DNA Complexes by the Covalent

Recent advances in rational gene transfer vector design based on ... Hydrophobized dextran-spermine conjugate as potential vector for in vitro gene tr...
1 downloads 0 Views 377KB Size
492

Bioconjugate Chem. 2000, 11, 492−501

Steric Stabilization of poly-L-Lysine/DNA Complexes by the Covalent Attachment of Semitelechelic poly[N-(2-Hydroxypropyl)methacrylamide] David Oupicky´,*,† Kenneth A. Howard,† C ˇ estmı´r Konˇa´k,‡ Philip R. Dash,† Karel Ulbrich,‡ and Leonard W. Seymour† CRC Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TA, U.K., and Institute of Macromolecular 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)methacrylamide] (pHPMA). We have examined the effect of coating of the complexes with pHPMA on their physicochemical stability, phagocytic uptake in vitro, and biodistribution in vivo. The coated complexes showed stability against aggregation in 0.15 M NaCl and reduced binding of albumin, chosen as a model for the study of the interactions of the complexes with plasma proteins. The presence of coating pHPMA had no effect on the morphology of the complexes as shown by transmission electron microscopy. However, results of the study of polyelectrolyte exchange reactions with heparin and pLL suggested decreased stability of the coated complexes in these types of reactions compared to uncoated pLL/DNA complexes. Coated complexes showed decreased phagocytic capture by mouse peritoneal macrophages in vitro. Decreased phagocytosis in vitro, however, did not correlate with results of in vivo study in mice showing no reduction in the liver uptake and no increase in the circulation times in the blood. We propose that the rapid plasma elimination of coated pLL/DNA complexes is a result of binding serum proteins and also of their low stability toward polyelectrolyte exchange reactions as a consequence of their equilibrium nature.

INTRODUCTION

Polyelectrolyte polycation/DNA complexes are attracting considerable attention as a promising synthetic vector for gene delivery. However, despite substantial progress and considerable versatility and transfectional activity in vitro, simple polyelectrolyte gene delivery systems are quickly eliminated from the bloodstream following intravenous injection (plasma half-life typically less than 5 min) (Dash et al., 1999). The complexes are usually cleared quickly into the liver or spleen. Patterns of distribution in vivo indicate significant phagocytic capture by components of the reticuloendothelial system (RES), notably by tissue macrophages such as Kupffer cells (Dash et al., 1999). Rapid localization of DNA complexes in the RES (liver, spleen) prevents their targeting to non-RES tissues in a specific manner and results, therefore, in a severe limitation of their therapeutic potential. For targeted systemic delivery, a more prolonged plasma circulation of the vector is essential. It is, therefore, a central aim to decrease uptake by the RES in order to develop polymer-based DNA complexes with improved circulation times. In the design of long circulating DNA/polyelectrolyte complexes for gene delivery, we can be inspired by experiences obtained in the fields of similar particulate systems for drug delivery, notably liposomes and polymeric nanoparticles (Bazile et al., 1992; Illum et al., 1987; Lasic and Needham, 1995). * To whom correspondence should be addressed. Phone: +44121-414-3291. Fax: +44-121-414-3263. E-mail: davido@ cancer.bham.ac.uk. † CRC Institute for Cancer Studies. ‡ Institute of Macromolecular Chemistry.

The interaction of similar particulate drug delivery systems with the cells of the RES is determined to a great extent by physicochemical properties of their surface and their size with larger particles being taken up faster (Moghimi, 1995; Monfardini and Veronese, 1998). The initial stages of phagocytosis involve the physical attachment of the particulate to the surface of the macrophage. An increase in particulate hydrophobicity is known to increase uptake by forming hydrophobic interactions between the particulate and the cell surface (Monfardini and Veronese, 1998), while particles with polar and charged surfaces have an increased circulation time and reduced uptake by RES organs (Gabizon and Papahadjopoulos, 1992). To avoid RES capture, it is, therefore, important to reduce the hydophobicity of the particles; however, it is known that materials with strong positive surface charge bind biological membranes nonspecifically, while those with strong negative charge can be substrates for phagocytosis via the macrophage polyanion receptor (Roser et al., 1998). Hence, it may also be important to avoid significant surface charge of the DNA complexes to prolong blood circulation. Apart from nonspecific uptake of the particles by RES simply as a result of their physicochemical properties, adsorption of blood components onto particles also plays an important role. The blood contains a large number of proteins and glycoproteins that can function as opsonins (Moghimi, 1995). Rapid binding of one or more of these materials onto the complexes can occur and may have significant influence on the fate of the particulate. The first component adsorbed to the surface of positively charged complexes will probably be albumin, but other materials present in lower concentrations with higher

