Biophysical Journal Volume 84 May 2003 3307–3316
`
`3307
`
`Three-Dimensional Imaging of Lipid Gene-Carriers: Membrane
`Charge Density Controls Universal Transfection Behavior
`in Lamellar Cationic Liposome-DNA Complexes
`
`Alison J. Lin,* Nelle L. Slack,* Ayesha Ahmad,* Cyril X. George,
`and Cyrus R. Safinya*
`*Materials Department, Physics Department, and Biomolecular Science and Engineering Program, University of California,
`y
`Molecular, Cellular, and Developmental Biology Department, and Biomolecular Science
`Santa Barbara, California 93106; and
`and Engineering Program, University of California, Santa Barbara, California 93106
`
`y
`
`Charles E. Samuel,
`
`y
`
`ABSTRACT Cationic liposomes (CLs) are used worldwide as gene vectors (carriers) in nonviral clinical applications of gene
`delivery, albeit with unacceptably low transfection efficiencies (TE). We present three-dimensional laser scanning confocal
`
`C and inverted hexagonal HIIC
`microscopy studies revealing distinct interactions between CL-DNA complexes, for both lamellar La
`C complexes in cells identified two regimes. For low membrane
`nanostructures, and mouse fibroblast cells. Confocal images of La
`charge density (sM), DNA remained trapped in CL-vectors. By contrast, for high sM, released DNA was observed in the
`cytoplasm, indicative of escape from endosomes through fusion. Remarkably, firefly luciferase reporter gene studies in the
`C-mammalian cell system revealed an unexpected simplicity where, at a constant cationic to anionic charge
`highly complex La
`ratio, TE data for univalent and multivalent cationic lipids merged into a single curve as a function of sM, identifying it as a key
`C complexes climbs exponentially over  four decades with
`universal parameter. The universal curve for transfection by La
`increasing sM below an optimal charge density (sM* ), and saturates for sM [s
`M at a value rivaling the high transfection efficiency of
`
`
`HIIC complexes. In contrast, the transfection efficiency of HIIC complexes is independent of sM. The exponential dependence of TE
`
`on sM for LaC complexes, suggests the existence of a kinetic barrier against endosomal fusion, where an increase in sM lowers the
`C complexes and HIIC, confocal microscopy reveals the dissociation of lipid and DNA.
`
`barrier. In the saturated TE regime, for both La
`However, the lipid-released DNA is observed to be in a condensed state, most likely with oppositely charged macro-ion
`condensing agents from the cytoplasm, which remain to be identified. Much of the observed bulk of condensed DNA may be
`transcriptionally inactive and may determine the current limiting factor to transfection by cationic lipid gene vectors.
`
`INTRODUCTION
`
`The unrelenting research activity involving gene therapy with
`either synthetic vectors (carriers) or engineered viruses is
`currently unprecedented (Alper, 2002; Chesnoy and Huang,
`2000; Clark and Hersh, 1999; Ferber, 2001; Henry, 2001;
`Mahato and Kim, 2002; Miller, 1998). After the initial
`landmark studies (Felgner et al., 1987; Nabel et al., 1993;
`Singhal and Huang, 1994), cationic liposomes (CLs; closed
`bilayer membrane shells of lipid molecules) have emerged
`worldwide as the most prevalent synthetic vectors (carriers)
`(Ferber, 2001) whose mechanisms of action are investigated
`extensively in research laboratories, concurrently with on-
`going, mostly empirical, clinical trials to develop cancer vac-
`cines (Alper, 2002; Chesnoy and Huang, 2000; Clark and
`Hersh, 1999; Ferber, 2001; Henry, 2001; Mahato and Kim,
`2002; Miller, 1998). Primary among the advantages of CL
`over viral methods is the lack of immune response due to the
`absence of viral peptides and proteins. Moreover, while viral
`capsids have a maximum DNA-carrying capacity of ;40 kbp,
`CLs (which, when combined with DNA, form, in most
`
`Submitted October 3, 2002, and accepted for publication December 18,
`2002.
`
`Alison J. Lin and Nelle L. Slack contributed equally to this work.
`
`Address reprint requests to C. R. Safinya, MRL Rm. 2208, University of
`California, Santa Barbara, CA 93106. Tel.: 805-893-8635; Fax: 805-893-
`7221; E-mail: safinya@mrl.ucsb.edu.
