Nonlamellar Phases in Cationic Phospholipids, Relevance to Drug

Jan 23, 2015 - College of Pharmacy, The Ohio State University, 517 Parks Hall, 500 W. 12th Avenue, Columbus, Ohio 43210, United States. ‡. Departmen...
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Nonlamellar Phases in Cationic Phospholipids, Relevance to Drug and Gene Delivery Rumiana Koynova,*,†,‡ Boris Tenchov,†,§ and Robert C. MacDonald‡ †

College of Pharmacy, The Ohio State University, 517 Parks Hall, 500 W. 12th Avenue, Columbus, Ohio 43210, United States Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, United States § Department of Medical Physics and Biophysics, Medical University−Sofia, Sofia, Bulgaria ‡

ABSTRACT: Lipid aggregates have been used as drug carriers for several decades. Recently, nonlamellar liquid crystalline lipid systems have attracted attention as possible drug-delivery vehicles because of their unique nanostructure and physicochemical properties. Here we summarize data on the nonlamellar phase-forming propensity of the cationic phosphatidylcholines (cationic PCs). The class of cationic PCs has been specifically designed and explored for the purpose of nonviral gene delivery. These lipids were found to comprise an attractive cationic lipid class because they are biodegradable, have low toxicities, and in a number of cases, display high transfection activity. Lipids of this class form a variety of polymorphic and mesomorphic phases−lamellar and nonlamellar, depending on the structure of their hydrocarbon chains and especially on the third hydrocarbon chain used to alkylate the PC phosphate group and convert the zwitterionic PC headgroup into a cation. Here we characterize the phase behavior and transfection activity of eight cationic PCs that have been identified as forming nonlamellar phases−inverted hexagonal and cubic. We then demonstrate that those cationic PCs that also form nonlamellar lipoplexes are notably less efficient gene nanocarriers in comparison with the cationic PCs forming lamellar phase lipoplexes. KEYWORDS: cationic phospholipid, cubic phase, hexagonal phase, lipoplex, transfection, drug delivery

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INTRODUCTION Possible applications of lipid structures as drug delivery vehicles have been investigated for some decades, at least since the discovery of liposomes.1 Along with the lamellar lipid systems forming liposomes, nonlamellar liquid crystalline systems, such as inverted bicontinuous cubic, hexagonal, and sponge mesophases, have attracted attention as possible drug-delivery vehicles as well, because of their unique microstructure and physicochemical properties.2 Specifically, synthetic cationic lipids and lipidlike compounds are being developed as nonviral carriers of nucleic acids into cells,3−6 and they currently represent one of the most widely used strategies for in vivo gene delivery. They readily form complexes (lipoplexes) with polyanionic nucleic acids, protecting the latter until entry into cells. Because systemic circulation and cellular uptake of free DNA are obstructed by nuclease degradation and by the size and negative charge of DNA, therapeutic practices including gene transfection and gene silencing require efficient delivery vectors in order to condense, protect, and chaperone the genetic material to the target cells. Two principal carriers include viral and nonviral vectors. Viral vectors are most effective,7,8 but their application is unfavorable for their immunogenicity and oncogenicity. Synthetic cationic lipids are presently the most widely used constituents of nonviral gene carriers.3−5 However, a critical obstacle for the clinical applications of lipid-mediated gene delivery is its unsatisfactory efficiency for many cell types. For this reason, lipofection still accounts for only 5.4% of gene therapy clinical trials (113 trials for the period 1989−2014), whereas viral vectors are applied in © XXXX American Chemical Society