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

Steric Stabilization of poly-L-Lysine/DNA Complexes

affinities for the surface will displace the first adsorbed layer as a function of time (Davis and Illum, 1988). One of the most successful strategies for obtaining longcirculating nanoparticles and liposomes has been polymer attachment [usually poly(ethylene glycol) (pEG)] at the particle surface, for creating a hydrophilic cloud reducing interactions with proteins and cells (steric stabilization). This so-called stealth technology has been successful in liposomes and nano- and microparticulate drug delivery systems (Lasic, 1997; Monfardini and Veronese, 1998). The concept of using block or graft copolymers of cationic and hydrophilic nonionic monomers has been introduced by several groups as a new potential way in development of self-assembling polyelectrolyte gene delivery vectors (Katayose and Kataoka, 1997; Toncheva et al., 1998; Wolfert et al., 1996). The aim was to increase the hydrophilic character of the complexes and at the same time introduce a surface hydrophilic layer acting in a similar way as in stealth liposomes. However, in vivo biodistribution studies showed that these complexes are removed very quickly from the murine bloodstream similar to the complexes between DNA and simple polycations (Oupicky et al., 1999a). Unsuitable morphology of the complexes and protein binding at serum protein concentrations were proposed as the major reasons for their rapid removal (Oupicky et al., 1999a). A particular problem with this approach may reflect thermodynamic instability of the complexes resulting from necessary entrapment of hydrophilic chains of the block and graft copolymers within the polyelectrolyte core of the complex. To address this possibility and to examine its effect on the stability of the otherwise-hydrophobic core, in this study, we have allowed simple polyelectrolyte complexes to self-assemble before covalent linkage of the hydrophilic polymer, only to the surface of the polyelectrolyte particle. Although significant attention is paid to the development of nonviral polyelectrolyte systems for gene delivery, most of the published works concentrate on in vitro study of the properties of the complexes. Relatively little information is available about suitability of these complexes for in vivo use (Chemin et al., 1998; Hashida et al., 1998; Laurent et al., 1999; Ogris et al., 1999). In this work, we therefore report the study of stability of the polyelectrolyte complexes of DNA with poly-L-lysine coated with hydrophilic pHPMA chains in aqueous NaCl solutions and their interaction with albumin. The results of the phagocytic uptake of pLL/DNA complexes by mouse peritoneal macrophages are discussed together with the biodistribution analysis in mice performed in order to obtain more information on the effect of the hydrophilic pHPMA coating on biological properties of complexes. EXPERIMENTAL PROCEDURES

Materials. A circular 5.2 kb expression vector containing a SV40 promoter-driven luciferase reporter gene and ampicilin resistance was used in most experiments. This was prepared by growth in Escherichia coli DH5R and purified using Qiagen columns (Dorkin, U.K.). Following the final ethanol precipitation, the purity of the DNA was checked by agarose gel electrophoresis and 260/280 nm absorption. In the rest of the experiments, calf thymus DNA (ct-DNA) (sodium salt) was used. ct-DNA and polyL-lysine hydrobromide (pLL) (molecular weight 19 600) were from Sigma Chemical Co. Semitelechelic poly[N(2-hydroxypropyl)methacrylamide]s with carboxylic acid end group were prepared by radical polymerization as described in (Oupicky et al., 1999b).

