The Role of Organ Vascularization and Lipoplex-Serum Initial Contact in Intravenous Murine Lipofection
2003; Elsevier BV; Volume: 278; Issue: 41 Linguagem: Inglês
10.1074/jbc.m302232200
ISSN1083-351X
AutoresDmitri Simberg, Sarah Weisman, Yeshayahu Talmon, Alexander Faerman, Tzipora Shoshani, Yechezkel Barenholz,
Tópico(s)Hepatitis B Virus Studies
ResumoFollowing intravenous administration of cationic lipid-DNA complexes (lipoplexes) into mice, transfection (lipofection) occurs predominantly in the lungs. This was attributed to high entrapment of lipoplexes in the extended lung vascular tree. To determine whether lipofection in other organs could be enhanced by increasing the degree of vascularization, we used a transgenic mouse model with tissue-specific angiogenesis in liver. Tail vein injection of N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP)/cholesterol lipoplexes resulted in increased lipoplex entrapment in hypervascularized liver but did not boost luciferase expression, suggesting that lipoplex delivery is not a sufficient condition for efficient organ lipofection. Because the intravenously injected lipoplexes migrated within seconds to lungs, we checked whether the effects of immediate contact with serum correlate with lung lipofection efficiency of different DOTAP-based formulations. Under conditions mimicking the injection environment, the lipoplex-serum interaction was strongly dependent on helper lipid and ionic strength: lipoplexes prepared in 150 mm NaCl or lipoplexes with high (>33 mol%) cholesterol were found to aggregate immediately. This aggregation process was irreversible and was inversely correlated with the percentage of lung cells that took up lipoplexes and with the efficiency of lipofection. No other structural changes in serum were observed for cholesterol-based lipoplexes. Dioleoyl phosphatidylethanolamine-based lipoplexes were found to give low expression, apparently because of an immediate loss of integrity in serum, without lipid-DNA dissociation. Our study suggests that efficient in vivo lipofection is the result of cross-talk between lipoplex composition, interaction with serum, hemodynamics, and target tissue "susceptibility" to transfection. Following intravenous administration of cationic lipid-DNA complexes (lipoplexes) into mice, transfection (lipofection) occurs predominantly in the lungs. This was attributed to high entrapment of lipoplexes in the extended lung vascular tree. To determine whether lipofection in other organs could be enhanced by increasing the degree of vascularization, we used a transgenic mouse model with tissue-specific angiogenesis in liver. Tail vein injection of N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP)/cholesterol lipoplexes resulted in increased lipoplex entrapment in hypervascularized liver but did not boost luciferase expression, suggesting that lipoplex delivery is not a sufficient condition for efficient organ lipofection. Because the intravenously injected lipoplexes migrated within seconds to lungs, we checked whether the effects of immediate contact with serum correlate with lung lipofection efficiency of different DOTAP-based formulations. Under conditions mimicking the injection environment, the lipoplex-serum interaction was strongly dependent on helper lipid and ionic strength: lipoplexes prepared in 150 mm NaCl or lipoplexes with high (>33 mol%) cholesterol were found to aggregate immediately. This aggregation process was irreversible and was inversely correlated with the percentage of lung cells that took up lipoplexes and with the efficiency of lipofection. No other structural changes in serum were observed for cholesterol-based lipoplexes. Dioleoyl phosphatidylethanolamine-based lipoplexes were found to