gene transfer by dna–gelatin nanospheres

10
Gene Transfer by DNA–Gelatin Nanospheres Vu L. Truong-Le,* Scott M. Walsh,² Erik Schweibert,‡ Hai-Quan Mao,² William B. Guggino,‡ J. Thomas August,* , § ,1 and Kam W. Leong² *Department of Pharmacology and Molecular Sciences, ²Department of Biomedical Engineering, Department of Physiology, and §Department of Oncology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205 Received April 6, 1998, and in revised form October 5, 1998 A DNA and gelatin nanoparticle coacervate contain- ing chloroquine and calcium, and with the cell ligand transferrin covalently bound to the gelatin, has been developed as a gene delivery vehicle. In this study, the coacervation conditions which resulted in the forma- tion of distinct nanoparticles are defined. Nano- spheres formed within a narrow range of DNA concen- trations and achieved incorporation of more than 98% of the DNA in the reaction. Crosslinking of gelatin to stabilize the particles does not effect the electro- phoretic mobility of the DNA. DNA in the nanosphere is partially resistant to digestion with concentrations of DNase I that result in extensive degradation of free DNA but is completely degraded by high concentra- tions of DNase. Optimum cell transfection by nano- sphere DNA required the presence of calcium and nanospheres containing transferrin. The biological in- tegrity of the nanosphere DNA was demonstrated with a model system utilizing DNA encoding the cystic fi- brosis transport regulator (CFTR). Transfection of cul- tured human tracheal epithelial cells (9HTEo) with nanospheres containing this plasmid resulted in CFTR expression in over 50% of the cells. Moreover, human bronchial epithelial cells (IB-3-1) defective in CFTR-mediated chloride transport were comple- mented with effective transport activity when trans- fected with nanospheres containing the CFTR trans- gene. © 1999 Academic Press Synthetic gene delivery vehicles that have the re- quired efficiency and safety for use in human gene therapy are being widely investigated as possible al- ternatives to biological vectors of viral and bacterial origin (1, 2). While the current synthetic systems are less efficient than viral vectors, rapid advances in cat- ionic lipid designs (3, 4), amphiphilic DNA-binding peptides (5, 6), and polymers (7, 8) suggest that this gap may be closing. Moreover, synthetic DNA delivery systems that incorporate biological properties such as ligands for receptor-mediated endocytosis (9), agents that promote endosomal disruption (10), and cytoplas- mic self-replication (11, 12) have achieved efficient lev- els of gene transfer. We previously introduced a novel gene delivery ve- hicle made of crosslinked DNA– gelatin nanosphere coacervates (13, 14). Nanosphere formation is driven by a combination of electrostatic and entropic forces with sodium sulfate employed as a desolvating reagent to facilitate greater charge– charge interactions be- tween plasmid DNA and gelatin (15, 16). Sodium sul- fate effects phase separation by influencing the degree of hydration of the two ionic species and thus increas- ing the degree of inter- and intracoulombic (attractive) forces between the ion pairs (17, 18). In addition, it has been suggested that sodium sulfate exerts a charge- shielding effect on oppositely charged sites of gelatin, which reduces the repulsive forces among the gelatin molecules and their intramolecular segments (15–17). Sodium sulfate was also crucial in forming chitosan– DNA nanoparticles (19). Because DNA is employed as the limiting reagent in the coacervation reaction, high DNA encapsulation efficiency (.98%) and loading level (25–30%, w/w) were obtained. The condition for nano- sphere formation is mild, requiring neither the contact with high temperatures nor organic solvents, and per- mits the coencapsulation of labile agents. Cellular up- take of nanospheres was greatly enhanced by conjugat- ing transferrin on the nanosphere surface, a phenom- enon that may be attributed to endocytosis of the nanospheres. In addition, transfection of transferrin- coated nanospheres was increased by the inclusion of chloroquine, presumably by its interference with the acidification of endosomal compartments, and thereby 1 To whom correspondence should be addressed. 0003-9861/99 $30.00 47 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 361, No. 1, January 1, pp. 47–56, 1999 Article ID abbi.1998.0975, available online at http://www.idealibrary.com on

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Page 1: Gene Transfer by DNA–Gelatin Nanospheres

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Archives of Biochemistry and BiophysicsVol. 361, No. 1, January 1, pp. 47–56, 1999Article ID abbi.1998.0975, available online at http://www.idealibrary.com on

ene Transfer by DNA–Gelatin Nanospheres

u L. Truong-Le,* Scott M. Walsh,† Erik Schweibert,‡ Hai-Quan Mao,† William B. Guggino,‡. Thomas August,*,§,1 and Kam W. Leong†Department of Pharmacology and Molecular Sciences, †Department of Biomedical Engineering, ‡Department ofhysiology, and §Department of Oncology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205

eceived April 6, 1998, and in revised form October 5, 1998

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A DNA and gelatin nanoparticle coacervate contain-ng chloroquine and calcium, and with the cell ligandransferrin covalently bound to the gelatin, has beeneveloped as a gene delivery vehicle. In this study, theoacervation conditions which resulted in the forma-ion of distinct nanoparticles are defined. Nano-pheres formed within a narrow range of DNA concen-rations and achieved incorporation of more than 98%f the DNA in the reaction. Crosslinking of gelatin totabilize the particles does not effect the electro-horetic mobility of the DNA. DNA in the nanosphere