Bioconjugate Chem., Vol. 11, No. 4, 2000 493

Formation of pLL/DNA Complexes and Their Coating with poly(HPMA). All complexes in this study were prepared in water at DNA concentration 20 µg/mL (phosphate group concentration 61.5 nmol/mL). pLL was added to DNA solution in a single addition so the N:P ratio was 1.2 (pLL amino group concentration 73.8 nmol/ mL). Freshly prepared solution of N-hydroxysuccinimidyl ester of carboxylic acid group terminated pHPMA (pHPMA-NHS) in water was then added to the complex solution followed by addition of Hepes buffer (pH 7.8) to reach final concentration 10 mM. The reaction was carried out at room temperature for at least 3 h before any other experiment. Two different molecular weights (number average) of pHPMA (8500 and 5000) and two different concentrations of them were used in the coating reaction (24.6 and 74 nmol/mL). For the study of the dose effect on in vivo biodistribution, the coated complexes (pHPMA-pLL/DNA) were concentrated on Centricon-100 filters (Millipore). The increase of concentration to 120 µg/mL (determined by UV absorption) was achieved without any significant change in the size of the complexes. Albumin-Induced Turbidity Assay. The ability of albumin (bovine serum albumin, Sigma) to cause the aggregation of the complexes was examined using a Perkin-Elmer (Buckinghamshire, U.K.) LS50B fluorimeter. The coated or uncoated complexes were both in 10 mM Hepes (pH 7.8). Albumin solution (30 mg/mL) was added stepwise to the complexes and changes in turbidity were monitored. Studies were performed at emission and excitation wavelengths set to the same value of 600 nm. Polylysine-Induced Aggregation of Coated Complexes. The ability of pLL (19.6 kg/mol) to cause the aggregation of pHPMA-coated pLL/DNA complexes was monitored using commercial light-scattering equipment (Zetasizer, Malvern Instruments, U.K.). To the solution of coated complexes [prepared 24 h before the experiment using two different concentrations of pHPMA(8500)], two different amounts of pLL (final total concentration of pLL 30 and 75 µg/mL, i.e., 144.2 and 361 nmol/mL of pLL amino groups) were added to the solution immediately after an addition of NaCl (final concentration 0.15 M) and the changes in the size of the complexes were monitored. Heparin Release of DNA. Heparin solution (12 µL, Heparin Sodium, Multiparin, 1000 units/mL; CP Pharmaceuticals Ltd. Wrexham, U.K.) was added to 15 µL of the solution of coated or uncoated complexes and left to incubate for 1 h. The complexes were then analyzed on 0.8% agarose gel (TBE buffer, 0.5 µg/mL ethidium bromide, 90 V, 45 min). To permit also quantitative analysis, DNA was spiked with 32P-radiolabeled DNA as described below. The amount of DNA released was quantified using a Phosphoimager (Molecular Dynamics, Chesham, U.K.). Transmission Electron Microscopy. The complexes were prepared in water at final DNA concentration of 20 µg/mL. A total of 10 µL of the solution was dispensed onto paraffin, and a piece of freshly cleaved mica (Agar Scientific, 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 mica was then washed twice in double-distilled water and allowed to dry before the sample was visualized by rotary shadowing 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 into sections and floated on water to release the sample that

494 Bioconjugate Chem., Vol. 11, No. 4, 2000

was then picked up using sticky grids (carbon 200 mesh) and allowed to dry. The samples were then viewed using a JEOL 1200 EX Transmission Electron Microscope. Static Light Scattering (SLS). Static light-scattering measurements were performed with a commercial Fica 40 apparatus in vertically polarized light at wavelength 632.8 nm and temperature 25 °C. The apparatus was calibrated with benzene at 90°. The intensities of scattered light at 45° (I45), 90° (I90), and 135° (I135) have been measured for all the samples studied. Since the concentrations of particles and their refractive increments are not known, the intensity I90 normalized with intensity scattered by benzene at 90° is given for a qualitative discussion of results (I90 is roughly proportional to the weight-average molecular-weight of particles). Disymmetries I45/I135 are used as a qualitative measure of particle sizes (radius of gyration). Dynamic Light Scattering (DLS). Polarized DLS measurements were made in the angular range 30-135° using a light-scattering apparatus equipped with an HeNe (632.8 nm) and Ar-ion laser (514.5 nm) and an ALV 5000, multibit, multi-tau autocorrelator covering approximately 10 decades in delay time. Most of the measurements were realized at the scattering angle 90°. The inverse Laplace transform using the REPES (Jakes, 1995) method of constrained regularization is similar in many respects to the inversion routine CONTIN (Provencher, 1979). However, REPES directly minimizes the sum of the squared differences between the experimental and calculated intensity time correlation functions using nonlinear programming. This method uses an equidistant logarithmic grid with fixed components (here a grid 10 components/decade) and determines their amplitudes. As a result, a distribution function A(τ) of decay times is obtained. From the characteristic decay time, τ [the peak positions of A(τ)], the corresponding apparent average diffusion coefficient, D(90°), was calculated. The apparent average hydrodynamic radius, RHa, was calculated from D(90°) using the Stokes-Einstein equation (viscosity of water at 25 °C 0.894 cP), and the distribution of decay times A(τ) was recalculated on the distribution of hydrodynamic radii A(RHa). 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 mW external laser was used. In Vitro Association of Complexes with Mouse Macrophages. Adult female BALB/c mice (25 g) were killed by cervical dislocation and injected intraperitoneally, under sterile conditions, with 5 mL of medium 199 containing 20% fetal calf serum (Gibco, Paisley, U.K.). The abdomen was then agitated gently, the peritoneum exposed and breached, and the medium removed using a syringe. The medium was then centrifuged (500g, 10 min) and the pellet resuspended in medium 199 containing 50% fetal calf serum. The cell suspension was then plated out into multiwell 6-well plates (480 000 cells/well). The macrophages were allowed to adhere for 2 h before medium (containing nonadherent cells) was removed. Fresh medium containing Amphotericin B (Sigma Chemical Co., Poole, U.K., 2.5 µg/mL), Penicillin, and Streptamycin (Sigma Chemical Co., Poole, U.K., 100 units/mL, 0.1 mg/mL, respectively) was then added to the cells. After 40 h, the cells were used in cellular association studies. The complexes were formed with YOYO-1 labeled plasmid DNA (one YOYO-1 molecule/300 bp) using the same protocol as described above. Complexes were added

