steric stabilization of poly- l -lysine/dna complexes by the covalent attachment of semitelechelic...

Steric Stabilization of poly-L-Lysine/DNA Complexes by the CovalentAttachment of Semitelechelicpoly[N-(2-Hydroxypropyl)methacrylamide]

David Oupicky,*,† Kenneth A. Howard,† Cestmır Konak,‡ Philip R. Dash,† Karel Ulbrich,‡ andLeonard W. Seymour†

CRC Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TA, U.K., and Institute ofMacromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2,162 06 Prague 6, Czech Republic. Received October 21, 1999; Revised Manuscript Received February 25, 2000

The concept of steric stabilization was utilized for self-assembling polyelectrolyte poly-L-lysine/DNA(pLL/DNA) complexes using covalent attachment of semitelechelic poly[N-(2-hydroxypropyl)methacry-lamide] (pHPMA). We have examined the effect of coating of the complexes with pHPMA on theirphysicochemical stability, phagocytic uptake in vitro, and biodistribution in vivo. The coated complexesshowed stability against aggregation in 0.15 M NaCl and reduced binding of albumin, chosen as amodel for the study of the interactions of the complexes with plasma proteins. The presence of coatingpHPMA had no effect on the morphology of the complexes as shown by transmission electronmicroscopy. However, results of the study of polyelectrolyte exchange reactions with heparin and pLLsuggested decreased stability of the coated complexes in these types of reactions compared to uncoatedpLL/DNA complexes. Coated complexes showed decreased phagocytic capture by mouse peritonealmacrophages in vitro. Decreased phagocytosis in vitro, however, did not correlate with results of invivo study in mice showing no reduction in the liver uptake and no increase in the circulation timesin the blood. We propose that the rapid plasma elimination of coated pLL/DNA complexes is a resultof binding serum proteins and also of their low stability toward polyelectrolyte exchange reactions asa consequence of their equilibrium nature.

INTRODUCTION

Polyelectrolyte polycation/DNA complexes are attract-ing considerable attention as a promising synthetic vectorfor gene delivery. However, despite substantial progressand considerable versatility and transfectional activityin vitro, simple polyelectrolyte gene delivery systems arequickly eliminated from the bloodstream following in-travenous injection (plasma half-life typically less than5 min) (Dash et al., 1999). The complexes are usuallycleared quickly into the liver or spleen. Patterns ofdistribution in vivo indicate significant phagocytic cap-ture by components of the reticuloendothelial system(RES), notably by tissue macrophages such as Kupffercells (Dash et al., 1999). Rapid localization of DNAcomplexes in the RES (liver, spleen) prevents theirtargeting to non-RES tissues in a specific manner andresults, therefore, in a severe limitation of their thera-peutic potential. For targeted systemic delivery, a moreprolonged plasma circulation of the vector is essential.It is, therefore, a central aim to decrease uptake by theRES in order to develop polymer-based DNA complexeswith improved circulation times. In the design of longcirculating DNA/polyelectrolyte complexes for gene de-livery, we can be inspired by experiences obtained in thefields of similar particulate systems for drug delivery,notably liposomes and polymeric nanoparticles (Bazileet al., 1992; Illum et al., 1987; Lasic and Needham, 1995).

The interaction of similar particulate drug deliverysystems with the cells of the RES is determined to a greatextent by physicochemical properties of their surface andtheir size with larger particles being taken up faster(Moghimi, 1995; Monfardini and Veronese, 1998). Theinitial stages of phagocytosis involve the physical attach-ment of the particulate to the surface of the macrophage.An increase in particulate hydrophobicity is known toincrease uptake by forming hydrophobic interactionsbetween the particulate and the cell surface (Monfardiniand Veronese, 1998), while particles with polar andcharged surfaces have an increased circulation time andreduced uptake by RES organs (Gabizon and Papahad-jopoulos, 1992). To avoid RES capture, it is, therefore,important to reduce the hydophobicity of the particles;however, it is known that materials with strong positivesurface charge bind biological membranes nonspecifically,while those with strong negative charge can be substratesfor phagocytosis via the macrophage polyanion receptor(Roser et al., 1998). Hence, it may also be important toavoid significant surface charge of the DNA complexesto prolong blood circulation.

Apart from nonspecific uptake of the particles by RESsimply as a result of their physicochemical properties,adsorption of blood components onto particles also playsan important role. The blood contains a large number ofproteins and glycoproteins that can function as opsonins(Moghimi, 1995). Rapid binding of one or more of thesematerials onto the complexes can occur and may havesignificant influence on the fate of the particulate. Thefirst component adsorbed to the surface of positivelycharged complexes will probably be albumin, but othermaterials present in lower concentrations with higher

* To whom correspondence should be addressed. Phone: +44-121-414-3291. Fax: +44-121-414-3263. E-mail: [email protected].

† CRC Institute for Cancer Studies.‡ Institute of Macromolecular Chemistry.

492 Bioconjugate Chem. 2000, 11, 492−501

10.1021/bc990143e CCC: $19.00 © 2000 American Chemical SocietyPublished on Web 06/16/2000

affinities for the surface will displace the first adsorbedlayer as a function of time (Davis and Illum, 1988). Oneof the most successful strategies for obtaining long-circulating nanoparticles and liposomes has been polymerattachment [usually poly(ethylene glycol) (pEG)] at theparticle surface, for creating a hydrophilic cloud reducinginteractions with proteins and cells (steric stabilization).This so-called stealth technology has been successful inliposomes and nano- and microparticulate drug deliverysystems (Lasic, 1997; Monfardini and Veronese, 1998).

