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Cationic copolymers nanoparticles for nonviral gene vectors:Synthesis, characterization, and application in gene delivery
Giovanna Gomez d’Ayala,1* Anna Calarco,2* Mario Malinconico,1 Paola Laurienzo,1 OrsolinaPetillo,2 Angela Torpedine,2 Gianfranco Peluso2
1Institute of Polymers Chemistry and Technology, CNR, Via Campi Flegrei 34, 80078 Pozzuoli, Naples, Italy2Institute of Protein Biochemistry, CNR, Via Pietro Castellino 111, 80131 Naples, Italy
Received 19 May 2009; revised 9 September 2009; accepted 4 December 2009
Published online 2 March 2010 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32752
Abstract: The major aim of nonviral delivery systems for
gene therapy is to mediate high levels of gene expression
with low toxicity. Nowadays, one of the most successful syn-
thetic polycations used in gene delivery research is poly(ethy-
lenimine) (PEI) in its high-molecular weight (HMW) branched
form. However, PEI is not the ideal transfection agent in vivo
because of its overwhelming cytotoxicity. To overcome its
toxic effects with a minimal impact on transfection efficiency,
PEI has been conjugated with several nonionic biocompatible
polymers. Here, we describe the synthesis of nanosized par-
ticles consisting of HMW PEI (25 kDa) crosslinked with poly-
(e-caprolactone) (PCL, 50–60 kDa), a biodegradable aliphatic
polyester. PCL was modified by the insertion of glycidyl
groups able to condense with the amines of PEI to chemically
bind PEI onto PCL. The nanoparticles obtained have been
characterized in relation to their physicochemical and biologi-
cal properties, and the results are extremely promising in
terms of low cell toxicity and high transfection efficiency.
These biological effects might be related to the peculiar DNA
binding to covalently connected polymeric nanoparticles,
without the formation of entangled DNA/polymer-soluble
aggregates. VC 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part
A: 94A: 619–630, 2010
Key Words: polycaprolactone, poly(ethylenimine), gene deliv-
ery, nanoparticles, nonviral DNA vectors
INTRODUCTION
Poly(ethylenimine) (PEI) is a synthetic cationic polymer thatis largely known in literature for its applications in nonviralvector systems for gene delivery.1,2 PEI spontaneously asso-ciates with phosphates of DNA through electrostatic interac-tions because of the protonated amine groups forming sta-ble DNA/PEI polyplexes.3,4 Highly branched, high-molecularweight (HMW) PEI shows a good transfection efficiencybecause of its buffering capacity, but it has also beenreported to be toxic for many cellular lines. To avoid thisadverse effect, low-molecular weight (LMW) PEI has beencopolymerized with nonionic biocompatible polymers.Diblock copolymers of PEI with hydrophilic macromoleculesforming DNA polyplexes with neutral surface charge as wellas ternary block copolymers obtained by grafting diblockcopolymers of poly(ethylene glycol) (PEG) and poly(e-capro-lactone) (PCL) onto branched PEI have been extensivelyinvestigated.5,6 Recently, it has been reported that copoly-mers obtained by the reaction between PCL diacrylate andLMW PEI showed effective and stable DNA condensationwith particle sizes below 200 nm.7
A different approach resides in the combination of HMWPEI with biopolymers, either natural or synthetic, thatallows for an easier manufacturing of the materials toobtain devices such as nanocarriers. Examples of the real-
ization of vectors based on solid nanoparticles of blends ofPEI with biodegradable polymers are reported in litera-ture.8,9 In nanoparticle-based gene delivery systems, DNAcan be encapsulated inside the nanoparticle10,11 or adsorbedonto nanoparticle cationic surface.12,13 This last system hasthe advantage of ease binding with DNA, DNA protection,avoidance of the direct contact of plasmid with organic sol-vents during particles preparation; furthermore, a rapidrelease of DNA inside the target cells is facilitated. More-over, DNA/nanoparticle complexes have a much lower tend-ency to aggregate when compared with soluble DNA/polyca-tion polyplexes.
The objective of this work is to prepare and characterizegraft copolymers of PCL and PEI suitable for their applica-tions in gene delivery. PCL was previously modified by theinsertion of glycidyl groups able to condense with theamines of PEI. Chemical bonds with PCL avoid the releaseof toxic free PEI in the living cell after delivery of the DNA.Nanoparticles of controlled size and persistent shape havebeen obtained by an emulsion method and have been char-acterized by scanning electron microscopy (SEM). The nano-particles have been characterized in relation to their physi-cochemical and biological properties. Some experimentalresults are extremely promising in terms of low cell toxicityand high transfection efficiency.
*These authors contributed equally to this work.
Correspondence to: P. Laurienzo; e-mail: [email protected] or G. Peluso; e-mail: [email protected]
Contract grant sponsor: Italian Ministry of University and Research; contract grant number: 2006-prot. RBIP06ZJ78_002
VC 2010 WILEY PERIODICALS, INC. 619
MATERIALS AND METHODS
MaterialsPCL (CAPA 650, molar mass 50–80 kDa) was kindly giftedfrom Solvay (Belgium); PEI (PEI25K, highly branched, MW25 kDa), triethylamine (TEA), glycidyl methacrylate (GMA),and dibenzoylperoxide (DBPO) were purchased fromAldrich. TEA was distilled at reduced pressure and wasstored under nitrogen atmosphere; GMA was anhydrified onan aluminum oxide column before use. All solvents were ofanalytical degree and were used as received.
