Materials Science and Engineering C 31 (2011) 224–229

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Materials Science and Engineering C

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Formulation and characterization of naked DNA and complexed DNA loadedpolymer films

Debasish Mondal ⁎, Subbu S. VenkatramanSchool of Materials Science and Engineering, Nanyang Technological University, Blk N4.1-02-06, 50, Nanyang Avenue, Singapore 639798, Singapore

⁎ Corresponding author. Biomaterials Laboratory, ScEngineering, Nanyang Technological University, Block N4.Singapore 639798, Singapore. Tel.: +65 67904044; fax: +

E-mail address: dmondal@ntu.edu.sg (D. Mondal).

0928-4931/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.msec.2010.08.021

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 April 2010Received in revised form 2 July 2010Accepted 20 August 2010Available online 21 September 2010

Keywords:Sustained releaseLipoplexPCL filmBurst effectTransfectionIn vitro

Sustained release of DNA from polymeric films is of considerable interest for enhanced and prolonged genetherapy. This report describes the detailed studies of the formulation of naked plasmid DNA (pDNA) andcomplexed DNA-incorporated polymer films and in vitro release of the DNA. The effect of hydrophilicpolymers (PEG, HA) also studied to modulate the release of pDNA and complexed DNA (lipoplex) fromslow biodegradable polymer (PCL) films. The polymer system consists of a biodegradable semi-crystallinepolymer (PCL) blended with a hydrophilic polymer such as poly (ethylene glycol) (PEG) or hyaluronic acid(HA). For the release of pDNA (naked DNA), a burst effect was always seen, and the addition of HA and PEGdid not suppress the burst release of pDNA from PCL films. For complexed pDNA (lipoplex), the release wasslow, but it could be accelerated using additives such as PEG or HA. The transfection efficiency of thecomplexed DNA and the naked pDNA was determined in vitro using COS 7 cells to evaluate the bioactivity ofthe released DNA. Transfection was observed from released lipoplexes samples from PCL/HA film. Overall,this work suggested that these polymeric DNA delivery systems are promising for the local sustained releaseof DNA from implanted films.

hool of Materials Science and1, #B1-01, 50 Nanyang Avenue,65 67909081.

ll rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Currently, most of the DNA delivery work has been devoted ondeveloping new nonviral carrier system that can match the efficacyrates exhibited by viral vectors. Another major prospect of DNAdelivery research is that the localization of DNA and sustaining itsrelease. This characteristic is also vital as it increases the availability ofpDNA at the site of action and sustaining its release over time, but ithas not received as much as attention. Sustained release polymericgene delivery systems have been shown to provide DNA withprotection before it is released, thus increasing gene transfer relativeto bolus delivery and prolonging gene expression. Additionally, sitespecific delivery can be achieved by simple implantation or directinjection [1,2].

Polymers provide an attractive alternative for the long-termdeliveryof a variety of drug molecules such as proteins, peptides, oligonucleo-tides, and DNA. Although, in recent years a wide range of biocompatibleor biodegradable polymers including microspheres, nanospheres havebeen explored for the sustained release of DNA, existing studies mainlyfocus on microspheres, nanospheres formulation and not implantablefilmsor coatings. The successof therapeutic genedelivery systems is still

limited due to some practical limitations such as DNA damage, lowencapsulation efficiency, bolus delivery, and low expression.

Therefore, itwouldbehighlybeneficial to thepatient if thedurationofaction of the injectable or implantable sources of genes could beextended. This extensionmaybeachievedby sustaining the releaseof thegene vector over several days or months because prolonged andcontinuous DNA delivery to tissues will enhance gene expression [3].Earlier, we reported the sustained delivery of complexed and nakedpDNA from a PCL/gelatin matrix [4,5]. The work was mainly focused onpDNAandcomplexedDNArelease andon thecomparisonof lipoplexandpolyplex release from PCL/gelatinmatrix. The objective of this work is tocompare pDNA and complexed DNA release from blends of otherhydrophilic polymers (PEG, HA) in PCL. This report investigates the effectof hydrophilic polymers (PEG, HA) to modulate the release profile ofpDNA and complexed DNA (lipoplex) from PCL films. Poly ( -caprolac-tone) (PCL) is a semi-crystalline biocompatible and biodegradablepolyester with a low melting point (~57 °C) and a low glass transitiontemperature (~−60 °C). It degrades slowly relative to the PLA/PLGpolymers. PCL is rubbery at room temperature, and this characteristiccontributes to the very high permeability of PCL for many therapeuticdrugs. PCL exhibits certain desirable characteristics for drug-deliveryapplications, including biodegradability, biocompatibility, and commer-cial availability [6–8].

