J O U R N A L A publication of the American Pharmaceutical Association and the American Chemical Society
O F
Pharmaceutical
Sciences
November 1995 Volume 84. Number 11
RESEARCH ARTICLES
Physicochemical and Pharmacokinetic Characteristics of Plasmid DNA/ Cationic Liposome Complexes RAM I. MAHATO,KENJIKAWABATA, TAKEHIKO NOMURA, YOSHINOBU TAKAKURA, AND MITSURUHASHIDA~ Received June 23, 1995, from the Depattment of Drug Delivety Research, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-01,Japan. Accepted for publication August 25, 1995@. Abstract 0 The objectives of this study were (i) to characterize the plasmid DNA encoding the chloramphenicol acetyltransferase reporter gene (pCAT) complexed with cationic liposomes (Lipofectin and LipofectACE) in terms of particle size and 5 potential, (ii) to compare pharmacokinetic characteristics, and (iii) to study the hepatic uptake mechanisms. DNAlLipofectACEcomplexes showed a negative 5 potential of -36 mV at 1:5 wlw ratio, but a positive 5 potential of 19 mV at 1:lO w/w ratio. Lipofectin samples showed a positive 5 potential of 21-28 mV at these ratios. These preparations showed a wide particle size distribution ranging from 600 to 1200 nm. Following intravenous injection of 1:lO w/w ratio [3zP]pCAT/liposomecomplexes at a dose of 0.1 mg DNAlkg into the tail vein of mice, radioactivity was rapidly eliminated from the plasma and almost 50-60% of the dose was taken up by the liver within 5 min after administration. Plasmid DNAfliposome complexes were predominantly taken up by the liver nonparenchymal cells. The hepatic uptake was inhibited by preceding administration of dextran sulfate (DS), but not by polycytidic acid (poly[C]) and polyinosinic acid (poly[l]), suggesting the involvement of a phagocytic process. We suggest that these complexes are preferentially taken up by the liver nonparenchymal cells mainly via Kupffer cell phagocytosis.
Introduction In vivo delivery of plasmid DNA encoding therapeutic proteins to patients, via parenteral or direct injection to the diseased tissues, represents an ideal methodology in gene t h e r a ~ y . l - ~Cationic liposomes are preferable carriers to transport plasmid DNA to specific cells in a controlled manner. These liposomes containing gene sequences interact spontaneously with negatively charged cell surfaces, delivering associated polynucleotide t o the cell's interior.s @Abstractpublished in Advance ACS Abstracts, October 1, 1995.
0 1995, American Chemical Society and American Pharmaceutical Association
We have been systematically studying the pharmacokinetic properties of macromolecules in relation to their physicochemical properties, such as molecular weight and electric charge. In particular, these studies have demonstrated the importance of electric charge in the disposition of macromolecules of molecular weight greater than 70 000 Da after intravenous injection. Cationic macromolecules are rapidly taken up by the liver due to electrostatic interaction, whereas macromolecules having weak anionic charge, such as bovine serum albumin and (carboxymethyl)dextran, show prolonged plasma retention. On the basis of these findings, we have developed various kinds of macromolecular carrier systems for drugsg and proteins1°-12 of small molecular weight, and have demonstrated their therapeutic usefulness. Physicochemical properties play an important role in the interaction of lipid vesicles with biological membranes. Although gene transfection of plasmids complexed with cationic liposomes are being studied extensively, little attention seems to be paid to understanding their physicochemical characteristics and cellular uptake mechanisms. Recently we studied the disposition and gene expression of pCAT alone or complexed with cationic 1ip0somes.l~We used Lipofectin and LipofectACE as model cationic carriers, because they are most widely used commercially available liposomes. Following iv injection of [32PlpCAT alone or complexed with cationic liposomes at 1:5 wlw ratio, radioactivity was eliminated from plasma and accumulated mainly in the liver and the lung. Both DNMiposome complex samples showed in vivo gene expression in the lung, heart, kidney, and spleen, but not in the liver. In the present study, we characterized the plasmid DNMiposome complexes in terms of particle size and 5 potential, and studied their hepatic uptake mechanisms after iv injection into mice.