Dulbecco’s modified Eagle’s medium (DMEM), fetal bo-vine serum (FBS), penicillin–streptomycin, trypsin, and Dul-becco’s phosphate-buffered saline (PBS) were purchasedfrom Hyclone (Milan, Italy); plastic tissue cultures wereobtained from Falcon (Milan, Italy); pEGFP-N2 plasmid wasobtained from BD Biosciences Clontech (Milan, Italy); Pure-YieldTM Plasmid Maxiprep System, 1-kb DNA ladder, and6�DNA loading buffer (30% glycerol, 125 mM EDTA, 0.25%bromophenol blue, and 0.25% xylene cyanol) were obtainedfrom Promega (Milan, Italy); and Triton X-100, sulforhod-amine B (SRB), and LDH were obtained from Sigma (Milan,Italy).
Synthesis of copolymerThe grafting of PEI onto PCL was performed in two steps.In the first step, PCL was functionalized by the insertion ofGMA molecules in the melt by using a Rheocord mixer fromHaake (USA), equipped with two roller blades. PCL (40 g)was introduced into the chamber of the Rheocord mixer at100�C and at a mixing speed of 32 rpm until melting, andthen a solution of 0.8 g of DBPO in 8.0 g of GMA was added.The grafting reaction was carried out for 20 min. ModifiedPCL (PCL-g-GMA) was purified to remove unreacted GMA,free GMA oligomers, and traces of initiator by dissolution in500 mL of chloroform followed by precipitation in a largeexcess of methanol. The recovered white precipitate waswashed twice with fresh methanol and then dried undervacuum until constant weight (1H NMR (CDCl3): d 1.1[ACH3, GMA], 1.3–1.8 [AOACH2A(CH2)4A, PCL], 2.3[ACH2ACOA, PCL], 2.65–2.85 [ACH2A of epoxy ring ofGMA], 3.25 [ACHA of epoxy ring of GMA], 3.8–4.3[ACH2AOCOA, GMA], 4.05 [ACH2AOCOA, PCL]). Graftingdegree (d 2.3/d 3.25) ¼ 7.4%.
For the grafting reaction (second step), 1.50 g of PCL-g-GMA was dissolved in 30 mL of chloroform in a three-necked glass flask equipped with a magnetic stirrer, a nitro-gen inlet, and a condenser. Then, 0.15 mL (1.08 mmol) ofTEA was added. A solution consisting of 1.50 g of PEI in 10mL of chloroform was added dropwise. The temperaturewas raised to 70�C, and the solution was stirred for 1 h.The final product was recovered by precipitation with meth-anol (which is a good solvent for PEI, but not for PCL). Theresulting white precipitate was repeatedly washed withmethanol, and then dried under vacuum until constantweight to give PCL-g-GMA-g-PEI copolymer (hereafter, sim-ply coded as PCL–PEI).
The grafting degree (GD), defined as the ratio betweengrafted PEI and the initial amount of PCL-g-GMA, and the
grafting efficiency (GE), defined as the ratio between initialamount of PEI and grafted PEI, were determined throughgravimetric measurements according to the following equa-tions:
GD ¼ ðPEIÞg=ðPCL-g-GMAÞ � 100
GE ¼ ðPEIÞg=ðPEIÞ � 100
The results reported are a mean of three experiments.
Characterization of the copolymerFTIR spectra were obtained by using a Perkin-Elmer spec-trometer, model Paragon 500 (average of 20 scans, at a re-solution of 4 cm�1). Polymer samples were ground withKBr and compressed to obtain disks.
For 1H NMR spectroscopy, the sample (PCL-g-GMA,about 20 mg) was dissolved in deuterated chloroform, andone-dimensional spectra were recorded with a BrukerAvance DPX300 apparatus operating at 300 MHz.
For solid-state NMR analysis, samples (PCL-g-GMA, PEI,PCL–PEI) were finely cut and packed into 4-mm zirconiarotors and sealed with Kel-F caps. Solid-state 13C single-pulse excitation MAS (SPE MAS) NMR spectra were acquiredat 100.47 MHz on a Bruker Avance II 400 spectrometeroperating at a static field of 9.4 T. Experiments were per-formed using a recycle delay of 10 s, a 13C p/2 pulse widthof 3.80 ls, and a spin rate of 6.3 kHz. For each sample,1000–2000 scans were collected.
Thermograms were recorded with a differential scanningcalorimeter Mettler DSC 30. Samples, sealed in an aluminumpan, underwent runs at an heating/cooling rate of 10�C/min according to the following protocol: 1� run ¼ heatingfrom �100 to 150�C, followed by 2 min in isotherm at150�C; 2� run ¼ cooling from 150 to �100�C; 3� run ¼heating from �100 to 150�C.
