.9 22.7 ± 1.3 −9.8 ± 1.3 10 15 5.39

LPN-4 192.8 ± 6.0(0.265)

175.6 ± 9.2(0.261)

97.7 ± 6.7(0.197)

23.2 ± 2.9 20.1 ± 1.6 −11.6 ± 1.5 10 20 7.19

LPN-5 151.1 ± 3.6(0.198)

143.5 ± 6.5(0.107)

95.8 ± 7.4(0.296)

22.0 ± 1.7 18.8 ± 1.4 −10.6 ± 1.6 10 30 11.17

LPN-6 133.2 ± 2.5(0.186)

126.3 ± 7.3(0.192)

86.4 ± 8.3(0.106)

20.5 ± 2.0 16.8 ± 2.1 −9.4 ± 1.4 10 40 14.38

LPN-7 121.3 ± 3.9(0.136)

108.7 ± 8.2(0.106)

67.2 ± 7.0(0.194)

18.7 ± 1.1 16.1 ± 1.4 −9.7 ± 1.6 10 50 17.98

LPN-8 109.5 ± 4.8(0.283)

100.8 ± 0.5(0.162)

65.2 ± 8.2(0.199)

16.5 ± 2.3 10.1 ± 1.2 −9.1 ± 1.3 10 60 21.72

lPEI - 425.8 ± 12.2(0.579)

348.4 ± 1.7(0.390)

41.4 ± 0.2 35.5 ± 2.3 −13.5 ± 1.8 20 - -

bPEI - 365.5 ± 15.8(0.498)

268.5 ± 1.4(0.372)

43.5 ± 2.1 38.9 ± 0.5 −14.2 ± 1.3 10 - -

12

10

8

6

4

2

01 4 7 10 13 16 19 22 25 28 31

pH

Volume of 0.1N HCI (units)

0.1M NaCl

LPN-5

LPN-8

LPN-2

lPEI

Figure 1. Buffering capacity of lPEI and LPNs. Error bars represent ±standard deviation (SD) from the mean. X-axis, 1 Unit = 25 μl.

170 R. Goyal et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 167–175

increased with percent cross-linking with LPN-8 showing themaximum buffering capacity. It was also observed that all theLPNs showed increased buffering capacity in comparison withthe native lPEI (data not shown).

DNA retardation assay

The amount of NPs required to retard a fixed amount ofpDNA increased as the percent cross-linking increased.Complete retardation of 0.3 μg of pDNA occurred at N/P ratioof 4 in case of lPEI and bPEI, whereas LPN retarded at slightlyhigher N/P ratios ranging from 5 – 6 (Figure S2).

In vitro transfection studies

The study was carried out at N/P ratios ranging from 4 to30 (see gel retardation assay). After 36 hours of transfection,cells showed fairly high level of GFP gene expression (FigureS3). The transfection efficiency of LPN-1 to LPN-8 / pDNAnanoplexes first increased with increasing N/P ratio and thendecreased beyond an optimal value (Figure S4). Thefluorescent intensity of GFP expression in case of LPN-5/pDNA formulation at N/P ratio of 10 varied significantly (P b0.01) with the fluorescent intensity of lPEI (at N/P ratio 20)and bPEI (at N/P ratio of 8). A similar effect was observedwith percentage of cross-linking (Figure S5). Among all thetested LPNs, the highest level of gene expression wasobserved with LPN-5 formulation. Further, the transfectionefficiency was found to be cell-line dependent showingmaximum efficiency in HEK293, followed by HeLa andHepG2. LPN-5/DNA nanoplex enhanced the gene transferefficacy ∼1- to 15-fold in the absence of serum in comparisonwith bPEI (25 kDa, gold standard), lPEI and commercially

available transfection reagents in HEK293 cells (Figure S6).Likewise, the transfection efficiency of LPN-5/DNA nanoplexwas found to be ∼seven-fold and ∼four-fold in comparisonwith lPEI in HeLa and HepG2 cells, respectively. Thepresence of 10% serum did not alter the transfection efficiencyof LPN/DNA nanoplexes significantly (Figure 2).