give low expression, apparently because of an immediate loss of integrity in serum, without lipid-DNA dissociation. Our study suggests that efficient in vivo lipofection is the result of cross-talk between lipoplex composition, interaction with serum, hemodynamics, and target tissue "susceptibility" to transfection. Cationic lipids, which are extensively employed as in vitro transfection agents, are promising agents for in vivo delivery of nucleic acids for a variety of applications from functional proteomics (1Shoshani T. Faerman A. Mett I. Zelin E. Tenne T. Gorodin S. Moshel Y. Elbaz S. Budanov A. Chajut A. Kalinski H. Kamer I. Rozen A. Mor O. Keshet E. Leshkowitz D. Einat P. Skaliter R. Feinstein E. Mol. Cell Biol. 2002; 22: 2283-2293Crossref PubMed Scopus (476) Google Scholar) to therapeutics (2Dass C.R. Int. J. Pharm. 2002; 241: 1-25Crossref PubMed Scopus (48) Google Scholar). Obviously, the applications of cationic lipid-DNA complexes (lipoplexes) as in vivo delivery vehicles are useful only when the lipoplex-mediated transfection (lipofection) is high enough to modify the function of the protein/gene of interest. The most challenging route of in vivo administration of lipoplexes is the intravenous (i.v.) 1The abbreviations used are: i.v., intravenous(ly); DOTAP, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; VEGF, vascular endothelial growth factor; CF, carboxyfluorescein; CFPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein); LRPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl); DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine; HCPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-hydroxycoumarin); EYFP, enhanced yellow fluorescent protein; UHV, unsized (non-extruded) heterogeneous vesicles; LUV, large unilamellar vesicles; cryo-TEM, cryogenic transmission electron microscopy; FRET, fluorescence resonance energy transfer; CMV, cytomegalovirus; FACS, fluorescence-activated cell sorter. route. Following mouse tail vein injection, most of the transgene expression occurs in the lungs, producing more than 80% of the total expressed protein in the animal (3Liu F. Qi H. Huang L. Liu D. Gene Ther. 1997; 4: 517-523Crossref PubMed Scopus (238) Google Scholar, 4Mahato R.I. Anwer K. Tagliaferri F. Meaney C. Leonard P. Wadhwa M.S. Logan M. French M. Rolland A. Hum. Gene Ther. 1998; 9: 2083-2099Crossref PubMed Scopus (162) Google Scholar, 5Song Y.K. Liu F. Chu S. Liu D. Hum. Gene Ther. 1997; 8: 1585-1594Crossref PubMed Scopus (281) Google Scholar). Despite significant efforts to optimize lipoplex performance following i.v. administration (4Mahato R.I. Anwer K. Tagliaferri F. Meaney C. Leonard P. Wadhwa M.S. Logan M. French M. Rolland A. Hum. Gene Ther. 1998; 9: 2083-2099Crossref PubMed Scopus (162) Google Scholar, 6Li S. Huang L. Gene Ther. 1997; 4: 891-900Crossref PubMed Scopus (453) Google Scholar), the "built-in" biological factors determining the efficiency of lung lipofection remain unclear. It has been shown that within minutes after injection, 60–80% of the lipoplexes are entrapped in the lungs (4Mahato R.I. Anwer K. Tagliaferri F. Meaney C. Leonard P. Wadhwa M.S. Logan M. French M. Rolland A. Hum. Gene Ther. 1998; 9: 2083-2099Crossref PubMed Scopus (162) Google Scholar, 7Niven R. Pearlman R. Wedeking T. Mackeigan J. Noker P. Simpson-Herren L. Smith J.G. J. Pharm. Sci. 1998; 87: 1292-1299Abstract Full Text PDF PubMed Scopus (99) Google Scholar, 8Li S. Tseng W.C. Stolz D.B. Wu S.P. Watkins S.C. Huang L. Gene Ther. 1999; 6: 585-594Crossref PubMed Scopus (335) Google Scholar). This "first-pass" entrapment was ascribed to the highly extended lung capillary bed and was proposed to explain the predominant expression in the lungs (8Li S. Tseng W.C. Stolz D.B. Wu S.P. Watkins