Tensile tests were performed using a dynamometer Ins-tron Mod.4301, which conforms to the iso 5893 standard,equipped with a personal computer for data acquisition.Sheets were obtained by casting the reaction solution, after1 h at 70�C, in a glass Petri dish placed onto a carefully lev-eled platform to guarantee a homogeneous thickness, andthen washing the cast film with methanol. The samples fortensile analysis were cut from the sheets in dog-bone shapeusing a cutter. Specimens of 1-cm wide, 4-cm long, andaround 0.2-mm thick were used. For each sample, six speci-mens were tested. All the measurements were achieved atroom temperature, at crosshead speed of 10 mm/min, atgauge length of 22 cm, and at nominal strain rate of 0.45mm/min.
Preparation of nanoparticlesNanoparticles of PCL–PEI copolymer were prepared directlyfrom the reaction solution by a simple emulsion procedure.The reaction was stopped after 1 h by ice cooling, and 2 mLof the chloroform solution was homogenized with 20 mL of0.5% PVA aqueous solution by means of an ultrasonic proc-essor (Vibra Cell mod. VC 505; Sonics and Materials,
620 GOMEZ d’AYALA ET AL. CATIONIC COPOLYMERS NANOPARTICLES FOR NONVIRAL GENE VECTORS
Danbury, CT). The particle suspension was stirred at 200rpm rate for 24 h for chloroform evaporation. The obtainednanospheres were collected by centrifugation in a high-speed centrifuge (Hermle Z323) at 8000 rpm for 15 min,carefully washed to remove unreacted PEI with a water/methanol mixture, and then freeze dried. Nanoparticleswere stored at �4�C.
Scanning electron microscopyNanoparticles were sputter coated with a gold/palladiumalloy before examination with a Philips Mod. XL20 SEM.
Preparation of plasmid DNApEGFP-N2 plasmid (4.7 kb) was transformed in E. coli DH5aand amplified in terrific broth media at 37�C overnight. Theplasmid was purified by Promega maxiprep system accord-ing to the manufacturer’s protocol. The purity of plasmidDNA (pDNA) was certified by the absorbance ratio atOD260/OD280, and by distinctive bands of DNA fragments atcorresponding base pairs in gel electrophoresis after restric-tion enzyme treatment of DNA. pDNA was stored at �20�Cuntil used.
Characterization of nanoparticles/plasmid complexMeasurement of the interactions between DNA and nano-particles. Complexes of nanoparticles and pDNA (PCL–PEI/DNA) were formed by first diluting plasmid and the appro-priate amount of nanoparticles separately with 150 mMNaCl, pH 7.4, to equal volumes. The nanoparticles suspen-sion was then added to the EGFP-encoded plasmid solution,vortexed immediately at room temperature, and allowed tostand for 30 min to attain complexes. Then, the complexeswere electrophoresed on an agarose gel containing 1% ofethidium bromide (EtBr) and with Tris-acetate EDTA (TAE)running buffer at 80 V for 90 min. Images were acquiredusing a GelDoc 2000 gel documentation system (Bio-Rad,Milan, Italy) equipped with a UV transluminator. QuantityOne, version 1.1 software (Bio-Rad) was used for band inte-gration and background correction. EtBr, a DNA intercalingdye, was used to examine the association of DNA with thenanoparticles to determine the complex formation.
Particle sizes and zeta potential measurements. Dynamiclight scattering (DLS) with a Zetasizer Nano ZS90 instru-ment (Malvern Instruments, Worcestershire, UK) was usedto measure the diameter and zeta potential of the PCL–PEI/DNA complexes at different nanoparticles/DNA weight ratiosprepared as described earlier. The hydrodynamic diameterof the freshly prepared complexes was measured at 25�Cwith a scattering angle of 90� (10 mW He–Ne Laser, 633nm), and the zeta potential was determined by the standardcapillary electrophoresis cell of Zetasizer Nano ZS at posi-tion 17.0 and at 25�C. Polystyrene nanospheres (220 6 6nm and �50 mV) were used to verify the performance ofthe instrument. All the average values were performed withthe data from six separate measurements. Arithmetic meanand standard deviation were calculated from six consecutive
runs, and samples were analyzed using Malvern PCSsoftware.
DNAse I degradation assay. Naked pDNA and PCL–PEI/DNA complex at different weight ratios were prepared in asolution of Tris buffer (pH 7.9) containing 60 mM MgCl2.Samples were incubated with 100 lL of DNase I (12.7 U/lg; Sigma). The absorbance of DNA at 260 nm was meas-ured continuously for 1 h, and the values are plotted in acurve (relative absorption vs. time).14
Cell line experimentsCell culture. Human cervix epithelial carcinoma (HeLa)cells, human colon carcinoma (Caco-2) cells (passage 15–20), and human hepatocellular liver carcinoma (HepG2)cells were cultured in DMEM supplemented with 10% FBS,streptomycin at 100 lg/mL, and penicillin at 100 U/mL.Cells were maintained at 37�C in humidified and 5% CO2 in-cubator. The medium was replenished every other day, andcells were subcultured after reaching confluences.