more than 66% (http://www.wiley.com//legacy/wileychi/ genmed/clinical/). Progress in improving the efficacy of lipidaided transfection has been impeded because its mechanism is still largely unknown and critical stages in the process of intracellular delivery are not well distinguished. To better understand phenomena underlying gene and drug delivery by cationic lipids, we examined some relevant physical properties of cationic phospholipids and their complexes, with special focus on their phase behavior, more specifically, the ability of some of them to form nonlamellar phases. Our work is focused on a particularly noteworthy cationic lipid class, cationic phosphatidylcholines.9,10 They are attractive principally because they are derived from the natural phosphatidylcholines (PCs), the zwitterionic phosphocholine headgroup of which is converted into a cation by esterification of the phosphate group (Figure 1A). These lipids are slowly metabolized and exhibit low toxicities.9,10 Several lipids of this type have demonstrated excellent transfection efficiencies for plasmid DNA, both in vitro and in vivo, as well as in antitumor and anticystic fibrosis gene therapies.11−17 They have been found to be efficient siRNA carriers as well, exceeding in some cases the gold standard in gene silencing, Lipofectamine RNAiMAX.18 They also exhibit a wide variety of stable phases other than lamellar. It is this characteristic that allows us in this Received: November 30, 2014 Accepted: January 23, 2015

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Figure 1. (A) Structure of cationic phosphatidylcholines (PCs) (1,2-diacyl-sn-glycero-3-alkylphosphocholines).; (B) 1,2-dioleoyl-sn-glycero-3ethylphosphocholine (diC18:1-EPC; EDOPC), the best studied representative of the cationic PCs; (C) structures of nonlamellar phase-forming cationic phosphatidylcholines.

Nonlamellar liquid crystalline lipid systems, such as inverted bicontinuous cubic, hexagonal, and sponge mesophases, have often been considered as possible drug delivery vehicles because of their unique microstructure and distinctive physicochemical properties (Figure 2).2 Studies related to the application of nonlamellar lipid phases in drug delivery can be traced back to reports describing the application of inverted cubic and hexagonal liquid crystalline phases in the field of controlled release formulations for inhalation drug delivery.25,26 The introduction of a method for fragmentation of the cubic and hexagonal lipid phases to reproducible and stable nanosized particles known as cubosomes and hexosomes27 further stimulated the search for nonlamellar lipid pharmaceutical carriers. Various bioactive molecules can be solubilized and protected from hydrolysis and/or oxidation in aqueous as well as the lipid hydrophobic phase. Nonlamellar liquid crystalline lipid aggregates have demonstrated high encapsulation capacity and hence applications involving controlled release of drugs. Valuable properties such as a wide solubilization spectrum, high drug payloads, effective encapsulation, stabilization, and protection of sensitive drug substances suggest the nonlamellar lipid aggregates as promising pharmaceutical carriers. Here we summarize data on the nonlamellar phase-forming propensities of the cationic PCs, collected by small-angle synchrotron X-ray diffraction. To date, eight cationic PCs have been identified as forming nonlamellar phases−inverted hexagonal and cubic−and their phase behavior has been characterized. Cationic PCs that form nonlamellar phases and either lamellar or hexagonal phase lipoplexes have been found to be rather inefficient gene carriers, particularly in comparison to certain cationic PCs that reside in lamellar phases and form lamellar phase lipoplexes. Still, summarizing data on their phase behavior and understanding the specifics of their mesomorphic self-assembly is worthwhile because they may have a promise as alternative (to liposomes) drug delivery agents.

communication to focus on how the transfection efficiency of these lipids depends on the characteristics of their phases. Polar lipids are known for their ability to form an impressive variety of polymorphic and mesomorphic phases−lamellar and nonlamellar−when dispersed in aqueous media19,20 (Figure 2),

Figure 2. Liquid crystalline lipid phases. (A) Lamellar phase; (B) hexagonal phase HI; (C) inverted hexagonal phase HII; (D) inverted micellar cubic phase QIIM; (E) bilayer cubic (QII) phase Im3m; (F) bilayer cubic phase Pn3m; (G) bilayer cubic phase Ia3d; (H) sponge (L3) phase.