The concept of using block or graft copolymers ofcationic and hydrophilic nonionic monomers has beenintroduced by several groups as a new potential way indevelopment of self-assembling polyelectrolyte gene de-livery vectors (Katayose and Kataoka, 1997; Tonchevaet al., 1998; Wolfert et al., 1996). The aim was to increasethe hydrophilic character of the complexes and at thesame time introduce a surface hydrophilic layer actingin a similar way as in stealth liposomes. However, in vivobiodistribution studies showed that these complexes areremoved very quickly from the murine bloodstreamsimilar to the complexes between DNA and simplepolycations (Oupicky et al., 1999a). Unsuitable morphol-ogy of the complexes and protein binding at serumprotein concentrations were proposed as the major rea-sons for their rapid removal (Oupicky et al., 1999a). Aparticular problem with this approach may reflect ther-modynamic instability of the complexes resulting fromnecessary entrapment of hydrophilic chains of the blockand graft copolymers within the polyelectrolyte core ofthe complex. To address this possibility and to examineits effect on the stability of the otherwise-hydrophobiccore, in this study, we have allowed simple polyelectrolytecomplexes to self-assemble before covalent linkage of thehydrophilic polymer, only to the surface of the polyelec-trolyte particle.

Although significant attention is paid to the develop-ment of nonviral polyelectrolyte systems for gene deliv-ery, most of the published works concentrate on in vitrostudy of the properties of the complexes. Relatively littleinformation is available about suitability of these com-plexes for in vivo use (Chemin et al., 1998; Hashida etal., 1998; Laurent et al., 1999; Ogris et al., 1999). In thiswork, we therefore report the study of stability of thepolyelectrolyte complexes of DNA with poly-L-lysinecoated with hydrophilic pHPMA chains in aqueous NaClsolutions and their interaction with albumin. The resultsof the phagocytic uptake of pLL/DNA complexes bymouse peritoneal macrophages are discussed togetherwith the biodistribution analysis in mice performed inorder to obtain more information on the effect of thehydrophilic pHPMA coating on biological properties ofcomplexes.

EXPERIMENTAL PROCEDURES

Materials. A circular 5.2 kb expression vector contain-ing a SV40 promoter-driven luciferase reporter gene andampicilin resistance was used in most experiments. Thiswas prepared by growth in Escherichia coli DH5R andpurified using Qiagen columns (Dorkin, U.K.). Followingthe final ethanol precipitation, the purity of the DNA waschecked by agarose gel electrophoresis and 260/280 nmabsorption. In the rest of the experiments, calf thymusDNA (ct-DNA) (sodium salt) was used. ct-DNA and poly-L-lysine hydrobromide (pLL) (molecular weight 19 600)were from Sigma Chemical Co. Semitelechelic poly[N-(2-hydroxypropyl)methacrylamide]s with carboxylic acidend group were prepared by radical polymerization asdescribed in (Oupicky et al., 1999b).

Formation of pLL/DNA Complexes and TheirCoating with poly(HPMA). All complexes in this studywere prepared in water at DNA concentration 20 µg/mL(phosphate group concentration 61.5 nmol/mL). pLL wasadded to DNA solution in a single addition so the N:Pratio was 1.2 (pLL amino group concentration 73.8 nmol/mL). Freshly prepared solution of N-hydroxysuccinimidylester of carboxylic acid group terminated pHPMA (pHP-MA-NHS) in water was then added to the complexsolution followed by addition of Hepes buffer (pH 7.8) toreach final concentration 10 mM. The reaction wascarried out at room temperature for at least 3 h beforeany other experiment. Two different molecular weights(number average) of pHPMA (8500 and 5000) and twodifferent concentrations of them were used in the coatingreaction (24.6 and 74 nmol/mL).

For the study of the dose effect on in vivo biodistribu-tion, the coated complexes (pHPMA-pLL/DNA) wereconcentrated on Centricon-100 filters (Millipore). Theincrease of concentration to 120 µg/mL (determined byUV absorption) was achieved without any significantchange in the size of the complexes.

Albumin-Induced Turbidity Assay. The ability ofalbumin (bovine serum albumin, Sigma) to cause theaggregation of the complexes was examined using aPerkin-Elmer (Buckinghamshire, U.K.) LS50B fluorim-eter. The coated or uncoated complexes were both in 10mM Hepes (pH 7.8). Albumin solution (30 mg/mL) wasadded stepwise to the complexes and changes in turbiditywere monitored. Studies were performed at emission andexcitation wavelengths set to the same value of 600 nm.

Polylysine-Induced Aggregation of Coated Com-plexes. The ability of pLL (19.6 kg/mol) to cause theaggregation of pHPMA-coated pLL/DNA complexes wasmonitored using commercial light-scattering equipment(Zetasizer, Malvern Instruments, U.K.). To the solutionof coated complexes [prepared 24 h before the experimentusing two different concentrations of pHPMA(8500)], twodifferent amounts of pLL (final total concentration of pLL30 and 75 µg/mL, i.e., 144.2 and 361 nmol/mL of pLLamino groups) were added to the solution immediatelyafter an addition of NaCl (final concentration 0.15 M) andthe changes in the size of the complexes were monitored.

Heparin Release of DNA. Heparin solution (12 µL,Heparin Sodium, Multiparin, 1000 units/mL; CP Phar-maceuticals Ltd. Wrexham, U.K.) was added to 15 µL ofthe solution of coated or uncoated complexes and left toincubate for 1 h. The complexes were then analyzed on0.8% agarose gel (TBE buffer, 0.5 µg/mL ethidiumbromide, 90 V, 45 min). To permit also quantitativeanalysis, DNA was spiked with 32P-radiolabeled DNA asdescribed below. The amount of DNA released wasquantified using a Phosphoimager (Molecular Dynamics,Chesham, U.K.).