Cell viability assay. The cytotoxicity of PCL–PEI nanopar-ticles, complexed or not with DNA, and of PCL-g-GMA wasdetermined in a separate set of experiments using both theLDH assay, to measure immediate perturbation of mem-brane integrity after 4 h of incubation, and the SRB assay,to detect changes in cell viability after an incubation time of24 h. Cells were seeded in 24-well plates at an initial den-sity of 1 � 105 cells/well for HeLa cells, 8 � 104 cells/wellfor HepG2, and Caco-2 cells in 0.5 mL of growth mediumand incubated for 24 h prior to the addition of PCL-g-GMAand PCL–PEI/DNA at different nanoparticles/DNA weight ra-tio. Untreated cells were taken as control with 100% viabil-ity, and cells without addition of reagent were used as blankto calibrate the spectrophotometer to zero absorbance. Tri-ton X-100 1% was used as positive control of cytotoxicity.SRB assay was used as previously described,15 and LDHassay was used according to the manufacturer’s instruc-tions. Cytotoxicity of polymer was compared with PEI. Theresults were expressed as mean values 6 standard devia-tion of four measurements.
Cellular uptake. For nanoparticle uptake study, 24-wellplates were seeded with different cell lines at 5 � 104/welldensity, and the cells were allowed to attach for 24 h. Themedium in the wells was replaced with the suspension offreshly prepared or lyophilized nanoparticles, labeled or notwith the fluorescent dyes, such as coumarin-6, and incu-bated for 1 h. In a separate experiment, to study the effectof incubation time on nanoparticle uptake, the medium wasreplaced with 1 mL per well of a 50 lg/mL suspension oflabeled nanoparticles in complete growth medium, and theplate was incubated for 30 min, 1 h, and 2 h. At the end ofthe incubation period, the cells were washed three timeswith PBS to remove the nanoparticles which were not inter-nalized, and then lysed by culture lysis reagent (Promega,Milan, Italy). The dye from the nanoparticles was extractedand analyzed by a high-performance liquid chromatography
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(HPLC), following a previously described method.16 A stand-ard plot with different concentrations of nanoparticles wasconstructed simultaneously under similar conditions todetermine the amount of nanoparticles in cell lysates. Thedata were normalized to per milligram cell protein.
In vitro gene expression assay. One day before EGFPtransfection assay, cells were seeded in 24-well plates at adensity dependent of the cell line in DMEM with 10% FBS.When the cells were at 50–70% confluence, the medium ineach well was replaced with free-serum medium or normalmedium containing naked pEGFP or PCL-PEI/DNA complexat different nanoparticles/DNA weight ratios and incubatedfor 4 h under standard incubator conditions. After 4 h, themedium was replaced with 1 mL of complete medium andincubated until 44-h post-transfection and was observed byconfocal microscope.
The analysis of transfection efficiency was performedusing a flow cytometer (FACS, BD Biosciences, MountainView, CA). Cells were first washed with PBS and detachedwith 0.3 mL of trypsin. Growth medium was then added,and the cells suspension was centrifuged at 14,000 rpm for10 min. Two further cell-washing cycles of resuspensionand centrifugation was carried out in PBS before fixation in0.4 mL of 1% paraformaldehyde. The percentage of cellsexpressing GFP was then determined from 10,000 eventsand reported as a mean 6 standard deviation of at leastfour samples.
RESULTS AND DISCUSSION
Grafting reactionThe grafting of PEI onto PCL was performed in a two-stepprocedure, as illustrated in Scheme 1.
PCL was first functionalized through radicalic insertionof GMA molecules, and then the obtained PCL-g-GMA wasreacted with PEI by the condensation of glycidyl groupswith amines using TEA as catalyst. The first step was per-formed according to a procedure slightly modified withrespect to the literature.17 Viscosity measurements on poly-mer solutions showed a value of ginh very close to that ofstarting PCL (1.07 dL/g). The second step involved theepoxies of GMA molecules grafted onto PCL, which reactedwith both primary and secondary amines of PEI. As PEI hasmany primary and secondary amines, and PCL as well hasmany glycidyl molecules grafted on, the reaction betweenPEI and PCL-g-GMA may result in a crosslinked product.The reaction was monitored, and copolymers obtained atdifferent reaction times were isolated and characterized byseveral techniques. The relative amount of PEI with respectto PCL was fixed at 50 wt %. PEI was added dropwise tofavor the grafting on PCL of single PEI molecules. The reac-tion was stopped at regular time intervals of 30 min within2 h, and the GD and GE were determined through gravimet-ric measurements, on the removal of the unreacted PEI byselective solvents (Table I). It was found that the GD levelsoff at �65% already after 30 min, with a GE of around68%; this is an indication of a high reactivity of the amines
SCHEME 1. Proposed reaction scheme for the synthesis of PCL–PEI. (A–D) Proposed structures of copolymers (backbone coarse line refers to
PCL, fine branched lines to PEI). (E) Proposed structure of the PCL–PEI nanoparticles.