S.C. Huang L. Gene Ther. 1999; 6: 585-594Crossref PubMed Scopus (335) Google Scholar, 9Song Y.K. Liu F. Liu D. Gene Ther. 1998; 5: 1531-1537Crossref PubMed Scopus (75) Google Scholar). Another factor thought to affect lung lipofection is the interaction of lipoplexes with serum. Incubation in pure serum or in serum-containing media can decrease the lipoplex delivery and alter intracellular processing (10Escriou V. Ciolina C. Lacroix F. Byk G. Scherman D. Wils P. Biochim. Biophys. Acta. 1998; 1368: 276-288Crossref PubMed Scopus (189) Google Scholar, 11Yang J. Chen S. Huang L. Michalopoulos G.K. Liu Y. Hepatology. 2001; 33: 848-859Crossref PubMed Scopus (101) Google Scholar), lower or reverse the zeta (ζ)-potential (12Zelphati O. Uyechi L.S. Barron L.G. Szoka Jr., F.C. Biochim. Biophys. Acta. 1998; 1390: 119-133Crossref PubMed Scopus (300) Google Scholar), and even cause lipoplex disintegration upon prolonged exposure (6Li S. Huang L. Gene Ther. 1997; 4: 891-900Crossref PubMed Scopus (453) Google Scholar, 7Niven R. Pearlman R. Wedeking T. Mackeigan J. Noker P. Simpson-Herren L. Smith J.G. J. Pharm. Sci. 1998; 87: 1292-1299Abstract Full Text PDF PubMed Scopus (99) Google Scholar, 12Zelphati O. Uyechi L.S. Barron L.G. Szoka Jr., F.C. Biochim. Biophys. Acta. 1998; 1390: 119-133Crossref PubMed Scopus (300) Google Scholar). However, the importance of the above mentioned serum effects for lung transgene expression following i.v. administration is in question because: (a) the injected lipoplexes are rapidly cleared from the circulation (4Mahato R.I. Anwer K. Tagliaferri F. Meaney C. Leonard P. Wadhwa M.S. Logan M. French M. Rolland A. Hum. Gene Ther. 1998; 9: 2083-2099Crossref PubMed Scopus (162) Google Scholar, 7Niven R. Pearlman R. Wedeking T. Mackeigan J. Noker P. Simpson-Herren L. Smith J.G. J. Pharm. Sci. 1998; 87: 1292-1299Abstract Full Text PDF PubMed Scopus (99) Google Scholar) and internalized in lungs (13Barron L.G. Gagne L. Szoka Jr., F.C. Hum. Gene Ther. 1999; 10: 1683-1694Crossref PubMed Scopus (97) Google Scholar), thus ruling out long-term exposure to serum; (b) the pre-incubation of DOTAP/cholesterol lipoplexes with serum for as long as 30 min does not significantly impair lipofection in the lungs (8Li S. Tseng W.C. Stolz D.B. Wu S.P. Watkins S.C. Huang L. Gene Ther. 1999; 6: 585-594Crossref PubMed Scopus (335) Google Scholar, 14Sakurai F. Nishioka T. Saito H. Baba T. Okuda A. Matsumoto O. Taga T. Yamashita F. Takakura Y. Hashida M. Gene Ther. 2001; 8: 677-686Crossref PubMed Scopus (152) Google Scholar). In this work, we focused on formulations based on DOTAP, the most commonly used cationic lipid for various in vivo applications. Our goal was to clarify the mechanisms underlying murine lipofection, especially the role of organ vascularization and lipoplex-serum initial contact. To do this, we first checked whether the extent of vascularization is critical for the process of lipofection in the liver. We injected DOTAP/cholesterol lipoplexes into transgenic mice with VEGF-induced, liver-specific angiogenesis. Our results show that hypervascularization in the liver does not increase level of lipofection, despite increased entrapment of the lipoplexes. The fact that enlarging the vascular bed and increasing vascular permeability in liver are not sufficient to promote efficient lipofection leads to the conclusion that lungs might be an organ that is just more susceptible than liver to this process. Next, we checked the relevance of immediate lipoplex-serum interaction to the process of lung lipofection. We found that the first contact between injected lipoplexes and serum affects lipoplex structure, the mode of delivery, and lipofection efficiency in the lungs. Lipids and Fluorescent Probes—DOTAP, carboxyfluorescein-PE (CFPE), and lissamine rhodamine-PE (LRPE) were obtained from Avanti Polar Lipids (Alabaster, AL). DOPE was purchased from Lipoid (Ludwigshafen, Germany). Cholesterol and spermidine base were purchased from Sigma. The pH-sensitive probe 7-hydroxycoumarin-PE (HCPE) was prepared in our laboratory. 