Experimental Section Materials-Lipofectin (DOTMA,N-(2,3-bis(oleyloq)propyl)-N,N,Ntrimethylammonium chloride, and DOPE, dioleoylphosphatidyleth-
0022-3549/95/3184-1267$09.00/0
Journal of Pharmaceutical Sciences / 1267 Vol. 84, No. 11, November 1995
anolamine, 1:1,w/w ) and LipofectACE (DDAB, dimethyldioctadecylammonium bromide, and DOPE, L2.5, w/w) were purchased from Gibco BRL, Life Technologies, Inc. [a-32P]dCPT (800 Ciimmol) and pCAT control vectoir were obtained from NEN Research Products, Boston, MA, and Pmmega, Madison, WI, respectively. Soluene-350 and Clear-sol I were purchased from Packard and Nacalai Tesque, Tokyo, Japan, respectively. Poly[Il, poly[C], and dextran sulfate (DS, mw 500 kDa) were purchased from Sigma Chemicals, St. Louis, MO. All other chemicals were obtained commercially a s reagent-grade products. Preparation of Plasmid DNA-The plasmid was amplified i n the HB 101 strain of Escherichia coli, extracted by t h e alkaline lysis technique and purified by the precipitation with polyethylene glyc01.l~ The purified plasmid was diluted with sterilized phosphate-buffered saline. The purity was confirmed by 1%agarose gel electrophoresis followed by ethidiuni bromide staining and DNA concentration was measured by W absorption at 260 nm. For disposition studies in mice, the plasmid was labeled with [CI-~~PI~CPT by a nick translation technique.l* Particle Size a n d g Potential Measurement-[ potential and particle size of plasmid DNMiposome complexes were measured using procedures and equipment similar to those described by Oka et al.15 Plasmid DNA and cationic liposomes (Lipofetcin or LipofectACE) were diluted separately with saline and mixed together a t the ratios of 1:5 and 1:l'O w/w (pCAT: liposomes) a t room temperature, and complex formation was allowed to proceed for 15 min a t room temperature. The tlatal dilution factor was maintained at 8 for all liposomal samples. The electrophoretic mobility of these liposomal samples was determined with a ELS-800 Zetasizer (Otsuka Electronics Co. Ltd., Osaka, Japan). All experiments were performed at 26 "C using normal saline. The sample compartment was connected t o a glass capillary cell. Under a constant voltage, DNMiposome complexes started to migrate across the capillary on which the beam of a helium-neon laser was focused a t a constant angle of 15". The same light source (110 mW He-Ne laser) and wavelength (6.37 mm) were used in all exiperiments. The 5 potential was automatically calculated from the electrophoretic mobility based on the Smoluchowski formula. Following the determination of electrophoretic mobility, the samples were subjected to mean particle size measurement by the same equipment using the same light source and wavelength. In Vivo Distribution Experiment-Saline containing a 1 : l O w/w ratio pCAT/liposome complexes was administered to mice (Shizuoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka, Japan) via the taiI vein a t a dose of 0.1 mg DNAkg. Blood was collected from the vena cava under ether anesthesia at 1, 5, 15 and 30 min, and the mice were killed. Tissues (liver, spleen, kidney, and lung) were isolatled, washed with saline, blotted dry, and weighed. Blood was centrifuged and plasma was separated. Prior to the collection of 30 min postinjection blood samples, 100 pL of urine was collected directly from the urinary bladder using a 26-gauge needle syringe. Two hundred microliters of plasma, 100 pL of urine, and a small amount of each tissue were digested with Soluene-350 (0.5 mL for plasma and urine and 0.7 mL for tissues) through incubation overnight at 45 "C. Following digestion, 0.2 mL of 2 N HC1 (for neutralization) and !j mL of Clear-sol I (scintillation medium) were added to each tissue, plasma, and urine sample, the samples were stored overnight, and radioactivity was measured using a scintillation counter (LSA-500, Bteckman, Tokyo, Japan). D a t a Analysis-The tissue distribution data of [32P]pCAT/liposome complexes were analyzed in terms of a clearance and a tissue uptake rate index using monoexponential equations as described by Takakura et a1.I6 The change in the amount of radioactivity in a tissue with time can be described as follows:
dT(t)/dt = CLi,C(t) - Ko,,T(t) where T ( t )(70of dose/g) represents the amount of radioactivity in 1 g of the tissue, C ( t ) (% of dose/mL) is the plasma concentration of radioactivlty, CL,, (mUh-g)is the tissue uptake rate index from the plasma to the tissue, Kout(lh)is the rate constant for efflux from the tissue. In the present study, the efflux process can be considered negligible during the initial time points. Hence, eq 1integrates to
Table 1-Particle Size and g Potential of pCAT Cornplexed with Liposornesa Samples
Ratio (w/w)
Particle Size (nm)
Potential (mV)
pCAT/LipofectACE pCAT/LipofectACE pCATlLipofectin pCAT/Lipofectin
1 :5
867 ?c 280 1234 508 1135i235 600 k 122
-36.8k 1.96 18.95?c 8.62 20.51 +_ 1.80 28.02 0.41
1:lO
1 :5 1:lO
*
*
a V a l u e ~are expressed as the mean *SD of three measurements of single samples.
where tl (h)is the sampling time. According to eq 2, the tissue uptake rate index is calculated using the amount of radioactivity in the tissue a t a n appropriate interval of time and the area under the plasma concentration-time curve (AUC) up to the same time point. Then, the organ clearance (CL,,,) is expressed as follows:
CL,, = CL,,W
(3)
where W(g) is the total weight of the organ. When the tissue uptake process followed nonlinear kinetics, CL,, values would represent a n average value for the overall experimental period. Total body clearance (CLt,t,l) was calculated from AUC for infinite time (AUC,) by the following equation:
CLtotal= dose/AUC,
(4)
The organ uptake clearance and the tissue uptake clearance index were calculated using the values up to 30 min after injection, assuming that the degradation of pCAT complexed with liposomes is negligible within this period. Hepatic Cellular Localization of [32PlpCAT Complexed with Liposomes-Mice were anesthetized with pentobarbital sodium (4060 mgkg) and injected intravenously with the 1:5 or 1:lO wlw ratio [32P]pCAT:LipofectACEcomplexes at a dose of 0.1 mg DNAkg. The mouse body temperature was kept a t 37 "C by a heat lamp during the experiment. At 10 min after administration, the liver was perfused first with preperfusion buffer (Ca2+-and Mg2+-freeHEPES solution, pH 7.2) for 10 min and then with HEPES solution containing 5 mM CaCl2 and 0.05% (w/v) collagenase (type I) (pH 7.5) for 10-20 min. As soon as the perfusion was started, the vena cava and aorta were cut, and the perfusion rate was maintained a t 3-4 mumin. Following the discontinuation of perfusion, the liver was excised and deprived of the capsule membranes. The cells were dispersed in icecold Hank's-HEPES buffer containing 0.1% BSA by gentle stirring. The dispersed cells were filtered through cotton mesh sieves, followed by centrifugation at 50g for 1min. The pellets containing parenchymal cells (PC) were washed twice with Hank's-HEPES buffer by centrifuging at 50g for 1min. The supernatant containing nonparenchymal cells (NPC) was similarly centrifuged two more times. The resulting supernatant was then centrifuged twice a t 200g for 2 min. PC and NPC were separately resuspended in ice cold Hank's-HEPES buffer (2 mL for PC and 0.8 mL for NPC). The cell number and viability were determined by the trypan blue exclusion method. The cells (0.2 mL) were digested with Soluene-350 (0.7 mL) through incubation overnight at 45 "C. Following digestion, 0.2 mL of 2 N HC1 and 5 mL of Clear-sol I were added, the samples were stored overnight, and radioactivity was measured using a scintillation counter. The amount of radioactivity in each cell fraction was calculated as the percentage of dose per lo7 cells. Inhibitory Effect of Polyanions on Hepatic U p t a k e of [32PlpCATLiposome Complexes-Unlabeled polyanion (polyiC1, poly[I], or dextran sulfate) was injected into the tail vein of mice at a dose of 20 mgkg. One minute after polyanion administration, 1:5or 1 : l O w/w ratio [32PIpCATLipofectACE complexes was also injected similarly at a dose of 0.1 mg DNA/kg. At 10 min following DNA administration, the mouse was killed, and the liver was harvested and subjected t o radioactivity assay as mentioned before.
Results 6 Potential and Particle Size Measurement-Table 1 summarizes the 5 potential and mean particle size of DNN
1268 / Journal of Pharmaceutical Sciences Vol. 84, No. 1I, November 1995
8or
plexes. However, coadministration of poly[C] and poly[I] failed to inhibit the hepatic uptake of pCAT when administered in the complexed forms (Figure 4).
60
Discussion 0
10
20
30
0
10
20
30
Time (mid
Figure 1-Tissue accumulation of radioactivity after iv injection of [32P]pCAT/ liposome complexes at a dose of 0.1 mg of DNNkg in mice. (A) 1:lO w/w pCAT/ LipofectACE, (B) 1:lO w/w pCATILipofectin: plasma (0),liver (O), spleen (A), kidney (A),and lung (0).Values are expressed as the mean +SD of three or four animal experiments.
liposome complexes. This result illustrates that pCATLipofectin complexes had a 5 potential of 21 mV at 1 5 wlw ratio and 28 mV at 1:lO wlw ratio. Unlike Lipofectin samples, DNALipofectACE complexes showed a negative 5 potential (-36 mV) at 1 5 wlw ratio, but a positive one (19 mV) a t 1:lO wlw ratio. These preparations showed an increase in 5 potential as the amount of liposome increased. The particle size distribution of these samples was quite wide, ranging from 600 to 1200 nm. Due to the heterogeneous nature of these DNAAiposome complex samples, some variations in mean particle size and 5 potential are expected from different measurements and preparations. Tissue Distribution of [32P]pCAT after Intravenous Injection-Figures 1 and 2 respectively illustrate the time course and tissue uptake of radioactivity in plasma, kidney, spleen, liver, and lung after intravenous injection of [32P]pCATAiposome complexes into mice. These preparations showed rapid elimination of radioactivity from plasma and initial accumulation in the liver and lung; however, the lung accumulation did not sustain beyond 5 min after administration. No radioactivity was found in the urine, and kidney accumulation for all these samples was ve.y low (less than 1%of dose). Pharmacokinetic Analysis-Table 2 summarizes the AUC, total body and hepatic clearances, and tissue uptake rate indices for representative organs. The data for 1:5 wlw ratio pCAT/liposome complex samples, which were examined previously,13 are also shown for comparison. The pharmacokinetic analysis revealed a small AUC and a large total body clearance of all DNAfliposome complex samples. Tissue uptake indices of these preparations were quite similar: the spleen uptake index was the largest, leaving the hepatic uptake index and the lung index as second and third largest. However, 1:5 wlw pCAT/LipofectACE complex samples showed the lung uptake index much larger than that of spleen uptake index. Hepatic Cellular Localization of [32PlpCAT Complexed with Liposomes-Figure 3 shows the intrahepatic distribution of radioactivity in PC and NPC a t 10 min after iv injection of 1:5 and 1:lO wlw ratio [32P]pCAT/LipofectACE complexes into mice at a dose of 0.1 mgkg. Our previously published data12J7Jsfor naked pCAT and galactosylated and succinylated bovine serum albumin (Gal-BSA and Suc-BSA) are also shown in this figure for comparison. Like naked pCAT and Suc-BSA, the radioactivity derived from [32P]pCATl LipofectACE complexes preferentially accumulated in NPC. Inhibitory Effect of Polyanions on Hepatic U p t a k e of [32PlpCAT&iposome Complexes-To determine the uptake mechanism of [32PlpCAT/liposomecomplexes by hepatic NPC, we performed competition studies using unlabeled DS, poly[I], and poly[Cl. Strongly negatively charged DS significantly blocked the hepatic uptake of [32P]pCAT/LipofectACEcom-
Our intent in this study was to characterize plasmid DNA/ liposome complexes in terms of 5 potential and particle size and to see whether these physicochemical properties have any influence on their disposition characteristics and hepatic uptake process. Electrophoretic light scattering is an important technique for characterization of charged macromolecules (such as proteins, nucleic acids, etc.) and microlnanoparticulate~.1~~20 In the present study, 5 potential and particle size of plasmid DNMiposome complexes were measured in normal saline using this technique. All the preparations except 1 5 wlw pCAT/LipofectACE samples had a positive 5 potential. On the basis of the composition, we also calculated the number of positive charges borne by the cationic liposomes and the negative charges borne by the plasmid. The molecular weight of a nucleotide is about 315 Da and hence 1pg of pCAT should have 3.17 nmol of negatively charged molecules. Lipofectin is composed of 1:l wlw cationic lipid DOTMA (mw -644 Da) and neutral lipid DOPE (mw -744). Therefore, 5 and 10 pg of Lipofectin (i.e. 2.5 and 5 pg of cationic lipids) should have approximately 3.9 and 7.8 nmol of positively charged molecules, respectively. LipofectACE is composed of 1:2.5 wlw cationic lipid DDAB (mw -631 Da) and DOPE. Therefore, 5 and 10 pg of LipofectACE (i.e. 1.43 and 2.86 pg of cationic lipids) should have approximately 2.27 and 4.54 nmol of cationic molecules, respectively. This calculation suggests that the net charge density of 1 5 wlw pCAT:LipofectACE samples should be negative, which is in good agreement with the data obtained from 5' potential measurement. Rolland et a1.21 also reported that the 5 potential of the different DNMiposome complexes was dependent on the cationic liposome/DNA ratio. For a constant amount of DNA, the net negative charge of the complex gradually decreased and then turned into positive as the amount of liposome increased. The particle size of DNMipofectin complexes are smaller (256 f 82 nm) than our data (Table 1). This discrepancy might be due to the fact that these DNMiposome complexes are heterogenous and hence they filtered the complex through 0.2 pm pore size filter papers to exclude the large particles, whereas we did not filter our samples. This may be also due to the different types of plasmid DNA and measurement conditions used in these two studies. The variability in particle size obtained in our study should not be due to the instrument, but due to the heterogeneity of the complexes as the particle size distribution obtained from a laser light scattering technique is affected by the sample's geometry. Hence, samples need to be homogeneous and spherical to avoid the effect of their movement. The particle size of plasmid DNMiposome complexes was also measured by Gershon et This research group characterized the structural feature of the plasmid DNMiposome complexes by electron microscopy. The region along the DNA molecules became covered by liposome aggregates as the amount of liposomes increased for a constant amount of DNA. The mean particle size of the complexes was in the range of 0.5-1 pm. Following intravenous injection of 1:10 wlw ratio [32P]pCAT/ liposome complexes into mice, radioactivity was rapidly eliminated from plasma due to the extensive uptake by the liver (Figure 1). No radioactivity was found in the urine, and the renal accumulation of radioactivity for all these samples was less than 1%of the dose and did not increase with time. However, in a previous study17using naked pCAT we observed an increase in both renal accumulation (percentage of dose Journal of Pharmaceutical Sciences / 1269 VoL 84, No. I f , November 1995
Table 2-Pharrnacokinetic Parameters of pCATlLiposome Complexes after Intravenous Injection at a dose of 0.1 mg DNAikg into Mice
Samples
Ratio (WW
pCAT/LipofectACEa pCATlLipofectACE pCATlLipofectina pCAT/Lipofectn
1 :5 1:lO 1 :5 1:lO
Clearance k U h )
AUC (% of dose-hlmL)
Total
Tissue Uptake Index bUhlg)
Liver
Liver
0.625 159987 41 958 38336 0.569 175805 128250 85118 0.756 132240 58196 47759 0.701 142675 74588 49550 a Data for 1:5 wlw ratio samples of pCAT/LlpofectACE and pCATlLipofectin Complexes are published
Lq
30
0
25
50
75
Luna
4904 2507 4504 5578
89033 248589 105797 120800
417532 4fi224 ___ 12533 35179
_I
I
5
200 300
Soleen
30
E
I
Kidnev
0
25 50
75
200 300
Tissue concentration (% of doselmg or mL)
DS
Figure 2-Tissue distribution of radioactivity after iv injection of [32P]pCAT/liposome complexes at a dose of (1.1 mg DNAlkg in mice. (A) 1:IO wlw pCATlLipofectACE, (6) 1 :I0 wlw pCATAipofectin: plasma (solid line), liver (dotted), spleen (thin stripes), kidney (thick stripes), and lung (unfilled). Values are expressed as the mean ?SD of three or four animal experiments.
pCATLF( 1 5 )
npc
pCAT
NPC
I
Suc-BSA
Gal-BSA 0
3
6
9
Recovery (% of dose/ 1 x 107 cells)
Figure 3-Hepatic localization of radioactivity at 10 rnin after iv injection of [32P]pCATlLipofectACE complexes at a dose of 0.1 mg DNAfkg for parenchymal and nonparenchymal cells. Values are expressed as the mean kSD of three animal experiments. Data for Suc-BSA, Gal-BSA, and pCAT were taken from published result^.^^,^*
a t 1 and 30 min were 2% and 6%,respectively) and urinary excretion of radioactivity (no radioactivity at 1 min, but 6% of dose at 30 min) with time due to the excretion of degraded [32P]pCATvia the kidney. This suggests that unlike naked pCAT, DNA complexed with cationic liposomes was fairly stable and excretion of degradation products via the kidney was too low to be detected. The 5 potential seems to influence the lung and spleen accumulations of pCATAiposome complexes and this is more predominant in the case of LipofectACE samples. In our previous study,13 we observed sustainable lung accumulation for 1 5 w/w ratio pCAT/LipofectACE samples. There was a sharp decrease in lung uptake but an increase in spleen and liver uptakes as the 5 potential of these preparations changed from negative to positive values. Unlike LipofectACE preparations, pCAT/Lipofectin complexes had similar disposition patterns at these two ratios, probably due to the fact that these preparations had similar, positive 5 potentials. Although no aggregation was seen in the plasma samples, we cannot rule out the possibility of aggregatiodentrapment 1270 / Journal ofPharniaceutical Sciences Vol. 84, No. 11, November 1995
0
20
40
60
80
100
Hepatic Uptake (% of control)
Figure 4-Inhibitory effects of polyanions on the hepatic uptake of radioactivity of [32P]pCAT/LipofectACEcomplexes administered intravenously at a dose of 0.1 mglkg in mice at 1 rnin after iv injection of 20 mglkg of polyanions. Hepatic accumulation was determined at 10 min after DNA administration. The results < 0.001, Key: are expressed as the mean f S D of three mice. " P c 0.01, ***f (gray) pCAT; (white) 1.5 wlw pCAT/LipofectACE, (black) 1:IO wlw pCATl LipofectACE. Data for pCAT were taken from published results.17
occurring in the pulmonary capillaries as a possible reason for lung accumulation in the 1:5 w/w pCATLipofectACE sample. Hence, a substantial number of different ratio DNA/ liposome complex samples need to be tested before any definitive statements can be made. The uptake of pCATAiposome complexes by hepatic NPC is in good agreement with our previous observation for naked pCAT.17 However, total recovery of radioactivity of DNA/ liposome complex samples in the liver cells following collagenase perfusion was higher than that observed for naked pCAT (Figure 3). This suggests that cationic liposomes improved the stability of pCAT in the liver cells, resulting in negligible efflux of degraded pCAT from the liver to the blood circulation or the other tissue compartments. Scavenger receptors recognize a wide range of polyanions, including acetylated low-density lipoprotein, Suc-BSA, DS, poly[Il, and bacterial lipopolysa~chrides,2~ whereas some other polyanions including poly[C] do not seem to be ligands for the scavenger receptors.24 In a previous study,17we demonstrated that prior administration of polyanions such as poly[I], SucBSA, Gal-BSA, and dextran sulfate caused a substantial decrease in the hepatic uptake of [32PlpCAT,but poly[C] had no significant effect. From this finding, we concluded that the hepatic uptake of [32P]pCATis mediated by the scavenger receptors on the NPC. Involvement of scavenger receptors in the hepatic uptake of pCAT was further supported by our subsequent work using the single-pass rat liver perfusion system, in which unlabeled polyanions were introduced through the portal vein prior to DNA a d m i n i ~ t r a t i o n .Emlen ~~ et aLZ6also reported that single-stranded DNA from calf thymus was bound to the liver NPC. Because 1:5 w/w pCAT:LipofectACE complexes had a negative 5 potential, we speculated that the hepatic uptake mechanism of this DNMiposome complex sample should be
similar to that of naked pCAT. Hence, we performed competition studies using unlabeled dextran sulfate (DS),poly[Il, and poly[Cl. Preceding administration of DS caused a substantial decrease in the hepatic uptake of [32P]pCATcomplexed with LipofectACE, but no significant effect was observed by prior administration of poly[C] and poly[I] (Figure 4). Poly[I] is a well-known ligand for the scavenger receptors and hence this result suggests that unlike for naked [32P]pCAT,scavenger receptors on the NPC are not involved in the hepatic uptake of [32PlpCAT/liposomecomplexes. DS is an aspecific inhibitor of phagocytotic pathways and could manage to suppress 4050%of the hepatic uptake of pCAT complexed with LipofectACE (Figure 3). This is in good agreement with the findings of Patel et al.,27who demonstrated that prior administration of DS caused 40-50% suppression in the hepatic uptake of cationic liposomes. All the pCAT/LipofectACE samples were taken up by a similar mechanism regardless of their 5 potential, suggesting that the 5 potential does not play a significant role in the hepatic uptake of pCATAiposome complexes. However, the 5 potential seems likely to play an important role in the hepatic uptake of pCAT complexed with cationic macromolecules when a portion of DNA may be recognized by the scavenger receptors. Although we checked the effect of charged polymers (i.e., DS, poly[I], poly[C]) only on the hepatic uptake of the complexes, on the basis of a simple clearance concept, we speculate that the prior administration of DS may increase both plasma concentration and the lung and spleen uptakes of the complex, whereas that of poly[I] and poly[C] should not play any significant roles in the uptake process by these tissues. In a previous study,13 following iv injection, weak but significant gene expression for both Lipofectin and LipofectACE samples was observed in most of the tissues examined except the liver. For gene expression, intact pCAT should be delivered to the nucleus. Many processes such as transport into the cells, intracellular trafficking, degradation, etc. should also be involved before pCAT can function as a gene. Hepatic uptake of pCATAiposome complexes via Kupffer cell phagocytosis might have resulted in poor gene expression efficiency by the liver. The findings obtained here suggest that Kupffer cell phagocytosis are mainly responsible for the hepatic uptake of pCAT after complex formation. Unlike for naked pCAT, the scavenger receptors on NPC do not seem to play a significant role in the hepatic uptake of pCATAiposome complexes. Further studies are ongoing to isolate Kupffer and endothelial cells by centrifugal elutriation from perfused rat livers to study the uptake process by these cells.
References and Notes 1. Friedmann, T. Science. 1989,244, 1275-1281. 2. Felgner, P. L. Adu. Drug Del. Res., 1990, 5, 163-187. 3. Rose, J. K.; Buonocore, L.; Whitt, M. A. Biotechniques 1991,10, 520-525. 4. Legendre, J.-Y.; Szoka Jr., F. C. Pharm. Res. 1992, 9, 12351242. 5. Tolstoshev, P. Ann. Rev. Pharmacol. Toxicol. 1993, 32, 573596. 6. Trapnell, B. C . Adu. Drug Del. Rev. 1993, 12, 185-199. 7. Zhou, X.; Huang, L. Biochim. Biophys. Acta. 1994, 1154, 327340. 8. Bertling, W. M.; Gareis, M.; Paspaleeva, V.; Zimmer, A,; Kreuter, J.; Nurnberg, E.; Harper, P. Biotech. Appl. Biochem. 1991, 13, 390-405. 9. Imoto, H.; Sakamura, Y.; Ohkouchi, K.; Astumi, R.; Takakura, Y.; Sezaki, H.; Hashida, M. Cancer Res. 1992, 52, 4396-4401. 10. Fujita, T.; Nishikawa, M.; Ohtsubo, Y.; Ohno, J.; Takakura, Y.; Sezaki, H.; Hashida, M. J . Drug Targeting 1994,2, 157-165. 11. Takakura, Y.; Masuda, S..; Tokuda, H.; Nishikawa, M.; Hashida, M. Biochem. Pharmacol. 1994,47, 853-858. 12. Takakura, Y.; Fujita, T.; Furitsu, H.; Nishikawa, M.; Sezaki, H.; Hashida, M. Int. J . Pharm. 1994,105, 19-29. 13. Mahato, R. I.; Kawabata, K.; Takakura, Y.; Hashida, M. J . Drug Targeting 1995,3, 149-157. 14. Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Lab. Press: Plainview, NY,1989. 15. Oka, K.; Otani, W.; Kameyama, K.; Kidai, M.; Takagi, T. App. Theor. Electronics 1990, 1, 273-278. 16. Takakura, Y.; Takagi, A.; Hashida, M.; Sezaki, H. Pharm. Res. 1987, 4, 293-300. 17. Kawabata, K.; Takakura, Y.; Hashida, M. Pharm. Res. 1995, 12, 825-830. 18. Nishikawa, M.; Ohtsubo, Y.; Ohno, J.; Fujita, T.; Koyam, Y.; Yamashita, F.; Hashida, M.; Sezaki, H. Int. J . Pharm. 1993,85, 75-85. 19. Miiller, R. H. Colloidal Carriers for Controlled Drug Delivery and Targeting; CRC Press: Boca Raton, 1991; pp 59-82. 20. Yoshioka, H. Biomaterials 1991, 12, 861-864. 21. Rolland, A.; Duguid, J.; Barron, M.; Gong, L.; Levin, J.; Eastman, E. Proceed. Intern. Symp. Controlled Release Bioact. Muter. 1994,21, 240-241. 22. Gershon, H.; Ghirlando, R.; Guttman, S. B.; Minsky, A. Biochemistry 1993,32, 7143-7151. 23. Krieger, M.; Acton, S.; Ashkenas, J.; Pearson, A.; Penman, M.; Resnick, D. J . Biol. Chem. 1993,268,4569-4572. 24. Pearson, A. M.; Rich, A,; Krieger, M. J . Biol. Chem. 1993,268, 3546-3554. 25. Yoshida, M.; Mahato, R. I.; Kawabata, K.; Takakura, Y.; Hashida, M. Pharm. Res. (submitted). 26. Emlen, W.; Rifai, A.; Magilavy, D.; Mannik M. Am. J . Pathology. 1988,133, 54-60. 27. Patel, K. R.; Li, M. P.; Baldeshwieler J. D. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 6518-6522.
JS950264X
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