622 GOMEZ d’AYALA ET AL. CATIONIC COPOLYMERS NANOPARTICLES FOR NONVIRAL GENE VECTORS
of PEI toward GMA molecules. In the first stages of the reac-tion, the number of molecules of PEI grafted onto PCL, aswell as the number of reacted amines for each PEI molecule,gradually increases [Scheme 1(A,B)]. With the continuousaddition, as the GD remained constant, some changesoccurred in the structure of the copolymers. Still, unreactedamines can react with free glycidyl molecules on the sameor on different PCL macromolecules. The structure graduallyevolves from a physical cluster to a chemical network. Aftermore than 1 h, the solution becomes viscous but still wellstirrable [a coarse crosslinking may have developed at thistime, as sketched in Scheme 1(C)]; at higher times, the solu-tion becomes more and more viscous due to increasedcrosslinking until gelation with precipitation of a solid prod-uct occurs [Scheme 1(D)]. It is worth noticing that therecovered dried copolymers are insoluble even if the reac-tion was stopped before gelation, probably due to the for-mation of a very entangled and stable structure. Followingthis observation, it was realized that a possible strategy toobtain, for example, microspheres and nanospheres, consistsin stopping the reaction by ice cooling when the mixture isstill well stirrable and emulsifying the reaction solution
with water, without isolating the copolymer. According tothis procedure, in the chosen experimental conditions, 1 hof reaction is a good compromise between solubility of thereacting species, viscosity of the solution, and GD of theobtained copolymer. So, if not otherwise specified, the co-polymer simply coded as PCL–PEI is referred to 1-h reactiontime in the following. Homogeneous nanoparticles with asurface consisting of hydrophobic/hydrophilic segmentswith a net positive charge [Scheme 1(E)] were reallyobtained in this way. The oil-in-water emulsion procedureaccounts for a prevalent concentration of PEI at or near thenanoparticle surface.18
Spectroscopic characterizationThe PCL–PEI copolymer has been characterized by FTIRspectroscopy to confirm the reaction. Diagnostic bands ofPEI at 1580 cm�1 (CAN stretching) and at 3350–3290cm�1 (primary and secondary amines NAH stretching,respectively) are detected, along with the typical ester bandof PCL (1727cm�1), and an additional band at 3446 cm�1,attributed to stretching of AOAH which are generated dur-ing the reaction. A comparison between the spectra of prod-ucts obtained at different reaction times evidenced adecrease of the amine bands with time. This is in agreementwith the proposed schematic structures of the copolymer[Scheme 1(A,B)], as the number of unreacted aminesdecreases with time.
The occurrence of chemical reactions has also beenchecked and highlighted through solid-state NMR. 13C single-pulse experiments were performed either on reference reac-tants (PCL-g-GMA and PEI) or on the PCL–PEI copolymer.The relative 13C spectra are shown in Figure 1 (curves a–c).
TABLE I. DSC Parameters Relative to PCL, PEI, and PCL–PEI
Copolymer (65% PEI by Weight) Detected in the Second
Heating Run
SampleDHm
(J/g)DHc
(J/g)Xc
(%)Tc
(�C)Tm
(�C)Tg
(�C)
PCL 53 49 37.3 13 62 �68PCL–PEI copolymer 23 19 16.5 33 57 �52PEI – – – – – �52
FIGURE 1. 13C SPE MAS spectra: (curve a) PCL-g-GMA, (curve b) PEI, and (curve c) PCL–PEI. The inset shows the spectral subtraction of PCL-g-
GMA from PCL–PEI 13C SPE MAS spectrum. The peaks marked with an asterisk correspond to spinning speed sidebands.
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The more resolved 13C spectrum of neat PEI (Fig. 1, curve b)is indicative of a viscous substance having a higher chain mo-bility with respect to PEI in a solid-phase network (Fig. 1,curve c). In the spectrum of the reaction product, the follow-ing signals are present: typical resonances of methylenegroups in 10–30 ppm region, essentially attributable to PCLand GMA;19,20 a low resolved area ranged from 35 to 60ppm, attributed to the resonances of ACH2A moieties ofPEI21 superimposing on those of residual ACH2A and ¼¼CHAepoxy groups of GMA (centered at 43 and 48 ppm, respec-tively); a main peak at 173 ppm corresponding to PCL car-bonyl, overlapped to a smaller signal at 177, due to C¼¼Ogroups of GMA. Interestingly, in the copolymer spectrum, thesignal centered at 65 ppm, assignable to the methylenoxy ofPCL and GMA (ACH2OAC¼¼O), shows a clear shoulder, whichis not present in the reference reactant spectra (Fig. 1, curvesa and b). Accordingly, the 13C spectrum of reaction productafter PCL-g-GMA spectral subtraction, shown in the inset ofthe figure, underlines this new resonance at 68 ppm besidestypical PEI signals. This resonance is characteristic of aACHAOH group, thus confirming that the reaction doesoccur, according to Scheme 1. The presence of unreacted ep-oxy groups is an indication of the fact that the reaction is notcomplete, probably due to the gelification of the system thatreduces the chain mobility and the accessibility to GMA resid-ual groups.