FACS analysis was performed on HEK293 cells toevaluate the reporter-gene expression using flow cytometry.It was observed that lPEI showed the highest transfectionefficiency (16% ± 1.5%) at N/P ratio of 30, and bPEItransfected ∼23% ± 2.3% cells at N/P ratio of 10. LPN-5NPs showed the highest transfection efficiency (67% ± 3.2%)at N/P ratio of 10. The transfection efficiency of LPN-5 wasfound to be significantly higher (P b 0.05) than that ofLipofectamine™ (14% ± 1.4% transfected cells). From this

Figure 2. GFP fluorescence intensity in HEK293, HeLa and HepG2 cells in presence of serum, transfected with LPN/DNA, lPEI/DNA, bPEI/DNA, Superfect™/DNA, Fugene™/DNA, GenePORTER 2™/DNA and Lipofectamine™/DNA complexes. *P b 0.01 vs. lPEI, bPEI and Lipofectamine™.

171R. Goyal et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 167–175

study, it was also observed that on increasing the N/P ratio,the transfection efficiency first increased and then decreasedbeyond an optimal value (Figure 3).

DNA release assay

We observed that LPN-5 NPs released 85% ± 4.1% pDNAwith 2 units of heparin (Figure 4). With the same amount ofheparin, the amount of pDNA released from lPEI and bPEI was96 ± 3.6%, and 55% ± 3.1%, respectively (Figure 4).

Delivery of siRNA using LPN-5

We observed ∼86% knockdown of GFP expression usingLPN-5/pDNA/siRNA as a carrier in comparison with 65% and58% knockdown by lPEI/pDNA/siRNA and Fugene™/pDNA/siRNA complexes, respectively (Figure S7a, see SupportingInformation). In the case of PLK 1 siRNA, after 36 hours oftreatment, the observed knockdown of PLK 1 by LPN-5/siRNAand siPORT NeoFx™/siRNA formulation was ∼53% and∼50%, respectively (Figure S7b).

Cell viability assay

Cell viability was assessed on HEK293, HeLa and HepG2cells, when exposed to LPNs, and found to range between∼85% – 99%. In contrast, cells exposed to bPEI orLipofectamine™ showed a significantly reduced viability (Pb 0.01) (Figure 5). For the other commercial reagents, the cellviability was found to be in the range of 68%–88%(Superfect™), 72%–94% (Fugene™) and 81%–98% (Gene-PORTER 2™) in the 3 cell lines tested.

In Drosophila-based in vivo assays, we did not observemortality either in control or in any of the treatment groupsduring 36 hours of exposure period. Of the 4 stress genes ofDrosophila analyzed in this study, transcript levels of hsp60,

hsp83 and hsp23 in larvae exposed to LPN-5, LPN-5/pDNAand Lipofectamine™ did not differ significantly from those ofsucrose controls (P N 0.05; Figure S8a, see SupplementaryInformation). In addition, the transcript levels of hsp70 inlarvae exposed to LPN-5 or LPN-5/pDNA were similar tothose in their respective controls (P N 0.05; Figures S8 a andb). The transcript levels of hsp70 in larvae exposed toLipofectamine™ were significantly higher in comparison withthose in their controls (P b 0.05; Figures S8 a and b).Interestingly, when larvae treated with LPN-5 and Lipofecta-mine™ for 36 hours were put back on normal food andobserved for their development to adult, LPN-5 treatedorganisms did not show any significant (P N 0.05) increasein mortality (∼71% survival) and Lipofectamine™-treatedones showed ∼56% mortality in comparison with theircontrols. Moreover, neither a delay in fly emergence nor anymorphological defect was observed in LPN-5 treated organ-isms (Figure S9).

Intracellular localization of nanoplexes

We observed red fluorescence (LPN-5 NPs) (Figure S10,see Supplementary Information) and green fluorescence(YOYO-1 labeled DNA) (Figure S10) near the plasmamembrane of HeLa cells within 30 minutes of the adminis-tration of complexes to cells, which indicates that complexesmay dissociate after entering the cell. Nuclear staining wasdone using DAPI (blue). NP/DNA complex formation wasevidenced by the yellow fluorescence in the overlaid images(Figure S10) at some places. LPNs (LPN-5) were found tocarry pDNA inside the cytoplasm within 1 hour and to thenucleus within 2 hours of the addition of complexes to thecells, as evidenced by the red and green fluorescence insidethe cytoplasm and nucleus (Figure S10).