Oupicky´ et al. Table 1. Characteristics of Coated and Uncoated pLL/ DNA Complexes complex

I90a

I45/I135b

RHa (nm)c

pLL/DNA pHPMA(5500)-pLL/DNA pHPMA(8500)-pLL/DNA

29 44 45

1.79 1.77 1.64

46 53 54

a Intensity of scattered light at 90° normalized with the intensity scattered by benzene. b Ratio of intensities of scattered light at 45 and 135°. c Hydrodynamic radius.

at a DNA concentration of 20 µg/mL to plates containing adherent mouse macrophages in 50% 199 media, 50% FCS (75 µL of complexes/well). The complexes were added for various periods of time (0.5-1.5-3 h) after which the cells were removed by standard trypsin treatment, washed, and resuspended in PBS. Association of the complexes with cells was determined by the amount of YOYO-1/DNA detected with a Coulter EPICS XL flow cytometer using an argon ion laser set for excitation 488 nm and emission 520 nm. Cell groups were defined to exclude debris and duplets, and each cell type was gated individually against a negative control of cells without addition of complexes. Radiolabeling of DNA Expression Vectors. Plasmid DNA was linearized with HindIII and then radiolabeled with [32P]dCTP using the Ready-to-Go Oligolabeling kit (Pharmacia Biotech, St. Albans, U.K.). Unincorporated nucleotides were removed using MicroSpin columns (Pharmacia Biotech), and the purity of the labeled DNA was checked following agarose gel electrophoresis and quantitative analysis with a PhosphoImager (Molecular Dynamics, Chesham, U.K.). In Vivo Biodistribution Study. pLL/DNA and pHPMA-pLL/DNA complexes were formed in water 12 h before the experiment using either ct-DNA or plasmid DNA containing a spike of 32P-labeled DNA. Before injection, glucose was added to a final concentration of 5% (w/v) to ensure iso-osmolarity. Complexes (0.2 mL) were administered via the tail vein to briefly anaesthetized 6-week-old female BALB/c mice. After 30 min, the animals were killed and dissected to permit determination of radioactivity distribution. The blood, lungs, liver, spleen, kidneys, intestines, and carcass (with tails being removed) were isolated, weighed, and dissolved in 10 M sodium hydroxide at 75 °C. Scintillation medium (20 mL) was added to each blood and tissue sample (1 mL) before geometry-corrected analysis of their contained radioactivity in a Packard scintillation counter. A bloodstream volume of 8.64 mL/100 g body weight was assumed (Seymour et al., 1991). RESULTS

Coating of DNA Complexes and Their Morphology. The complexes were formed using only a slight excess of pLL (N:P ratio 1.2; ζ potential +20 mV) in order to limit the presence of free pLL, which would also react with pHPMA-NHS in the coating reaction. It has been shown that pLL/DNA complexes of this composition contain only a negligible amount of free pLL (Ward et al., 1999). On the other hand, using such a relatively low charge ratio results in complexes that are more susceptible to salt-induced aggregation. The coating reaction was therefore conducted in 10 mM Hepes buffer, pH 7.8. Under these conditions, only negligible aggregation of the complexes was observed during the coating reaction. The characteristics of the pLL/DNA complexes coated with pHPMA-NHS having number-average molecular weight 5500 and 8500, respectively, are given in Table 1. The

Steric Stabilization of poly-L-Lysine/DNA Complexes

Bioconjugate Chem., Vol. 11, No. 4, 2000 495

Figure 1. Transmission electron micrographs showing the morphology of plasmid and ct-DNA complexes coated with semitelechelic pHPMA(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) pHPMApLL/plasmid DNA (74 nmol/mL pHPMA-NHS); (50000× magnification).

coating slightly increased the hydrodynamic radius of complexes (1.17 times), and the scattered intensity (I90) (proportional to molecular weight of particles) was also correspondingly increased (1.55 times). This increase shows that the molecular weight of pLL/DNA complexes is roughly proportional to RH2.7 of the complexes which is close to the model of solid spheres (exponent 3). Incubation of pLL/DNA complexes with pHPMA containing a nonreactive carboxylic acid end group did not cause any change in their size or molecular weight, confirming that the observed changes of particle parameters (Table 1) indicate covalent attachment of pHPMA-NHS to the pLL/DNA complexes. The covalent attachment of pHPMANHS was confirmed also by the decrease of amino group content [determined by TNBSA assay (Snyder and Sobocinski, 1975)] in pLL/DNA complexes after their coating with pHPMA-NHS. The data in Table 1 also demonstrate higher sensitivity of static light scattering methods compare to DLS and shows its importance in the study of these high-molecular-weight systems.

The idea of modifying pLL/DNA complexes with semitelechelic pHPMA-NHS assumes binding of the polymer to the surface of the complexes with little or no disturbance of the actual pLL/DNA complexes. However, the coating (especially when using high excess of coating polymer) may induce undesired changes in the morphology of the complexes. Figure 1 shows TEM pictures of pLL/DNA before and after coating with two different amounts of pHPMA(8500)-NHS. In no case was there a sign of any change in the morphology of the complexes. The complexes retained their spherical shape and also their diameter of about 100 nm. Stability of Complexes in Salt. A combination of static and dynamic light scattering techniques was also used to examine the stability of complexes in 0.15 M NaCl. Table 2 shows the results of the stability measurements. The uncoated pLL/DNA complexes precipitated very fast, forming large aggregates almost immediately after the addition of NaCl, while the coated complexes were stable for at least 24 h. The scattered intensity I90

496 Bioconjugate Chem., Vol. 11, No. 4, 2000

Oupicky´ et al.

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

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

complex

I90b

I45/I135c

RHa (nm)d

complex

I90b

I45/I135c

RHa (nm)d

pLL/DNA after 2 h pHPMA(5500)-pLL/DNA After 2 h After 5.5 h After 24 h pHPMA(8500)-pLL/DNA after 2 h after 5.5 h after 24 h

29

1.79

44 44 46 47 45 45 47 47

1.77 1.79 1.76 1.76 1.64 1.57 1.56 1.60

46 827 53 53 55 55 54 53 58 57

pLL/DNA +albumin +albumin + NaCl pHPMA(5500)-pLL/DNA +albumin +albumin + NaCl pHPMA(8500)-pLL/DNA +albumin +albumin + NaCl

29 86 327 44 56 74 45 58 60

1.79 1.69 5.1 1.77 1.73 3.5 1.64 1.54 1.65

46 53 199 53 55 61 54 53 57

a

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 90 normalized with the intensity scattered by benzene. c Ratio of intensities of scattered light at 45 and 135°. d Hydrodynamic radius.

a Complexes were formed in water and coated in 10 mM Hepes (pH 7.8). After 12 h, albumin was added (final concentration 1 mg/mL). Then NaCl (final concentration 0.15 M) was added to the solution of the complexes and albumin and changes of size and scattering intensities were measured. b Intensity of scattered light at 90° normalized with the intensity scattered by benzene. c Ratio of intensities of scattered light at 45 and 135°. d Hydrodynamic radius.

Figure 2. Albumin induced turbidity in the solution of pLL/ ct-DNA complexes coated with pHPMA(8500) at the concentration 24.6 nmol/mL. Albumin solution (30 mg/mL) was added stepwise to the complexes and changes in turbidity were monitored at 600 nm.

of the coated complexes increased only by a few percent showing practically negligible aggregation. Neither types of parameters used for characterization of size of the complexes showed any significant change. This observation not only suggests the absence of aggregation but also indicates that no change in morphology of the complexes was induced at this stage (Provencher, 1979). Interaction of Complexes with Albumin. The addition of albumin to pLL/DNA complexes in water results in significant turbidity that may be utilized as a convenient and simple measure of the stability of the complexes toward albumin interaction. Figure 2 compares pLL/DNA and pHPMA-pLL/DNA in this assay. While the addition of low concentration of albumin (