and phase state has been recognized as potentially important for the transfection activity of lipoplexes.21−24 A variety of key parameters that are relevant to drug and gene delivery are quite clearly dependent upon the phase organization of delivery vehicles. These include: encapsulation capacity, release kinetics, external charge density and interactions with target cells, permeability, stability in serum, susceptibility to fusion with cellular membranes and phase evolution thereafter, and the ability to circumvent specific biological barriers and avoid capture by particular tissues. In addition, it has been recognized that the lipoplex lipids can undergo phase changes upon interaction with cellular lipids and that this process may even be of paramount importance. B

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NONLAMELLAR PHASES IN CATIONIC PHOSPHATIDYLCHOLINES

The cationic PCs are triesters, specifically alkyl phosphate PC derivatives, in which the headgroup zwitterion has been converted into a cation (Figure 1A).9,28 Their synthesis comprises reaction of an alkyl triflate with the respective phosphatidylcholine, resulting in the triflate salt of Osubstituted phosphatidylcholine.9,29 Cationic PCs with short R3 chains, in particular the ethyl phosphatidylcholines (ePCs), arrange into bilayers and readily form dispersions of uni- or oligolamellar liposomes in water.10 At electrolyte concentrations as high as those of physiological solutions in which electrostatic repulsion is largely suppressed, and at higher lipid contents, these lipids form well-correlated lamellar phases.10 The structures of cationic PCs found to form nonlamellar phases are shown in Figure 1B. Next we describe the phase behavior of these lipids. Dioleoyl Cationic PC Derivatives with Different R3 Chain Length. Increasing the length of R3 chain in the cationic PC derivatives shifts the hydrophilic−lipophilic balance and generally results in replacement of the lamellar Lα phase by nonlamellar phases. A good illustration of this behavior is given by a set of dioleoyl PC derivatives with methyl, ethyl (Figure 1B), propyl, hexyl (Figure 1C-d), decyl (Figure 1C-f) and octadecyl (Figure 1C-g) R3 chains. The first three, with an R3 chain that is 1, 2, or 3 C atoms long (diC18:1-mePC, diC18:1ePC and diC18:1-C3PC), form the Lα phase (Figure 3A shows the first in this group). The diC18:1-C6PC lipid, with an R3 chain of 6 C atoms long, forms the cubic Pn3m phase (Figure 3B), whereas with an R3 chain 10 or 18 C atom long, the respective lipids−diC18:1-C10PC and diC18:1-C18PC−form inverted hexagonal phases (illustrated for the latter compound in Figure 3C).29−34 The sequence of A to B to C shown in Figure 3 can be taken to illustrate the evolution of the propensities of set of differently O-substituted dioleoylphosphatidylcholines to change from lamellar to cubic to hexagonal phase as shape of the molecule is changed, such molecular shape changes being responsible for changes in molecular packing, which then subsequently cause an increase in the curvature of the polar-water interface.20,35 That is, when the substituent is lengthened from 2 carbons (A) to 6 carbons (B) to 18 carbons (C), the molecular shape of the cationic PC transforms from nearly cylindrical to more truncated conelike and the phase propensity changes from lamellar to bilayer cubic to inverted hexagonal. The same progression of phases is often seen for a single molecule upon heating, that is many lamellar molecules are converted to cubic and then to hexagonal phases as the temperature is raised, as an increase in temperature raises the cross-sectional area of the lipid tails. Small-angle X-ray diffraction patterns recorded upon heating and cooling of an aqueous dispersion of diC18:1-C6PC (Figure 1C-d) showed that this lipid resides in the cubic phase at all temperatures in the range −10 to 90 °C.30 Up to 12 maxima were visible on the diffraction pattern (Figure 4), indexing as the full set of the initial 12 reflections characteristic of the cubic Pn3m phase (cubic aspect #4),36 with a ∼8.8 nm unit cell size at 20 °C. This highly ordered structure remained unchanged on storage at room temperature for at least 24 h and upon cooling to −10 °C.