Thermal characterizationA typical DSC curve relative to the copolymer obtained after60�C reaction time in second heating run is reported in Fig-ure 2. In the first run, the endotherm of PCL melting at62�C was always followed by the evolution of water around100�C, totally absent in pristine PCL. Thermal parametersrelative to PCL phase in the copolymer compared with thoseof plain PCL and PEI are summarized in Table I. The evalua-tion was based on the reported enthalpy of fusion of 139.5J/g for 100% crystalline PCL.22 Melting and crystallizationenthalpies showed a large decrease with respect to plainPCL, as a consequence of entanglements and crosslinks. TheTc was 20�C higher for the copolymer, suggesting a nucleat-ing role played by the PEI in the PCL melt crystallization.
PEI exhibits only one glass transition step at �52�C; infact, because of its highly branched structure, it is com-pletely amorphous. Only one Tg can be detected in the co-polymer, which corresponds to the PEI phase, whereas theTg of PCL phase is not detectable.
These characterizations (NMR and DSC, respectively)demonstrate the formation of a stable covalent linkagebetween PEI and PCL and the absence of any significantchange of the chemical–physical properties of the two poly-mers in the copolymer. In addition, the characterization ofdifferent batches of PCL–PEI proves the reproducibility ofour synthesis approach, which is an important issue for bio-medical materials.
Tensile propertiesAlong with chemical–physical characteristics, the tensileproperties of PCL–PEI copolymer were also considered,keeping in mind that the deformability under flow condi-tions of corresponding nanoparticles is an important param-eter to be considered in view of applications as carriers inthe blood. Tensile parameters of pure PCL, PCL-g-GMA, andPCL–PEI are reported in Table II. The tensile strength atbreak of PCL-g-GMA was comparable with those of theunmodified PCL; this indicates that there was no reductionin the molecular weight and no significant changes in thecrystallinity. For the PCL–PEI copolymer, the elongation atbreak was much lower, due to crosslinking and/or to thehigh number of hydrogen bonds that results in a veryentangled structure, and also tensile strength at break andelastic modulus were reduced with respect to PCL and PCL-g-GMA; these last results were probably related to the intro-duction of a completely amorphous, low Tg PEI as bridgingsegments. A similar trend was also detected by Nagata andSato23 for photocured polyesters based on PCL macrodiolswith 4,40-(adipoyl)dioxydicinnamic acid as a photosensitivechain extender. The decrease in E0 indicates a more pro-nounced linear deformability of the obtained copolymerunder low stress. As a consequence, the relative nano-spheres will have a good deformability under flow condi-tions (low stress). This may be beneficial for some in vivoapplications.24
Nanoparticles characterizationNanoparticles have been characterized by SEM (Fig. 3). Par-ticles are spherical and very regular in size (around 200nm), with smooth surface; although some nanospheresappeared fused together, particles were easily redispersedin water by sonication (0.1 mg/mL). As known from litera-ture,13,25 freeze-dried PCL-based nanoparticles can be
FIGURE 2. DSC curve of PCL–PEI (PCL melting at 60�C and Tg of PEI
can be noticed).
TABLE II. Young’s Modulus (E), Strength at Break (rB), and
Elongation at Break (eB) of Plain PCL, PCL-g-GMA, and
PCL–PEI Copolymers
Sample E (MPa) rB (MPa) eB (%)
PCL 318 6 27 33.0 1,330PCL-g-GMA 325 6 27 34.3 1,300PCL–PEI 131 6 7 5.4 7.17
624 GOMEZ d’AYALA ET AL. CATIONIC COPOLYMERS NANOPARTICLES FOR NONVIRAL GENE VECTORS
stored for a period of months without undergoing signifi-cant particle size changes and molecular weight decrease.
Characterization of nanoparticles/pDNA complexA prerequisite of a polymeric gene carrier is DNA condensa-tion. To confirm the copolymer/DNA complex formation, a
retardation assay and a EtBr exclusion assay were per-formed. The electrophoretic mobility of pDNA was retardedwith increasing amount of copolymer and even remained atthe top of the gel. As shown in Figure 4(A, lanes 6–7), thenanoparticles are able to completely bind pDNA at a weightratio higher than 5. The ability of the copolymer to effec-tively condense DNA was evaluated by measuring the reduc-tion in fluorescence intensity of EtBr due to its exclusionfrom DNA. The copolymer showed a good condensing activ-ity for DNA, reaching a maximal quenching of about 88%.The maximal condensation of DNA was observed at nano-particles/DNA weight ratio 6.7 (data not shown).
To demonstrate the ability of nanoparticles to protectDNA from nucleases, the nanoparticles were exposed toDNase I, and the absorbance was measured at 260 nm. Noincrease in absorbance could be observed in the case ofnanoparticles [Fig. 4(B)], whereas degradation of DNA wasevident with naked pDNA. The result indicates that the DNAwas significantly protected from nuclease degradation bybinding to nanoparticles.
The diameter of the nanoparticles/DNA complexesranged between 350 and 230 nm at the determined nano-particles/DNA weight ratios [Fig. 4(C)]. The differences innanoparticle size were not statistically significant. Zeta
FIGURE 3. SEM micrograph of a centrifuged nanoparticles dispersion.
FIGURE 4. Characterization of PCL–PEI/DNA complex. (A) PCL–PEI/DNA complexes with increasing amounts of PCL–PEI nanospheres. Lane 1:
DNA molecular weight marker; lane 2: plasmid DNA alone (0.5 lg); lanes 3–7: DNA (0.5 lg) with progressively increasing amount of nanopar-
ticles (nanoparticles/DNA weight ratio: 1, 1.7, 3.3, 6.7, 10). (B) Degradation assay with DNAse I on PCL–PEI/DNA (n; nanoparticles/DNA weight
ratio 6.7) in comparison with naked pDNA (~), measured by spectrophotometric continuous detection at k ¼ 260 nm of released nucleotides.
The same experiments were carried out for nanoparticles/DNA weight ratio 3.3 and 10 (data not shown). (C, D) Distribution of particle size and
Z-potential measured by dynamic light scattering (mean 6 SD, n ¼ 6).
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potential is an indicator of the surface charge of nanopar-ticles/DNA complexes, and a positive surface charge allowsan electrostatic interaction between cellular membrane andcomplex. As shown in Figure 4(D), the zeta potential of thecomplexes increased in parallel with the rinsing nanopar-ticles/DNA weight ratio, ranging from �6.6 mV to þ12.5mV, which results in a good affinity for cell surface.
In vitro cytotoxicitySRB protein staining has been widely used for cell prolifera-tion and chemosensitivity testing, substituting for tetrazo-
lium-based assays.15 The results of SRB assay were rela-tively stable and not influenced by some factors, such asdetecting time which induce the fluctuation of data by MTTassay. Thus, in this study, SRB assay was used to investigatethe cytotoxicity of nanoparticles/DNA complexes followingincubation on HeLa, HepG2, and Caco-2 cells. Nanoparticles/DNA complexes were applied to the cells at weight ratiofrom 1.7 to 10. The effect of PCL–PEI/DNA on the cell via-bility was compared with PEI. Figure 5(B,D,F) shows thatPCL–PEI/DNA nanoparticles have remarkably lower cytotox-icity on three different cell lines than soluble PEI. Indeed,
FIGURE 5. The effect of PCL–PEI/DNA complex on membrane integrity and viability on Caco-2 (A, B), HeLa (C, D), and HepG2 (E, F) cells. Cells
were treated with different nanoparticles/DNA weight ratios for 4 h (LDH assay: A, C, and E) or 24 h (SRB assay: B, D, and F). Control cells cul-
tured in nanoparticle-free media were run in parallel to treatment groups. Data are expressed as percentage of untreated cells and are the mean
6 SD of four separate experiments.
626 GOMEZ d’AYALA ET AL. CATIONIC COPOLYMERS NANOPARTICLES FOR NONVIRAL GENE VECTORS
PCL–PEI/DNA showed above 90% cell viability at nanopar-ticles/DNA weight ratio 6.7. By contrast, cell viabilitydropped by about 20% in the presence of PEI, confirmingthe hypothesis that HMW PEI aggregates on the cell surfaceand impairs important membrane functions.
In addition to SRB assay, the extracellular concentrationof LDH was also carried out. The toxic effect of PCL–PEI/DNA complexes on the cultured cells was determined bymeasuring the release of LDH into the culture medium. Asshown in Figure 5(A,C,E), the addition of PCL–PEI/DNAnanoparticle complexes to cell cultures did not induce anysignificant LDH release in comparison with PEI in all thecell types tested. Overall, LDH assay indicates that PCL–PEIis not cytotoxic to cells tested. Moreover, no toxicity wasevident in case of incubation of cells with PCL-g-GMA andPCL–PEI nanoparticles without DNA (data not shown).
Cellular uptakeIt is clear that cell transfection is dependent on DNA/vectoruptake efficiency. Nonviral vectors labeled with fluorescentdyes, such as coumarin-6, are frequently used to study thiscellular uptake. Recent studies have demonstrated that lessthan 0.6% of the incorporated coumarin-6 could leach outfrom the nanoparticles over 48 h under in vitro sink condi-tions and that the raw coumarin-6 cannot be directly inter-nalized by the cells.26 Thus, the fluorescence measured fromour uptake experiments reflects the fluorescent nanopar-ticles taken up by the cells but not the released fluores-cence. Coumarin-6 was incorporated into PCL–PEI nanopar-ticles, and the corresponding cellular uptake efficiency wasmonitored by HPLC.
The uptake of nanoparticles by cells was dependent onthe concentration of the nanoparticles in the medium. Theuptake increased with increase in the concentration, show-ing almost a first-order kinetics [Fig. 6(A)]. It can be clearlyobserved that the efficiency of nanoparticles uptake by cells,assayed on 1 h of incubation, was higher at lower nanopar-ticles concentration, whereas it decreased at greater concen-tration, which indicates the saturated and limited capabilityof cellular uptake of the nanoparticles [Fig. 6(B)]. The nano-particle uptake was also dependent on the incubation time.The uptake was seen as early as at 5 min, which increasedgradually with the incubation time [Fig. 6(C)].