80

70

60

50

40

30

20

10

0lPEI bPEI LPN-5 Lipofectamine

N/P Ratio5 10 20 30

Tran

sfec

tio

n E

ffic

ien

cy (

%)

*

Figure 3. Percent transfection efficiency of LPN-5 /DNA nanoplexdetermined using FACS at various N/P ratios and compared with lPEI,bPEI, and Lipofectamine™. *P b 0.01 vs. lPEI, bPEI and Lipofectamine™ atrespective N/P ratio.

120

100

80

60

40

20

00 0.1 0.3 0.5 0.6 0.7 0.8 0.9 1 1.5 2

DNA release assay

% o

f D

NA

rel

ease

d

Units of Heparin

LPN-5bPEIlPEI

Figure 4. DNA release assay of lPEI, bPEI and LPN-5 NPs. To a 20 μl ofLPN-5/DNA nanoplex, heparin, in increasing concentration, was added andincubated for 20 min at 25 ± 2°C. The samples were run on 0.8% agarose gelat 100 V for 45 min. Error bars represent ± SD from the mean.

172 R. Goyal et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 167–175

DNase I protection assay

In contrast with the free pDNA (0.3 μg), which was degradedcompletely by DNase I within 15 minutes, LPN-5 effectivelyprovided protection to ∼88% after 30 minutes and 82% after 2hours (Figure S11, see Supplementary Information).

In vivo gene expression in Balb/c mice

In vivo gene expression studies were performed to extend thein vitro studies on the use of LPN-5 as a gene-delivery vehicle.The comparisons were made with bPEI and naked DNA aloneusing firefly luciferase expression as a reporter system. Out of allthe groups tested for gene expression, the naked DNA groupperformed the least in all. LPN-5 showed best expression in thespleen, followed by heart and brain. The luciferase expression inspleen and heart was ∼1.5-fold and in brain, it was ∼5-fold incomparisonwith bPEI. In lungs, liver and kidney, little expressionwas observed with all of the groups tested (Figure 6). Theobserved superiority of LPN-5 over bPEI, in vivo, is consistentwith our in vitro results.

Discussion

Gene therapy presents a promising approach for thetreatment of several genetic and acquired disorders.26,27

Among the viral and nonviral vectors used for gene delivery,the later offers certain advantages in terms of safety, stabilityand cost-effectiveness leading to efficient gene delivery.Among the nonviral vectors, bPEI, in particular, has beenshown to condense plasmids effectively into colloidalparticles that effectively transfect DNA both in vitro and invivo.28,29 The bPEI/DNA complexes, however, are very toxicto the cells and are prone to aggregation.30 Linear PEI, onthe other hand, possesses insignificant toxicity but lowtransfection efficiency.31

In this study, we have explored linear PEI cross-linked withvarying amounts of BDE as a new gene carrier for possibleclinical applications based on high transfection efficiency andlow toxicity in cells. The modification leads lPEI intonanosized particles (size ranging from 100 – 250 nm), therebyincreasing the chance of bioavailability.32 These NPs were

subjected to physical characterization and their superiority overnative PEIs (linear and branched) and the standard transfectionreagents were evaluated through various biological assays. Thesize of the NPs was inversely proportional to the percent cross-linking and we observed that the size reduced further oncomplexing with plasmid DNA (pDNA). This is contrary to thecase of bPEI, where size of NPs increases on complexationwith pDNA.33 This difference can be explained by consideringthe fact that, on complexation with pDNA, the positive chargeon the particles becomes partially neutralized, which makesthese particles less water dispersible, resulting in the decreasein the size of complexes, which is also in accordance with aprevious study.34 Moreover, in the presence of 10% serum(FBS), the pDNA complexed LPNs showed an additionaldecrease in the size, which might be due to the fact that serumcauses the dehydration of the NP/DNA complexes, resulting inthe decrease of hydrodynamic diameter, ultimately leading totheir compaction.33 Further, the observed stability of LPN-5/pDNA nanoplex's size in the presence and absence of serumindicates the nonaggregation properties of the projected NPs.We observed spherical-shaped NPs under TEM and AFM butthe particles' size was smaller than that determined by DLS.This discrepancy in size might be attributed to the principles ofDLS and TEM: DLS measures hydrodynamic diameter of theNPs, whereas TEM gives the size of NPs in dehydrated form.23

The decrease in size and surface charge (zeta potential) ofLPNs on increasing the percent cross-linking might be due to thecompactness of the polymer and partial charge buried within LPNpore, which was not available for measurement. Zeta potential ofNPs also decreased upon complexing with DNA, which isconsistent with the gel-retardation assay observations. Moreover,these particles still have positive values, i.e., the presence ofbuffering amines, which is sufficient to allow them to promoteendosomal disruption. However, in 10% serum, the zeta potentialof the LP nanoplexes, lPEI and bPEI polyplexes was found to benegative, which is consistent with a previous study.33

The interaction between the positively charged NPs andnegatively charged pDNA plays an important role in determiningthe transfection efficiency of the system.35,36 The interactionsshould be strong enough to compactly pack the complex and

LPN-1

LPN-2

LPN-3

LPN-4

LPN-5

LPN-6

LPN-7

LPN-8lP

EIbPEI

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ct

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ty %

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Figure 5. Cell viability profile of LPN/DNA nanoplexes at an N/P ratio of 10 and lPEI/DNA polyplex at an N/P ratio of 20, and Superfect™/DNA, Fugene™/DNA, GenePORTER 2™/DNA and Lipofectamine™/DNA complexes in HEK293, HeLa and HepG2 cells. *P b 0.01 vs. bPEI and Lipofectamine™.

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Figure 6. In vivo gene expression analysis using LPN-5, bPEI and control(naked DNA) in Balb/c mice 7d post intravenous injection using pGL3control vector as a reporter gene. *P b 0.05 vs. bPEI and control in therespective organs.

173R. Goyal et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 167–175

carry it proficiently into the cell, but slack enough to release itinto the intracellular milieu, resulting in an increased geneexpression.35,36 The results of heparin release assay indicate thatthe interaction of LPN with pDNA is relatively loose incomparison with that of bPEI and is stronger than that of lPEI(Figure 4), which is in agreement with a recent report.34 Thenative lPEI, with its loose complexing with pDNA, canpotentially release pDNA before it reaches the cell and thebPEI, forming a very tight complex with DNA, is quite unlikelyto release all the pDNA into the cell. However, LPN having theirinteraction levels with pDNA between bPEI and lPEI may carryDNA across the cellular barriers and be loose enough to release itonce it reaches inside the cell. The increased transfectionefficiency of the LPNs in comparison with lPEI or bPEI indeedsupports this finding. These results suggest the efficient carryingof DNA by LPNs.

lPEI exhibits considerable buffering capacity over a pH range3 to 10 and helps in release of pDNA from endosomes/lysosomes(Proton sponge effect).37,38 Therefore, we determined thebuffering capacity of the LPNs and found that the proton-capturing tendency of the NPs increased with increasing thepercent cross-linking. Upon determination of the bufferingcapacity, we evaluated the cytotoxicity of the synthesizedLPNs because cytotoxicity is one of the most critical issues ingene delivery. The observed lower toxicity of LPNs is notsurprising given the fact that their parent compound lPEI itselfpossesses very low cytotoxicity due to the absence of primaryamines across the whole length of the polymer. Recently, thetransfection efficiency of PEIs was shown to rely partly on theircapacity to buffer endolysosomes.32 We, therefore, analyzed thetransfection efficiency of these particles.39

The transfection efficiency of the LPN series together withbPEI, lPEI and commercial transfection reagents was evaluatedin 3 different cell lines, i.e., HEK293, HeLa and HepG2 in thepresence and absence of serum. Of all the LPNs tested, LPN-5exhibited highest transfection efficiency in all the cell lines. Inaddition, LPN-5 showed significantly higher (P b 0.05)transfection efficiency than lPEI, bPEI and Lipofectamine™.N/P ratio was also found to play an important role indetermining the transfection efficiency. It was observed thattransfection efficiency first increased with increase in N/P ratio,attained a maximum value and decreased thereafter. Further, inthe presence of serum, transfection efficiency followed the sametrend as in the absence of serum with insignificant decrease intransfection efficiency. These results strengthen the practicalutility of the synthesized LPNs for in vivo gene delivery. LPN-5and bPEI both showed highest transfection efficiency at N/Pratio 10 while lPEI showed at 20. These results suggest that theprocess of crosslinking not only increased the transfectionefficiency of the synthesized NPs but also reduced the dose ofthe same required to achieve significantly higher transfection

174 R. Goyal et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 167–175

efficiency. Further, the initial increase in transfection efficiencywith increasing buffering capacity and subsequent decreasingtrend after attaining a maximum efficiency, suggested thatbuffering capacity is not the only parameter in deciding thetransfection efficiency of the system. Consistent with this, thetransfection efficiency was shown to depend on the topology ofthe polymeric backbone, too,40 and a polymer despite having ahigher buffering capacity might not necessarily exhibit highertransfection efficiency.41

From the above experiments, it is clear that LPN-5 is the bestamong the LPNs synthesized in the present study. Therefore, wefocused on LPN-5 and assessed LPN-5′s toxicity in vivo and itsefficiency to deliver DNA/siRNA in vitro and in vivo. In vivotoxicity assays were carried out using Drosophila melanogasterand the results are consistent with those of in vitro assays. Heatshock proteins (hsps) have been shown as excellent bio-indicators of cellular stress/damage.40 Lack of induction ofhsps above the control level in Drosophila larvae, exposed toLPN-5, LPN-5/pDNA, suggested that these materials did notinduce cellular stress in vivo. Conversely, a recent study ofcellular toxicity of a different NP (silver NPs) using Drosophilashowed the induction of hsp70.42 This further supports ourfinding on in vivo noncellular toxicity of LPN-5.

An efficient gene delivery system is the one that providessufficient protection against nucleases present in the cellularenvironment to the gene of interest to be delivered into thecell from degradation. LPN-5 protected DNA against DNase Ifor a considerable time period, as seen by DNase protectionassay, which implicates its practical utility for in vivoadministration of pDNA. However, sequence-specific genesilencing using small interfering RNA (siRNA) is an emergingtechnology that is now being evaluated in clinical trials as apotentially novel therapeutic strategy. Therefore, we tested theefficiency of LPN-5 in delivering siRNA. The observedknockdown of GFP or PLK 1 suggested that LPN-5 efficientlydelivered GFP siRNA and PLK 1 siRNA into cells andknocked down the targeted gene. Hence, delivery of differenttypes of nucleic acids increases the potent application of LPN-5 in gene therapy.

The intracellular localization of DNA is an important factorfor successful gene therapy. Intracellular trafficking studiessuggested the presence of both DNA and LPN-5 inside thenucleus. This observation is in agreement with a reportedstudy where lPEI delivered nucleic acids to the nucleus.43 Thisfinding clearly demonstrates efficient intracellular delivery ofDNA using LPN-5. As a proof of this principle, we complexedLPN-5 with pGL3 and injected intravenously into Balb/c miceand tracked the expression of the reporter gene, luciferase.Significantly increased luciferase expression in the spleen ofanimals injected with LPN-5 complexed with pGL3 incomparison with their controls (bPEI and naked DNA)suggested that LPN-5 has the potential to carry DNA/genein vivo. Although maximum expression in the spleen is quiteintriguing, it parallels the findings of a previous study.44 Verylittle gene expression observed with naked DNA may be dueto degradation of the same in the biological systems by thenucleases present in the blood.45 Some organs, such as theliver, spleen, bone marrow and certain tumors, have endothelia

with large meshes, and others have tight endothelia.46 Thisdifference may explain the lower expression levels of thereporter gene in the remaining tissues. Nevertheless, our resultsclearly demonstrate the potential of LPN-5 to deliver the genein vivo.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.nano.2011.06.001.

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