Figure 3. Effect of R3 chain length on the phase behavior of the cationic dioleoyl PC. Small-angle X-ray diffraction patterns recorded at 20 °C of (A) lamellar Lα, (B) cubic Pn3m, and (C) inverted hexagonal HII phases formed by dioleoyl cationic PCs with ethyl, hexyl, and octadecyl R3 chains, respectively, diC18:1-EPC,33 diC18:1-C6PC,30 and diC18:1-C18PC.31 Reproduced with permission from ref 34. Copyright 2009 The Royal Society of Chemistry.

Figure 4. SAXD patterns of a diC18:1-C6PC dispersion recorded during heating−cooling scans. Inset: diffraction pattern of diC18:1C6PC dispersion recorded at 20 °C at the beginning of the heating scan. Reproduced with permission from ref 30. Copyright 2008 American Chemical Society.

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ACS Biomaterials Science & Engineering Diffraction patterns recorded upon heating and cooling scans of diC18:1-C18PC (Figure 1C-g) reveal an inverted hexagonal phase throughout the whole temperature interval 0−90 °C.31 Several other PC derivatives were also found to form wellordered nonlamellar phases, either in the entire temperature range 20−90 °C, or subsequent to an initial phase transition from the lamellar phase: C18:1/C4−C10PC. Similarly to diC18:1-C6PC, C18:1/C4− C10PC (Figure 1C-b) resides in the cubic Pn3m phase at all temperatures in the range 20−100 °C (Figure 5A).

Figure 6. SAXD patterns of diC22:1-EPC dispersions recorded on heating. Inset: diffraction pattern taken at 20 °C after cooling. Reproduced with permission from re f30. Copyright 2008 American Chemical Society.

a transient QIa3d cubic phase in the temperature interval 25−30 °C. On cooling, the QPn3m cubic phase transforms directly into the gel Lβ phase at 17 °C (Figure 7). DiC16:4me-C16PC. Similarly to diC18:1-C18PC, the branched-chain compound diC16:4me-C16PC (Figure 1C-h) resides in the inverted hexagonal phase throughout the whole temperature interval 0−90 °C.31 Mixtures of Cationic Lipids with Helper Lipids. It has been reported for many cationic lipid formulations that their transfection efficacy is improved by the presence of certain neutral lipids (so-called helper lipids). These helper lipids are typically molecules that have long been known to facilitate membrane fusion. Because fusion of the lipoplex membrane with a cell membrane appeared to be necessary for effective transfection, these molecules were tested for their effect on transfection of a number of different cationic lipid-like transfection agents. These helper lipids are also known to enhance the propensity of the cationic lipid to form nonlamellar phases. The most common and widely used helper lipids are phosphatidylethanolamines, especially dioleoylphosphatidylethanolamine (DOPE), and cholesterol. The class of cationic PCs is no exception with respect to the enhanced ability to form nonlamellar phases in mixtures with helper lipids. Thus, cholesterol, which is known as a membrane “fusogen”40 and has also been reported to promote hemifusion between vesicles,41 induces the formation of cubic phase when added to the lamellar di18:1-ePC (EDOPC) (Figure 8). Another common helper lipid, DOPE, has also been reported to promote formation of cubic phase in di18:1-ePC (EDOPC).32 It is remarkable, however, that in marked contrast to other cationic lipid carriers, the transfection efficiency of the class of cationic PCs does not benefit from the presence of helper lipids.10,32 Transfection Efficiency of Inverted Hexagonal Phase Lipoplexes. Similarly to other cationic lipids, the cationic PCs interact electrostatically and form stable complexes (lipoplexes) with the polyanionic nucleic acids. The structure of most lipoplexes is a multilamellar sandwich in which lipid bilayers

Figure 5. SAXD patterns of (A) C18:1C4−C10PC dispersions recorded on heating−cooling scans; (B) of diC10-C8PC dispersion recorded at 20 °C.