`Ó 2003 by the Biophysical Society
`0006-3495/03/05/3307/10 $2.00
`
`lamellar LC
`a or
`instances, self-assemblies with distinct
`inverted hexagonal HC
`II nanostructures; see Raedler et al.,
`1997; Koltover et al., 1998; Lasic et al., 1997), place no limit
`on the size of the DNA. Thus, if complexed, for example,
`with human artificial chromosomes (Wilard, 2000), optimally
`designed CL-carriers offer the potential of potent vectors
`comprised of multiple human genes and regulatory sequences
`extending over hundreds of thousands of DNA base pairs.
`Despite all the promise of CLs as gene vectors, their
`transfection efficiency (TE; ability to transfer DNA into cells
`followed by expression), compared to viral vectors, remains
`notoriously low, resulting in a flurry of research activity
`aimed at enhancing transfection (Alper, 2002; Chesnoy and
`Huang, 2000; Clark and Hersh, 1999; Ferber, 2001; Henry,
`2001; Mahato and Kim, 2002; Miller, 1998). A further sense
`of urgency for developing efficient synthetic carriers stems
`from the recent tragic events associated with the use of
`engineered adenovirus vectors leading to the death of a patient
`due to an unanticipated severe immune response (Marshall,
`2000). In addition, in the latest gene therapy trials using
`modified retrovirus vectors to treat children with severe
`combined immunodeficiency, a French gene therapy team
`has reported a major setback where one patient (out of eleven)
`developed a blood disorder similar to leukemia which is
`confirmed to have resulted from insertion of the modified
`retrovirus in the initial coding region of a gene related to the
`early development of blood cells (Marshall, 2002).
`
`Moderna Ex 1006-p. 1
`Moderna v Arbutus
`
`

`

`3308
`
`Lin et al.
`
`Our in vitro studies should apply to TE optimization in ex
`vivo cell transfection, where cells are removed and returned
`to patients after transfection. In particular, our studies, aimed
`at understanding the chemically and physically dependent
`mechanisms underlying TE in continuous (dividing) cell
`lines, should aid clinical efforts to develop efficient CL-vector
`cancer vaccines in ex vivo applications. The vaccines are
`intended to induce transient expression of genes coding for
`immunostimulatory proteins in dividing cells (Chesnoy and
`Huang, 2000; Nabel et al., 1993; Rinehart et al., 1997;
`Stopeck et al., 1998); thus, the nuclear membrane, which dis-
`solves during mitosis, is not considered a barrier to expression
`of DNA.
`A critical requirement for enhancing transfection via
`synthetic carriers is a full understanding of the different
`nanostructures of CL-DNA complexes and the physical
`and chemical basis of interactions between complexes and
`cellular components. Toward that end, we used a combination
`of three-dimensional laser scanning confocal microscopy
`(LSCM), which closely followed complexes across the
`plasma membrane and inside the cytoplasm, and reporter
`gene TE studies that gave a statistically meaningful measure
`of the total amount of protein synthesized by cells from deli-
`vered DNA. Furthermore, we examined the structure-de-
`pendent basis of transfection through imaging of CL-DNA
`complexes exhibiting either the LC
`a (cationic lipid DOTAP
`mixed with neutral lipid DOPC) or HC
`II (DOTAP mixed with
`neutral lipid DOPE) nanostructure (Koltover et al., 1998;
`Raedler et al., 1997). Previous to our report, TE studies had
`shown that in mixtures of DOTAP and neutral lipids, typically
`at a wt.:wt. ratio of between 1:1 and 1:3, DOPE aided, while
`DOPC severely suppressed,
`transfection (Farhood et al.,
`1995; Hui et al., 1996), hence suggesting that HC
`II complexes
`transfect more efficiently than LC
`a complexes.
`
`MATERIALS AND METHODS
`
`Materials
`
`Lipids included univalent cationic lipids DOTAP (1,2-dioleoyl-3-trimethyl-
`ammonium-propane) and DMRIE (n-(2-hydroxyethyl)-n,n-dimethyl-2,3-
`bis(tetradecyloxy)-1-propanaminium), multivalent cationic lipid DOSPA
`(2,3-Dioleyloxy-n-(2-(sperminecarboxamido)ethyl)-n,n-dimethyl-1-propan-
`iminium penta-hydrochloride), and neutral lipids DOPC (1,2-dioleoyl-sn-
`glycero-3-phosphocholine) and DOPE (1,2-dioleoyl-sn-glycero-3-phos-
`phoethanolamine). DOTAP, DOPC, DOPE were purchased from Avanti
`Polar Lipids, Inc., and DMRIE and DOSPA were gifts from Vical Inc. Plas-
`mid DNA containing the Luciferase gene and SV40 promoter/enhancer ele-
`ments was used (pGL3-control vector, Promega, Cat. E1741).