Transmission Electron Microscopy. The complexeswere prepared in water at final DNA concentration of20 µg/mL. A total of 10 µL of the solution was dispensedonto paraffin, and a piece of freshly cleaved mica (AgarScientific, Stanstead, U.K.) was placed on top for 2 min.The mica was then removed and placed on a drop of 2%solution of uranyl acetate for a further 2 min. The micawas then washed twice in double-distilled water andallowed to dry before the sample was visualized by rotaryshadowing with platinum (5° angle, 6 cm distance,rotation at 60 rpm) in a High Vacuum Coating Unit(Edwards, Crawley, U.K.) The sheet of mica was cut intosections and floated on water to release the sample that

Steric Stabilization of poly-L-Lysine/DNA Complexes Bioconjugate Chem., Vol. 11, No. 4, 2000 493

was then picked up using sticky grids (carbon 200 mesh)and allowed to dry. The samples were then viewed usinga JEOL 1200 EX Transmission Electron Microscope.

Static Light Scattering (SLS). Static light-scatteringmeasurements were performed with a commercial Fica40 apparatus in vertically polarized light at wavelength632.8 nm and temperature 25 °C. The apparatus wascalibrated with benzene at 90°. The intensities of scat-tered light at 45° (I45), 90° (I90), and 135° (I135) have beenmeasured for all the samples studied. Since the concen-trations of particles and their refractive increments arenot known, the intensity I90 normalized with intensityscattered by benzene at 90° is given for a qualitativediscussion of results (I90 is roughly proportional to theweight-average molecular-weight of particles). Disym-metries I45/I135 are used as a qualitative measure ofparticle sizes (radius of gyration).

Dynamic Light Scattering (DLS). Polarized DLSmeasurements were made in the angular range 30-135°using a light-scattering apparatus equipped with an He-Ne (632.8 nm) and Ar-ion laser (514.5 nm) and an ALV5000, multibit, multi-tau autocorrelator covering ap-proximately 10 decades in delay time. Most of themeasurements were realized at the scattering angle 90°.

The inverse Laplace transform using the REPES(Jakes, 1995) method of constrained regularization issimilar in many respects to the inversion routine CON-TIN (Provencher, 1979). However, REPES directly mini-mizes the sum of the squared differences between theexperimental and calculated intensity time correlationfunctions using nonlinear programming. This methoduses an equidistant logarithmic grid with fixed compo-nents (here a grid 10 components/decade) and determinestheir amplitudes. As a result, a distribution function A(τ)of decay times is obtained. From the characteristic decaytime, τ [the peak positions of A(τ)], the correspondingapparent average diffusion coefficient, D(90°), was cal-culated. The apparent average hydrodynamic radius, RH

a,was calculated from D(90°) using the Stokes-Einsteinequation (viscosity of water at 25 °C 0.894 cP), and thedistribution of decay times A(τ) was recalculated on thedistribution of hydrodynamic radii A(RH

a).The experimental error of radius determination for the

complexes was typically about 3%.Alternatively, for the routine measurements, Malvern

Instrument’s Zetasizer 1000 system with a 70 mWexternal laser was used.

In Vitro Association of Complexes with MouseMacrophages. Adult female BALB/c mice (25 g) werekilled by cervical dislocation and injected intraperito-neally, under sterile conditions, with 5 mL of medium199 containing 20% fetal calf serum (Gibco, Paisley,U.K.). The abdomen was then agitated gently, theperitoneum exposed and breached, and the mediumremoved using a syringe. The medium was then centri-fuged (500g, 10 min) and the pellet resuspended inmedium 199 containing 50% fetal calf serum. The cellsuspension was then plated out into multiwell 6-wellplates (480 000 cells/well). The macrophages were al-lowed to adhere for 2 h before medium (containingnonadherent cells) was removed. Fresh medium contain-ing Amphotericin B (Sigma Chemical Co., Poole, U.K.,2.5 µg/mL), Penicillin, and Streptamycin (Sigma Chemi-cal Co., Poole, U.K., 100 units/mL, 0.1 mg/mL, respec-tively) was then added to the cells. After 40 h, the cellswere used in cellular association studies.

The complexes were formed with YOYO-1 labeledplasmid DNA (one YOYO-1 molecule/300 bp) using thesame protocol as described above. Complexes were added

at a DNA concentration of 20 µg/mL to plates containingadherent mouse macrophages in 50% 199 media, 50%FCS (75 µL of complexes/well). The complexes were addedfor various periods of time (0.5-1.5-3 h) after which thecells were removed by standard trypsin treatment,washed, and resuspended in PBS. Association of thecomplexes with cells was determined by the amount ofYOYO-1/DNA detected with a Coulter EPICS XL flowcytometer using an argon ion laser set for excitation 488nm and emission 520 nm. Cell groups were defined toexclude debris and duplets, and each cell type was gatedindividually against a negative control of cells withoutaddition of complexes.

Radiolabeling of DNA Expression Vectors. Plas-mid DNA was linearized with HindIII and then radio-labeled with [32P]dCTP using the Ready-to-Go Oligola-beling kit (Pharmacia Biotech, St. Albans, U.K.). Unincor-porated nucleotides were removed using MicroSpin col-umns (Pharmacia Biotech), and the purity of the labeledDNA was checked following agarose gel electrophoresisand quantitative analysis with a PhosphoImager (Mo-lecular Dynamics, Chesham, U.K.).