DiC10-C8PC. DiC10-C8PC (Figure 1C-a) also forms a cubic phase of the Pn3m type (unit cell size 8.61 nm) at all temperatures in the range 20−90 °C (Figure 5B). DiC22:1-ePC. Small-angle X-ray diffraction patterns recorded upon heating and cooling of an aqueous dispersion of diC22:1ePC (Figure 1C-e) over the temperature range 20−90 °C show that this lipid forms both lamellar and cubic phases upon hydration, and fully converts into and remains as a cubic phase after heating.30 The diffraction pattern after hydration at room temperature, before heating, reveals a lamellar phase, coexisting with a mixture of cubic Im3m and Pn3m phases. Upon heating, the coexisting lamellar phase (repeat period d = 5.86 nm) and mixture of cubic Im3m and Pn3m phases convert into a cubic Pn3m phase at temperatures between 50 and 70 °C (Figure 6). This transition is not reversible−the cubic phase remains stable on cooling and on subsequent storage at room temperature up to 24 h; it indexes as the full set of the initial 11 Pn3m reflections, with an 11.65 nm unit-cell size (Figure 6, inset). It is worth noting that cubic phase formation in diC22:1-ePC seems likely to be induced by the headgroup positive charge, since its zwitterionic analog, the phosphatidylcholine with two C22:1 chains (diC22:1-PC) does not form nonlamellar phases.37 It is assumed that the charged lipids facilitate the transformation into cubic phases by increasing the electrostatic repulsion between the lipid bilayers and reducing in this way the unbinding energy required for dissipation of the lamellar phase prior to its conversion into bilayer cubic phase.38 DiC10-C14PC. Similarly to diC22:1-ePC, diC10-C14PC (Figure 1C-c) exhibits an irreversible lamellar−cubic transition on heating, at temperatures between 25 and 30 °C (Figure 7).39 Thus, on heating, the phase sequence is Lβ → Lα → QIa3d → QPn3m, with a chain melting Lβ → Lα transition at 18 °C, and conversion of the Lα phase into the cubic QPn3m phase through D

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Figure 7. SAXD patterns recorded upon heating and cooling scans of diC10-C14PC dispersions. Adapted with permission from ref 39. Copyright 2008 Libertas Academica.

C18PC (cf. a = 6.1 nm for lipid dispersion vs a = 6.5 nm for the lipoplexes) (Figure 9A). The lower intensity of the (11) and (20) lipoplex diffraction peaks relative to the HII pattern of the pure lipid is known to result from the higher electron density of DNA relative to water.48 It is thus an indication of the presence of DNA in the core of the hexagonal phase cylinders.31 DiC18:1-C6PC represents the sole example to date of a cationic PC that forms a cubic phase by itself and forms HII phase lipoplexes when the lipid/DNA weight ratio is 4:1 (Figure 9B).30 At higher DNA content (3:1 lipid/DNA weight ratio), it was found to form lamellar phase lipoplexes.29,32 Mixtures of lamellar and nonlamellar phase-forming lipids, such as diC18:1-ePC/diC18:1-C18PC and diC18:1-ePC/ diC16:4me-C16PC, form cubic phases at certain ratios. Their lipoplexes have been found to arrange into coexisting lamellar and hexagonal phases, epitaxially matched (ahex ≈ dlam) (Figure 10).31 In Table 1, the hydrophilic−lipophilic balance (HLB) has been calculated for each lipid according to the Griffin formula.47 Although it is generally known that HLB gives a rather imprecise measure of the distribution of oil−water affinities within molecules not accounting for any specific features of molecular architecture, it nevertheless provides some predictive guidance to the phase propensities and transfection activities of the cationic phospholipids. All but one of the six lipids with an HLB above 8.5 formed both lamellar lipid phases and lamellar lipoplexes. Four of those six lipids had the highest transfection activity of all compounds tested. All but one of the five lipids with an HLB between 7 and 8.5 formed cubic lipid phases and at least partially lamellar lipoplexes. The compounds with HLB’s below 7 formed HII lipid phases and also had the lowest transfection activities. The cationic phospholipids with the highest transfection efficiency have the highest HLB of 9.3 (however, lipids with similarly high HLB were also found to have much lower transfection efficiencies, e.g., diC10:0-C8PC and diC14:0-ePC (EDMPC) in Table 1).