`
`Liposome preparation
`
`Neutral lipids (DOPE, DOPC) were dissolved in chloroform and cationic
`lipids (DOTAP, DOSPA, DMRIE) were dissolved in a chloroform/methanol
`mixture. The lipid solutions were mixed in required ratios and the solvent
`was evaporated, first under a stream of nitrogen and then in vacuum over
`night, leaving a lipid film behind. The appropriate amount of millipore water
`was added to the dried lipid film, resulting in the desired concentration (0.1
`mg/ml–25 mg/ml) and incubated at 378C for at least 6 h to allow formation
`of liposomes. To form small unilamellar vesicles, liposome solutions were
`vortexed for 1 min, tip-sonicated to clarity (for 5–10 min), and filtered with
`0.2 mm filters to remove metal particulates arising from the sonicator tip.
`Liposome solutions were then stored at 48C.
`
`Transfection
`
`L-cells were transfected at a confluency of 60–80%. Using liposome (0.5
`mg/ml) and DNA (1 mg/ml) stock solutions, CL-DNA complexes were
`prepared in DMEM, which contained 2 mg of pGL3-DNA at a cationic-to-
`anionic charge ratio of 2.8 and allowed to sit for 20 min for complex
`formation. The cells were then incubated with complexes for 6 h, the
`optimized time of transfer into cells before removal, rinsed three times with
`phosphate-buffered saline (Gibco BRL), and incubated in supplemented
`DMEM for an additional 24 h (sufficient for a complete cell cycle) to allow
`expression of the luciferase gene. Luciferase gene expression was measured
`with the Luciferase Assay System from Promega. Each transfection ex-
`periment was repeated between 3–6 times over a short period of a few days
`yielding the error bars (Slack, 2000; Lin et al., 2002). In addition, the
`experiments were repeated four times over a 12-month period using different
`cell batches. While the absolute value of the average of each transfection
`measurement between the different experiments (done with several months
`separating experiments) varied by as much as a decade (which is also
`commonly found by other groups; Boussif et al., 1995), the observed trend
`in the transfection data was completely reproducible. Transfection efficiency
`was normalized to mg of total cellular protein using the Bio-Rad Protein
`Assay Dye Reagent Concentration solution (Bio-Rad) and is expressed as
`relative light units per mg of total cellular protein 6 1 SD. The transfection
`protocol is commonly used by others (Boussif et al., 1995).
`
`Laser scanning confocal microscopy (LSCM)
`
`L-cells were seeded on coverslips in six-well plates and allowed to grow,
`reaching a confluency of 60–80%. DNA was labeled following the Mirus
`Label IT (PanVera Corporation) protocol, which is fluorescent at 492 nm.
`Lipids were labeled with 0.2% (wt) Texas Red DHPE (Molecular Probes),
`which is fluorescent at 583 nm. Using labeled liposome (0.5 mg/ml) and
`DNA (0.1 mg/ml) stock solutions, CL-DNA complexes were prepared in
`DMEM using 2 mg of pGL3-DNA (at a cationic to anionic charge ratio of
`2.8), allowed to sit for 20 min for complex formation, and incubated with
`cells for the optimal 6-h transfer time. Cells were rinsed three times with
`phosphate-buffered saline (Gibco BRL), fixed by soaking in a fixing solution
`(3.7% formaldehyde in PBS) for 20–30 min and mounted using SlowFade
`Light Antifade Kit (Molecular Probes) for microscopy. Confocal images
`were taken with a Leica DM IRBE confocal microscope. The images of Figs.
`3 and 5 (repeated more than 10 times) are representative of the typical
`behavior in a given field of view (Lin, 2001).
`
`Cell culture
`
`X-ray diffraction (XRD)
`
`Mouse fibroblast L-cell lines were subcultured in DMEM (Dulbecco’s
`modified Eagle’s medium, Gibco BRL) supplemented with 1% (vol/vol)
`penicillin-streptomycin (Gibco BRL) and 5% (vol/vol) fetal bovine serum
`(HyClone Lab) at 378C and 5% CO2 atmosphere every 2–4 days to maintain
`monolayer coverage.