2D. Hirsch-Lerner and Y. Barenholz, manuscript in preparation. DNA—pCMV-EYFPmito, a plasmid coding for enhanced yellow fluorescent protein (EYFP) carrying mitochondrial localization signal was purchased from Clontech (Palo Alto, CA). pCMV-Luc coding for the luciferase gene was constructed by insertion of a 875-bp CMV promoter-enhancer fragment into pGL3-enhancer (Promega, Madison, WI). pCMV-p53, based on pcDNA3 was provided by Dr. Moshe Oren, Weizmann Institute of Science, Rehovot, Israel. All plasmids were propagated in Escherichia coli and purified in sterile endotoxin-free form using the Qiagen EndoFree Plasmid Mega kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol and were analyzed for purity and topology as described previously (15Even-Chen S. Barenholz Y. Biochim. Biophys. Acta. 2000; 1509: 176-188Crossref PubMed Scopus (49) Google Scholar). Liposome Preparation—For all preparations we used water purified with the WaterPro PS HPLC/UV Ultrafilter Hybrid model (LABCONCO, Kansas City, MO), which delivers Type I 18.2-megaohm/cm sterile (pyrogen-free to 0.06 endotoxin unit/ml) water. Unsized (nonextruded) heterogeneous vesicles (UHV) were prepared by mixing lipids in tert-butyl alcohol (Baker, Deventer, Holland) in a sterile isopropylene tube. The lipids were freeze-dried overnight and rehydrated in sterile water with gentle vortexing to make a 20 mm DOTAP concentration. Extruded large unilamellar vesicles (LUV) were prepared from UHV by stepwise extrusion through polycarbonate filters of 400- and 30-nm pore size, as described elsewhere (16Zuidam N.J. Barenholz Y. Biochim. Biophys. Acta. 1997; 1329: 211-222Crossref PubMed Scopus (137) Google Scholar). For fluorescent labeling, a fluorescently labeled phospholipid was added at concentrations of 0.25–0.5 mol % to the lipids before the freeze-drying step. Preparation of Lipoplexes—The lipoplexes were prepared under aseptic conditions. Briefly, volumes of DNA and cationic liposomes were made equal with sterile pure water, and DNA was added dropwise to the cationic liposomes with gentle agitation. The lipoplexes were prepared in 90% of the final volume, allowing for 9% NaCl or 50% dextrose in the remaining 10% of the volume to be added just before injection, to obtain an isotonic concentration of 5% dextrose or 0.9% NaCl in the formulations. When lipoplexes were prepared in different final volumes (200 or 800 μl), the volumes of DNA and lipid were correspondingly adjusted before the last mixing step, but the total dose of the components per animal was kept constant. When spermidine was used for pre-condensation of plasmid, it was added to plasmid solution at an amine/phosphate charge ratio of 0.5 10 min before adding the plasmid to the cationic liposomes. The lipoplexes were allowed to form for at least 15 min before injection. Transgene Expression and Function—The protocols for the experiments were approved by the Ethics Committee of the Hebrew University's Authority for Animal Facilities. For p53 efficacy study, B16-F10.9 melanoma cells (250,000 cells/mouse) were injected into the tail veins of C57BL syngeneic mice on day 1 at a constant volume of 0.25 ml/mouse, and pCMV-p53-based lipoplexes were administrated i.v. on day 5. Lipoplexes with pcDNA3 served as control. Twenty days after tumor cell injection the animals were sacrificed. The lungs were excised and weighed, and metastases were counted under a binocular microscope. For study of EYFP or