s partially resistant to digestion with concentrationsf DNase I that result in extensive degradation of freeNA but is completely degraded by high concentra-

ions of DNase. Optimum cell transfection by nano-phere DNA required the presence of calcium andanospheres containing transferrin. The biological in-egrity of the nanosphere DNA was demonstrated with

model system utilizing DNA encoding the cystic fi-rosis transport regulator (CFTR). Transfection of cul-ured human tracheal epithelial cells (9HTEo) withanospheres containing this plasmid resulted inFTR expression in over 50% of the cells. Moreover,uman bronchial epithelial cells (IB-3-1) defective inFTR-mediated chloride transport were comple-ented with effective transport activity when trans-

ected with nanospheres containing the CFTR trans-ene. © 1999 Academic Press

Synthetic gene delivery vehicles that have the re-uired efficiency and safety for use in human geneherapy are being widely investigated as possible al-ernatives to biological vectors of viral and bacterialrigin (1, 2). While the current synthetic systems areess efficient than viral vectors, rapid advances in cat-

a1 To whom correspondence should be addressed.

003-9861/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

onic lipid designs (3, 4), amphiphilic DNA-bindingeptides (5, 6), and polymers (7, 8) suggest that thisap may be closing. Moreover, synthetic DNA deliveryystems that incorporate biological properties such asigands for receptor-mediated endocytosis (9), agentshat promote endosomal disruption (10), and cytoplas-ic self-replication (11, 12) have achieved efficient lev-

ls of gene transfer.We previously introduced a novel gene delivery ve-

icle made of crosslinked DNA–gelatin nanosphereoacervates (13, 14). Nanosphere formation is driveny a combination of electrostatic and entropic forcesith sodium sulfate employed as a desolvating reagent

o facilitate greater charge–charge interactions be-ween plasmid DNA and gelatin (15, 16). Sodium sul-ate effects phase separation by influencing the degreef hydration of the two ionic species and thus increas-ng the degree of inter- and intracoulombic (attractive)orces between the ion pairs (17, 18). In addition, it haseen suggested that sodium sulfate exerts a charge-hielding effect on oppositely charged sites of gelatin,hich reduces the repulsive forces among the gelatinolecules and their intramolecular segments (15–17).odium sulfate was also crucial in forming chitosan–NA nanoparticles (19). Because DNA is employed as

he limiting reagent in the coacervation reaction, highNA encapsulation efficiency (.98%) and loading level

25–30%, w/w) were obtained. The condition for nano-phere formation is mild, requiring neither the contactith high temperatures nor organic solvents, and per-its the coencapsulation of labile agents. Cellular up-

ake of nanospheres was greatly enhanced by conjugat-ng transferrin on the nanosphere surface, a phenom-non that may be attributed to endocytosis of theanospheres. In addition, transfection of transferrin-oated nanospheres was increased by the inclusion ofhloroquine, presumably by its interference with the

cidification of endosomal compartments, and thereby

47

Page 2: Gene Transfer by DNA–Gelatin Nanospheres

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48 TRUONG-LE ET AL.

mproving the intracellular half-life of nanosphereNA or by contribution to the stability of the DNA (20).The present report further characterizes the bio-

hemical features of this DNA delivery system. Therincipal questions addressed are the reaction condi-ions necessary for particle formation, the protection ofanosphere DNA against DNase I digestion, the effectf encapsulated calcium on transfection, and the bio-ctivity of the DNA and its encoded protein as indi-ated by use of DNA encoding the cystic fibrosis trans-ort regulator (CFTR).2

XPERIMENTAL PROCEDURES

Plasmid constructs, antibodies, and cell lines. pSA306 is a plas-id containing the truncated CFTR gene with a 26-amino-acid

pitope tag at the carboxy terminus (21). pRELuc contains a lucif-rase reporter gene inserted in the RSV promoter-driven InvitrogenREP7 plasmid vector (14). mLAMP-1pcDNA is a RSV promoter-riven pcDNA I plasmid (Invitrogen) with full-length murineAMP-1 gene (22). HIVgp160/LAMP is a pcDNA1 plasmid contain-

ng the HIV gp160/LAMP-1 insert (23). The AAV-CFTR is recombi-ant AAV virion containing the truncated CFTR cassette (24). Mono-lonal antibodies are 934, chicken anti-human CFTR epitope tagntibody (25); and CHA, a nonspecific IgG with no known in vivoouse epitope (26). 9HTEo is a human tracheal epithelial cell line