`
`the Stanford Synchrotron
`The XRD experiments were carried out at
`Radiation Laboratory at 10 KeV. To prepare CL-DNA samples liposome
`solutions (25 mg/ml) and DNA solutions (5 mg/ml) were each diluted in
`DMEM at 1:1 (vol.:vol.), then mixed at the desired cationic to anionic
`charge ratio of 2.8 and centrifuged before loading into for 1.5-mm x-ray. We
`
`Biophysical Journal 84(5) 3307–3316
`
`Moderna Ex 1006-p. 2
`Moderna v Arbutus
`
`

`

`Imaging and Efficacy of Lipid-DNA Complex
`
`3309
`
`note that similar to previous findings (Koltover et al., 1998; Raedler et al.,
`1997) the self-assembled structures of CL-DNA complexes does not change
`in the concentration range between the x-ray samples and the samples for
`confocal microscopy and transfection.
`
`RESULTS AND DISCUSSION
`
`The initial electrostatic attraction between positively charged
`CL-DNA complexes and mammalian cells is known to be
`mediated by negatively charged cell
`surface sulfated
`proteoglycans (Fig. 1 a, expanded view; also see Mislick
`and Baldeschwieler, 1996). Consistent with previous studies,
`we found that LC
`a complexes gain entry into the cell through
`endocytosis (Fig. 1, b and c; see also Labat-Moleur et al.,
`1996; Zabner et al., 1995). LSCM of LC
`a CL-DNA particles
`in cells revealed two distinct types of behavior. At low
`membrane charge density, sM  0.005 e/A˚ 2 ¼ e/(200 A˚ 2),
`mostly intact LC
`inside cells
`a complexes were present
`implying that DNA was trapped by the lipid vector. Further
`TE experiments confirmed that the intact complexes were
`themselves trapped in endosomes (Fig. 1 c). At high sM 
`
`FIGURE 1 Model of cellular uptake of LC
`a complexes. (a) Cationic
`complexes adhere to cells due to electrostatic attraction between positively
`charged CL-DNA complexes and negatively charged cell-surface sulfated
`proteoglycans (shown in expanded views) of mammalian plasma mem-
`branes. (b and c) After attachment, complexes enter through endocytosis. (d )
`Only those complexes with a large enough membrane charge density (sM)
`escape the endosome through activated fusion with endosomal membranes.
`(e) Released DNA inside the cell is observed by comfocal microscopy to be
`present primarily in the form of aggregates. The DNA aggregates must reside
`in the cytoplasm because oppositely charged cellular biomolecules able to
`condense DNA are not present in the endosome. Arrows in the expanded
`view of c indicate the electrostatic attraction between the positively charged
`membranes of the complex and the negatively charged membranes of the
`endosome (comprised of sulfated proteoglycans and anionic lipids), which
`tends to enhance adhesion and fusion. Arrows in the expanded view in
`d indicate that the bending of the membranes hinders fusion.
`
`0.012 e/A˚ 2  e/(83 A˚ 2), we observed released DNA inside
`cells consistent with the escape of CL-DNA complexes into
`the cytoplasm through fusion with anionic endosomal
`membranes (Fig. 1 d ). LSCM further determined that the
`released DNA was condensed (Fig. 1 e), most likely, with
`oppositely charged cytoplasmic condensing agents absent in
`endosomes. Unlike the endosomal environment, the cyto-
`plasm contains many multivalent cationic biomolecules such
`as spermine and histones, which become available during the
`cell cycle in millimolar concentration levels, and are able to
`condense DNA (Bloomfield, 1991).
`Corresponding TE measurements exhibited two remark-
`able features. First and foremost, an unexpected simplicity
`emerged from this highly complex LC
`a-cell system, where sM
`was found to be a universal parameter controlling TE. The TE
`data for LC
`a complexes containing DOPC mixed with the
`multivalent cationic lipid DOSPA or the univalent cationic
`lipids DOTAP and DMRIE, at a constant cationic to anionic
`charge ratio, merged onto a universal curve when plotted
`versus sM. Second, this universal TE curve increased ex-
`ponentially, over four decades, with increasing sM. This be-
`havior is consistent with a model describing a kinetic barrier
`for fusion of CL-DNA complexes with the endosomal mem-
`brane (Fig. 1 d), where an increase in sM lowers the barrier
`height. This new understanding of the fundamental role of the
`lipid carrier sM has led to redesigned LC
`a DOPC-based carriers
`with efficacy competitive with the TE of HC
`II DOPE-based
`carriers.
