Characterization and evaluation of the efficacy of ... · Characterization and evaluation of the efﬁcacy of cationic complex mediated plasmid DNA delivery in human embryonic palatal

Characterization and evaluation of the efficacy ofcationic complex mediated plasmid DNA deliveryin human embryonic palatal mesenchyme cellsSheetal D’Mello1, Aliasger K. Salem1*, Liu Hong2 and Satheesh Elangovan2*1Division of Pharmaceutics and Translational Therapeutics, College of Pharmacy, University of Iowa, IA, USA2Department of Periodontics, College of Dentistry, University of Iowa, IA, USA

Abstract

The purpose of this study was to develop and test a non-viral gene delivery system that can beemployed to deliver genes of interest into a pre-osteoblastic cell line. Human embryonic palatalmesenchymal (HEPM 1486) cells were transfected with vector-plasmid DNA (pDNA) complexes. Weexplored calcium phosphate and polyethylenimine (PEI) as non-viral vectors and compared theirrespective in vitro transfection efficacies. Plasmid DNA encoding luciferase protein (LUC) wascomplexed with PEI (with differing N:P ratios) and calcium phosphate (with differing Ca:P ratios),using established protocols. The complexes prepared were then characterized for size and surfacecharge, using a Malvern Zetasizer Nano-ZS. The transfection efficiency and cytotoxicity of theprepared complexes were evaluated in HEPM cells. The PEI–pDNA complexes over the whole rangeof N:P ratios were found to be<160 nm in size, while the calcium phosphate–pDNA complexes wererelatively bigger. The PEI–pDNA complexes prepared at a N:P ratio of 10 were found to have maxi-mum transfection efficiency at 4 h of treatment, with minimal cytotoxicity. The highest transfectionefficiency obtained with calcium phosphate–pDNA complexes (Ca:P 200) was nearly 12-fold lowerthan that obtained with PEI–pDNA complexes (N:P 10). Following this, transgene expression in theHEPM cells treated with complexes prepared at a N:P ratio of 10 was further examined, using pDNAcoding for enhanced green fluorescent protein (EGFP-N1) or therapeutically relevant platelet-derivedgrowth factor B (PDGF-B). In conclusion, PEI was a more effective vector for delivering genes ofinterest to pre-osteoblasts than calcium phosphate. Copyright © 2014 John Wiley & Sons, Ltd.

Received 23 August 2013; Revised 4 December 2013; Accepted 3 January 2014

Keywords branched polyethylenimine; calcium phosphate; non-viral vectors; gene delivery; plasmidDNA; HEPM cells; transfection efficiency; cytotoxicity

1. Introduction

Gene therapy involves treatment of diseases by thedelivery of foreign genetic material into specific cells ofthe host (Mulligan, 1993). The genetic material, in theform of plasmid DNA (pDNA) encoding a functional genefor a therapeutic protein, can be used to supplement or

alter the expression of existing genes or replace a mutatedgene within the affected host cells. Depending on the natureof the disease, either short-term or long-term gene expres-sion may be needed. The main prerequisites of a successfulgene therapy is that it should be safe, highly efficient, con-trollable and selective to the target cells (Verma et al., 2000).

Tissue-engineering approaches for bone regenerationhave included protein therapy to deliver osteogeniccytokines and growth factors such as bone morphogeneticproteins (BMPs) (Zegzula et al., 1997), delivery of genesencoding protein factors that promote bone growth(Fang et al., 1996), and implantation of osteogenic cellsat sites of bone defects (Lieberman et al., 1999). Theseapproaches may be combined with biomimetic scaffoldsto enhance the regeneration process (Shea et al., 2000).

*Correspondence to: Satheesh Elangovan, 801 NewtonRoad, S464, Department of Periodontics, University ofIowa College of Dentistry, Iowa City, IA 52242, USA.E-mail: [email protected] K. Salem, 15 S. Grand Avenue, S228 PHAR University ofIowa College of Pharmacy, Iowa City, IA 52242 USA.E-mail: [email protected]

Copyright © 2014 John Wiley & Sons, Ltd.

JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH ARTICLEJ Tissue Eng Regen Med (2014)Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.1873

The major drawback of protein therapy is that it requiressupra-physiological doses of growth factors, which isexpensive and runs the risk of inducing toxicity (Endoet al., 2006; Fang et al., 1996). In addition, exogenouslysupplied proteins are susceptible to instability, rapid clear-ance, due to short half-lives, and loss of bioactivity. Thealternative use of gene therapy is less expensive becausethe in vitro production of plasmid DNA is relatively simpleand economical as compared to commercial proteinproduction (Horn et al., 1995). In addition, local cell-mediated production of growth factors in situ would pro-mote efficient cell surface receptor targeting and requireless protein to achieve similar levels of therapeutic effect,when compared to protein therapy. Using implantedgene-activated matrices, prolonged plasmid gene expres-sion and continuous protein production is achieved thatstimulates osteogenesis and bone repair in vivo (Bonadioet al., 1999). Localized gene therapy also averts systemictoxicity that can occur as a result of dose dumping duringprotein therapy (Langer, 1998; Terrell et al., 1993).