25), and IB-3-1 is a human bronchial epithelial cell line bearing theelta508 mutation (21).Synthesis of nanospheres. Porcine type A gelatin (175 bloom,

igma) was boiled in distilled water (5% w/v) for 2 h, adjusted to pH.5 with NaOH, and then sterilized by filtering through a 0.22-mmlter. Gelatin–DNA nanosphere coacervates were synthesized byixing 100 ml of a solution containing 5% (w/v) gelatin and 4 mM

hloroquine with 100 ml of a solution containing 20 mg of plasmidNA and 4.3 mM Na2SO4. Nanospheres without chloroquine were

ynthesized similarly except for a higher Na2SO4 concentration (45M). The reaction mixture was vortexed for 1 min at the highest

peed at 55°C in a 0.5-ml Eppendorf tube. The unreacted componentsere removed by centrifuging the mixture on a 100-ml sucrose stepradient (35, 55, and 80%, w/w) at 40,000g for 7 min. The sucroseraction containing the nanospheres (55% layer) was diluted withater to 200 ml, and 1 ml human transferrin (Sigma) solution (20g/ml in H2O) and 22 ml of a 0.2 M 4-morpholineethanesulfonic acid

uffer solution, pH 4.5, containing 0.02 mg/ml 1-ethyl-3-(3-dimeth-laminopropyl)carbodiimide hydrochloride (EDC) were added. Theeaction was carried out at room temperature for 30 min and thentopped by the addition of sodium acetate, pH 6.0, to 0.1 M finaloncentration. CaCl2-treated nanospheres were generated by incu-ating freshly synthesized nanospheres for 12 h at 4°C with CaCl2

nd NaN3 to a final concentration of 0.5 M CaCl2 and 1% NaN3 andhen subsequently dialyzing against saline using a 300 kDa MWCOembrane for 12 h with two solution changes. Unless otherwise

tated, this formulation is referred to as “standard condition” orNsp/cq/Ca/trf.”

In vitro transfection. Nanospheres and the other DNA prepara-ions (2 mg total DNA/well) were incubated for 4 h at 37°C and 5%O2 with 1 3 105 human kidney epithelial 293 cells grown on 12-well

2 Abbreviations used: RSV, Rous sarcoma virus; AAV, adeno-asso-iated virus; CFTR, cystic fibrosis transmembrane regulator; Nsp,anospheres; CQ, chloroquine; trf, transferrin; DMEM, Dulbecco’sodified Eagle’s medium; EDC, 1-ethyl-3-(3-dimethylaminopropyl-

carbodiimide hydrochloride; PLL, poly-L-lysine; PBS, phosphate-

duffered saline.

lates in medium containing Dulbecco’s modified eagle’s mediumDMEM), 2% fetal bovine serum, 2 mM L-glutamine, 50 units/mlenicillin, 50 mg/ml streptomycin, and 10 mg/ml gentamycin. Un-ound nanospheres were removed by washing with DMEM, and theells were further incubated in fresh medium containing 10% serumor 3 days. Luciferase gene expression was measured by assaying fornzyme activity in permeabilized cell extracts with the use of auminometer (14). Transfection using Lipofectin and CaPO4–DNAoprecipitation was carried out as described by Felgner (27) and byraham and van der Eb (28), respectively.36Chloride efflux assay. Cells were seeded at 30–50% confluence

nd transfected as above. Cells were washed 33 with CaMg-freehosphate-buffered saline (Gibco-BRL) to remove serum. Thirty mi-roliters of 36Cl2 solution (NEN-Dupont; 1 mCi/ml) was diluted in 9l of Ringers solution, and 1.5 ml of this loading solution was added

o each well of a six-well plate. The plate was incubated for 2–4 h in37°C warm room. The Ringers solution for these experiments was

tandard HCO32-free, Hepes- and phosphate-buffered 140 mM NaCl

ingers solution supplemented with 5 mM glucose and titrated to pH.45 with 1 N NaOH. All efflux runs were performed in a 37°C warmoom. Each well served as its own control. At Time 0, Ringersithout cAMP agonists was added and removed immediately. A

resh aliquot of Ringers was added immediately and the efflux runas started. This process was repeated every 15 s until the timeoint at 1 min, at which time, Ringers with forskolin (2.5 mM), 8romo-cAMP (250 mM), and CPT-cAMP (250 mM) was added andemoved and the sampling continued for the remaining 4 min of thefflux run. At the end of the run, 0.5 N NaOH was added in twoliquots to lyse and recover all of the cell lysate to determine howuch labeled chloride had remained in the cells to standardize the

ata. Each sample was diluted in scintillation cocktail, counted in acintillation counter, and normalized on a Microsoft Excel spread-heet as the rate of labeled chloride lost from the cells per minute.

ESULTS

Complex coacervation of gelatin and DNA to formanospheres. Coacervation reactions carried out us-

ng a range of concentration combinations of DNA,elatin, and Na2SO4 showed that the formation of dis-inct DNA–gelatin nanospheres occurred over a nar-ow range of conditions. Diagrams of typical conditionsor nanosphere formation with plasmids in the sizeange of 7 to 12 kbp are shown in Fig. 1. For example,ith HIVgp160/LAMP pcDNA1, nanoparticles were

ormed with concentrations (w/v) of 0.0025 to 0.005%NA, 1.5 to 5% gelatin, and 0.2 to 1.2% Na2SO4. Ineneral, the required concentration of DNA was moreonfined than those of other components. Outside ofhese conditions, soluble gelatin–DNA complexes, ag-regates, or loosely associated flocculates were ob-ained. Other conditions important for particle forma-ion include a reaction temperature range of 55 6 7°C,he absence of other buffers or salts in the coacervationeaction, and the extent of mixing—a function both ofhe speed of vortexing and the dimensions of the reac-ion vessel. The conditions for coacervation mayhange if the mixing conditions are modified and ifther charged molecules are included or the mixingonditions are modified and must be independently

etermined for any variation in the reaction protocol.
Page 3: Gene Transfer by DNA–Gelatin Nanospheres