`We used positively charged CL-DNA complexes prepared
`at r ¼ DOTAP/DNA (wt./wt.) ¼ 6 (r ¼ 2.2 is the isoelectric
`point) from mixtures of cationic and neutral lipids complexed
`with plasmid DNA (pGL3). The weight ratio r ¼ 6, which
`gave a cationic-to-anionic charge ratio of 2.8, was chosen as it
`corresponded to the middle of a typical plateau region
`observed for optimal transfection conditions as a function of
`increasing r above the isoelectric point. X-ray diffraction
`(XRD) results elucidated the structures of these complexes in
`water and in DMEM, a common environment for in vitro
`studies of cells. Synchrotron XRD of DOTAP/DOPC com-
`plexes at the mole fraction FDOPC ¼ 0.67 (Fig. 2, left)
`showed sharp peaks at q001 ¼ 0.083 A˚ ÿ1, q002 ¼ 0.166 A˚ ÿ1,
`with a shoulder peak at q003 ¼ 0.243 A˚ ÿ1 (due to the form
`factor), and q004 ¼ 0.335 A˚ ÿ1, resulting from the layered
`a phase (d ¼ interlayer spacing ¼ dm1 dw ¼
`structure of the LC
`2p/q001 ¼ 75.70 A˚ ) with DNA intercalated between cationic
`left,
`inset). For DOTAP/DOPE
`lipid bilayers (Fig. 2,
`complexes at FDOPE ¼ 0.69, XRD (Fig. 2, right) revealed
`four orders of Bragg peaks at q10 ¼ 0.103 A˚ ÿ1, q11 ¼ 0.178
`A˚ ÿ1, q20 ¼ 0.205 A˚ ÿ1, and q21 ¼ 0.270 A˚ ÿ1, denoting the HC
`phase (Fig. 2, right, inset) with a unit cell spacing of a ¼
`p
`q10Þ ¼ 70:44 ˚A: Except for a difference in lattice
`4p=ð
`3
`constants, the structures of CL-supercoiled DNA complexes
`are analogous to the ones reported recently for CL-linear
`DNA complexes (Koltover et al., 1998, 1999; Raedler et al.,
`1997; Salditt et al., 1997).
`
`II
`
`ffiffiffi
`
`Biophysical Journal 84(5) 3307–3316
`
`Moderna Ex 1006-p. 3
`Moderna v Arbutus
`
`

`

`3310
`
`Lin et al.
`
`FIGURE 2 Comparison of structure and transfection efficiency (TE). Left (mole fraction FDOPC ¼ 0.67) shows a typical XRD scan of lamellar (inset) LC
`a
`complexes. Right (mole fraction FDOPE ¼ 0.69) shows a typical XRD scan of inverted hexagonal (inset) HC
`II complexes. Middle displays the corresponding
`TE, as measured by luciferase enzyme assays of transfected mouse L-cells.
`
`Transfection experiments were done using plasmid DNA
`(pGL3), which contains the firefly luciferase reporter gene, to
`measure TE and its correlation to the solution structures of
`complexes. Mouse L-cells were transfected with CL-DNA
`complexes and incubated on average for at least a full cell-
`cycle, during which expression occurred. A standard luci-
`ferase assay allowed us to evaluate quantitatively the amount
`of synthesized protein by measuring the bioluminescence
`(number of emitted photons) in relative light units per mg of
`cell protein. Fig. 2 (middle) clearly demonstrates the higher
`TE, by more than two decades, attained by complexes in the
`II phase at FDOPE ¼ 0.69 compared to LC
`HC
`a complexes at
`FDOPC ¼ 0.67.
`To further understand the structure-function correlation,
`we examined the transfer process of CL-DNA complexes into
`cells and the mechanism of the subsequent DNA release
`using LSCM, which provides an optical resolution of ;0.3
`mm in the x and y, and ;3/4 mm in the z. The complexes were
`doubly tagged with fluorescent labels, a red one for lipid and
`a green covalent one for DNA. Fig. 3 shows LSCM pictures
`of mouse L-cells after 6 h of transfer time. By comparing
`images of the x-y, y-z, and x-z planes, we were able to
`determine the position of an object relative to a cell. Fig. 3 A
`shows a typical confocal image of a mouse cell transfected
`II complexes at FDOPE ¼ 0.69. The lipid fluorescence
`with HC
`clearly outlines the plasma membrane, indicating fusion of
`lipid with the plasma membrane before or after entry through
`the endocytic pathway (Wrobel and Collins, 1995). An
`aggregate of complexes (yellow) was seen inside a cell as well
`as a clump of DNA (green) in the cytoplasm. The image
`shows that the interaction between HC
`II complexes and cells
`leads to the dissociation and release of DNA from the CL-
`vector consistent with the measured high TE.
`The corresponding confocal images with LC
`a complexes at
`FDOPC ¼ 0.67 are shown in Fig. 3 B. In striking contrast to
`transfection with HC
`II complexes, we observed no free DNA,
`
`Biophysical Journal 84(5) 3307–3316
`
`but rather many individual intact CL-DNA complexes inside
`cells. Fig. 3 B highlights one such typical complex. In the
`absence of fusion, complexes entered cells through endocy-
`tosis (Fig. 1). This was further substantiated in LSCM images
`of cells prepared at 48C, where endocytosis is inhibited,
`which showed complexes attached to the outside cell surface
`and none within the cell body (Lin et al., 2000; Lin, 2001;
`Safinya et al., 2002). At FDOPC ¼ 0.67, most of the DNA
`remained trapped by the CL-vector consistent with the
`measured low TE. As we discuss below, chloroquine-based
`experiments showed that the intact CL-DNA complexes were
`typically trapped within endosomes.
`As the concentration of DOPC decreased in the LC
`a CL-
`DNA complexes, we observed an unexpected enhancement
`in TE by two decades. Fig. 4 A (red diamonds) exhibits the
`nontrivial dependence of TE on FDOPC for DOPC/DOTAP-
`DNA complexes, which starts low for 0.5 \ FDOPC \ 0.7
`and increases dramatically to a value, at FDOPC ¼ 0.2,
`rivaling that achieved by DOPE/DOTAP-DNA complexes.
`Similar results were obtained for another univalent cationic
`lipid DMRIE (Fig. 4 A, black triangles). The key experi-
`ment, which led to a deeper understanding of TE, was
`a study done with the multivalent cationic lipid DOSPA (blue
`squares) replacing DOTAP. A qualitatively similar trend was
`observed with TE decreasing rapidly above a critical F
`DOPC,
`albeit with F
`DOPC shifted from ;0.2 (observed for DOTAP
`and DMRIE complexes) to 0.7 6 0.1 (DOSPA). The main
`difference between the cationic lipids is the notably larger
`charge density of DOSPA (Fig. 4 B, inset), with a larger head-
`group carrying potentially up to five cationic charges. Thus,
`at a given FDOPC, the membrane charge density (sM) is sig-
`nificantly larger in DOSPA compared to DOTAP or DMRIE
`containing complexes.
`We show in Fig. 4 B the same TE data of Fig. 4 A, now
`plotted versus the membrane charge density sM (i.e., the
`average charge per unit area of the cationic membrane):
`
`Moderna Ex 1006-p. 4
`Moderna v Arbutus
`
`

`

`Imaging and Efficacy of Lipid-DNA Complex
`
`3311
`
`area of the cationic to neutral lipid; scl ¼ eZ/Acl is the charge
`density of the cationic lipid with valence Z; and Fnl ¼ Nnl/
`(Nnl 1 Ncl) and Fcl ¼ Ncl/(Nnl 1 Ncl) are the mole fractions
`of the neutral and cationic lipids, respectively. For the plots in
`Fig. 4 B, we used Anl ¼ 70 A˚ (Langmuir trough data), rDOTAP
`¼ rDMRIE ¼ 1, rDOSPA ¼ 2, ZDOTAP ¼ ZDMRIE ¼ 1, and
`ZDOSPA¼ 3. We found that values of ZDOSPA between 3 and
`4 yield a good visual fit for the comparison between DOTAP
`and DOSPA while 2 and 5 clearly do not. For environments
`of neutral pH we expect ZDOSPA to be closer to 4. The value
`of ZDOSPA could be regarded as a fitting parameter in the
`range between 3 and 4. Remarkably, given the complexity of
`the CL-DNA-cell system, the data, spread out when plotted as
`a function of Fnl (Fig. 4 A), coalesce into a ‘‘universal’’ curve
`as a function of sM, with TE varying exponentially over
`nearly four decades as sM increases by a factor of  8 (Fig. 4
`B, sM between 0.0015 e/A˚ 2 and 0.012 e/A˚ 2), clearly demon-
`strating that sM is a key universal parameter for transfection
`with lamellar LC
`a CL-vectors. We now observe a single
`
`M  0:0104 6 0:0017 e= ˚A2  e=ð100 ˚A
`optimal s
`2
`) (Fig. 4
`B, arrow) where the universal TE curve saturates for
`sM [s
`M for both univalent and multivalent cationic lipid-
`containing CL-vectors. sM controls the average DNA
`interaxial spacing dDNA (Fig. 2, inset), which decreases as
`sM increases (Koltover et al., 1999; Raedler et al., 1997).