In Vivo Biodistribution Study. pLL/DNA and pHP-MA-pLL/DNA complexes were formed in water 12 hbefore the experiment using either ct-DNA or plasmidDNA containing a spike of 32P-labeled DNA.

Before injection, glucose was added to a final concen-tration of 5% (w/v) to ensure iso-osmolarity. Complexes(0.2 mL) were administered via the tail vein to brieflyanaesthetized 6-week-old female BALB/c mice. After 30min, the animals were killed and dissected to permitdetermination of radioactivity distribution. The blood,lungs, liver, spleen, kidneys, intestines, and carcass (withtails being removed) were isolated, weighed, and dis-solved in 10 M sodium hydroxide at 75 °C. Scintillationmedium (20 mL) was added to each blood and tissuesample (1 mL) before geometry-corrected analysis of theircontained radioactivity in a Packard scintillation counter.A bloodstream volume of 8.64 mL/100 g body weight wasassumed (Seymour et al., 1991).

RESULTS

Coating of DNA Complexes and Their Morphol-ogy. The complexes were formed using only a slightexcess of pLL (N:P ratio 1.2; ú potential +20 mV) in orderto limit the presence of free pLL, which would also reactwith pHPMA-NHS in the coating reaction. It has beenshown that pLL/DNA complexes of this compositioncontain only a negligible amount of free pLL (Ward etal., 1999). On the other hand, using such a relatively lowcharge ratio results in complexes that are more suscep-tible to salt-induced aggregation. The coating reactionwas therefore conducted in 10 mM Hepes buffer, pH 7.8.Under these conditions, only negligible aggregation of thecomplexes was observed during the coating reaction. Thecharacteristics of the pLL/DNA complexes coated withpHPMA-NHS having number-average molecular weight5500 and 8500, respectively, are given in Table 1. The

Table 1. Characteristics of Coated and Uncoated pLL/DNA Complexes

complex I90a I45/I135

b RHa (nm)c

pLL/DNA 29 1.79 46pHPMA(5500)-pLL/DNA 44 1.77 53pHPMA(8500)-pLL/DNA 45 1.64 54a Intensity of scattered light at 90° normalized with the intensity

scattered by benzene. b Ratio of intensities of scattered light at45 and 135°. c Hydrodynamic radius.

494 Bioconjugate Chem., Vol. 11, No. 4, 2000 Oupicky et al.

coating slightly increased the hydrodynamic radius ofcomplexes (1.17 times), and the scattered intensity (I90)(proportional to molecular weight of particles) was alsocorrespondingly increased (1.55 times). This increaseshows that the molecular weight of pLL/DNA complexesis roughly proportional to RH

2.7 of the complexes whichis close to the model of solid spheres (exponent 3).Incubation of pLL/DNA complexes with pHPMA contain-ing a nonreactive carboxylic acid end group did not causeany change in their size or molecular weight, confirmingthat the observed changes of particle parameters (Table1) indicate covalent attachment of pHPMA-NHS to thepLL/DNA complexes. The covalent attachment of pHPMA-NHS was confirmed also by the decrease of amino groupcontent [determined by TNBSA assay (Snyder and Sob-ocinski, 1975)] in pLL/DNA complexes after their coatingwith pHPMA-NHS. The data in Table 1 also demonstratehigher sensitivity of static light scattering methodscompare to DLS and shows its importance in the studyof these high-molecular-weight systems.

The idea of modifying pLL/DNA complexes with semi-telechelic pHPMA-NHS assumes binding of the polymerto the surface of the complexes with little or no distur-bance of the actual pLL/DNA complexes. However, thecoating (especially when using high excess of coatingpolymer) may induce undesired changes in the morphol-ogy of the complexes. Figure 1 shows TEM pictures ofpLL/DNA before and after coating with two differentamounts of pHPMA(8500)-NHS. In no case was there asign of any change in the morphology of the complexes.The complexes retained their spherical shape and alsotheir diameter of about 100 nm.

Stability of Complexes in Salt. A combination ofstatic and dynamic light scattering techniques was alsoused to examine the stability of complexes in 0.15 MNaCl. Table 2 shows the results of the stability measure-ments. The uncoated pLL/DNA complexes precipitatedvery fast, forming large aggregates almost immediatelyafter the addition of NaCl, while the coated complexeswere stable for at least 24 h. The scattered intensity I90

Figure 1. Transmission electron micrographs showing the morphology of plasmid and ct-DNA complexes coated with semitelechelicpHPMA(8500) at two different concentrations. (A) Uncoated pLL/ct-DNA; (B) pHPMA-pLL/ct-DNA (24.6 nmol/mL pHPMA-NHS);(C) pHPMA-pLL/ct-DNA (74 nmol/mL pHPMA-NHS); (D) pHPMA-pLL/plasmid DNA (24.6 nmol/mL pHPMA-NHS); (E) pHPMA-pLL/plasmid DNA (74 nmol/mL pHPMA-NHS); (50000× magnification).


of the coated complexes increased only by a few percentshowing practically negligible aggregation. Neither typesof parameters used for characterization of size of thecomplexes showed any significant change. This observa-tion not only suggests the absence of aggregation but alsoindicates that no change in morphology of the complexeswas induced at this stage (Provencher, 1979).