Human embryonic palatal mesenchymal (HEPM) cellsare osteogenic progenitors and therefore clinicallyrelevant in bone tissue regeneration. HEPM cells havebeen widely used as in vitromodel cells to study osteogen-esis. The HEPM cells are also a good cell type to studypalatal growth and closure (Yoneda and Pratt, 1982).In this study, as a proof of concept, HEPM cells wereevaluated for their ability to internalize cationic com-plexes of plasmid DNA, undergo transfection and produceproteins of interest. The long-term goal of this study wasto develop a safe and efficient non-viral gene deliverysystem that can deliver multiple genes in vivo for peri-odontal, bone and other orthopaedic applications. In thisreport, we show for the first time that HEPM cells canbe genetically manipulated, using cationic complexes ofplasmid DNA, to produce functional proteins.

2. Materials and methods

2.1. Reagents and plasmids

Branched polyethylenimine (PEI; mol. wt. 25 kDa) waspurchased from Sigma-Aldrich (St. Louis, MO, USA).Analytical grade calcium chloride dihydrate and dextrosemonohydrate was from Sigma-Aldrich, sodium chlorideand HEPES-free acid from RPI Corp. (Mt. Prospect, IL,USA), potassium chloride and sodium phosphate tribasicdodecahydrate from Fischer Scientific (Fair Lawn, NJ,USA). Plasmid DNA (6.4 kb) encoding the firefly lucifer-ase reporter protein (pLUC) driven by cytomegalovirus(CMV) promoter/enhancer (VR1255 plasmid DNA),plasmid DNA (4.7 kb) encoding the enhanced greenfluorescent protein (pEGFP-N1) driven by CMV promoter/enhancer, and plasmid DNA (4.9 kb) encoding platelet-derived growth factor B (pPDGF-B) were used in this study.The GenElute™ HP Endotoxin-free Plasmid Maxiprep Kitwas obtained from Sigma-Aldrich. The Luciferase Assay

System was purchased from Promega (Madison, WI,USA). The MicroBCA™ Protein Assay Kit was purchasedfrom Pierce (Rockford, IL, USA). The PDGF-BB ELISA Kitwas purchased from Quantikine® (R&D Systems, Minneap-olis, MN, USA). All the reagents used for transmissionelection microscopy (TEM) were from Electron MicroscopyServices (Ft. Washington, PA, USA). Agarose was obtainedfrom Bio-Rad Laboratories (Hercules, CA, USA). All otherchemicals and solvents used were of reagent grade. Humanpalatal mesenchyme (HEPM) stem cells were purchasedfrom the American Type Culture Collection (ATCC; Manas-sas, VA, USA). Eagle’s minimum essential medium (EMEM)was obtained from ATCC. Trypsin–EDTA (0.25%, 1�solution) and Dulbecco’s phosphate-buffered saline (PBS)was purchased from Gibco (Invitrogen, Grand Island, NY,USA). Fetal bovine serum (FBS) was obtained from AtlantaBiologicals (Lawrenceville, GA, USA). Gentamycin sulphate(50mg/ml) was purchased from Mediatech Inc. (Manas-sas, VA, USA). MTS cell growth assay reagent (Cell Titer96 AQueous One Solution Cell Proliferation Assay) waspurchased from Promega (Madison, WI, USA). AlexaFluor568 phalloidin was purchased from Invitrogen. TritonX-100was obtained from Sigma-Aldrich. Vectashield, Hardsetmounting medium with 4′,6-diamidino-2-phenylindole(DAPI) was obtained from Vector Laboratories (CA, USA).

2.2. Preparation of plasmid DNA (pDNA)encoding luciferase protein (LUC), enhancedgreen fluorescent protein (EGFP-N1) orplatelet-derived growth factor B (PDGF-B)

The chemically competent DH5α bacterial strain Escherichiacoli was transformed with the necessary pDNA to amplifythe plasmid. The pDNA in the transformed cultures was thenexpanded by amplification of the E. coli cells in Lennox Lbroth (LB broth) overnight at 37°C in an incubator shakerat 300 rpm. The pDNAwas extracted using GenElute HP En-dotoxin-free Plasmid Maxiprep Kit. The extracted pDNAwasanalysed for purity, using a NanoDrop 2000 UV-Vis Spectro-photometer (Thermoscientific, Wilmington, DE, USA), bymeasuring the ratio of absorbance (A260 nm: A280 nm).The concentration of pDNA solution was determined by UVabsorbance at 260nm. The size and quality of the extractedpDNA was determined by agarose gel electrophoresis.

2.3. Fabrication of calcium phosphate–pDNAcomplexes

Calcium phosphate–pDNA (LUC) complexes were preparedby a standard method, as described previously (Elangovanet al., 2013; Olton et al., 2007). Briefly, 500μl of a calciumprecursor solution inwater, comprisedof 62μl 2MCaCl2

.2H2Oand 50 μg pDNA (LUC) was added dropwise, whileslowly vortexing, to an equal volume of a phosphate pre-cursor solution in water, pH 7.5, containing varyingamounts of Na3PO4

.12H2O in the range 0.83–2.48mM ofphosphate to formulate complexes with different Ca:P

S. D’Mello et al.

Copyright © 2014 John Wiley & Sons, Ltd. J Tissue Eng Regen Med (2014)DOI: 10.1002/term

ratios (Table 1). The calcium concentration in the calciumprecursor solutionwas fixed at 248mM. Themixturewas in-cubated at room temperature for 20min. The final volumeof the complexes used in the transfection experiments was20μl, containing 1μg pDNA.