`Future designs of CL-vectors, which further enhance the
`packing of DNA based on the recent theoretical understand-
`ing of
`intermolecular
`interactions within the complex
`(Bruinsma, 1998; Harries et al., 1998; O’Hern and Lubensky,
`1998), may be expected to enhance TE.
`The TE data suggest vastly diverse behaviors of LC
`a CL-
`DNA complexes between low and high sM. As we discussed
`earlier, for low sM ¼ e/(200 A˚ 2) where TE is low, confocal
`images show DNA locked within complexes after endocy-
`tosis (Fig. 3 B). To test the idea that it is the endosomal vesicle
`that traps the complex, we carried out transfection experi-
`ments in the presence of chloroquine, a well-established bio-
`assay known to enhance the release of trapped material within
`endosomes by osmotically bursting the vesicle. The endo-
`cytic pathway involves the fusion of endosomes with lyso-
`somes (vesicles containing enzymes for degradation of
`material within endosomes) leading to late-stage endosomes,
`limiting the time available for CL-DNA complexes to escape.
`Chloroquine, a weak base, penetrates the lysosome and accu-
`mulates in a charged state; thus, lysosomes and late-stage
`endosomes tend to rupture due to increased osmotic pressure
`caused by counterions rushing in (Voet and Voet, 1995).
`The fractional increase (TEchloroquine/TE) for the DOSPA/
`DOPC and DOTAP/DOPC systems with added chloroquine
`as a function of sM (Fig. 4 C) shows the large increase in TE
`by as much as a factor of 60 as sM decreases and indicates that
`at low sM lamellar LC
`a complexes are trapped within endo-
`somes, consistent both with the confocal images (Fig. 3 B)
`and the measured low TE without chloroquine. At high sM,
`chloroquine has a much smaller effect on TE with the frac-
`
`Biophysical Journal 84(5) 3307–3316
`
`FIGURE 3 Laser scanning confocal microscopy (LSCM) images of
`transfected mouse L-cells. Red denotes lipid; green, DNA; and yellow, the
`overlap of the two denotes CL-DNA complexes. For each set, middle is the
`x-y top view at a given z; right is the y-z side view along the vertical dotted
`line; bottom is the x-z side view along the horizontal dotted line. Objects in
`circles are indicated by arrows in the x-z and y-z plane side views. (A) Cells
`II complexes (FDOPE ¼ 0.69), show fusion of lipid (red )
`transfected with HC
`with the cell plasma membrane and the release of DNA (green in the circle)
`within the cell. Thus, HC
`II complexes display clear evidence of separation of
`lipid and DNA, which is consistent with the high transfection efficiency of
`such complexes. The released exogenous DNA is in an aggregated state,
`which implies that
`it has been condensed with oppositely charged
`biomolecules of the cell, which remain to be identified. (B) Cells transfected
`a complexes at FDOPC ¼ 0.67, which results in a low membrane
`with LC
`charge density sM  0.005 e/A˚ 2 and low transfection efficiency (as plotted
`in Fig. 4 B). First, no fusion is observed. Second, intact CL-DNA complexes
`are observed inside cells (one such yellow complex is shown in the circle).
`The intact complex implies that DNA remains trapped within the complex,
`which is consistent with the observed low transfection efficiency of such low
`sM=LC
`a complexes. Because of the lack of fusion (which aided observation
`of the cell outline in A), we achieved cell outline by observation in reflection
`mode, which appears as blue. Bars ¼ 5 mm applies to all planes.
`sM ¼ eZNcl=ðNnlAnl1NclAclÞ
`¼ ½1 ÿ Fnl=ðFnl1rFclފscl:
`Here, Nnl and Ncl are the number of neutral and cationic
`lipids, respectively; r ¼ Acl/Anl is the ratio of the headgroup
`
`(1)
`
`Moderna Ex 1006-p. 5
`Moderna v Arbutus
`
`

`

`3312
`
`Lin et al.
`
`D. At high FDOPE [ 0.56, DOTAP/DOPE-DNA complexes
`are in the HC
`II phase (green open squares) and exhibit high
`TE. We further see that in contrast to DOPC containing
`complexes, which show a strong dependence on the mole
`fraction of neutral lipid (or equivalently sM), TE of DOPE
`containing complexes is independent of sM. The data show
`unambiguously that sM is a critical parameter for TE by LC
`a
`complexes and not so for HC
`II complexes. The mechanism
`of transfection by DOPE containing HC
`II complexes is in-
`dependent of sM and dominated by other effects; possibly,
`for example, the known fusogenic properties of inverted
`hexagonal phases. In contrast, for LC
`a complexes, high TE
`requires sM [s
`to produce a high TE of LC
`M: Thus,
`a
`complexes with large mole fraction of neutral lipid ;0.70
`(i.e., similar to those mole fractions where DOPE containing
`HC
`II complexes show high TE) requires the incorporation of
`multivalent cationic lipids (e.g., DOSPA) to ensure that
`sM [s
`M.
`LSCM images of cells transfected with LC
`a complexes
`at high sM displayed a path of complex uptake and DNA
`release distinct from transfections done with both LC
`a com-
`plexes at low sM (Fig. 3 B) and HC
`II complexes (Fig. 3 A).
`a complexes at FDOPC ¼
`A typical confocal image with LC
`0.18 (sM  0.012 e/A˚ 2) is shown in Fig. 5. Intact complexes
`were found inside the cell (Fig. 5, label 2 in x-y plane; box 2
`shows the equal green (DNA) and red (lipid) fluorescence
`intensity along the dotted line in x-y; see inset), but more
`interestingly, a mass of exogenous DNA successfully trans-
`ferred into the cytoplasm was also clearly evident (Fig. 5,
`label 1 in x-y plane; box 1 shows the much larger green
`fluorescence (DNA) intensity along the x-y direction). In the
`absence of fusion, high sM complexes enter cells through
`endocytosis. The integrated fluorescence intensity of the
`observed DNA (Fig. 5, box 1) is comparable to that of
`DNA complexed with lipids (Fig. 5, box 2), indicating that
`the released DNA is in the form of aggregates. Because
`endosomes contain no known DNA-condensing agent, these
`aggregates must reside in the cytoplasm (Fig. 1 e). The
`presence of lipid-released DNA in the cytoplasm after
`endocytic uptake of complexes agrees with the measured
`high TE, and moreover, implies fusion between CL-DNA
`lipids and endosomal membranes (Fig. 1 d), enabling escape
`from the endosomes. This is consistent with our finding that
`chloroquine has a small effect at high sM because endosomal
`escape is no longer a major obstacle.
`The confocal image also captured an aggregate of comp-
`lexes in contact with a large area of the cell surface,
`displaying no tendency toward fusion with the plasma mem-
`brane (Fig. 5, label 3 in x-y plane). Comparing the changes
`in fluorescence intensity along the x-y (Fig. 5, box 3) and z
`(Fig. 5, box 4) directions, from the outside toward the inside
`of the cell, we clearly see an aggregate of complexes caught in
`the process of dissociation after endocytosis, with released
`DNA toward the inside of the cell.
`
`(A) Transfection efficiency (TE) expressed as relative light
`FIGURE 4
`units per mg total cellular protein plotted as a function of varying mole fraction
`DOPC with cationic lipids DOSPA, DOTAP, and DMRIE. (B) TE plotted
`versus the membrane charge density sM, demonstrating universal behavior of
`CLs containing cationic lipids with different molecular charges and head-
`group areas (inset). For all three systems, TE increases with sM until an
`M (arrow at  0.0104 e/A˚ 2; determined by the intersection of two
`optimal s
`straight lines fit to the data (dashed lines) above and below the ‘‘knee’’), at
`which TE plateaus. (C ) The fractional increase TEchloroquine/TE for the
`DOSPA/DOPC and DOTAP/DOPC systems with added chloroquine as
`a function of sM. (D) TE plotted as a function of mole fraction of neutral lipid
`for the DOTAP/DOPC and the DOTAP/DOPE systems. The DOTAP/DOPC
`system is LC
`a . The DOTAP/DOPE system goes through two phase transitions:
`
`
`LCa ( filled green square) to coexisting LCa 1 HC
`II (green square with cross) to
`HC
`II (open green square). All transfection measurements were done with 2 mg
`of plasmid DNA at a constant cationic to anionic charge ratio of 2.8. (2.8 was
`chosen as it corresponded to the middle of a typical plateau region observed
`for optimal transfection conditions as a function of increasing cationic to
`anionic charge ratio above the isoelectric point of the complex.) Thus, every
`TE data point of A and B (which is found to vary by nearly four orders of
`magnitude) used the same amount of total charged species (anionic charge
`from DNA, cationic charge from cationic lipid) and t