luciferase expression, each BALB-C male mouse, weighing 20–30 g (Harlan Laboratories, Rehovot, Israel) received 40 μg of cationic lipid-complexed plasmid in different volumes of aqueous vehicle via the tail vein. Mice were sacrificed 10 s to 24 h post-injection by cervical displacement. When the animals were killed at 6 h or more post-injection, the organs were excised with prior perfusion with 5 ml of cold phosphate-buffered saline to remove blood from the tissue. Luciferase activity was assayed and quantified as described (17Hong K. Zheng W. Baker A. Papahadjopoulos D. FEBS Lett. 1997; 400: 233-237Crossref PubMed Scopus (293) Google Scholar). For analysis of fluorescent cells, the freshly excised tissue was digested at 37 °C and prepared for FACS as described (18Lavnikova N. Prokhorova S. Helyar L. Laskin D.L. Am. J. Respir. Cell Mol. Biol. 1993; 8: 384-392Crossref PubMed Scopus (66) Google Scholar). The lung cell suspensions were analyzed on a FACScan cell sorter (BD Biosciences). Lung digests of mice treated with non-fluorescent lipoplexes were used for baseline fluorescence determination. A transgenic mouse system for tetracycline-dependent switching of VEGF expression in the adult liver was kindly provided by the laboratory of Dr. E. Keshet, Department of Molecular Biology, Hebrew University, and is described elsewhere (19Dor Y. Djonov V. Abramovitch R. Itin A. Fishman G.I. Carmeliet P. Goelman G. Keshet E. EMBO J. 2002; 21: 1939-1947Crossref PubMed Scopus (342) Google Scholar). Fluorescent Lipid Quantification—For CFPE or LRPE quantification, the lipids were extracted from the homogenized tissues with 4 volumes of isopropanol/30% NH4OH (300:1, v/v), and the fluorescence intensity was measured on a PerkinElmer Life Sciences LS50B spectrofluorimeter. Stock dilutions of fluorescent lipids in the extracts of untreated mice lungs served for calibration. The extracted fluorescent lipids were analyzed for integrity by thin layer chromatography as described elsewhere (20Simberg D. Hirsch-Lerner D. Nissim R. Barenholz Y. J. Liposome Res. 2000; 10: 1-13Crossref Scopus (37) Google Scholar). Light and Fluorescence Microscopy—For fluorescence microscopy, the formaldehyde-fixed tissues were frozen, embedded at –25 °C in Jung tissue-freezing medium (Leica Instruments, Nussloch, Germany), and cut into 5–10-μm thin sections. The slides were viewed on an Olympus FV300 laser-scanning microscope (Olympus Optical, Japan). For study of lipoplex aggregation in different media and in serum, lipoplexes were mixed with mouse serum at different ratios, and then immediately a drop of the lipoplex dispersion was placed on a slide, covered with a cover glass, and viewed in transmitted mode using Nomarsky contrast. For estimating size of aggregates, 3-μm latex beads (Sigma) were added to the formulations. Lipoplex-Serum Interaction—For comparison of serum protein association with different lipoplexes, the latter were added to the mouse serum at a ratio of 1:2 (v/v). After a 5-min incubation, the mixtures (total 150 μl) were centrifuged at 14,000 rpm for 10 min. At the end of the procedure, 98% of the lipoplexes were in the pellet according to TLC analysis of fluorescent phospholipid or DOTAP in the supernatant (20Simberg D. Hirsch-Lerner D. Nissim R. Barenholz Y. J. Liposome Res. 2000; 10: 1-13Crossref Scopus (37) Google Scholar). The lipids from the pellet were extracted with 200 μl of isopropanol, and the pellet was redissolved in 100 μl of 20% sodium dodecyl sulfate. The protein was quantified by a modified Lowry method (21Peterson G.L. Methods Enzymol. 1983; 91: 95-119Crossref PubMed Scopus (1142) Google Scholar). For free (non-esterified) cholesterol determination, the lipid extract from the previous procedure was assayed using the Free Cholesterol Determination kit (Wako Chemicals, Richmond, VA) (22Shmeeda H. Petkova D. Barenholz Y. Am. J. Physiol. 1995; 268: H759-H766PubMed Google Scholar). Cryogenic Transmission Electron Microscopy (Cryo-TEM)—The cryo-TEM work was performed at the Hannah and George Krumholz Laboratory for Advanced Microscopy at the Technion, Haifa, Israel. Lipid dispersions and lipoplexes were prepared in exactly the same concentrations and conditions as throughout our study. For preparation of lipoplex-serum mixtures, lipoplexes were incubated in serum at a lipoplex/serum ratio of 1:2 for ∼5 min. The cryo-specimens were prepared, imaged, and processed as described (23Simberg D. Danino D. Talmon Y. Minsky A. Ferrari M.E. Wheeler C.J. Barenholz Y. J. Biol. Chem. 2001; 276: 47453-47459Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Physical Parameters of the Lipoplexes Before and After Incubation with Serum—The mass-weighted size distribution of the liposomes and lipoplexes in buffer/serum was determined at 25 °C using a non-invasive back-scattering particle size analyzer (ALV-NIBS, ALV, Linden, Germany). This technique allows one to measure size without diluting the sample. The ζ-potential of the lipoplexes and of lipoplex-serum aggregates was measured in 10 or 150 mm NaCl solution on a Zetasizer 2000 (Malvern Instruments, Malvern, UK). Viscosity of lipoplex dispersions was determined at 25 °C with a capillary semimicro viscometer (Cannon Instrument, State College, PA). Fluorescence Resonance Energy Transfer (FRET)—The plasmid DNA for FRET experiments was labeled covalently with carboxyfluorescein (CF). 3D. Simberg and Y. Barenholz, manuscript in preparation. Such labeling only minimally affects plasmid topology and physical properties. Liposomes were labeled with LRPE. First, the emission spectra (excitation 490 nm, emission 510–600 nm) of DNA-CF or LRPE-labeled liposomes were measured separately on the spectrofluorimeter. In the next step, cationic liposomes with or without LRPE were added to DNA-CF at a charge ratio of 5 (+/–), and the spectra were recorded again. Finally, the spectra were recorded after the addition of various amounts of mouse serum to the lipoplexes. Monitoring Membrane Surface Electrical Potential (ψ 0)—The surface potential of both HCPE-labeled DOTAP/DOPE (1/1) and DOTAP/cholesterol (1/1) liposomes was calculated from dissociation curves of the 7-hydroxycoumarin moiety of HCPE by recording excitation of HCPE at 405 nm and 380 nm, using emission at 450 nm, as described (16Zuidam N.J. Barenholz Y. Biochim. Biophys. Acta. 1997; 1329: 211-222Crossref PubMed Scopus (137) Google Scholar). The change of surface potential after the formation of lipoplexes and addition of serum was calculated from the change in degree of dissociation of the 7-hydroxycoumarin moiety of HCPE. Details and calculations are explained elsewhere (16Zuidam N.J. Barenholz Y. Biochim. Biophys. Acta. 1997; 1329: 211-222Crossref PubMed Scopus (137) Google Scholar, 24Zuidam N.J. Barenholz Y. Biochim. Biophys. Acta. 1998; 1368: 115-128Crossref PubMed Scopus (167) Google Scholar). We started our work using LUV DOTAP/cholesterol (1:1)-based lipoplexes (with pre-condensation of the plasmid with spermidine at an amine/phosphate charge ratio of 0.5) because these have been shown to exhibit high transfection efficiency after i.v. administration (17Hong K. Zheng W. Baker A. Papahadjopoulos D. FEBS Lett. 1997; 400: 233-237Crossref PubMed Scopus (293) Google Scholar). Throughout the study, to decrease the number of variables and to avoid toxicity inherent in the dose of cationic lipid and DNA (2Dass C.R. Int. J. Pharm. 2002; 241: 1-25Crossref PubMed Scopus (48) Google Scholar), we kept the plasmid dose constant at 40 μg/mouse (121 nmol DNA phosphate/mouse) and the DOTAP-to-DNA +/–charge ratio constant at 5 (605 nmol of DOTAP/mouse). We used a p53-coding plasmid to demonstrate that the gene phenotype and function of p53, which is a pro-apoptotic protein, indeed can be characterized in the lungs by lipoplex-mediated transfection. For this purpose, we injected the spermidine-based formulation, as described above, into mice with advanced lung metastases. Such treatment led to a significant decrease in the number of metastases (27.0 ± 1.4) compared with untreated animals (120.5 ± 13.4). The control group, in which the empty cassette pcDNA3 was used, also showed some regression of disease (78.5 ± 0.7), probably because of nonspecific, bacterial plasmid-dependent mechanisms (2Dass C.R. Int. J. Pharm. 2002; 241: 1-25Crossref PubMed Scopus (48) Google Scholar). Tail vein injection of pCMV-EYFPmito-based lipoplexes into normal mice produced visible EYFP expression predominantly in the lungs (Fig. 1A) and to a much smaller extent in the heart (Fig. 1B). The EYFPmito gene expression in lungs was confirmed by Northern blot (Fig. 1A, inset) and quantified by FACS (1.4 ± 0.3% EYFP-positive cells). On the other hand, in the liver no detectable EYFP expression was observed at 6–24 h post-injection (Fig. 1C). To determine whether transgene expression in the liver could be boosted by means of increased vascularization, we injected LUV DOTAP/cholesterol (1:1)-based lipoplexes, containing either EYFP- or luciferase-expressing plasmid, into mice with VEGF-overexpressing livers (19Dor Y. Djonov V. Abramovitch R. Itin A. Fishman G.I. Carmeliet P. Goelman G. Keshet E. EMBO J. 2002; 21: 1939-1947Crossref PubMed Scopus (342) Google Scholar). Such transgenic animals are characterized by extensive neovascularization and high vascular permeability, associated with greatly enlarged organ weight (up to 2.5 times). The results show that following tail vein injection, there was an increased liver accumulation of fluorescently labeled lipoplexes (Fig. 1D). Thus, as soon as 6 h post-injection, the normal livers contained on average 0.616 nmol of LRPE/organ, whereas VEGF-overexpressing livers contained on average 2.033 nmol of LRPE/organ. Despite more than a 3-fold boost in lipoplex entrapment, no visible EYFP expression was detected at 6–12 h post-injection. To increase the sensitivity of detection, we also measured luciferase expression levels. The expression was very low, 600 fg/mg tissue protein (about 100 times less than the corresponding values in the lungs) and did not differ from the normal transfected liver tissue. We varied the following formulation parameters. Presence and Nature of Helper Lipid— Table I shows the transfection efficiencies of the formulations prepared with different mole ratios of cholesterol or with DOPE instead of cholesterol. The optimal cholesterol mole fraction in the lipoplexes is ∼33%. Further increasing cholesterol content decreases transfection. Formulations based on DOTAP alone also showed inferior transfection efficiency (not shown). Replacing cholesterol with DOPE drastically decreases transfection (Table I).Table ITransfection efficiency, aggregation in serum, and delivery to lung of different DOTAP-based formulationsFormulationaFormulation numbers are boldface and enclosed in parentheses.Lung luciferase expressionInitial lipoplex sizeSize under "low serum" conditionsAggregation by microscopy, "high serum"Aggregation by microscopy, "low serum"Lipoplex-positive cells in lungsbQuantified by FACS.pg/mg of tissue proteinnmnm%200 dext UHV DOTAP/cholesterol(2:1)/pLuc (1)85 ± 3.36041194 (2.0)c-Fold change from original size is shown in parentheses.++11.2200 dext UHV DOTAP/cholesterol(1:1)/pLuc (2)57 ± 2.76201096 (1.8)++++++9.3200 dext UHV DOTAP/cholesterol(1:2)/pLuc (3)6.6 ± 1.213665000 (3.7)+++++++3.4200 NaCl UHV DOTAP/cholesterol(1:1)/pLuc (4)5.5 ± 2.38306850 (8.3)++++++++3.1800 dext UHV DOTAP/cholesterol(1:1)/pLuc (5)79.5520472 (1.0)NMNM10.2200 dext LUV DOTAP/cholesterol(1:1)/pLuc (6)11.3 ± 10.4142310 (2.2)NMNM4.4200 dext UHV DOTAP/DOPE(1:1)/pLuc (7)1.62 ± 0.24201190 (2.8)++++8.4a Formulation numbers are boldface and enclosed in parentheses.b Quantified by FACS.c -Fold change from original size is shown in parentheses. Open table in a new tab Lipoplex Size—To determine the effect of lipoplex size in the "pre-injection state," we kept lipid composition (DOTAP/cholesterol (1:1)) constant to avoid independent effects. We prepared lipoplexes of different sizes by: 1) starting from the ∼500-nm UHV or from the 120-nm LUV; and 2) preparing lipoplexes from UHV in a final volume of 200 μl ("high concentration") or 800 μl ("low concentration"). According to Table I, the high concentration UHV-derived lipoplexes (Formulation 2) were ∼600 nm in size, whereas the corresponding LUV-derived lipoplexes (Formulation 6) were ∼160 nm. The low concentration UHV-based lipoplexes (Formulation 5) were of intermediate size, ∼380 nm. Lung transgene expression was highest for the high concentration UHV-based formulation and lowest for the LUV-based formulation (Table I). High Versus Low Ionic Strength Medium for Preparation of Lipoplexes—The effects of media of various ionic strengths on the size and transgene expression efficiency of UHV DOTAP/cholesterol (1:1)-based lipoplexes were investigated. The presence of electrolyte resulted in partial aggregation of lipoplexes, although the aggregates could be redispersed by pipetting. Transgene expression in lungs (Table I) with 150 mm NaCl-based lipoplexes (Formulation 4) was 10-fold less efficacious than with 5% dextrose-based lipoplexes (Formulation 2). This effect of lipoplex medium on transfection was not a consequence of viscosity differences between the formulations, given that those differences are small (Table II) and the viscosity values are lower than that of mouse serum (1.5–2 centipoise).Table IIPhysicochemical parameters of selected lipoplex formulationsFormulationViscosityψ0 potential before addition of serumψ0 potential in serumBound serum proteinsResidual cholesterol after incubation in serumcPmVmVμg/nmol DOTAP% of initial200 dext UHV DOTAP/cholesterol(1:1)/pLuc (2)1.07169.2170.81.3395.6200 dext UHV DOTAP/cholesterol(1:2)/pLuc (3)NMNMNM1.0593.8200 NaCl UHV DOTAP/cholesterol(1:1)/pLuc (4)NMNMNM1.14NM200 dext LUV DOTAP/cholesterol(1:1)/pLuc (6)NM169.2170.81.22NM200 dext UHV DOTAP/DOPE(1:1)/pLuc (7)1.19157.3122.0NMNM Open table in a new tab We tracked the delivery and distribution of fluorescently labeled lipoplexes in the lung tissue at different times post-injection. For all UHV-based formulations, the greater part of the lipid dose (about 70%) reached the lung within the first 10 s post-injection, indicating a considerable first-pass effect. In the case of LUV-based lipoplexes, however, only 30% of the fluorescence was recovered in the lungs by this time. The reason for this might be that the smaller, LUV-based, lipoplexes are poorly entrapped in the lung vasculature. With time, the amount of fluorescence in lungs steadily decreased (40% after 30 min and 2% after 12 h for all UHV-based formulations; 15% after 30 min and 0.5% after 12 h for LUV-based lipoplexes). On the other hand, there was a significant dependence of UHV DOTAP/cholesterol-based lipoplex lung distribution on formulation parameters as early as 10 s post-injection. Thus, according to histological tissue examination, lipoplexes administered in 150 mm NaCl produced a very het
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