Interaction of Complexes with Albumin. The ad-dition of albumin to pLL/DNA complexes in water resultsin significant turbidity that may be utilized as a conve-nient and simple measure of the stability of the com-plexes toward albumin interaction. Figure 2 comparespLL/DNA and pHPMA-pLL/DNA in this assay. While theaddition of low concentration of albumin (<100 µg/mL)caused significant aggregation in uncoated complexes, inthe case of coated complexes, no turbidity was observedup to 11 mg/mL of albumin. This shows that the presenceof pHPMA stabilizes the complexes against albumininduced aggregation. However, the observed lack ofincreased turbidity of the coated complexes does notdirectly imply that albumin does not interact with thecomplexes. If albumin were simply to adsorb to thesurfaces of the complexes, no aggregation would beobserved, but it still could have an important effect onin vivo properties of the complexes.

Light-scattering techniques can contribute to betterevaluating the effect of albumin on the DNA complexes.Table 3 demonstrates the effect of an addition of albumin(1 mg/mL) on characteristics of uncoated and coated

complexes. The normalized intensity of scattered lightI90 from the solution of complexes is again used insteadof customary molecular weights because the concentra-tion of albumin possibly incorporated into DNA com-plexes is unknown. Comparing the data in Tables 1 and3, it can be seen that I90 increases several times and alsothe corresponding particle hydrodynamic radii increaseaccordingly in the case of uncoated pLL/DNA complexes.The average hydrodynamic radius of the uncoated com-plexes in the presence of 1 mg/mL of albumin increasedby about 8 nm compared with the original complex (Table3), but I90 increased almost 3 times, suggesting a largeincrease in molecular weight of the complexes due to theirinteraction with albumin. Dynamic light scattering offersalso a possibility of determining directly the distributionof hydrodynamic radii of the complexes, which is morecomplex information than average values. The effect ofalbumin on the distribution of hydrodynamic radii of theuncoated complexes is given in Figure 3. An addition ofalbumin causes a shift of whole radii distribution tohigher RH

a values. We assume that the shift of the sizedistribution is caused predominantly by albumin bindingon the surface of the complexes. NaCl addition results

Table 2. Effect of NaCl on Characteristics of Uncoatedand Coated pLL/DNA Complexesa

complex I90b I45/I135

c RHa (nm)d

pLL/DNA 29 1.79 46after 2 h 827pHPMA(5500)-pLL/DNA 44 1.77 53After 2 h 44 1.79 53After 5.5 h 46 1.76 55After 24 h 47 1.76 55pHPMA(8500)-pLL/DNA 45 1.64 54after 2 h 45 1.57 53after 5.5 h 47 1.56 58after 24 h 47 1.60 57a Complexes were formed in water and coated in 10 mM Hepes

(pH 7.8). NaCl was added after 12 h (final concentration 0.15 M).Changes of size and scattering intensities were measured 2, 5.5,and 24 h after NaCl addition. b Intensity of scattered light at 90normalized with the intensity scattered by benzene. c Ratio ofintensities of scattered light at 45 and 135°. d Hydrodynamicradius.

Figure 2. Albumin induced turbidity in the solution of pLL/ct-DNA complexes coated with pHPMA(8500) at the concentra-tion 24.6 nmol/mL. Albumin solution (30 mg/mL) was addedstepwise to the complexes and changes in turbidity weremonitored at 600 nm.

Table 3. Effect of Albumin and NaCl on Characteristicsof Coated and Uncoated pLL/DNA Complexesa

complex I90b I45/I135

c RHa (nm)d

pLL/DNA 29 1.79 46+albumin 86 1.69 53

+albumin + NaCl 327 5.1 199pHPMA(5500)-pLL/DNA 44 1.77 53

+albumin 56 1.73 55+albumin + NaCl 74 3.5 61

pHPMA(8500)-pLL/DNA 45 1.64 54+albumin 58 1.54 53

+albumin + NaCl 60 1.65 57a Complexes were formed in water and coated in 10 mM Hepes

(pH 7.8). After 12 h, albumin was added (final concentration 1mg/mL). Then NaCl (final concentration 0.15 M) was added to thesolution of the complexes and albumin and changes of size andscattering intensities were measured. b Intensity of scattered lightat 90° normalized with the intensity scattered by benzene. c Ratioof intensities of scattered light at 45 and 135°. d Hydrodynamicradius.

Figure 3. Effect of albumin and NaCl on distribution ofhydrodynamic radii A(RH

a) of uncoated pLL/DNA complexes: (1)complexes in water, (2) 0.3 h after addition of albumin (1 mg/mL), (3) 24 h after addition of albumin, (4) 5 h after addition ofNaCl (0.15 M) to albumin containing solution of complexes.


in aggregation of particles, which reflects in a shift ofradii distribution to higher values and broadening ofdistribution. The presence of free albumin molecules canbe identified with a small peak on A(RH

a) at low RHa

values.Only a minor increase in the scattering intensity and

size of the complexes was observed after albumin additionto the solution of coated complexes (Table 3 and Figure4). The scattering intensity and sizes of the complexeswere further increased when NaCl was added to thesolution of complexes in the presence of albumin. How-ever, whereas a marked increase was observed for theuncoated complexes, complexes coated with pHPMA-(8500) show only minor changes.

A small aggregation effect was observed, particularlyin the presence of both albumin and NaCl, in the solutionof the complexes coated with pHPMA(5500) demon-strated by an increase of both the intensity and hydro-dynamic radius in Table 3. The hydrodynamic radius ofcomplexes coated with pHPMA(8500) exhibited a notice-ably lower dependence on the albumin (1 mg/mL) andNaCl addition (Figure 4). Only small amount of ag-gregates could be detected after NaCl addition at highvalues of Rh

a (a small peak at high Rha values). Also

the increase of I90 was significantly lower than in caseof pHPMA(5500)-pLL/DNA. Thus, the pHPMA(8500)-pLL/DNA complexes were chosen for further study asthey appear to be more stable than these coated withpHPMA(5500).

Exchange Reaction between the Complexes, He-parin, and pLL. The effect of polymer coating on thestability of the complexes against polyelectrolyte ex-change reactions was studied in two systems. The abilityof DNA to be released from complexes was studied byincubation of the complexes with heparin and the abilityof pLL or pLL grafted with pHPMA to be replaced bycompeting polycation was studied by incubation with pLLof the same molecular weight.

The ability of the polyanion heparin sulfate to releaseplasmid DNA from its complexes with pLL was testedby monitoring appearance of free DNA by agarose gel

electrophoresis. Figure 5 shows that heparin was ableto release free DNA in both cases studied, i.e., in case ofboth uncoated and pHPMA-coated pLL/DNA complexes.Ethidium bromide staining did not allow quantificationof the amounts of DNA released in the two cases. Toaccomplish the quantitative analysis of DNA released,the DNA was spiked with 32P-labeled plasmid DNA andthe gel was analyzed with PhosphoImager. The analysisrevealed that 83% of DNA was released from the un-coated complexes. Under the same experimental condi-tions, 97% of DNA was released from the coated com-plexes.

Addition of pLL to the coated complexes in the presenceof 0.15 M NaCl resulted in immediate aggregation(Figure 6), although the coated complexes manifestedvery good stability against aggregation in salt (Table 2).The rate of the observed aggregation was found to be

Figure 4. Effect of albumin and NaCl on distribution ofhydrodynamic radii A(RH

a) of coated pHPMA(8500)-pLL/DNAcomplexes (24.6 nmol/mL pHPMA(8500)-NHS): (1) complexesin distilled water; (2) 0.3 h after addition of albumin (1 mg/mL); (3) 24 h after addition of albumin, (4) 5 h after addition ofNaCl (0.15 M) to albumin containing solution of complexes.

Figure 5. Agarose gel electrophoresis (0.8% gel; 0.5 µg/mLethidium bromide) of pLL/plasmid DNA and pHPMA(8500)-pLL/plasmid DNA complexes incubated with heparin. Lane 1, freeDNA; lane 2, pLL/DNA complexes; lane 3, pLL/DNA + heparin;lane 4, pHPMA-pLL/DNA; lane 5, pHPMA-pLL/DNA + heparin.

Figure 6. Aggregation of pLL/ct-DNA and pHPMA-pLL/ct-DNA complexes induced by an addition of pLL. pLL/DNAcomplexes were coated with pHPMA(8500) at two concentra-tions: 24.6 nmol/mL (pHPMA-pLL/DNA[1]) and 74 nmol/mL(pHPMA-pLL/DNA[2]). The coating reaction was left to proceedovernight. Then to the solution of the coated complexes NaClwas added (final concentration 0.15 M) followed by the additionof pLLsfinal concentration of pLL including pLL alreadypresent in the complexess35 µg/mL pLL(a); 70 µg/mL pLL(b).


dependent on the amount of pHPMA(8500) used for thecoating and the concentration of free pLL added to thecoated complexes (Figure 6). In this assay, the moststable complexes were those coated with higher concen-tration of pHPMA and the aggregation was also morerapid in the presence of higher concentrations of pLL.

In Vitro Association of the Complexes with Mac-rophages. Mouse peritoneal macrophages were used asan in vitro model for the study of the effect of pHPMA-(8500) coating on the association with phagocytic cells.

Cell association of YOYO-1 labeled complexes wasmonitored using primary cultures of mouse peritonealmacrophages incubated in 50% fetal calf serum (Figure7). Two concentrations of pHPMA(8500) were used forthe coating; however, no obvious effect was observed.Analysis by FACS showed a significant decrease incomplex association (∼10 times) for both types of coatedcomplexes in the whole time course of the study.

In Vivo Biodistribution of the Complexes. Re-duced macrophage uptake in vitro encouraged furtherinvestigation of the coated complexes in vivo as it mightbe expected they show prolonged circulation times in theblood. The body distribution of the coated and uncoatedpLL/DNA complexes containing 32P-radiolabeled DNAwas determined following intravenous injection intoBALB/c mice using both plasmid and ct-DNA.

pLL/ct-DNA complexes were cleared rapidly from thebloodstream showing less than 10% of radioactivity inthe blood after 30 min (Figure 8). Coating of thesecomplexes with pHPMA(8500) resulted in even loweramount of radioactivity found in the blood (∼3%). In bothcases, there was some deposition in the carcass (∼15%)and spleen (∼10%), but the majority of radioactivity (over60% of the recovered dose) was found in the liver. Thecoating of the complexes promoted even higher uptakeby the liver, and consequently, reduced levels of radio-activity were found in all the organs isolated.

A similar experiment with plasmid DNA complexes(Figure 9) showed significantly higher levels of uncoatedcomplexes remaining in the blood after 30 min (24.9 (4.1%). Coating of these complexes led again to a drop ofthe level of radioactivity in the blood to the levels similarto those found for coated ct-DNA complexes. The coatingof the complexes led also to an increase in the liveruptake compared to uncoated complexes. Increasing theinjected dose from 4 to 24 µg of DNA (using concentrated

coated complexes) had no significant effect on the levelsof radioactivity found in the blood or the biodistributionpattern.

DISCUSSION

In this study, we have examined properties of pLL/DNA complexes coated with semitelechelic hydrophilicpolymer pHPMA. pHPMA is known to be a nontoxic andnonimmunogenic polymer that was previously used inthe field of drug delivery as a carrier of low-molecular-weight drugs (Duncan and Ulbrich, 1993), for surfacemodification of proteins (Oupicky et al., 1999c) and alsoin combination with cationic polymers for the formationof DNA complexes (Oupicky et al., 1999b). The propertiesof this polymer are in many ways similar to those of pEG.However, it has significant advantages in terms of itsstructural versatility enabling easy incorporation ofvarious functionalities.

pLL/DNA complexes are usually prepared in waterbecause of their tendency to aggregate in salt solutions.Several possible means of increasing stability of thesecomplexes in salt solutions have been reported so far.Among them, using block and graft copolymers with

Figure 7. Cellular association of pLL/plasmid DNA complexescoated with pHPMA(8500) with murine peritoneal macrophagesafter incubation in 50% fetal calf serum. The increase in themean fluorescence of the cells, assessed by flow cytometricanalysis, was used as the measure of cellular association. ThepLL/DNA complexes were coated at 24.6 and 74 nmol/mL ofpHPMA(8500) (mean ( sd; n ) 3).

Figure 8. Body distribution of radioactivity determined in mice30 min following intravenous injection of pLL/ct-DNA andpHPMA-pLL/ct-DNA [pHPMA(8500), 24.6 nmol/mL] coatedcomplexes containing 32P-labeled DNA. The complexes wereadministered at a dose of 4 µg of DNA/mouse in 5% glucose intothe tail vein (mean ( sd; n ) 3).

Figure 9. Body distribution of radioactivity determined in mice30 min following intravenous injection of pLL/plasmid DNA orpHPMA-pLL/plasmid DNA [pHPMA(8500), 24.6 nmol/mL] coatedcomplexes containing 32P-labeled DNA. The complexes wereadministered at a dose of 4 µg of DNA (pLL/DNA and pHPMA-pLL/DNA[1]) or 24 µg of DNA (pHPMA-pLL/DNA[2])/mouse in5% glucose into the tail vein (mean ( sd; n ) 3).


hydrophilic nonionic polymers found widespread use(Kabanov and Kabanov, 1995; Katayose and Kataoka,1997; Seymour et al., 1998; Wolfert et al., 1996).

Another way of increasing their stability, known incolloid science as a steric stabilization, is to provide thecomplexes with a surface polymer layer. Such a layer cansignificantly affect interparticle forces; first, it can influ-ence van der Waals attractive forces, and second, it cangive rise to repulsion between the particles. The magni-tude of repulsion arising from the presence of theadsorbed layer clearly depends on the density with whichit covers the surface; the more thinly it is spread, thesmaller its effectiveness in preventing the particlesfrom approaching one another (Everett, 1988). Theconcept of steric stabilization has been exploited in thefield of drug delivery as a way of preventing the particlesfrom aggregation but also as a way of reducing theirinteractions with opsonins and components of RES whicheffectively increased circulation times of these particles(Davis and Illum, 1988). A similar approach has beenreported only recently also for DNA complexes. It wasreported that surface modification of DNA/polyethylene-imine complexes with pEG (5000 g/mol) results in anincreased level of the complexes found in plasma 30 minafter injection (∼30% of the injected dose) (Ogris et al.,1999).

The coating reaction with succinimidyl reactive estersof pHPMA requires the presence of free amino groupsaccessible on the surface of the complexes. pLL/DNAcomplexes of the composition used in this study werealready shown to bear surface positive charge (Wolfertet al., 1996). The presence of free amino groups suitablefor reaction with acylating agents was also demonstratedby fluorescamine assay (Read et al., 1999).

The coating reaction proceeded under mild conditionsleaving the complexes with slightly increased size andmolecular weight due to the covalent binding of pHPMA.The morphology of the complexes was not changed asjudged by TEM, and the complexes were still of roughlyspherical shape.

Testing the stability of the coated complexes insalt showed steric stabilization using pHPMA of bothmolecular weights (5500 and 8500) and also usingpHPMA(8500) at two different concentrations. The coat-ing polymer reduced hydrophobic character of pLL/DNAcomplexes as demonstrated by their increased solubilityin salts and also by successful preparation of concen-trated solutions (containing 120 µg/mL DNA). DNAcomplexes designed for use as in vivo gene deliverysystems should be stable in the body fluids, especially inthe blood. We have studied the interaction of the com-plexes with serum albumin as a model serum protein.This can provide a useful indication of the changes inthe properties of the complexes achieved by their modi-fication with pHPMA. Studying the interaction withalbumin can be important as it is likely that albumin istemporally the first component of blood binding similarparticulate systems (Davis and Illum, 1988).

Albumin is able to bind to the pLL/DNA complexesforming ternary complexes (Dash et al., 1999). At acertain ratio, the ternary complexes are hydrophobic andexcessive aggregation could be observed. The albumin-induced turbidity assay showed that coating the pLL/DNA complexes with pHPMA(8500) prevented albuminfrom causing such an aggregation of the complexes. Moresensitive analysis of the interaction of the complexes withalbumin (1 mg/mL) by static light scattering demon-strated similar results for uncoated pLL/DNA complexes,but also showed an increase in molecular weight of the

coated complexes after incubation with albumin despiteno simultaneous significant increase in their size. Thisresult suggests that albumin can probably still bind tothe coated complexes, although the binding is signifi-cantly reduced. Comparison of pHPMA with two differentmolecular weights showed that the higher molecularweight analogue is more effective in stabilization of thecomplexes against interaction with albumin especially inthe presence of NaCl. Albumin was able to reduce theaggregation of pLL/DNA complexes, acting as a surfacestabilizer (compare Tables 2 and 3). This suggests thatalthough the uncoated pLL/DNA complexes showed onlya low stability in salt solution or in low concentrationsof albumin, their size could be well stabilized in thepresence of much higher concentrations of proteins in theblood plasma.

Coating of the complexes with pHPMA may affect therelease of free DNA in two ways. It might stabilize thecomplexes by preventing heparin from coming into acontact with pLL/DNA part of the coated complexes.However, study of the effects of the coating on polyelec-trolyte exchange reactions showed that the surfacepolymer layer of pHPMA did not stabilize the complexesagainst replacing of DNA from the complexes. Moreover,quantitative analysis suggested that the coated com-plexes are even more susceptible to the exchange reactionwith heparin than uncoated ones.

Studying similar type of exchange reaction using pLLadded to the solution of the coated complexes, we haveobserved fast aggregation of the coated complexes in saltsolution. The most likely explanation for the observedtype of behavior is the exchange reaction of the addedfree pLL with surface pLL molecules of the complexgrafted with pHPMA. As a result, complexes similar touncoated pLL/DNA complexes are formed. The pHPMAgrafted on pLL located on the surface of the complexesduring coating reaction provided the complexes withincreased stability against aggregation in salt but thesegrafted pLL molecules could be apparently replaced bycompeting pLL, demonstrating equilibrium nature of thepolyelectrolyte complexes and also their tendency toprecipitation, which is the preferred state of the com-plexes. These results show that while pHPMA is veryeffective in preventing the particles from aggregation, itcannot effectively block the access of single molecules(like heparin or pLL) to the core of the complexes asmanifested by the ease of the exchange reactions.

To address the possibility that the coated complexesmay be able to evade phagocytic capture, in vitro modelrepresenting phagocytic components of the RES wasemployed, in the form of mouse peritoneal macrophages.The substantial levels of cell-association shown by pLL/DNA complexes may reflect their net positive surfacecharge, promoting nonspecific association with cells andmembranes as well as their hydrophobic nature and easybinding of various proteins that can promote the uptake.The particulate nature of pLL/DNA complexes may alsopromote physical attachment and subsequent phagocy-tosis. Coated complexes showed significantly decreasedlevels of capture possibly due to their higher hydrophi-licity manifested by increased stability against saltinduced precipitation. This may inhibit complex-cellattachment leading to a decrease in cell capture. Coatingof the complexes by pHPMA may also create a physicalbarrier between the complex and cell surface resultingin steric hindrance analogous to stealth technology (Lasicet al., 1991) leading to reduced uptake.

In attempting to design systems capable of betterpharmacokinetics, we have examined the biodistribution


of the pHPMA-coated and uncoated pLL/DNA complexes.There are several possible mechanisms that might con-tribute to the rapid blood clearance of pLL/DNA com-plexes, including rapid degradation by nucleases, acti-vation of complement, opsonisation, and phagocytosis orfiltering by capillary beds. However, it has been shownpreviously that DNA in the polyelectrolyte complexes isnot degraded by nucleases and also no complementactivation was observed for pLL/DNA complexes. Bindingof plasma proteins was proposed as the most likely reasonfor the fast removal of pLL/DNA complexes from theblood (Dash et al., 1999). The injected complexes quicklybind albumin and other plasma proteins, forming anextended ternary complex with a net negative surfacecharge. This ternary complex still resists immediatedegradation by serum nucleases but may promotephagocytic capture, particularly by Kupffer cells. We haveshown in this study that complexes coated with semi-telechelic pHPMA have demonstrated certain improvedproperties (increased stability in salt, against albumininduced aggregation, decreased albumin binding, andreduced phagocytic uptake) but these complexes have not,however, showed any prolongation in the circulatorytimes (Figures 6 and 7). On the contrary, in both casesstudied (plasmid and ct-DNA), the polymer-coated com-plexes showed even lower levels in the blood after 30 minpostinjection. The most remarkable difference was ob-served in case of plasmid pLL/DNA complexessdecreasefrom 25% (uncoated) to 3% (pHPMA-coated) of thecomplexes found in the blood after 30 min. The high levelof plasmid pLL/DNA complexes found in the bloodcompare to ct-DNA complexes is a surprising finding andthe reasons for such a difference are currently underinvestigation.

Despite all the improvements of properties of thecomplexes observed in in vitro experiments their in vivobehavior has not improved correspondingly. This sug-gests that the in vivo fate of the complexes may besignificantly influenced by factors not reflected in the invitro cell culture model. Among them high concentrationsand wide variety of proteins and other molecules inplasma, together with contact with cell surfaces andenormous shear stress in the blood circulation may playan important role.

We propose that the equilibrium nature of the coatedpLL/DNA complexes (manifested in the polyelectrolyteexchange reactions) might be one of the main reasonsfor their fast removal from the blood. The morphology ofthe coated complexes can be changed, as the pLLmolecules grafted with pHPMA may be removed from thecomplexes quickly following entry serum leaving thecomplexes unprotected. An alternative explanation isconceivable, namely that higher doses [closer to the dosesused in other particulate drug delivery systems (Oja etal., 1996)] are necessary in in vivo experiments to at leastpartly saturate RES.

There are several possible approaches to furtherstabilizing pLL/DNA complexes against polyelectrolyteexchange reactions. Several are being currently devel-oped in our laboratories including enshrouding thecomplex with multifunctional hydrophilic polymer cross-linking the surface of the complexes (Dash et al., 2000).

ACKNOWLEDGMENT

The support of the EU by Grant ERBIC20CT97005 andof the Grant Agency of the Czech Republic by Grant 307/96/K226 is gratefully acknowledged.

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