Hyaluronic acid (HA) is a naturally occurring polysaccharide foundin connective tissues like the umbilical cord, synovial fluid, andvitreous. Unmodified and derivatized HAs have been extensivelyutilized in the fields of drug delivery, cell encapsulation, and tissue

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regeneration because of their unique viscoelastic properties and goodbiocompatibility [9–17].

Poly (ethylene glycol) (PEG) is a non-toxic and hydrophilicpolymer. PEG has been extensively used as a drug carrier or drugrelease modifier because of its capability to improve the wettabilityand solubility of water insoluble drugs [18–23].

Recently, various polymer matrices have been explored for thedelivery of plasmid DNA. However, DNA damage due to degradationproducts of PLGA has been observed in PLGA delivery devices. Moreover,the DNA encapsulation efficiency is very low. The degradation productsare thought to damage the encapsulated DNA [24]. Howard et al.observed a sustained release profile of PEI/DNA complex from PLGAmicroparticles, but a significant initial burst effect for the PEI/DNAcomplexwas found [25]. Although the encapsulation efficiency is high ina hydrogel, the release period is relatively fast, whichmakes it difficult touse as a sustained release system for long periods [26,27]. Sacks et al.observed a substantial 50% burst effect of lipofectamine-pDNA complexon day 1 from collagen films [28]. Huang et al. reported a sustainedrelease profile of pDNA from PCL-PEG-PCL (PCEC) nanoparticles, but asignificant initial burst releaseof pDNAwas foundwithin2 h [29]. Zhaoetal. observed a sustained release profile of pDNA from PCL-Pluronic-PCL(PCFC) nanoparticles for 24 h time period. The significant burst releasewas found within 1 h and about 53% of total pDNA was released in 24 htime [30]. Rives et al. formed layered porous poly (lactide -co-glycolide)(PLG) scaffolds for the in vivo plasmidDNAdelivery [31]. They found thatthe incorporation efficiency of plasmid DNA significantly increasedrelative to the scaffold form without layers and that the transgeneexpression over a period of 1–2 weeks had a peaked expression level forthefirst fewdays and thendeclined [31]. But, therewas also a large initialburst effect observed within the first 3 days [31]. Except for our earlierwork, PCL films as a matrix for sustained release system of pDNA andcomplexed DNA have not been studied. Therefore, in this preliminarystudy, we formulated pDNA and complexed (lipoplex) DNA-loaded PCLfilms. The effect of hydrophilic polymers (PEG, HA) to modulate therelease profile pDNA and complexed DNA (lipoplex) from PCL films andin vitro transfection was investigated.

2. Materials and methods

2.1. Materials

The dual vector pEGFPLuc, encoding a fusion protein of enhancedgreenfluorescent protein (EGFP) and luciferase from thefirefly photinuspyralis, was purchased from Clontech (supercoiled, 6.4 kb). It waspropagated according to the standardmethod using a gigafilter kit fromQiagen and was conditioned in an autoclaved TE buffer. Lipofectamine(LPF) was provided by Invitro Life Technologies. Sodium oleate salt wasprovided by Sigma Chemicals; it was dissolved in water at aconcentration of 1 mg/ml. Ethidium bromide was purchased fromSigma Chemicals; it was diluted inwater at a concentration of 80 μg/ml.Poly caprolactone (Mn 80,000, PDI 1.4) was purchased from SigmaChemicals. Hyaluronic acid (HA) sodium salt was purchased fromAldrich. Poly Ethylene Glycol (PEG) 4000 (Av. Mw=3000) waspurchased from TCI Chemicals, Tokyo. Chloroform (GR grade) waspurchased from Tedia. The films were cast with the Automatic Filmapplicator AG-2150, which was purchased from BKY Gardner. Dulbec-co's Minimum Essential Medium (DMEM) and Penicillin/Streptomycinwere purchased from Invitrogen. Luciferase Reporter Assay Kit wasprovided by BD Clontech. The Micro BCA TM Protein Assay Reagent kitwas provided by Pierce Biotechnology, Rockford, IL, USA.

2.2. Preparation of lipofectamine- pDNA complexes

pDNAwas diluted to a working range of 28 μg in 100 μl (280 μg/ml).Lipofectamine was diluted using serum-free medium (SFM: DMEMwithout the addition of FBS – fetal bovine serum). Lipofectamine was

added to the pDNA such that the lipofectamine was 8 times the weightof pDNA. The complexes were vortexed vigorously, and then they wereincubatedat roomtemperature for 30 min.Noagglomerationwasnotedin the sample preparation.

2.3. Preparation of PEG solution

PEG solution (5%w/v) was prepared in water and sterilized byfiltration using a 0.2-μm filter.

2.4. Preparation of HA solution

HA solution (5 mg/ml) was prepared in water and sterilized usinga 0.2-μm filter.

2.5. Incorporation of complex into the films

2.5.1. PCL filmA 6.6% (w/v) PCL solution was made by dissolving the polymer in

chloroform. Then, 300 μl of PCL solutionwasmixedwith 100 μl of SFMcontaining LPF/DNA or pDNA; the mixture was vortexed at 2200 rpmand was cast as a film.

2.5.2. PCL/HA filmHA-containing films were prepared by adding HA solution

(2.5% w/w of PCL) to SFM containing LPF/DNA or pDNA; 1% (w/w)PVA was added as an emulsion stabilizer.

2.5.3. PCL/PEG filmPEG-containing films were prepared by adding PEG solution

(5% w/w of PCL) to SFM containing LPF/DNA or pDNA; 1% (w/w)PVA was added as an emulsion stabilizer.

All films were dried in the laminar hood for 3 days to enable theevaporation of the solvent. The specific concentrations selected hereare based solely on the quality of the emulsion obtained. The choice ofchloroform is mandated by pDNA activity retention in chloroformcompared to solvents such as dichloromethane or acetone.

2.6. Study of film morphology

Field emission scanning electron microscopy (FESEM, JEOLJSM- 6340) and scanning electron microscopy (SEM, JEOL-5310) wereused to analyze the morphology of the polymeric films at acceleratingvoltage 5.0 kV. The dried film samples were cut in to small pieces andmounted on ametal holder by using a double sided copper tape. Sampleswere coated with platinum (Fine auto Coater JEOL JSM-6340) for FESEMand with gold for SEM before observation.

2.7. Quantification of the released pDNA/complexed DNA (lipoplex)

The naked pDNA concentration was determined using EthidiumBromide (EtBr). The measurement was carried out on a Bio-Tek, FLX800 multi-detection micro plate reader with filters set at Ex=485 nmand Em=590 nm. A standard curve was prepared with differentknown concentrations of pEGFPLuc (pDNA) in the presence andabsence of sodium oleate. DNA concentration range for the standardcurvewas 0.1–1 μg/ml. The value of the slope (m) is 217.6 (y=217.6x,R2=0.99) in absence of sodium oleate and 135 (y=135x, R2=0.985)in presence of sodium oleate.

DNA concentrations of unknown samples were determined bycomparison to the standard curve. The naked pDNA loaded filmsamples were collected and quantified directly. In the case of filmsloaded with complexed pDNA, the pDNA was disassociated from theLPF before quantification. Sodium oleate was used for the dissociationof the lipoplexes. The amount of sodium oleate added was about 10times higher than the LPF. The samples were incubated at 37 °C for

Fig. 1. SEM images of PCL film surface on day 0 (A), PCL film surface on day 9 (B), crosssectional view of PCL film on day 9 (C).

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4 h. To the decomplexed samples, about 50 μl of ethidium bromide(100 μg/ml) was added and mixed well, and the fluorescenceintensity was measured. Appropriate blank solutions of PCL withsodium oleate at different intervals were used to get the final value.Another determination of free pDNA (without the decomplexingstep) was also carried out on the released lipoplex samples to quantifythe amount of uncomplexed and complexed pDNA at each time point.

2.8. Polymer degradation study

Size Exclusion Chromatography (SEC) from Agilent Technologies1100 Series was used for the degradation study of all the polymerfilms before and after immersion in water. A PLgel 5 μm-mixed Ccolumn was used with the mobile phase chloroform. The flow ratewas set at 1 ml/min, and about 100 μl of sample was injected into thecolumn. A separate set of films were prepared and collected atdifferent intervals for the SEC studies. Wet films were dried beforedissolving in the mobile phase.

2.9. In vitro release study

The films were placed in a 12-well cell culture plates. About 0.8 mlautoclaved water was used for the in vitro studies. The plates wereincubated at 37 °C, and the released medium was assayed atstipulated time intervals by a microplate fluorescence reader. Atevery time point, the buffer was removed and replaced with freshbuffer. About 1% antibiotic (Ampicillin) was used to avoidcontamination.

2.10. In vitro bioactivity

Samples of pDNA/lipoplexes released from the polymer films werecollected at predetermined intervals, air dried in the laminar hood fora few hours, and resuspended in SFM (0.5 ml). COS-7 cells wereseeded in a 24-well tissue culture grade plates at 2×104 cells per welland were grown overnight to approximately 80% confluence undernormal growth conditions. On the following day, the cell culturemedium was removed, and SFM containing the released pDNA/lipoplexes was added to the cells. The cells were incubated withpDNA/complexes at 37 °C in a 5% CO2 incubator for 5 h. The mediumwas replaced with 0.5 ml of 2× medium and left overnight; 0.5 ml of1× medium was added on the following day. Luciferase activity wasdetermined using a Luciferase assay kit (BD Sciences) after 48 h oftransfection. The total protein was assayed using a Micro BCA proteinassay (Pierce, Rockford, IL, USA). The activity was normalized to thetotal protein content.

2.11. Particle size measurement

Particle size measurement of the LPF/pDNA complexes wasperformed by photon correlation spectroscopy (PCS) using ZetaSizer NS (Malvern Instruments Ltd., Malvern, UK). Samples werediluted in PBS andmeasured in glass cuvettes at 25 °C and a scatteringangle of 90°.

All the experiments were performed in triplicates and all the datawere presented as mean±standard deviations.

3. Results and discussion

3.1. Film appearance and SEM analysis for films morphology

The thickness of all dried films was around 80 μm, as measuredusing a micrometer. The morphology of the films was studied tounderstand the mode of release of pDNA and complexed DNA(lipoplex) from polymer films. Fig. 1 shows the surface morphologyof PCL film at different time interval. The figure shows the few larger

gaps and few numbers of pores on the polymer surface and their non-uniform distribution. Figs. 2 and 3 show the morphology of PCL filmwith PEG and PCL film with HA respectively. Figs. 2B and 3B show theappearance of pores on the film surface and Figs. 2C and 3C show thepores in the cross sectional area of the films. PCL/PEG and PCL/HAmatrix show the large number of pores dispersed in the whole matrixcompare to PCL alone. The SEM figure clearly shows that PEG (Fig. 2)creates larger number of pores compared to HA (Fig. 3).

3.2. Degradation studies of PCL/PEG and PCL/HA films by size exclusionchromatography

Degradation studies of different polymer films were evaluated bysize exclusion chromatography. Fig. 4 shows the molecular weightchanges with specific time intervals. Molecular weight changes ofthree different polymeric films do not show major differences fromday 0 to day 30. This leads to the conclusion that the release is mostly

Fig. 2. SEM images of PCL/PEG film surface on day 0 (A), PCL/PEG film surface on day 9(B), cross sectional view of PCL/PEG film on day 9 (C).

Fig. 3. SEM images of PCL/HA film surface on day 0 (A), PCL/HA film surface on day 9(B), cross sectional view of PCL/HA film on day 9 (C).

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due to diffusion rather than due to bulk degradation, with solubility orpartitioning also playing a key role.

3.3. In vitro release studies of naked pDNA and complexed DNA fromPCL/PEG and PCL/HA films

These studies focus on polymer blends, with incorporation ofpDNA and complexed DNA. The main objective is to understand, andthereby modulate, the release of these genes from blended systems.The basic biodegradable polymer, PCL, is hydrophobic, and is notexpected to act as a good reservoir for DNA. Addition of hydrophilicpolymers (PEG, HA) was expected to partition the DNA into a morefriendly environment.

Fig. 5 shows the release profile of pDNA from PCL, PCL/HA (2.5% ofhigh molecular weight HA) and PCL/PEG (5% of a low molecularweight PEG) films. The release profile shows a biphasic behavior, aburst phase followed by diffusion-controlled phase. The release isdominated by the burst release (50% by day 5) of undissolved pDNAfrom PCL film. The films prepared by blending PCL with HA and PEG

release around 50% and 28% of pDNA, respectively, at the end of30 days.

The addition of HA and PEG does not suppress the burst release ofpDNA from PCL films. Generally, the addition of a hydrophilic polymersuppresses the burst effect, due to the greater solubility of the pDNAin the hydrophilic component. Earlier we found that the addition ofhydrophilic polymer (gelatin) slows down the release of pDNA fromPCL film, as the predominantly hydrophilic pDNA partitions prefer-entially into the gelatin phase [5]. Thus the lack of an effect of theaddition of HA and PEG is unexpected. We believe that this is likelydue to the large size of the supercoiled pDNA that prevents it frompartitioning readily into any of the hydrophilic polymers (HA, PEG),and quite likely exists as a separate third phase. For HA and for PEG,the overall release is somewhat slower than for PCL indicatingperhaps that a small amount of partitioning of pDNA into the PEG orthe HA may have occurred (possibly due to the smaller amounts ofthese two polymers or due to pDNA-polymer immiscibility). Again,there is slower release of pDNA following the burst release. Poreformation does not appear to account for the release behavior seen in

Fig. 6. Release percentage (%) of lipoplexes from polymer films. Data represent themean±SD, n=3.Fig. 4. Degradation profile of PCL/PEG and PCL/HA Films. Data represent the mean±SD,

n=3.

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case of pDNA, so there is likely to be some partitioning or associationof the pDNA into either PEG or HA.

Fig. 6 shows the effect of HA and PEG on the release profile oflipoplexes from PCL films. In contrast to the pDNA loaded films, noburst effect is observed with the lipoplex loaded samples. From thefigure it is clear that the effect of HA and PEG is to increase the releaseof lipoplexes from PCL films and the higher accumulated releasewas observed for PCL film with PEG. PCL/HA and PCL/PEG films bothshow around 15% of release at day 5 as compared with 3% from thePCL film.

The lower release of lipoplex compared to pDNA is generally due tohigher solubility of lipoplex in the hydrophobic polymer (PCL) matrix.However, all these hydrophilic additives (HA, PEG) do modulate therelease of lipoplexes, which are condensed forms of the pDNA, wherethe pDNA is associated with the positively charged sections of thelipofectamine and therefore more hydrophobic compared to the“naked” pDNA. Thus the lipoplex dissolves more readily in PCL andshows no burst effect. Upon addition of the hydrophilic polymer (HA,PEG), lipoplex release is increased. The reason for the increased releaseis due to the addition of hydrophilic polymer reduces the PCL phasethat is available for dissolving the lipoplex; hence some of the lipoplexbecomes dispersed, rather than dissolved. Subsequent release isfacilitated by diffusion through the pores created by the leaching outof the additive (PEG, HA).

Earlier we found that the addition of hydrophilic polymer (gelatin)is to accelerate the release of lipoplexes from the PCL matrix. Inanother study, Lu et al. observed that the diffusion of protein iscontrolled by the presence of pores in a PCL/PEG matrix and protein(lysozyme) diffused very slowly through the films [32]. The diffusion

Fig. 5. Release percentage (%) of pDNA frompolymerfilms. Data represent themean±SD,n=3.

of protein was increased by water-filled micropores formed by theleaching of PEG. The permeation rate of proteins increased with anincrease in the pore fraction [32]. Similar results are observed here.The SEM pictures of the PCL film with PEG (Fig. 2B) and PCL film withHA (Fig. 3B) below show the presence of pores on the surface of thepolymer films as early as day 9. The higher earlier release obtainedwith PEG addition could be due to the higher diffusion rate throughthe large number of pores created on the PCL/PEG film surface(Fig. 2B).

3.4. In vitro bioactivity of released DNA

The bioactivity of the released DNA was determined by in vitro celltransfection efficiency using COS-7 cell lines. The transfection efficiencyof the DNA (pDNA/complex DNA) released from the polymer films wasobserved using inverted phase contrast microscopy. The transfectionwas quantified using enzyme (luciferase) activity normalized using thetotal protein content of cell extracts. The transfection efficiency ofreleased DNA from polymer films was studied at all the time points.

Fig. 7 shows the transfection efficiency of released lipoplexes onday 5 from the PCL/HA film; however, the transfection is notmeasurable from day 9 onwards. In particular, lipoplexes releasedfrom PCL films and PCL/PEG films do not show any transfection, evenon day 5.

In order to determine the reasons for the inability to transfect thecell, the extents of de-complexation of the released lipoplexes(Table 1) as well as the particle size of the released lipoplexes weredetermined. The lipoplexes released from PCL/HA and PCL/PEG films

Fig. 7. Transfection of released lipoplex from PCL/HA film. Data represent the mean±SD,n=3.

Table 1Particle size and percentage (%) of complexed form of released lipoplexes at each timepoint (data represent the mean±SD, n=3).

Samples Particle size (nm) Complexed form (%)

Day Day

5 9 18 5 9 18

PCL/lipoplex 442±11 531±9 590±13 100 100 100PCL/HA/lipoplex 846±8 877±12 1182±15 100 79 –

PCL/PEG/lipoplex 831±10 865±7 1176±9 100 75 –

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are decomplexed to some extent, as shown in Table 1. However, thedegree of complexation is still relatively high on day 9. On the otherhand, released lipoplexes from the PCL matrix are in fully complexedform (Table 1) up to day 18. Thus, the decomplexation cannot by itselfexplain the reason for no transfection. Particle size (Table 1) datashow that the particle size of the lipoplexes released from the PCLmatrix are smaller than the lipoplexes released from PCL/HA andPCL/PEG matrices. However, lipoplexes released from PCL/HA matrixshow transfection on day 5 in spite of their larger size and lipoplexesreleased from PCL are unable to transfect the cells.

From the particle size data (Table 1), it is clear that one of thereasons for the lack of transfection efficiency of lipoplexes releasedfrom PCL and PCL/PEG matrices is the aggregation of the releasedcomplexes, which reduces nuclear penetration. Secondly, largerparticles could be degraded inside the lysosomal compartment.

On the other hand, lipoplexes released from PCL/HA films showtransfection on day 5. When lipoplexes are released from PCL-HAfilms, they might be associated with HA. Such released lipoplexesappear to be in the cytoplasm in larger numbers, with some of themreaching into the nucleus, in spite of their large size. Thus, HAassociation of the lipoplex appears to protect them from endosomal/lysosomal degradation. However, the transfection is not measurablefrom day 9 onwards. This result could be due to the loss of thelipoplex-HA association. At this point, we are not sure of themechanism for this protection, and further studies are planned toaddress this.

4. Conclusions

Our group has reported earlier on both naked (pDNA) andcomplexed DNA release and bioactivity studies of the DNA releasedfrom a PCL/gelatin matrix. In this study, other hydrophilic polymers(HA and PEG) were used with PCL to evaluate the sustained release ofpDNA and complexed DNA. For pDNA, the release is dominated by theburst release of undissolved pDNA. The addition of HA and PEG doesnot suppress the burst release of pDNA from PCL films. In contrast tothe pDNA-loaded films, no burst effect is observed with the lipoplex-loaded samples. The addition of HA and PEG enhances the release ofthe lipoplexes. Lipoplexes released from PCL/HA filmswere associatedwith HA and were able to transfect cells on day 5. Lipoplexes released

from PCL and PCL/PEG matrices were unable to transfect cells, whichcould be due to the combination of insufficient nuclear penetrationand enhanced lysosomal degradation.

In summary, these preliminary results show the potentiality of thissystem as interesting vehicle for long term release of DNA. Our futurestudies are concentrating on the release of complexed DNA for anextended period, evaluating the long term bioactivity of released DNAand understanding the factors that affect the bioactivity of DNA.

References

[1] V. Labhasetwar, J. Bonadio, A.S. Goldstein, W. Chen, R.J. Levy, J. Pharm. Sci. 87(1998) 1347.

[2] Y.S. Jong, J. Control. Rel. 47 (1997) 123.[3] D. Luo, K. Woodrow-Mumford, N. Belcheva, W.M. Saltzman, Pharm. Res. 16

(1999) 1300.[4] Y. Ramgopal, D. Mondal, S.S. Venkatraman, W.T. Godbey, J. Biomed, Mater. Res.

Part B Appl. Biomater. 85B (2008) 496.[5] Y. Ramgopal, D. Mondal, S.S. Venkatraman, W.T. Godbey, G.Y. Yuen, J. Biomed,

Mater. Res. Part B Appl. Biomater. 89B (2009) 439.[6] J. Kohn, R. Langer, in: B. Ratner, A. Hoffman, F. Schoen, J. Lemons (Eds.),

Biomaterials Science, Academic Press, San Diego, 1996, pp. 64–72.[7] J.M. Pachence, J. Kohn, in: R. Lanza, R.J.V. Langer (Eds.), Principles of Tissue

Engineering, Academic Press, San Diego, 1997, pp. 273–294.[8] J.C. Middleton, A.J. Tipton, Biomaterials 21 (2000) 2335.[9] M.F. Saettone, D. Monti, M.T. Torracca, P. Chetoni, J. Ocular Pharm. 10 (1994) 83.

[10] A.R. Moore, D.A. Willoughby, Int. J. Tissue React. 17 (1995) 153.[11] K. Morimoto, H. Yamaguchi, Y. Iwakura, K. Morisaka, Y. Ohashi, Y. Nakai, Pharm.

Res. 8 (1991) 471.[12] G. Gowland, A.R. Moore, D.Willis, D.A.Willoughby, Clin. Drug Invest. 11 (1996) 245.[13] J.A. Miller, R.L. Ferguson, D.L. Powers, J.W. Burns, S.W. Shalaby, J. Biomed. Mater.

Res. 38 (1997) 25.[14] D. Campoccia, P. Doherty, M. Radice, P. Brun, G. Abatangelo, D.F. Williams,

Biomaterials 19 (1998) 2101.[15] G.D. Prestwich, D.M. Marecak, J.F. Marecak, K.P. Vercruysse, M.R. Ziebell, J. Control.

Rel. 53 (1998) 93.[16] J.W. Kuo, D.A. Swann, G.D. Prestwich, Bioconjug. Chem. 2 (1991) 232.[17] K. Tomihata, Y. Ikada, J. Biomed. Mater. Res. 37 (1997) 243.[18] D. Law, E.A. Schmitt, K.C. Marsh, E.A. Everitt, W. Wang, J.J. Fort, S.L. Krill, Y. Qiu, J.

Pharm. Sci. 93 (2004) 563.[19] M. Moneghini, I. Kikic, D. Voinovich, B. Perissutti, J. Filipovic-Grcic, Int. J. Pharm.

222 (2001) 129.[20] N. Passerini, B. Albertini, B. Perissutti, L. Rodriguez, Int. J. Pharm. 318 (2006) 92.[21] S.Herrmann,S.Mohl, F. Siepmann, J. Siepmann,G.Winter, Pharm.Res. 24 (2007)1527.[22] S. Herrmann, G. Winter, S. Mohl, F. Siepmann, J. Siepmann, J. Control. Rel. 118

(2007) 161.[23] E. Verhoeven, F. Siepmann, T.R.M. De Beer, D. Van Loo, G. Van Den Mooter, J.P.

Remon, J. Siepmann, C. Vervaet, Eur. J. Pharm. Biopharm. 73 (2009) 292.[24] S. Ando, D. Putnam, D.W. Pack, R. Langer, J. Pharm. Sci. 88 (1999) 126.[25] K.A. Howard, X.W. Li, S. Somavarapu, J. Singh, N. Green, K.N. Atuaha, Y. Ozsoyc, L.W.

Seymourb, H.O. Alpar, Biochim. Biophys. Acta 1674 (2004) 149.[26] L.D. Shea, E. Smiley, J. Bonadio, D.J. Mooney, Nat. Biotechnol. 17 (1999) 551.[27] J.M. Dang, K.W. Leong, Adv. Drug Deliv. Rev. 58 (2006) 487.[28] C.H. Sacks, V. Elazar, J. Gao, A. Golomb, H. Adwan, N. Korchov, R.J. Levy, M.R.

Berger, G. Golomb, J. Control. Rel. 95 (2004) 309.[29] M.J. Huang, M.L. Gou, Z.Y. Qian, M. Dai, X.Y. Li, M. Cao, K. Wang, J. Zhao, J.L. Yang, Y.

Lu, M.J. Tu, Y.Q. Wei, J. Biomed. Mater. Res. A 86A (2008) 979.[30] J. Zhao, M. Gou, M. Dai, X.Y. Li, M. Cao, M. Huang, Y.J. Wen, B. Kan, Z.Y. Qian, Y.Q.

Wei, J. Biomed. Mater. Res. A 90A (2009) 506.[31] C.B. Rives, A. Rieux, M. Zelivyanskaya, S.R. Stock, W.L.L. Jr, L.D. Shea, Biomaterials

30 (2009) 394.[32] C.H. Lu, W.J. Lin, J. Biomed, Mater. Res. Part B Appl. Biomater. 63B (2002) 220.