2.4. Fabrication of PEI–pDNA complexes

Complexes were prepared by adding 500μl PEI solution inwater dropwise to 500μl pDNA (LUC/EGFP-N1/PDGF-B)solution in water containing 50μg pDNA, and mixed byvortexing for 20 s. The mixture was incubated at roomtemperature for 30min to allow complex formation betweenthe positively charged PEI (amine groups) and the negativelycharged pDNA (phosphate groups). Complexes werefabricated using different N (nitrogen):P (phosphate) ratios(molar ratio of amine groups of PEI to phosphate groups inpDNAbackbone) by varying the PEI amounts, while keepingthe amount of pDNA constant (Table 2). The final volume ofthe complexes utilized in the transfection and biocompatibil-ity experiments was 20μl, containing 1μg pDNA.

2.5. In vitro characterization of pDNA (LUC)complexes

2.5.1. Size and polydispersity

Size measurements of the complexes in water were car-ried out using Zetasizer Nano-ZS (Malvern Instruments,Westborough, MA, USA) and the mean hydrodynamicdiameter of the samples was determined by cumulativeanalysis. The particle size and particle size distributionby intensity were measured by photon correlation spec-troscopy (PCS), using dynamic laser light scattering(4mW He–Ne laser with a fixed wavelength of 633 nm,173° backscatter at 25°C) in 10mm diameter cells. Allmeasurements were done in triplicate. Complexes with a

N:P ratio of 10 were placed on carbon-coated grids for1min and negatively stained with 1% uranyl acetate for20 s. After drying, the samples were imaged with a JEOLJEM-1230 TEM.

2.5.2. Surface charge

Zeta potential (surface charge) determinations of thecomplexes in water were based on the electrophoreticmobility of the complexes, using folded capillary cells in au-tomaticmode of measurement duration, using the ZetasizerNano-ZS. Surface chargemeasurements were performed bythe laser scatteringmethod, using the Smoluchowskimodel(Laser Doppler Micro-electrophoresis, He–Ne laser, 633nmat 25°C). The mean value was recorded as the average ofthree measurements.

2.6. Cell culture

The HEPM cells were maintained in EMEM supplementedwith 10% FBS and 50 μg/ml gentamycin in a humidifiedincubator (Sanyo Scientific Autoflow, IR direct heat CO2

incubator) at 37°C containing 95% air and 5% CO2. Thecells were plated and grown as a monolayer in 75 cm2

polystyrene cell culture flasks (Corning, NY, USA) andsubcultured (subcultivation ratio of 1:6) after 80–90%confluence was achieved. Cell lines were started fromfrozen stocks and the mediumwas changed every 2–3days.The passage number at which the cells were used in exper-iments was in the range 4–8.

2.7. In vitro evaluation of the transfectionefficiency of pDNA (LUC) complexes

PEI–pDNA (LUC) complexes were prepared using N:P ratiosof 1, 5, 10, 15 and 20. Calcium phosphate–pDNA (LUC)complexes were prepared using Ca:P ratios of 100, 150,200 and 300. Cells were seeded at a density of 80 000cells/well in 24-well plates. The following day, at~70% cellconfluency, the cell culture mediumwas changed to serum-free medium and the treatments were gently vortexedand added dropwise into the wells. Each well was treatedwith 20μl complexes containing 1μg pDNA. The complexeswere incubated with the cells for 4 h or 24h. At theend of each treatment period, the cells were washed with1� phosphate-buffered saline (PBS) and fresh completeme-dium was added to the cells until analysis. After atotal incubation time of 48h, the cells were washed with1� PBS, treated with 1� lysis buffer and subjected to twofreeze–thaw cycles. The cells were scraped and centrifugedat 14 000 rpm for 5min. Luciferase expression was detectedby a standard luciferase assay system using a luminometer.The relative light units (RLU) values/mg total cell protein,indicative of the transfection efficiency, were normalizedagainst the protein concentration in cell extracts, using aMicroBCA Protein Assay Kit. The values are expressed asmean±SD for each treatment (n=3).

Table 1. Ca:P ratios with corresponding phosphate concentra-tions used in preparing calciumphosphate–pDNA (LUC) complexes

Ca:P ratio Phosphate precursor (mM phosphate)

100 2.48150 1.65200 1.24300 0.83

Table 2. N:P ratios with corresponding PEI amounts used in pre-paring PEI–pDNA (LUC–EGFP-N1–PDGF-B) complexes

N:P ratio PEI amount (μg)

1 0.135 0.6510 1.3015 1.9520 2.6

Non-viral gene delivery system in HEPM cells


2.8. In vitro evaluation of cytotoxicity of PEI–pDNA (LUC) complexes

Cell survival assays were conducted to demonstrate theeffect of the N:P ratio of the PEI–pDNA (LUC) complexeson the biocompatibility of complexes in HEPM cells over aperiod of time. Cells were seeded in clear polystyrene, flatbottomed, 96-well plates (Costar, Corning, NY) at a densityof 10 000 cells/well and allowed to attach overnight. Thefollowing day, the complete medium was replaced withserum-free medium and the cells were treated with com-plexes containing 1μg pDNA. Untreated cells were usedas controls. Cells treated with PEI alone or uncomplexedpDNA alone served as additional controls. The complexeswere incubated with the cells for 4 h or 24h to mimic theconditions used in the transfection experiments. At theend of the treatment period, the cells were washed with1� PBS and fresh complete medium was added to the cells,followed by addition of 20μl MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium] cell growth assay reagent. The plates wereincubated at 37°C in a humidified 5% CO2 atmosphere for4 h. The amount of soluble formazan produced by reductionof MTS reagent by viable cells was measured spectrophoto-metrically using SpectraMax Plus384 (Molecular Devices,Sunnyvale, CA, USA) at 490nm. The cell viability wasexpressed by the following equation:

Cell viability %ð Þ ¼ ½absorbance intensity of treated cells=absorbance intensity of untreatedcells controlð ÞÞ� � 100

Values are expressed as mean±SD for each treatmentperformed in triplicate.