In vitro gene expression assayComplexes prepared at different nanoparticles/pDNA weightratios ranging from 1 to 10 were used. All the cell lineswere transfected in vitro by 1 lg of pDNA complexed withpolymer. Gene transfer efficiency was measured and eval-uated for their transfection efficiency in cells by using fluo-rescence microscope and FACS in the presence or absenceof serum. PEI/pEGFP-N1 complexes were used as control.Considering the results of mean particle size of complexes,it is suggested that larger particle size and insufficient sur-face potential of complexes at low nanoparticles/DNAweight ratios resulted to lower GFP expression, whereas athigh weight ratios, very strong interactions between nano-particles and DNA might prevent the release of the DNA in
the cytoplasm.27 Flow cytometry was used to quantify thetransfection efficiency of nanoparticles/DNA complex in theabsence [Fig. 7(A,C,E)] or presence [Fig. 7(B,D,F)] of serum.PCL–PEI nanoparticles showed transfection efficiency in adose-dependent manner, whereas PEI reached optimum atPEI/DNA weight ratio 6.7 and further decreased till PEI/DNA weight ratio 10. This is undoubtedly because of thetoxicity of PEI at high concentrations in which PCL–PEIremained much efficient because of their significant low
FIGURE 6. Cellular uptake of PCL–PEI nanoparticles. (A) The effect of
nanoparticle concentration on uptake of nanoparticles by Caco-2,
HeLa, and HepG2. (B) The efficiency of uptake of nanoparticles by
Caco-2, HeLa, and HepG2 cells with different concentration of nano-
particles in the medium. Values represent mean 6 SEM (n ¼ 3). (C)
The effect of incubation time on the uptake of nanoparticles by Caco-
2, HeLa, and HepG2 cells. Data are the mean 6 SD of four separate
experiments.
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JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | AUG 2010 VOL 94A, ISSUE 2 627
toxicity. These data strongly suggest that the transfection ef-ficiency of PCL–PEI nanoparticles is superior to that of PEI.The aforementioned results of transfection efficiency as ameasure of GFP expression clearly indicated the excellenceof PCL–PEI over PEI, thereby their potential as a genecarrier.
Confirmation of gene-delivery capability of the polymer/DNA complex was also obtained by confocal microscopy. Asshown in Figure 8, strong fluorescence signal could beobserved when transfection was mediated by PCL–PEI at ananoparticles/DNA weight ratio of 6.7. In contrast, GFP
expression could not be detected when transfection wasmediated by naked pDNA, which was used as negativecontrol.
CONCLUSIONS
A novel crosslinked copolymer based on HMW PCL func-tionalized by GMA molecules (PCL-g-GMA) and PEI hasbeen synthesized, and its physicochemical properties havebeen assessed. The reaction not only yielded high degree ofPEI grafting but also favored the formation of insoluble gels.
FIGURE 7. In vitro gene transfection efficiency of PCL–PEI/DNA at various nanoparticles/DNA weight ratios in HeLa (A, B), HepG2 (C, D), and
Caco-2 (E, F) cells in the presence and absence of serum. Data are the mean 6 SD of four separate experiments.
628 GOMEZ d’AYALA ET AL. CATIONIC COPOLYMERS NANOPARTICLES FOR NONVIRAL GENE VECTORS
In fact, because of the high degree of functionality, a singlePEI can react with a large number of GMA-grafted mole-cules, and thus brings together many PCL backbones toform a three-dimensional network. The novelty of the newnanoparticles mainly resides in their crosslinked chemicalstructure. Crosslinking of nanoparticles represents a tool toenhance their storage and dimensional stabilities and alsotheir manipulation in the preparation of devices for con-trolled delivery. Indeed, it is possible to obtain regularnanoparticles from the reaction mixture by simple emulsiontechnique. In our knowledge, there are just few scientificarticles describing solid preformed PEI-based nanoparticlesand their biological applications. In addition, in the most
part of these studies, PEI was complexed through coopera-tive electrostatic interactions with other anionic polymers,28
or simply blended with other polymers before nanoparticlepreparation by double-emulsion solvent evaporation,29 orconverted into nanoparticles by introducing ionic and cova-lent crosslinkers without any addition of other polymers.30
Moreover, the properties of our nanoparticles can be easilytuned. For example, their size can be controlled by modulat-ing experimental conditions of preparation, while the sur-face charge can be adjusted by varying the PEI amount.Finally, in vitro cytotoxicity studies showed that the PCL–PEI/DNA complex exhibited less cytotoxicity than HMW PEI,and in all cell lines, the transfection efficiencies mediated by
FIGURE 8. Confocal laser scanning microscopy of GFP expression inCaco-2 (A, B), HeLa (C, D) and HepG2 (E, F) cell lines transfected with PCL-PEI/
DNA complex (original magnification �20). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
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JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | AUG 2010 VOL 94A, ISSUE 2 629
PCL–PEI increased with the increase of nanoparticles/DNAweight ratio in the presence or absence of FBS.
ACKNOWLEDGMENTS
The authors thank Dr. Maria Emanuela Errico (ICTP, CNR) forsolid-state NMR analysis.
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