Figure 8. SAXD patterns of diC18:9-ePC (EDOPC)/cholesterol (55:45, mol/mol) dispersions recorded on heating−cooling scans.

alternate with layers of DNA strands.10,42−44 Although infrequent, nonlamellar structures have also been reported. In general, lipids that on their own prefer the lamellar phase also form lamellar lipoplexes, and lipids forming HII phase by themselves often form HII phase lipoplexes. Notable exceptions from this rule are the lipids forming a bilayer cubic phase. Their lipoplexes do not retain the cubic symmetry and instead convert predominantly to the lamellar phase (Table 1).29,30,32 Thus, certain cationic PCs, which are effectively triplechained and form HII phases by themselves, were found to form inverted hexagonal phase lipoplexes (Table 1). The HII phase lipoplexes consist of DNA coated by lipid monolayers and arranged on a two-dimensional hexagonal lattice. This arrangement is identified by small-angle X-ray reflections in the ratio 1:√3:√4 and HII lattice constant, a, slightly exceeding (by ∼0.4 nm) that of the pure lipid phase in the case of diC18:1E

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ACS Biomaterials Science & Engineering Table 1. Cationic PCs: Phase Behavior, Hydrophilic/Lipophilic Balance, and Transfection Efficiency lipid

C atoms in chains

HLBa

lipid phaseb

lipoplex phase

transfectione (%)

diC14:1c-ePC diC14:1t-ePC C14:1C2−C14:1PC diC14:0-ePC (EDMPC) diC16:0-ePC (EDPPC) diC18:1-ePC (EDOPC) diC10:0-C8PC C18:1C4−C10PC diC10:0-C14PC diC18:1-C6PC diC22:1c-ePC diC18:1-C10PC diC18:1-C18PC diC16:4me-C16PCd

30 30 30 30 34 38 28 32 34 42 44 46 54 56

9.3 9.3 9.3 9.2 8.6 8.3 9.2 8.5 8.2 7.1 7.3 6.7 6.0 5.8

Lα Lα Lα Lα Lα Lα cubicc cubicc cubicc cubicc cubicc HII HII HII

Lα Lα Lα Lα Lα Lα Lα Lα Lα HII Lα no data HII HII

100 100 83 8.3 4.2 8.3 2.5 17.5 5.8 1.7 2.5 1.7 3.3 2.5

ref 34,45,46 34,45,46 34,45,46 34 34 34

present work present work 39 29,30,32 30 29 29,31 31,34

HLB calculated according to the Griffin formula;47 b37 °C, lipid content ∼40 wt % in PBS. cPn3m cubic phase. dC16:4me denotes phytanoyl chain. Transfection units are % relative to the most efficient cationic PC, diC14:1-ePC.45,46

a e

Figure 9. Inverted hexagonal phase lipoplexes with cationic PCs forming (A) HII phase and (B) cubic Pn3m phase.30,31 Lipid/DNA 4:1 w/w, 37 °C. Reproduced with permission from ref 34. Copyright 2009 The Royal Society of Chemistry.