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49GENE TRANSFER BY DNA–GELATIN NANOSPHERES

A detailed synthesis procedure for making the opti-ized nanospheres formulation containing plasmid in

he 7- to 12-kb size range is shown in Fig. 2. Theormation of uncrosslinked nanoparticles occurredithin seconds as gelatin is added to DNA at a high

tirring rate. More than 98% of the DNA added to theeaction enters the coacervate. The nanoparticles,hich may be viewed by phase-contrast microscopy,re separated from the unreacted components by cen-rifuging the nanosphere solution on a three-step su-rose density gradient solution. Although relativelytable at acidic pHs, these particles will dissociate inigh-ionic-strength solutions at neutral pH, but aretabilized by crosslinking by the use of EDC, a wateroluble carbodiimide that conjugates primary aminoroups to carboxyl side chains of amino acids. Ligands

IG. 1. Three-dimensional contour plots of the combination of Htandard reaction mixture that resulted in nanosphere formation. ANA, gelatin, and Na2SO4 concentrations [all axis are given as pe

equired for the formation of distinct, aggregate-free particles. The fond 0.01% (w/v)]; gelatin [0, 1, 2, 4, and 5% (w/v)]; and Na2SO4 [1.4eaction mixture. Outside the delineated region, only gelatin–DNA

r other reagents may also be included in the crosslink- u

ng reaction at this point, after which sodium acetate orlycine is added to quench any unreacted carbodiimide.NA crosslinking to itself or to chloroquine under the

onditions of the reaction was ruled out because thelectrophoretic mobility of DNA remained unchangedrom that of untreated DNA when DNA was incubatedith EDC or with chloroquine and EDC (Fig. 2b).hen EDC was incubated with an equimolar mixture

f DNA and gelatin, a DNA/gelatin ratio at which noel retardation occurred, the electrophoretic mobility ofNA also remained unchanged.The DNA–gelatin complex as visualized by trans-ission electron microscopy in multiple fields was a

pherical particle of solid core structure with a roughurface texture and composed of a matrix of inter-eaved gelatin and DNA (Fig. 3). Nanospheres made

-1gp160/LAMP DNA, gelatin, and Na2SO4 concentrations in theermutation of coacervation reaction conditions employing differentntages (w/v)] was carried out to determine the reaction conditions

ing concentration range were used: DNA [0, 0.00125, 0.0025, 0.005,1.136, 0.0852, and 0.284% (w/v)]. All concentration are final in theplexes or loosely associated flocculates were observed.

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Page 4: Gene Transfer by DNA–Gelatin Nanospheres

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50 TRUONG-LE ET AL.

f 412 6 120 nm, as determined by light scattering sizenalysis, and a zeta potential of 4.5 mV at pH 7.4. Theize of the particle was influenced by the temperaturef the reaction, the size of the plasmid, the Na2SO4

oncentration, and the speed of mixing. Micro- as wells nanoparticle size ranges could be produced. In gen-ral, nano-size particles were obtained at a tempera-ure of 55°C or lower, a Na2SO4 concentration higherhan 10 mM, and with smaller plasmids (,12 kb).

Protection of DNA against nuclease digestion. Aajor barrier to gene delivery in vivo is the vulnera-

ility of DNA to nucleases that are found in abundancen serum and the extracellular matrix. A possible ad-antage of the encapsulated DNA approach to geneelivery is the protection against nucleases. We previ-usly showed that nanosphere-encapsulated DNA wasetter protected against degradation in serum thanas naked DNA (14). In this study the effect of DNaseon nanosphere DNA compared to other forms of DNAomplexes was analyzed. Free plasmid DNA, nano-

IG. 2. Schematic of the synthesis procedure for DNA–gelatin nanof nanospheres. (b) Agarose gel electrophoresis of LAMP-1 pcDNA: lDC 1 gelatin (1:1 mole ratio of DNA to gelatin).

pheres, poly-L-lysine–DNA complex (PLL–DNA), and a

ipofectamine–DNA complex were incubated for 15in with 10 mU/ml DNase I. After incubation, the

anosphere samples were pelleted to separate the par-icles from the supernatant and the samples were an-lyzed by agarose gel electrophoresis (Fig. 4). Nano-phere DNA, Lipofectamine–DNA, and, to a lessor ex-ent, PLL–DNA made at a 1:1 weight ratio wereartially resistant to degradation during incubationith DNase I for 15 min. In contrast, naked DNA wasxtensively degraded. This protection of DNA by nano-pheres was only partial, since nanospheres incubatedn a higher DNase I solution (200 U/ml) showed com-lete digestion of nanosphere DNA. Interestingly,anospheres crosslinked with twice the EDC concen-ration did not differ in DNA protection level (data nothown). The lack of complete protection against nucle-se digestion suggests that DNA degradation occurredy a form of surface erosion of DNA that was dynam-cally mobile in the nanosphere, or, possibly moreikely, that the nanosphere was porous enough to allow

eres. (a) Standard synthesis procedures for the optimal formulation1, DNA alone; lane 2, DNA 1 EDC 1 chloroquine; lane 3, DNA 1

sphane

ccessibility of DNase to the particle’s interior, or both.

Page 5: Gene Transfer by DNA–Gelatin Nanospheres

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51GENE TRANSFER BY DNA–GELATIN NANOSPHERES

Effect of nanosphere calcium on transfection. Diva-ent metal cations such as Mg21, Ba21, and Mn21 canorm ionic complexes with the helical phosphates onNA (29). Ca21 exhibits similar affinity to DNA (Kd of.1 3 1023 M21) and forms CaPO4 complexes with theucleic acid backbone and thus may impart a stabili-ation function to certain DNA structures (30, 31). Itas been suggested that CaPO4, when used in com-lexes with plasmid DNA, exerts its positive effect onene transfer by stimulating cellular uptake of DNA in

process involving either endocytosis of the mem-rane-bound DNA complex or enhanced permeabiliza-ion of the plasma membrane to facilitate DNA entry32). In addition, some studies have correlated intra-ellular elevation of calcium with transcription andene expression (33). These observations suggest theossible benefit on nanosphere gene transfer by theodelivery of calcium. Transferrin-coated nanospheresdded to 293 cells transfected better in the presence ofxogenously added CaPO4 (100 mM) (Fig. 5). Suchransfection enhancement required the presence ofransferrin because CaPO4 failed to stimulate trans-ection when nanospheres alone (without transferrin)ere added to 293 cells. Because the CaPO4 effect onanosphere-mediated transfection required the pres-nce of transferrin on nanospheres, presumably to ini-

IG. 3. The gelatin–DNA nanosphere as visualized by electronicroscopy. 9HTEo cells were incubated with nanospheres contain-

ng pSA306 pDNA (2 mg/well) in complete medium for 15 min. Theells were washed twice with PBS, fixed by incubation with a PBSolution containing 2.5% glutaraldehyde and 2% paraformaldehydeor 30 min at 25°C, washed with 0.1 M cacodylate buffer, incubatedith 1% osmium tetraoxide, treated with 1% uranyl acetate, and

hen dehydrated in graded ethanol. The cells were embedded inpon, thin sectioned, and viewed on a Jeol electron microscope. Therrow shows a nanosphere at the surface of an 9HTEo cell after 15in incubation (75,0003). Bar, 100 nm.

iate surface binding, the results suggested that the a

dded CaPO4 may act via stimulation of cellular up-ake of bound nanospheres. The CaPO4 effect could beimicked when the nanospheres were loaded with cal-

ium by incubating newly synthesized transferrin-oated nanospheres with a concentrated calcium solu-ion. Transfection of 293 cells with crosslinked nano-pheres made with different calcium loading levelshowed the highest transfection was achieved withhose containing a 1.8% (w/w) calcium loading level.

The possibility that the CaCl2-mediated increase inransfection was due to the extracellular release ofNA and calcium ions from the nanosphere was inves-

igated. In that case, the resultant free DNA andaPO4 (formed with PO4

2 in the medium) could haveomplexed so that the enhancement in transfectionight have resulted from formation of free DNA–aPO4 complexes and not from intact nanospheres.his possibility was tested by measuring transfectionf 293 cells by nanosphere supernatant in the presencer absence of CaPO4 (Table I). The level of transfection

IG. 4. Protection of DNA against nuclease digestion. PlasmidNA, DNA–gelatin nanospheres, poly-L-lysine–DNA complex (PLL),r Lipofectamine–DNA complex was incubated with 10 mU/ml ofNase I for 15 min at 37°C. The nanosphere samples were pelleted

o separate the nanospheres from the supernatant, the pellet frac-ion incubated with trypsin to release the DNA, and the resultingNA from the supernatant and pellet fractions was subjected to 1%garose gel electrophoresis. The DNA from other samples was ex-racted twice with phenol:chloroform:isoamyl alcohol (50:49:1) prioro gel electrophoresis. Lanes: 1, untreated control DNA; 2, plasmidNA treated with 10 mU/ml DNase; 3, Nsp–DNA treated with 10U/ml DNase, pellet fraction; 4, Nsp-DNA treated with 10 mU/mlNase, supernatant fraction; 5, PLL–DNA at 1:1 weight ratio

reated with 10 mU/ml DNase; 6, Lipofectamine–DNA at 2:1 weightatio treated with 10 mU/ml DNase; 7, Nsp–DNA incubated in 200/ml DNase I for 15 min at 37°C, pellet fraction. Degradation of DNAas apparent with naked DNA and the PLL–DNA complex treatedith 10 mU/ml DNase and with nanosphere DNA treated with 200/ml DNase I. In contrast, DNA in nanospheres (lane 3) and Lipo-

ectamine–DNA complex (lane 6) exhibited greater resistance

gainst nuclease degradation at a concentration of 10 mU/ml DNase.
Page 6: Gene Transfer by DNA–Gelatin Nanospheres

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f supernatant from complete, crosslinked nano-pheres was barely above background, indicating thathere was very little, if any, free DNA in the nano-phere sample. In comparison, cells transfected withupernatant of the uncrosslinked nanosphere sampleontaining free DNA released from the nanospherehowed a significant level of luciferase activity in theresence of added CaPO4. It was also possible thatroteases in the medium may partially degrade theanosphere during the period of cell culture medium

ncubation, releasing free DNA and calcium which

IG. 5. The effect of calcium on nanosphere transfection of 293ells in vitro. Nanospheres synthesized containing pRELuc DNANsp), containing transferrin coating (Nsp/trf ), and containing trans-errin and calcium (Nsp/Trf/Ca) were added to cells in a 12-well dish2 mg total DNA/5 3 104 cells per well) for 4 h, washed to removexcess unbound nanospheres, and then assayed for luciferase expres-ion at Day 3. Nsp 1 CaPO4 and Nsp/Trt 1 CaPO4 represent,espectively, Nsp and Nsp/Trf samples that were incubated withells for 2 h, washed, pulsed with CaPO4 (100 mM) for two additionalours, and assayed for luciferase expression at Day 3. Data areverage values from triplicate determinations and are representa-ive of two different experiments, 6SD.

TAB

Nanospherepreparation

L

Nsp pellet Nsp

omplete 101,800 1450oncrosslinked 380 550

Note. Complete nanospheres (Nsp/trf/CQ/Ca) or noncrosslinked na15,000g, 12 min) to separate the particulate fraction (Nsp pellet) frraction was resuspended in water and incubated with 293 cells (2 mgas incubated with 293 cells in the presence or absence of CaPO4. Inedium (without cells) for 4 h and then centrifuged to separate the

upernatant fraction was subsequently incubated with 293 cells in tto determine gene transfection.

a Background subtracted.

ould also form DNA–CaPO4 complexes. To addresshis, the nanospheres were incubated with culture me-ium (without cells) for 4 h, and the resultant mediumas used to transfect cells under similar conditionsescribed above for the nanosphere supernatant (Table). Again, there was a significant level of transfectionnly with the uncrosslinked nanosphere sample.igher transfection activity was observed in the un-

rosslinked nanosphere group (using CaPO4 complex-tion) than in the nanosphere pellet group, which sug-ested that either crosslinking decreased the transfec-ion efficiency or that the amount of DNA released fromhe crosslinked nanospheres by Day 3 was small. Thus,e concluded that little DNA was released from the

rosslinked nanospheres into the cell culture mediumuring the time of incubation, and that the transfectionan be attributed to intact nanospheres and not to freeNA and calcium contaminants.Transfection with nanosphere DNA encoding the hu-an CFTR protein. Our studies have shown that the

ptimal nanosphere formulation, as defined in vitroransfection studies carried out on 293 human kidneypithelial cells, contained calcium in addition to chlo-oquine and surface-bound transferrin (14). Furtherocumentation of the transfection with nanosphereNA using different cell lines and a therapeutically

elevant model gene was carried out.The bioactivity of DNA delivered to cells by nano-

pheres was studied by transfecting 9HTEo cells withlasmid pSA306 expressing a chimeric human CFTRrotein containing a 26-amino-acid epitope tag at thearboxy terminus. Gene expression analysis, as mea-ured by antibody binding to the epitope tag, showedpproximately 50 to 60% of the cells incubated with theanospheres were positive for the expression of the tagFig. 6). The frequency of stained cells following nano-phere transfection was significantly greater than thatf cells transfected by the equivalent DNA dose given

I

iferase activity (light units)a

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1CaPO4 1CaPO4

30 910 449013,830 450 418,010

pheres (Nsp/CQ) containing the pRELuc plasmid were centrifugedthe supernatant fraction (Nsp supernatant). The nanosphere pelletA/105 cells), while the nanosphere supernatant (2 mg DNA/105 cells)

eparate experiment, nanospheres were incubated with fresh cultureticulate fraction from the supernatant fraction (Nsp medium). The

presence or absence of CaPO4. Luciferase assay was carried at Day

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Page 7: Gene Transfer by DNA–Gelatin Nanospheres

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53GENE TRANSFER BY DNA–GELATIN NANOSPHERES

s Lipofectamide–DNA complex (.50% vs ;23%). Thisransfection frequency was the highest observed forny cell types tested using nanospheres as the trans-ectant agent. Other cells (293 human kidney epithe-ial, HeLa cells, IB-3-1 human bronchial epithelial,OS-7 monkey cells, and U937 human histiocytic–onocytic cells) typically showed a range of 2–15%

ransfection frequency. Variations in the transfectionrequency were also observed with Lipofectamine–NA and CaPO4–DNA transfection methods. None of

he nonviral gene transfection methods tested couldatch the results achieved by use of AAV. The major-

ty of HTE cells infected under similar conditions with09 AAV encoding the CFTR gene were transfected,epresenting an efficiency based on the amount of DNAhat was generally 10 to 100 times greater in cellransfection than that achieved with the DNA plasmidelivery systems (data not shown).Cells transfected with nanosphere DNA encoding theFTR gene form a functional chloride channel. Addi-

ional studies with plasmid pSA306 expressing a chi-eric human CFTR protein were conducted with IB-

-1 cells, a human bronchial epithelial cell line bearinghe delta508 mutation which results in a defectiveAMP-stimulated chloride transport phenotype. Thisystem was used to study the efficacy of transfectionith DNA–gelatin nanospheres in reconstituting chlo-

ide transport, comparing the effect of transfectionith free DNA, DNA–gelatin nanospheres, or infectionf the cells with an AAV CFTR vector system (Fig. 7).n this standard assay (34), the function of CFTR isssessed by cAMP stimulation of 36Cl2 efflux and is

IG. 6. Expression of the CFTR gene in 9HTEo human tracheal epiene with a 26-amino-acid epitope tag were prepared as described uarried out with 9HTEo cells plated on a cover glass in a 12-well pNA/3 3 105 cells/well, in nanospheres, as a Lipofectamine complex,

ells were then fixed with 4% paraformaldehyde, blocked by adding 1BS for 10 min, incubated with the anti-epitope tag antibody mAb

ncubated with a 1:1000 dilution of fluorescein isothiocyanate-labelef view shown contained cells at ;75–80% confluency (the correspohowed only background cellular autofluorescence; the occasional cellransfected cells showed specific staining in approximately 50% of tpproximately 20% of the cells.

xpressed as the percentage rate of efflux per minute t

ormalized to 100%, as a function of time after cAMPtimulation. Because the efflux is followed with multi-le samples and cAMP is added at the 1-min timeeriod after the beginning of the experiment, each cul-ure dish serves as its own control. Efflux of Cl2 fromhe nontransduced cells (background cells), was nottimulated by cAMP. Similarly, in cells treated withree DNA there is no detectable increase in the rate ofl2 release following cAMP addition. The small fluctu-tions in the chloride efflux rate as seen with theontransduced cells following cAMP addition are at-ributed to mechanical perturbations in the cells bydding cAMP. In contrast, when cAMP was added toells transfected with the gene encoding the CFTRarrier by nanosphere DNA or by the AAV CFTR vec-or, there was a rapid and transient increase in 36Cl2

fflux, indicative of the action of functional CFTR. Inhe nanosphere-treated cells, the rate jumped from lesshan 20% per minute just prior to cAMP addition topproximately 50% per minute after addition of cAMP.ikewise, in the AAV CFTR-treated cells the rate in-reased from about 20% per minute prior to 40% after.his rapid and transient increase in efflux demon-trated that CFTR was functioning in the transducedells compared to nontransfected controls. Because thebsolute magnitude of the response depends not onlyn the expression of CFTR in an individual cell but alson the number of cells transduced, a quantitative com-arison of the absolute magnitude of the responsemong groups is not possible. It may be noted, how-ver, that a comparable result in this assay was ob-ained with 109 AAV, which contains much less DNA

lial cells. Nanospheres containing pSA306 DNA encoding the CFTRr Experimental Procedures. Transfection with the CFTR gene was. The cells were transfected by the addition of pSA306, 2 mg total

as naked DNA, washed after 4 h, and then incubated for 3 days. Thebovine serum albumin for 30 min, permeabilized with 0.1% saponin

(1:100 dilution) for 1 h, washed three times with PBS, and thennti-chicken IgG antibody (Jackson Lab, Bar Harbor, ME). The fieldng bright-field image is not shown). (A) Free DNA transfected cellsith a higher level of staining were not investigated. (B) Nanosphere-cells. (C) Lipofectamine–DNA-transfected cells showed staining in

thendelateor%934d andis whe

han the 5 mg added as nanosphere DNA. We previ-

Page 8: Gene Transfer by DNA–Gelatin Nanospheres

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54 TRUONG-LE ET AL.

usly have shown that the transduction efficiency ofanosphere DNA in vitro, while greater than that ofaked DNA, was 10 to 1000 times less than that ofAV virions (14).

ISCUSSION

Complex coacervation has been widely used to for-ulate controlled release microspheres. We had previ-

usly described the use of gelatin–chondroitin sulfatend collagen–chondroitin sulfate complex coacervateso deliver therapeutic agents (26, 34). In this system,NA and gelatin are used as the oppositely chargediopolymers. Since there was no evidence of crosslink-ng of the DNA to gelatin or to any other nanosphereomponents, the nanosphere can be thought of as aolid core structure composed of gelatin entangled withNA. The spherical nature of the coacervate is a resultf the gelatin–DNA complex adopting the minimal en-rgy architecture in a process governed by the relativenterfacial tension.

One of the advantages of the nanosphere approach toene delivery is the enhanced protection of DNAgainst nuclease degradation, which should improve inivo bioavailability of the DNA. Our data showed theignificant improvement in DNA protection when theanospheres were incubated with a low concentrationf DNase I (10 mU/ml) compared to the rapid degrada-ion of free DNA and a DNA–PLL complex. Higheregrees of nanosphere crosslinking further stabilizedhe gelatin matrix, which in turn improved the half-lifef DNA treated with DNase; however, at highrosslinking density, the transfectability of the nano-phere was compromised, possibly because of a reducedate of release of the DNA (unpublished results). Inddition to increasing the crosslinking density of theelatin matrix, we have attempted to increase the re-istance of nanosphere DNA to DNase by coencapsu-ating DNase inhibitors. Encapsulation of sodium io-oacetate (35) and aurintricarboxylic acid (36), bothnown to be potent DNase I inhibitors, showed a minornhancement in DNA protection, but failed to showonsistent improvement in transfection level (data nothown).Other materials added to the coacervation reaction

an be coencapsulated in the nanosphere by eitherovalent conjugation, entrapment, or ionic interactionsith DNA or gelatin. However, these nanospheres areermeable to low-molecular-weight compounds ashown by the partial loss of chloroquine and calciumuring dialysis. This suggests that larger and moreharged compounds should enjoy a higher loadingevel. In principle, this may include peptides, proteins,nd a variety of smaller but charged compounds. Chlo-oquine was added to enhance transfection by DNA,

IG. 7. 36Chloride efflux assay of chloride transport across humanronchial epithelial IB-3-1 cells transfected with pSA306 DNA asree DNA, nanosphere–DNA, or AAV CFTR viral particles. TheB3-1 cells are a human CF bronchial epithelial cell line with defec-ive transport of Cl2. pSA306 DNA (5 mg total DNA/5 3 105 cells/per5-mm well of a multiwell plate), nanospheres containing pSA306NA (5 mg total DNA/5 3 105 cells/well), or AAV CFTR virions (109

iral particles/5 3 105 cells/well) were incubated with IB-3-1 cells forh as described above and then analyzed for chloride efflux at Dayas described in detail elsewhere (34). The transfected cells were

abeled with 36Cl2 for 4 h, washed to remove excess 36Cl2 (t 5 0 is theime after the last wash) and loaded with cAMP at t 5 1 min toctivate the CFTR 36Cl2 current. The medium was sampled for 36Cl2

ffluxed from the cells at various time points. The data for eachxperiment are averages of six separate assays from six individualishes of cells run on the same day and are representative of twoxperiments done on two separate occasions. The results are ex-ressed as the percentage rate of 36Cl2 efflux per minute normalizedo 100% and plotted as a function of time. Bkgd, background 36Cl2

resumably by interfering with the acidification of en-

Page 9: Gene Transfer by DNA–Gelatin Nanospheres

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55GENE TRANSFER BY DNA–GELATIN NANOSPHERES

osome compartments (37) or reducing DNase degra-ation (38). A chloroquine loading level of 2% (w/w)nto the nanospheres was achieved by reducing thea2SO4 concentration 10-fold in the coacervation reac-

ion. This high uptake of chloroquine was attributed tots affinity for polynucleotides (Kd of 1024 M21) viantercalation in the DNA helix and electrostatic inter-ctions with the phosphate backbone (39). The level ofransgene expression in 293 human kidney epithelialells incubated with nanospheres was also markedlyncreased by the presence of transferrin on the nano-phere and to a lesser extent by calcium. In the absencef transferrin there was no detectable transfection of93 cells with as much as 10 mg total DNA either aselatin–DNA nanospheres or gelatin–DNA complexes.ransferrin has been demonstrated to be efficient inediating binding and uptake of DNA–polymer or

protein complexes in several studies (40), presumablyy acting as a ligand to the corresponding cell surfaceeceptor and thereby enhancing the binding and/or theptake of the nanosphere into the cell. Transfectionnhancement by calcium was attributed to calciumhat was associated with the nanospheres and not tohose resulting from leakage. Our data also suggestedhat a possible role of calcium is to facilitate release ofNA from the gelatin matrix by its competition forlectrostatic interactions with the gelatin and DNA.owever, other explanations that account for the en-ancement in transfection such as stimulated uptakef nanospheres, perhaps as a result of the formation ofaPO4 complexes on the nanospheres, or specific intra-

ellular effects are also plausible.These in vitro transfection experiments using theFTR gene have shown that a large percentage ofHTEo cells could be transfected by use of the DNA–elatin nanospheres. In addition, successful comple-entation of defective chloride transport in mutant

B-3-1 cells was demonstrated. While similar resultsave been accomplished using a variety of viral andonviral gene delivery methods in vitro (41), the ther-peutic advance to successful in vivo CF gene therapyemains a great challenge. Our studies have shownhat under in vitro cell culture conditions, both generansfer efficiency and efficacy were poorer than withhe AAV viral counterpart. However, the microencap-ulated DNA approach may offer some advantages thatre unique compared to other approaches, such as tar-eted gene delivery by the use of monoclonal antibodiesr cell ligands and codelivery of therapeutic compoundsn a controlled fashion. Studies of the delivery of theFTR gene to rabbit airways by use of nanosphere–NA have shown a correspondingly high efficiency of

ransfection of airway epithelial cells as was observedn these in vitro studies, and an evaluation of theodelivery of sodium 4-phenylbutyrate, an agent that

as been found to enhance the CFTR function by stim-

lating the mutant DF508 CFTR activity (42), is un-erway.

CKNOWLEDGMENTS

This study was supported by NIH CA 68011, NIH 1 RO1 A141908,nd the Cystic Fibrosis Foundation.

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