2.9. In vitro visualization of transfection withPEI–pDNA (EGFP-N1) complexes

To determine the qualitative fluorescence expressed byEGFP-N1, HEPM cells were plated at a density of 50000 cells/well in clear, flat-bottomed, eight-chamberedglass slides with covers (Lab-Tek, Nunc™, NY) that hadpreviously been coated with 0.1% poly-L-lysine. Thecells were allowed to attach overnight and the followingday, the cell culture medium was removed. The cellswere treated with complexes containing 1μg pDNA (N:P 10) in serum-free medium. Untreated cells, cellstreated with uncomplexed pDNA and cells treated withPEI alone were used as controls. The complexes were in-cubated with the cells for 4 h or 24 h. At the end of eachtreatment period, the cells were washed with 1� PBSand fresh complete medium was added to the cells untilanalysis. After a total incubation time of 48 h, the cellswere washed with 1� PBS. The cells were then fixedwith 4% paraformaldehyde (Hatfield, PA, USA),followed by permeabilization of the cells with 0.2% Tri-ton X-100. The cells were later treated with phalloidinto fluorescently tag the cellular actin with AlexaFluor568 phalloidin. The specimen was finally mounted with

Vectashield Hardset mounting medium containing DAPI.The cells were washed with PBS during every step inthe process. The cellular fluorescence was observedusing confocal laser scanning microscopy (�63; CarlZeiss 710, Germany) equipped with Zen 2009 imagingsoftware. The images were processed using ImageJsoftware (National Institutes of Health, MD, USA).

2.10. In vitro evaluation of transfection with PEI–pDNA (PDGF-B) complexes

The transfection in HEPM cells was further evaluated byusing pDNA encoding for the PDGF-B protein. PEI–pDNAcomplexes were prepared using an N:P ratio of 10.The cells were plated at a seeding density of 80 000cells/well in 24-well plates. The following day, at ~70%cell confluency, the cell culture medium was changed toserum-free medium and the treatments were gentlyvortexed and added dropwise into the wells. Each wellwas treated with complexes containing 1μg pDNA. Thecomplexes were incubated with the cells for 4 h. At theend of the treatment period, the cells were washed with1� PBS and fresh complete medium was added to the cellsuntil analysis. After a total incubation time of 48 h,the cell culture supernatants were then collected bycentrifugation and analysed for PDGF-BB, using a PDGF-BB ELISA Kit. Untreated cells and cells treated withnaked, uncomplexed plasmids were employed as controls.The data are represented as mean±SD for each treat-ment carried out in triplicate.

2.11. Agarose gel retardation assay for PEI–pDNA(LUC) complexes

The condensation of pDNA by polycationic PEI polymerand the subsequent retention and stability of pDNAwithinthe complexes (N:P ratio 10) was assessed using gelelectrophoresis. Samples were loaded into the wells of a1% agarose gel (in 1� TAE buffer containing 5μg/mlethidium bromide) with BlueJuice gel loading buffer(2� final concentration). Electrophoresis was carried outat 80mA in 1� TAE running buffer. The pDNA bands werevisualized using a UV transilluminator.

2.12. Data presentation and statistical analysis

Numerical data were reported as mean± standard devia-tion (SD) from at least three replicate samples. Thegraphs were generated using Prism 5.0 (GraphPad Soft-ware Inc., San Diego, CA, USA). The data were comparedby ANOVA, followed by a Tukey post-test analysis. Thedifferences between the groups were considered to bestatistically significant at p< 0.05. Statistical analyseswere performed using Prism 5.0 software.

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3. Results and discussion

Plasmid DNA is non-toxic and relatively high doses can beadministered to achieve sustained gene expression andtherapeutic quantities of protein production (Parker et al.,1995). The main concern with the use of pDNA is the safetyand efficacy of the gene-delivery vehicles. Both viral andnon-viral vector systems can be used for gene transfer. Viralvectors, such as adenoviruses and retroviruses, are usuallycapable of high transfection efficiencies (Anderson, 1998).However, viral vectors have the potential to stimulate hostimmune responses, inflammatory and toxic reactions, ran-dom insertion of the viral genome and mutagenesis thathinders the clinical translatability of this approach (Endoet al., 2006). On the other hand, non-viral vectors lack suchdrawbacks and are easy to synthesize. Although non-viralgene delivery systems generally display lower transfectionefficiencies when compared to viruses, they neverthelesshave the potential to be applied to a wide array of applica-tions in the dental and craniofacial fields. In this study, weexplored the non-viral delivery of different genes to HEPMcells using PEI–pDNA complexes. Polyethylenimine hasproved to be an effective vehicle for pDNA in several appli-cations (Boussif et al., 1995; Intra and Salem, 2008; Kircheiset al., 2001). Branched PEI has yielded significantly highertransfection efficiencies than linear PEI, and is thereforecommonly used for polymer-mediated pDNA delivery.Attributes of branched PEI, such as effective protonabilityand buffering capacity, high cationic charge density poten-tial and tight condensation of pDNA, contribute to its hightransfection efficiency (Boussif et al., 1995; Godbey et al.,1999b). The transfection efficiencies of three different mo-lecular weights of PEI (25, 50 and 800kDa) were examinedin vivo and it was found that 25kDa PEI resulted in thehighest transfection efficiency of all. PEI–pDNA complexesmade using low molecular weight PEIs (≤ 10kDa) wouldreadily dissociate and result in lower transfection efficien-cies (Abdallah et al., 1996; Godbey et al., 1999c). Weused a systematic approach to identify the optimal amine:phosphate (N:P) ratio at which PEI–pDNA complexes couldgenerate maximum transfection in HEPM cells whilstmaintaining low cytotoxicity. The transfection efficiency ofPEI vector was also compared to an alternative non-viralcalcium phosphate vector that is widely used for pDNAdelivery due to its biocompatibility and biodegradability(Liu et al., 2011; Olton et al., 2007; O’Mahoney and Adams,1994). The calcium/phosphate (Ca:P) ratio of the calciumphosphate–pDNA complexes is a measure of the amountof calcium to phosphate used in synthesizing a particularprecipitate. In order to optimize the stoichiometry (N:Pand Ca:P ratios) of these complexes, formulations withvarying N:P and Ca:P ratios were fabricated and the influ-ence of ratio stoichiometry on complex size, charge andstability, transfection efficiency and toxicity in HEPM cellswas evaluated. The zeta potentials and sizes of the pDNAcomplexes are very critical parameters governing theinteraction of the complexes with the cells and their uptakeinto the cells via endocytosis. This ultimately affects the

maximum transfection efficiency attained and cell viability(Bettinger et al., 1999; Naoto et al., 1986). The optimizedcomplex ratio can improve the transfection efficiencies byovercoming the formulation and various other barriers inthe intracellular and intranuclear transport of pDNA. Thetransfection efficiencies of these complexes varied signifi-cantly in different cell types compared, and hence it wasnecessary that the transfection method be optimized foreach cell line used.

3.1. Generation of pDNA encoding LUC, EGFP-N1or PDGF-B proteins

The recovery and purity of the isolated pDNA wasdetermined by spectrophotometric analysis. The ratio of ab-sorbance at A260 nm: A280 nm (optical density of pDNAsolution) was within the range 1.8–2.0, as recommendedby the manufacturer’s protocol.

3.2. Size and surface charge of calciumphosphate–pDNA (LUC) complexes

In preparing these complexes, the amount of calciumprecursor (248mM) and pDNA (1μg) was maintained ata constant level, while the amount of phosphate precursorwas varied, so as to generate different Ca:P ratios: 100,150, 200 and 300 (Table 1). This was done to assess theeffect of Ca:P stoichiometry on the particle size of theresulting complexes. The size of the complexes was foundto directly correlate with the Ca:P ratio (Figure 1a). Thesize decreased from 2317±163 nm to 538±6nm, withan increase in the Ca:P ratios from 100 (2.48mM phos-phate) to 300 (0.83mM phosphate). With a decline inthe phosphate concentration in the phosphate precursorsolution from 2.48mM to 0.83mM (increasing Ca:P ra-tios), the pDNA bound and condensed more efficiently,forming smaller, more stable complexes. The surfacecharge of the complexes prepared at Ca:P ratio of 200was found to be –11±0.9mV.

3.3. In vitro gene expression by calciumphosphate–pDNA (LUC) complexes

Plasmid DNA was complexed with various Ca:P ratios(Table 1) to determine the influence of the Ca:P stoichi-ometry in achieving the optimum transfection efficiencyin HEPM cells. The cells were treated with complexescontaining 1μg pDNA for 4 h or 24 h, and processed asdescribed in Materials and methods. Untreated cells andcells treated with uncomplexed pDNA served as controls.The results demonstrated that the Ca:P ratios of thecomplexes significantly affected the expression of the lucgene in HEPM cells (p< 0.05). The amount of transfec-tion obtained was higher for complexes with a Ca:P ratioof 200 (1.24mM phosphate) than with Ca:P ratios of100 (2.48mM phosphate), 150 (1.65mM phosphate) or



300 (0.83mM phosphate) (Figure 1b). The different trans-fection efficiencies obtained with different Ca:P ratios maybe partly attributed to the sizes of these complexes. It hasbeen reported that the particle size of calcium phosphate–pDNA complexes significantly affects the extent of trans-fection obtained. Calcium phosphate complexes tend toaggregate with time, resulting in reduction in the level oftransfection attained (Sokolova et al., 2006; Welzel et al.,2004). As the Ca:P ratios increased from 100 to 200, thesizes of complexes obtained decreased almost four-fold,from an average size of 2317nm to an average size of605nm, respectively, with the latter consequently havingthe highest transfection efficiency among the groups. Ca:Pratio of 100 exhibited the lowest transfection efficiencyamong the various ratios tested. Although Ca:P 300 had asmall average particle size of 538nm, the drop in thetransfection achieved could be due to low pDNA-bindingcapacity or low pDNA-condensation capacity of Ca:P 300(Olton et al., 2007). The lower transfection capabilities of

Ca:P 100 and, in particular, Ca:P 150 (average size of823nm) could also be due to the reduced binding andcondensation of pDNA within the complexes. The geneexpression attained at the second treatment time point of24h was much lower than that attained after 4 h of treat-ment (data not shown).

3.4. Size and surface charge of PEI–pDNA (LUC)complexes

The PEI–pDNA (LUC) complexes were fabricated using aconstant amount (1μg) of pDNA and varying amounts ofPEI in order to generate N:P ratios of 1, 5, 10, 15 and 20(Table 2, Figure 2a). With increasing N:P ratios, the sizesof the complexes progressively decreased from 157±1nmfor N:P 1 to 82±2nm for N:P 20 (Figure 2b), whilstthe surface charge of these complexes increased from –

22±2mV for N:P 1 to+45±0.3mV forN:P 20 (Figure 2c).These findings were the results of complexation, coatingand condensation of the entire length of the pDNA by PEI(Dunlap et al., 1997; Godbey et al., 1999a; Hsu and Uludağ,2008; Ogris et al., 1998). Complexes prepared at a N:P ratioof 10 were typically in the 100nm size range, with a netsurface charge of~+ 35mV. The width of the particle sizedistribution is given by the polydispersity index (PDI).Lower values of PDI indicate narrow size distribution andhomogeneity of the sample. In our study, the PDI was< 0.3,confirming the relative uniformity of the particle sizedistribution. Transmission electron microscopy images ofcomplexes prepared at a N:P ratio of 10 showed the forma-tion of discrete, spherical particles of ~50–100nm in size(Figure 2d).

3.5. In vitro gene expression by PEI–pDNA (LUC)complexes

Plasmid DNA was complexed with various amounts of PEI(Table 2) to determine the N:P ratio that would yieldmaximum transfection efficiency and optimum levels ofgene expression in HEPM cells. The cells were treatedwith complexes containing 1μg pDNA for 4 h or 24 hand processed as described before. Untreated cells werethe controls, while cells treated with PEI alone were thenegative controls. Cells treated with uncomplexed pDNAserved as a control comparison with complex-treatedcells. The luc gene expression levels were dependent onthe transfection efficiencies of the N:P ratios used in thisexperiment (Figure 3a). The transfection efficiencyincreased as the N:P ratio increased from 1 (0.13μg PEI)to 10 (1.30μg PEI). Transfection efficiencies then droppedwhen the N:P ratios increased to≥ 15. The different trans-fection efficiencies obtained with different N:P ratioscould be attributed to the sizes and surface charges ofthe complexes, the extent of pDNA binding and condensa-tion and the toxicity of the complexes. Complexesprepared with a N:P ratio of 1 had a size of 157 nm anda surface charge of –22mV. The pDNA-binding and -

Figure 1. (a) Effect of different Ca:P ratios on size of calciumphosphate–pDNA (LUC) complexes; (inset) table displaying aver-age sizes of the complexes (with SDs) corresponding to differentCa:P ratios; and (b) and transfection efficiency of calcium phos-phate–pDNA (LUC) complexes in HEPM cells at 4 h of incubation;*p<0.05; **p<0.01; ***p<0.001

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condensation capacities of complexes with N:P ratio 1were also lower than complexes prepared at higher N:Pratios, thus contributing to the instability of the complex(Dunlap et al., 1997; Hsu and Uludağ, 2008). Thesefactors resulted in low transfection efficiencies of thecomplexes. Although complexes prepared at a N:P ratioof 5 had a net positive surface charge, the sizes of thecomplexes remained larger than complexes prepared ata N:P ratio of 10. Incubation of the complexes preparedat a N:P ratio of 5 with the cells for 24 h showed anincrease in the level of transgene expression obtainedrelative to that obtained after 4 h of incubation. Thismay be attributed to higher uptake and entry of the com-plexes into the cells over time. After 4 h of treatment,complexes prepared at a N:P ratio of 10 were able to trans-fect HEPM cells more efficiently than complexes preparedat other N:P ratios. This may be a result of their small size(100 nm) and high positive surface charge (+35mV)compared to complexes with N:P ratios< 10, and the

negative impact on cell viability due to PEI (see below)for N:P ratios> 10. However, the transgene expressiondecreased in cells treated with complexes prepared at a N:P ratio of 10 at 24h, due to induction of cytotoxicity byPEI (see below). For the same reason, even though com-plexes prepared at N:P ratios of 15 and 20 had small sizesand highly positive surface charges, the amount of trans-gene expression in the cells declined with these treatments.For complexes prepared at a N:P ratio of 15, the geneexpression generated was found to be lower than geneexpression generated by complexes prepared at a N:P ratioof 10 at both 4h and 24h of treatment. The transfectionresulting at 4 h of treatment from complexes prepared at aN:P ratio of 20 was lower than the transfection generatedfrom complexes prepared at a N:P ratio of 15, whichdecreased further at 24h of incubation time. The optimaltransfection efficiency obtained at 4 h of treatment withPEI–pDNA complexes at a N:P ratio of 10was nearly 12-foldhigher than that obtained with calcium phosphate–pDNA

Figure 2. Characterization of PEI-based gene delivery system. (a) Schematic showing the formation of PEI–pDNA complexes; (b) ef-fect of the various N:P ratios on size of PEI–pDNA (LUC) complexes; (inset) table displaying average size of the complexes (with SD)corresponding to different N:P ratios; and (c) surface charge of PEI–pDNA (LUC) complexes; (inset) table displaying average surfacecharge of the complexes (with SD) corresponding to different N:P ratios; (d) TEM images of PEI–pDNA (LUC) complexes prepared at aN:P ratio of 10; *p<0.05; ***p<0.001



complexes (Ca:P ratio of 200). Possible reasons for theseobservations are the high positive surface charge andsmaller sizes of the PEI–pDNA complexes, accompaniedby strong pDNA binding and condensation capacity of PEI,which are factors that favour high transfection efficiencyof gene delivery vectors.

Polyethylenimine is able to condense pDNA efficiently,delivers pDNA into the cells by ionic interactions withthe cell membrane and releases pDNA into the cytoplasmfrom endocytic vesicles. Within the acidic endo-lysosomalcompartments, protonation of PEI causes PEI to act as a‘proton sponge’, thus triggering osmotic swelling andvesicle disruption (Boussif et al., 1995). In contrast, calciumphosphate–pDNA complexes are reported to enter the cellsthrough the cell membrane calcium ion channel-mediatedendocytosis (Bisht et al., 2005). When the complexes areprepared by the calcium phosphate co-precipitation

reaction technique, they aggregate rapidly with time andincrease in particle size, and consequently the transfectionreproducibility and efficiency is decreased (Sokolova et al.,2006). Transfection efficiencies of the calcium phosphate–pDNA complexes strongly depend on the formulationparameters, including pH, temperature, and standing timebetween preparation and application, therefore resultingin inherently poor method reproducibility comparedwith PEI-based transfection methods (Jordan and Wurm,2004; Seelos, 1997; Sokolova et al., 2006; Urabe et al.,2000). In addition, inefficient pDNA binding and condens-ing capacities, reduced cellular and nuclear uptake, limitedendosomal escape and very little protection of pDNAfrom nuclease degradation can all lead to lower levels ofgene expression while using calcium phosphate as vector,when compared to PEI (Orrantia and Chang, 1990).Polyethylenimine–pDNA complexes were therefore moreeffective for delivering genes of interest to HEPM cells thanthe calcium phosphate–pDNA complexes.

3.6. In vitro cell viability assay for PEI–pDNA(LUC) complexes

Cell viability assays were performed to assess the effect ofco-culture of PEI–pDNA (LUC) complexes with HEPMcells on cell survival at 4 h or 24 h of incubation time. Plas-mid DNA (1 μg) was complexed with different amounts ofPEI (Table 2) to generate complexes with varying N:Pratios. The cells were treated with complexes for 4 h or24 h and processed as described in Materials andmethods. Untreated cells, cells treated with uncomplexedpDNA and PEI-treated cells served as controls. We quanti-fied the percentage cell viability at both the treatmenttime points of 4 h and 24 h and we observed a PEI dose-and time-dependent cytotoxicity (Figure 3b). The fact thatcomplexes prepared at a N:P ratio of 10 were able to trans-fect HEPM cells more efficiently than complexes preparedat N:P ratios≥15 at 4 h is consistent with the cell viabilitydata (Figure 3a, b). The cell viability results at 4 h showedrelatively low cellular toxicity (13%) for complexes pre-pared at a N:P ratio of 10 when compared to the cytotox-icity induced by complexes prepared at N:P ratios≥ 15.Approximately 87% of the cells were viable at 4 h whenincubated with complexes prepared at a N:P ratio of 10.However, the cell viability decreased to 20% at 24 h forcells incubated with N:P 10 complexes. This effect on cellviability explains the corresponding decrease in transfec-tion efficiency observed for N:P 10 complexes at 24 h.Similarly, the amount of PEI used in complexes preparedat N:P ratios of 15 and 20 caused a dramatic decrease incell survival at both 4 h and 24 h, and consequently hada significant negative impact on their transfection efficien-cies (p< 0.05) (described above). These data clearlyindicate that the amount of PEI used for transfecting theHEPM cells and the incubation time are critical factorsdetermining the degree of transfection efficiency andtoxicity. Complexes prepared at N:P ratios of 1 and 5 wererelatively non-toxic to the cells, but they displayed low

Figure 3. (a) Transfection efficiency and (b) and biocompatibil-ity (PEI toxicity) of PEI–pDNA (LUC) complexes fabricated at dif-ferent N:P ratios in HEPM cells at 4 h or 24h (*p<0.05;**p<0.01; ***p<0.001)

S. D’Mello et al.


transfection efficiencies. Of all the complexes prepared,only the PEI–pDNA complexes prepared at a N:P ratio of10 demonstrated high transfection efficiencies at 4 h oftreatment time, with minimum cytotoxicity. Hence, onlythe complexes prepared at a N:P ratio of 10 were used forcell transfections in the subsequent in vitro experiments.

3.7. In vitro gene expression by PEI–pDNA(EGFP-N1) complexes

Transfection in HEPM cells was further investigated byconfocal microscopy. Plasmid DNA encoding for theEGFP-N1 protein (1μg) was complexed with 1.3μg PEIto form complexes at a N:P ratio of 10. Cells were exposedto the complexes for 4 h or 24 h and further processed asdescribed above. Using fluorescent markers, we detectedsuccessful transfection occurring in cells at both the treat-ment time points of 4 h and 24 h (Figure 4). The confocalimages (Z-series, �63) revealed the transfected cellsappearing green as a whole, as evidenced by expressionof the EGFP-N1 fluorescent protein throughout thecytoplasm. In these fixed cells, phalloidin permeated the

plasma membrane to stain the cytoplasmic F-actin red.The cell nuclei were stained blue by DAPI. The controlcells (untreated, or incubated with naked pDNA or PEIalone) did not show any green fluorescence, thus rulingout transfection with naked pDNA or autofluorescencefrom the cells, pDNA or PEI. Confocal microscopyimaging, along with the quantitative results obtainedearlier, confirmed the ability of PEI–pDNA complexes totransfect HEPM cells efficiently.

3.8. In vitro gene expression by PEI–pDNA(PDGF-B) complexes

Since our future goals are ultimately targeted towardsstimulation of new bone tissue formation, we investigatedthe transgene nuclear delivery and expression of growthfactor protein, PDGF-B. Platelet-derived growth factorplays an important role in angiogenesis, is chemotacticand mitogenic for osteoblasts and vascular endothelialcells, and stimulates osteoblast type-1 collagen synthesisand extracellular matrix (Battegay et al., 1994; Bolander,1992). Based on the prior N:P ratio optimizationexperiments, we evaluated the efficiency of PEI–pDNAcomplexes prepared at a N:P ratio of 10; 1 μm pDNAwas complexed with 1.3μg PEI to form complexes at aN:P ratio of 10. Cells were exposed to the complexes for4 h and processed as described for other transfection exper-iments. The PDGF-BB ELISA test demonstrated that thePDGF-BB levels (PDGF-BB concentration 56pg/ml) were3.5-fold higher in cells treated with complexes prepared ata N:P ratio of 10 than with uncomplexed, naked pDNA(PDGF-BB concentration 16pg/ml) (Figure 5). Theseresults confirmed the ability of PEI–pDNA complexes totransfect HEPM cells with genes that are therapeuticallyrelevant to bone regeneration.

Figure 4. Confocal laser scanning microscopic images (Z-series,�63) demonstrating: (upper row) EGFP-N1 protein expression inHEPM cells at 4h or 24h after transfection with plasmids alone;PEI–pDNA (EGFP-N1) complexes prepared at a N:P ratio of 10 after(middle row) 4h or (lower row) 24h of transfection: nuclei, blue,DAPI-stained; cytoplasm and cell membrane, red, phalloidin-stained; and EGFP-N1, green. Scale bar=20μm

Figure 5. Expression of PDGF-BB protein in HEPM cells at 4 h af-ter transfection of the cells with PEI–pDNA (PDGF-B) complexesprepared at a N:P ratio of 10; **p<0.01



3.9. Gel retardation assay for PEI–pDNA (LUC)complexes

We evaluated the structural integrity of the complexesprepared at a N:P ratio of 10 by determining the electro-phoretic mobility of the complexes on a 1% agarose gel.To establish conditions similar to those used in the trans-fection experiments, complexes with N:P 10 were incu-bated with cell culture growth medium (containing 10%FBS) for 1 h at 37°C. This was performed to assess the ef-fect of degradative enzymes, such as DNases, and proteinspresent in the growth medium on complex dissociationand pDNA degradation. Uncomplexed pDNA was used asthe control. The gel retardation assay demonstrated thestability of pDNA within the complexes (Figure 6). Theuncomplexed pDNA moved freely in the gel (towardsthe positive terminal) as discrete bands, whereas thecomplexed pDNA was retarded in the loading well of thegel. This result suggests that PEI entraps pDNA and re-stricts its mobility in the gel. Upon treatment with the cellculture growth medium, the free plasmid appeared toundergo enzymatic digestion, as visualized by fragmenta-tion, smear and band dislocation. By contrast, thecomplex structure and the incorporated pDNA werecompletely unaffected. The complexes containing pDNA

remained in the wells with no sign of decomplexation.The presence of serum DNases and proteins in the growthmedium had no adverse effect on the complex conformationor the entrapped pDNA. Thus, PEI was able to effectivelybind, package and condense the pDNA, consequentlyforming strong and stable complexes with pDNA. The con-densation of DNA by PEI enables the formation of compactparticles for endocytosis and also protects the entrappedpDNA from intracellular nuclease digestion (He et al., 2003).

4. Conclusions

Cationic complexes incorporating pDNAs encoding theLUC, EGFP-N1 or PDGF-B proteins were successfully pre-pared. Two different non-viral vectors, PEI and calciumphosphate, were empirically assessed as gene deliveryvectors. It was found that PEI is a more effective vectorthan calcium phosphate for delivering genes of interestto pre-osteoblastic HEPM cells. The cell complex incuba-tion time, amount of PEI in complexes, size and surfacecharge of the complexes were critical parameters forachieving efficient transfections. In vitro experimentsdemonstrated that PEI–pDNA complexes prepared at aN:P ratio of 10 were able to transfect LUC, EGFP-N1 andPDGF-B genes efficiently into HEPM cells with relativelylower cytotoxicity than complexes prepared at other N:Pratios. Future studies will explore the in vivo transfectionefficacy of PEI–pDNA complexes prepared at a N:P ratioof 10 for the purpose of bone regeneration.

Conflict of interest

The authors have declared that there is no conflict of interest.

Acknowledgements

This study was supported by a University of Iowa Start-upGrant, the ITI Foundation for the Promotion of Implantology,Switzerland (ITI Research Grant No. 855 2012), the AmericanCancer Society (Grant No. RSG-09-015-01-CDD) and theNational Cancer Institute at the National Institutes of Health(Grant Nos 1R21CA13345-01, R21CA128414-01A2 and UI MayoClinic Lymphoma SPORE).

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