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RELEVANCE TO DRUG AND GENE DELIVERY

considerably for different types of cells, which further complicates assessments. A recent comprehensive review of the cationic PC lipid class concluded that the lipids of the highest efficacy consistently form stable, well-correlated Lα phases under physiological conditions.34 The accumulated data regarding the transfection efficacy of cationic PC lipoplexes show rather low activities of the hexagonal phase lipoplexes (Table 1).30,31,34 Although these studies were focused specifically on cationic PCs, they were designed to include definitive identification of lipid and lipoplex phases in addition to extensive transfection measurements, and comparison of the data illustrate that hexagonal diC18:1-C6PC

It was concluded in some earlier studies that hexagonal phase lipoplexes are more efficient in transfection than lamellar lipoplexes.21,23 In later studies, various ambiguous and contradictory estimates of the transfection efficiency of the hexagonal phase were published.21,23,24,32,49,50 The situation is further complicated by the diversity of lipid phases, which extends considerably beyond the lamellar/hexagonal alternatives and encompasses variety of polymorphic and mesomorphic phase structures, particularly for lipid mixtures. There is also plenty of evidence that transfection varies F

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Figure 11. Lamellar cationic lipid carriers that promote nonlamellar structures upon mixing with membrane lipids easily release DNA and exhibit superior transfection efficiency. Adapted with permission from ref 52. Copyright 2007 National Academy of Sciences U.S.A.

Figure 10. (A) Coexisting lamellar and inverted hexagonal phases in lipoplexes of EDOPC/diC18:1-C18PC 60:40 at 37 °C as revealed by their SAXD patterns, and (B) cartoon representation of the suggested aggregate morphology. Reproduced with permission from ref 31. Copyright 2006 Biophysical Society.

protection of sensitive drugs in vivo against rapid degradation by endogenous enzymes, and the reported plentiful ability of the cationic phospholipids to form nonbilayer phases, these lipids can be outlined as promising pharmaceutical carriers.

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lipoplexes exhibit lower transfection potency than lamellar lipoplexes formed by a cationic phospholipid of a rather similar chemical structure, diC18:1-ePC (EDOPC).30 Likewise, rather low transfection activity was reported for two other cationic phospholipids that form hexagonal lipoplexes−diC18:1-C18PC and diC16:4me-C16PC.31 Induction of hexagonal lipoplex formation by the addition of DOPE to cationic O-alkylphosphatidylcholines was also found generally nonbeneficial for transfection.32 We thus conclude that for the class of the cationic PCs, the lipids forming hexagonal phase lipoplexes are notably less efficient gene carriers than the lipids forming lamellar phase lipoplexes. Although hexagonal phase lipoplexes seem to be generally associated with low transfection activity, the organization of a lipoplex in the lamellar phase is no guarantee, in itself, of high transfection potency, for rather low activity was also observed with the lamellar lipoplexes formed by some representatives of the cationic PCs.34 Nevertheless, it should be recognized that these two observations are not inconsistent, for, as will be pointed out next, it appears that those lamellar complexes that stand out as effective transfection agents readily undergo change of phase to the bilayer cubic phase.34 These kind of cubic phases have been shown to be composed of a continuous lipid bilayer.51 A viewpoint has emerged from our previous research that the critical factor in lipid-mediated transfection is the phase evolution of lipoplex lipids upon interaction and mixing with membrane lipids. Such interactions appear to be decisive for transfection success. Thus, lamellar lipoplex formulations, which are readily susceptible to undergoing lamellar-tononlamellar phase transitions upon mixing with cellular lipids, were found rather consistently associated with superior transfection potency, presumably as a result of facilitated DNA release (Figure 11).31,45,46,52−54 In addition to gene delivery, cationic phospholipids have been used as carriers for delivery of other drugs as well. For example, liposomes of cationic ePCs have been found efficient in delivery of anticancer derivatives of ATP to multiple myeloma cells.55 Applications of non-bilayer-forming cationic phospholipids in drug delivery have not been reported so far. With regard to the important issues addressed by using nonlamellar liquid crystalline phases and their corresponding nanoparticles such as solubilization, drug payload, and

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 1-614-268-1443. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS B.T. acknowledges support from the Medical University−Sofia, Grant 1-I/2012. REFERENCES

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DOI: 10.1021/ab500142w ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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DOI: 10.1021/ab500142w ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX