Tuning the Self-Assembling of Pyridinium Cationic Lipids for Efficient

Jul 8, 2013 - Department of Pharmaceutical Sciences and Moulder Center for Drug Discovery Research, School of Pharmacy, Temple University,...
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Tuning the Self-Assembling of Pyridinium Cationic Lipids for Efficient Gene Delivery into Neuronal Cells Sushma Savarala,†,‡ Eugen Brailoiu,§ Stephanie L. Wunder,† and Marc A. Ilies*,‡ †

Department of Chemistry, College of Science and Technology, Temple University, 130 Beury Hall, 1901 North 13th Street, Philadelphia, PA-19122 ‡ Department of Pharmaceutical Sciences and Moulder Center for Drug Discovery Research, School of Pharmacy, Temple University, 3307 North Broad Street, Philadelphia, PA-19140 § Department of Pharmacology and Center for Translational Medicine, School of Medicine, Temple University, 3500 North Broad Street, Philadelphia, PA-19140 S Supporting Information *

ABSTRACT: We are reporting a new set of biocompatible, low-toxicity pyridinium cationic lipids based on a dopamine backbone on which hydrophobic alkyl tails are attached via an ether linkage. Due to their optimized hydrophilic/hydrophobic interface and packing parameter, the new lipids are able to strongly self-assemble either alone or when coformulated with colipids DOPE or cholesterol. The supra-molecular assemblies generated with the novel pyridinium amphiphiles were characterized in bulk and in solution via a combination of techniques including DSC, nanoDSC, SAXS, TOPM, TEM, DLS, zeta potential, and electrophoretic mobility measurements. These cationic bilayers can efficiently condense and deliver DNA to a large variety of cell lines, as proven by our self-assembling/ physicochemical/biological correlation study. Using the luciferase reporter gene plasmid, we have also conducted a comprehensive structure−activity relationship study, which identified the best structural parameters and formulations for efficient and nontoxic gene delivery. Several formulations greatly surpassed established transfection systems with proved in vitro and in vivo efficiency, being able to transfect a large variety of malignant cells even in the presence of elevated levels of serum. The most efficient formulation was able to transfect selectively primary rat dopaminergic neurons harvested from nucleus accumbens, and neurons from the frontal cortex, a premise that recommends these synthetic vectors for future in vivo delivery studies for neuronal reprogramming.

1. INTRODUCTION The success of gene therapy as a revolutionary method to treat diseases at their core level relies on finding efficient and safe vectors for nucleic acid delivery.1−3 The use of viral vectors guarantees a good efficiency, but is associated with major safety concerns related with the immunogenicity, mutagenicity, and uncontrolled tropism of the viral vector in the patient.4,5 Cationic lipids constitute promising alternatives to the use of viruses for delivering genes therapeutically due to their reduced immunogenicity and cytotoxicity, which allows safe repeated administration of the nucleic acid-based therapeutic agent(s).4,6−8 This is an essential factor in siRNA-related therapies, where multiple administrations are required9 and explains the investment of pharmaceutical companies interested in siRNA technology toward novel cationic amphiphiles for siRNA delivery. When formulated alone, or in the presence of colipids such as cholesterol (Chol, C) or dioleoylphosphatidylethanolamine (DOPE), the positively charged amphiphiles can self-assemble forming cationic lipid bilayers or other threedimensional assemblies.4,6−8,10,11 These supra-molecular assemblies can associate and compact DNA or RNA, masking their negative charge and protecting the nucleic acid from the action © 2013 American Chemical Society

of endogenous and exogenous nucleases. The association process is triggered by electrostatic attraction between the positively charged lipid assemblies and negatively charged nucleic acids. The counterions of both entities are released, together with water molecules from the hydration shell, in a process with substantial entropic gain. A comprehensive threedimensional reorganization of both materials occurs, generating cationic lipid−DNA complexes (lipoplexes10,12−15). Other major advantages associated with the use of cationic lipids in gene delivery are the practically unlimited size of the gene to be compacted and delivered, and the possibility to manufacture them under good manufacturing practice (GMP) conditions in existing facilities of the pharmaceutical industry. Entire artificial chromosomes were successfully delivered using this technology.16,17 However, the efficiency of cationic lipidmediated delivery must be increased through a better understanding of the delivery barriers in vitro and in vivo and Received: April 24, 2013 Revised: June 17, 2013 Published: July 8, 2013 2750

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Chart 1. Representative Cationic Lipids Used in Synthetic Gene Delivery Systems

parameters P ∼ 1 in combination with standard hydrophobic anchors comprising two alkyl chains (Chart 1), thus enabling the formation of well-packed lamellar structures that will confer stability to lipoplexes under a wide range of conditions and disruptive natural amphiphiles encountered in vivo. Mention must be made that similar benefic effects were observed when substituting the small tetraalkylammonium polar head with larger phosphonium and especially arsonium congeners, as in the case of lipid EG372 (Chart 1).44−46 Other self-assembling structural elements such as aromatic groups can be included in the structure of the cationic amphiphiles in order to enhance the assembly stability under harsh, disruptive environments encountered in vivo.30,37,38,47−49 In a previous study38 we compared the biological properties of cationic lipids having a trimethylpyridinium polar head connected to two hydrophobic tails via aliphatic or aromatic linkers and ester bonds. Interestingly, the transfection efficiency and cytotoxicity of representatives based on aliphatic linkers such as SPYRIT-2 and SPYRIT-7 were found to be superior to their congeners with aromatic backbones such as SPYRIT-13. Formulations of these pyridinium lipids with cholesterol at 1:1 molar ratio have surpassed the efficiency of standard transfection system DOTAP/Chol 1:1 both in vitro38 and in vivo,39 while displaying reduced cytotoxicity. Transfection data also revealed that in the case of dopamine-derived series, the aromatic ring clearly contributed to the self-assembling process, with the C12 member (SPYRIT-13) being the most efficient in the aromatic series, while the C14 representatives SPYRIT-2 and SPYRIT-7 displayed the highest transfection efficiency for the aliphatic series. Importantly, SPYRIT-7 was more efficient than SPYRIT-2, proving that a cylindrical shape of the cationic lipid is superior to an angled design.38 These observations prompted us to hypothesize that the lower transfection activity of dopamine-derived SPYRIT-13 and congeners as compared with SPYRIT-7 might be due to a bent conformation adopted by the former cationic lipid at the water/oil interface, positioning that is dictated by the pyridinium polar head and the two ester groups (Figure 1). This conformation may be actually favored by the lipophilic PF6− counterion. Con-

through adapting the structure of the lipoplex to these delivery barriers.4,6−8,10,18−20 For systemic delivery, the cationic lipid assemblies must condense the nucleic acid cargo strongly enough to resist the interaction with various proteins, cells and other figurative elements of the blood. Once the target tissue is reached and the complexes are endocytosed, the lipid component of the lipoplex must fuse with endosomal vesicle and quickly release the nucleic acid into the cytoplasm, where it can exert its therapeutic function (RNAs, siRNAs) or from where it will travel to the nucleus (DNA) to be internalized, transcribed and eventually translated into therapeutic proteins back into the cytoplasm of the target cell.4,6−8,10,18,19 The structural constraints imposed by these processes trigged a substantial effort from the synthetic gene delivery community. More than two decades after the introduction of DOTMA in the seminal article of Felgner et al.21 a large number of cationic lipids bearing various polar heads (tetraalkylammonium, polyamines, amidinium, guanidinium, heterocyclic) and hydrophobic tails (alkyl, cholesteryl) were synthesized and tested as gene delivery vectors.6−8,22−25 Many cationic lipids and cationic-lipid-based formulations are commercially available for in vitro gene delivery, such as Lipofectamine (DOSPA/DOPE 3/1 w/w). Some of them were used successfully for in vivo delivery of nucleic acids in experimental animals, and a select few,26 such as DOTAP/cholesterol,27,28 were advanced to clinical trials for treatment of lung cancer and cystic fibrosis in humans.26 Heterocyclic cationic lipids including imidazolium29−32 and pyridinium33−43 representatives (Chart 1) introduced in recent years by several groups proved to be particularly well balanced for the antagonistic processes of nucleic acid packing and releasing required for efficient gene delivery in vitro29,33−41 and in vivo.39 In these lipids the positive charge is delocalized on the heterocyclic moiety, thus increasing the lipophilicity and decreasing hydration of the polar head, with benefic effects on self-assembling ability of the amphiphiles. An enhanced stability of supramolecular assemblies can constitute an important advantage for systemic delivery in vivo. Moreover, the relatively large (heterocyclic) polar head can easily generate packing 2751

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Figure 1. Self-assembling of cationic lipids in bulk: DSC traces (5 °C/min) representing the first cooling from isotropic liquids (panels a,b) and second heating (panels c,d) of the cationic lipids 4 (panels a,c) and 5 (panels b,d), together with SAXS dynamic for 4Ste (panel e) and TOPM texture for 4Ste and 5Ste (panels f,g). Transition temperatures (°C) and enthalpy changes (in parentheses, J/g) are indicated for each lipid.

sequently, we have decided to replace the ester groups with the more lipophilic ether ones, thus allowing the aromatic linker to move into the hydrophobic phase and thus to ensure a fully extended conformation for the entire molecule, effectively moving the water/oil interface at the level of the pyridinium polar head. The new position of the hydrophilic/hydrophobic interface would be further stabilized by a more hydrophilic counterion such as Cl−. The new design incorporating ether linkages was expected to confer an elongated linear shape for the hydrated amphiphile with a P ∼ 1, allowing better packing into the horizontal dimension and enhanced self-assembling into robust lamellar phases. Moreover, we hypothesized that a nontoxic, efficient, and robust synthetic transfection system based on pyridinium lipids with dopamine backbone might be advantageous toward transfecting primary cells that display receptors for this catecholamine on their surface such as neurons. Toward these goals, we are reporting herein the synthesis, self-assembling, physicochemical, and biological properties of this new set of pyridinium cationic lipids.

Chart 2. The Design Rationale for the New Pyridinium Amphiphilesa

a

Changing the ester groups to more lipophilic ether one allows an extended conformation of amphiphile backbone in the oil phase, reducing the cross-section of the molecule and enhancing its selfassembling.

Materials. 3-Hydroxytyramine hydrochloride, triethylamine, acetic acid, fatty acids, salts, and so on were from Acros and/or Fisher Scientific (Pittsburgh, PA) and were used without further purification. Dowex 1X8-200 was from Sigma-Aldrich (St Louis, MO) or BioRad

2. MATERIALS AND METHODS Please refer to the Supporting Information for additional details. 2752

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Scheme 1. Synthesis of the New Pyridinium Cationic Lipids, As Hexafluorophosphates (4) and Chlorides (5)

(Hercules, CA). Solvents (HPLC quality) were from Fisher Scientific (Pittsburgh, PA), EMD (Gibbstown, NJ), and VWR International (West Chester, PA). Cholesterol, DOTAP, and DOPE were from Avanti Polar Lipids (Alabaster, AL) and were used as received. Tris Acetate EDTA (TAE) buffer, Lambda DNA/Hind III markers, and Blue juice−Blue/Orange Loading dye were from Promega (Madison, WI). DNA plasmidsgWiz Luc plasmid encoding the firefly luciferase gene and gWiz GFP encoding the green fluorescent proteinwere from Aldevron (Fargo, ND). The GelStar Nucleic acid gel stain was from Lonza (Rockland, ME). Agarose (ultrapure), neurobasal-A media, and fura-2AM were from Invitrogen (Carlsbad, CA). Techniques. The purity and the structure identity of the intermediary and final products were assessed by a combination of techniques that includes thin-layer chromatography (TLC), high performance liquid chromatography (HPLC), differential scanning calorimetry (DSC and nano-DSC), 1H- and 13C NMR, and elemental analysis. TLC was carried out on SiO2-precoated aluminum plates (silica gel with F254 indicator; layer thickness 200 μm; pore size 60 Å, from Sigma-Aldrich), eluted with MeOH/CHCl3 10/90 (v/v) unless specified otherwise. The melting points and/or transition temperatures for cationic lipids in bulk were determined by DSC, using a TA Instruments Q200 MDSC (New Castle, DE) and a heating/cooling rate of 5 °C/min. A Thermolyne heating stage microscope (Dubuque, IA), equipped with an Olympus 5X objective, was also used for this purpose. Nanodifferential scanning calorimetry (nano-DSC) measurements for the hydrates samples were obtained on a TA Instruments (New Castle, DE) NanoDSC-6300. Samples were scanned at heating/ cooling rates of 1 °C/min, using 1−2 mg lipid. NMR spectra were recorded at ≈300 K with a Bruker Avance III 400 Plus spectrometer equipped with a 5 mm indirect detection probe, operating at 400 MHz for 1H NMR, at 100 MHz for 13C NMR, and at 376 MHz for 19F-NMR. Chemical shifts are reported as δ values, using tetramethylsilane (TMS) as the internal standard for proton spectra and the solvent resonance for carbon spectra. Assignments were made based on chemical shifts, signal intensity, COSY, HMQC, and HMBC sequences. HPLC was performed using a Shimadzu Prominence UFLC, equipped with an LC-20AD pump, vacuum degasser, column oven, and UV detector, using a Zorbax RX-C18 column (4.6 mm x 25 cm), following the method of Mayer et al.63 Briefly, cationic lipids 4−15 (as PF6− or Cl−) were dissolved in methanol, and 100 μL of the homogeneous solution was injected into the system. Lipids were eluted at 0.5 mL/min constant flow using linear gradient elution from 50% solution A (0.15% trifluoroacetic acid (TFA) in H2O) and 50% solution B (0.05% TFA in iPrOH) to 100% solution B in 10 min. This gradient was followed by a 10 min plateau at 100% solution B, before going back to the initial solvent mixture in 2 min. UV detection was done at 205 nm.

Elemental analyses were performed by combustion, using a PerkinElmer 2400 Series II CHNS analyzer. Small-angle X-ray scattering (SAXS) experiments were performed on a Bruker AXS Nanostar U using Cu Kα radiation from a turbo Xray source (TXS, focal spot =0.1 mm × 1 mm) running at 1.2 kW, coupled with Montel-P multilayer optics and a Vantec-2000 position sensitive area detector. The beam was collimated using three pinholes with apertures of 750, 400, and 1000 μm. The distance from source to first pinhole was 200 mm, from first pinhole to second pinhole was 925 mm, from second pinhole to third pinhole was 482 mm, from third pinhole to sample was 57 nm, and from sample to detector was 1132 mm. Sample was mounted in the middle of an O-ring and then sandwiched between mylar (or similar) windows in the powder/gel holder. The diameter of the beamstop was 4 mm. Experimental data was worked up using SAXS for Windows XP software. The calibration of the system was performed using silver behenate.

3. RESULTS AND DISCUSSION Synthesis of Pyridinium Lipids. The synthesis of the new cationic lipids is depicted in Scheme 1 and was achieved through direct alkylation of the 3,4-dihydroxyphenylethylpyridinium common intermediate 3, easily accessible from the reaction of 2,4,6-trimethylpyrylium hexafluorophosphate 1 with dopamine 2.38 This original convergent synthetic strategy50 has at its core the generation of pyridinium polar head and linker through the reaction of pyrylium salts with primary amines in a single, high yield step. Variations in pyrylium salt substitution and in the structure of the primary amine allow for the generation of a wide variety of pyridinium cationic lipids, gemini surfactants, and lipophilic polycations.37−40 In the present case, alkylation of pyridinium diphenol 3 with decyl(Dec), lauryl- (Lau), myristyl- (Myr), palmityl- (Pal), stearyl(Ste), or oleyl (Ole) bromide, in the presence of dry K2CO3 in dimethylformamide (DMF), generated lipids 4Dec−4Ole (all hexafluorophosphates) in fair yields, after thorough purification via flash chromatography and recrystallization from ethanol and/or hexane/ethyl acetate mixtures (Scheme 1). The counterion of the cationic amphiphiles plays an important role in the self-assembling of these charged molecules and was shown to have a decisive impact on the physicochemical and biological properties of their DNA complexes.38,51−53 Hence, two counterions, namely hexafluorophosphate and chloride, were investigated in the present study. These anions were selected due to their proven optimum biological properties (transfection efficiency/cytotoxicity ratio) manifested in conjunction with the pyridinium polar head.38 In this context, lipids 5Dec-5Ole, bearing the chloride counterion, 2753

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Chart 3. (a) Schematic Representation of Inlayer Packing of Individual Molecules in the Two Possible Smectic Phases Formed by Lipids 4 and 5 at Low and High Temperature and (b) Different Conformations Assumed to Be Responsible for the Packing Differences between the Two Smectic Phases, Revealing the Mismatch between the Cross-Section of the Polar Head and the Cross-Section of the Hydrophobic Part of the Amphiphiles

The broken-fan texture displayed by 4Ste (Figure 1f) also indicates an SC phase. We hypothesize that the difference in cross-section areas of polar head and hydrophobic tail in the most stable conformation of the molecules at low temperature (with pyridinium and phenyl rings in the same plane) favors interdigitation of the alkyl chains and generates a more compact smectic phase. The formation of this phase is also favored by the bulky and lipophilic PF6− counterions strongly associated with polar heads that reduce the mobility and increase the steric bulk of pyridinium cationic heads. At very high temperature these interactions are destroyed, and the pyridinium moiety can access conformations that are more compact, yielding noninterdigitated structures with a higher d-spacing (Chart 3). In terms of LC phase dynamics, one may consider that the lipophilic counterion templates the microphase segregation and the supra-molecular association very fast, so only a small (about 10 °C) supercooling is observed in all cases (4Lau−4Ste, Figure 1a). Further ordering through crystallization of the hydrophobic chains is observed, and Tc decreases with decreasing chain length. Interestingly, the difference between chain crystallization (Figure 1a) and chain melting (Figure 1c) is almost constant (∼10 °C), proving the preordering effect of the counterion and its strength. Unsaturation of chains destabilizes the self-assembly, translating into a shift of peak positions, while d spacing remains about the same (41.7 nm for 4Ole versus 41.2 nm for 4Ste; SAXS difractograms not shown). The whole assembling/disassembling process is highly reproducible and can be repeated several times with identical results (data not shown). The Cl− counterion in lipids 5 is less lipophilic than PF6−, and it does not localize fast at the polar/nonpolar interface. As a consequence, the LC phases of lipids 5 (Figure 1b,d) are templated with more difficulty (slower kinetics), as proven by the huge supercooling interval observed in all Cl− compounds upon cooling their isotropic liquid phases. Interestingly, the amount of supercooling increases from 5Lau to 5Ste. A possible explanation is that the more hydrophilic counterion Cl− tends to locate away from the polar/nonpolar interface and therefore will buffer less the electrostatic repulsions between the positively charged polar heads. The main ordering element in this case is the hydrophobic part of the molecule, which drives the microphase segregation. Elongation of the chain from 5Lau to 5Ste triggers the templation of LC with increased efficiency. At room temperature, the LC phases are less organized as compared with PF6− congeners as evidenced by

were synthesized from their hexafluorophosphate congeners 4 through anion exchange on Dowex resins and crystallization from ethyl acetate (Scheme 1). 3.1. Self-Assembling of Novel Amphiphiles in Bulk and in Solution and the Physicochemical Properties of Their Supramolecular Assemblies. The self-assembling properties of novel cationic lipids 4 and 5 were studied in bulk, as well as in hydrated form, through a combination of analytical methods that involved DSC, thermal optical polarized microscopy (TOPM), SAXS experiments, nanoDSC, dynamic light scattering (DLS), and zeta potential experiments. Our goal was to establish how the main structural elements such as the length of hydrophobic tail, the alkyl chain unsaturation, or the counterion are influencing the thermotropic and lyotropic supramolecular assemblies of these amphiphiles (Figures 1−3). The self-assembling in bulk of lipids 4 and 5 is presented in Figure 1. The analysis of this assembling behavior is important because it allows one to evaluate the strength of the supramolecular assemblies of lipids in the dehydrated form, which is relevant for easiness of liposomal formulation and lipoplex stability. While examining Figure 1 one may observe two different self-assembling dynamics for hexafluorophosphates 4 and chlorides 5 as revealed by DSC. In the case of lipids 4 (Figure 1a,c) the isotropization temperatures for saturated compounds are very close (within a small interval of less than 10 °C). We attribute this behavior to the strong cohesive effect of the lipophilic PF6− counterion, which was shown to localize preferentially at the polar/nonpolar interface, bridging together two adjacent lipid polar heads.54 Upon cooling from isotropic liquid melts, liquid crystalline (LC) smectic assemblies are formed for chain lengths longer than 10 C atoms (compounds 4Lau−4Ste and 4Ole, Figure 1a), proven by SAXS and TOPM (Figure 1e,f, done for 4Ste). Analysis of SAXS difractograms of 4Ste revealed a d-spacing of about 41 Å (2Θ ∼ 2.14) at low and medium temperatures, which increased to about 55 Å (2Θ ∼ 1.60) at 102 °C (Figure 1g). Based on the DSC/TOPM/SAXS analysis and considering the previously elucidated X-ray crystal structure of a structurally related pyridinium cationic lipid,54 we considered a self-assembling model with two interconverting smectic phases that differ only in the degree of chain interdigitation. Since the d-spacing in the high temperature phase (55 Å) is smaller than double the calculated molecular length of the one lipid molecule in the fully extended conformation (about 37 Å), we assume that the bilayers are tilted and we are dealing with smectic C phases (SC) (Chart 3). 2754

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Figure 2. Nano-DSC traces of lipids 4 (panel a) and 5 (panel b), in MLV form (hydrated), performed at 1 °C/min in deionized water. Second cooling is shown in both cases, together with the transition temperature for each lipid.

Figure 3. Size (nm, panels a,b) and zeta potential (mV, panels c,d) of liposomes generated from lipids 4 and 5, either alone, or coformulated with cholesterol (C) or DOPE (D) at 1:1 molar ratio. The standard cationic lipid transfection system DOTAP/cholesterol (1:1 molar ratio, DOTAP/C) was added as reference, in orange.

TOPM (compare Figure 1g with Figure 1f) and SAXS (data not shown). The smaller volume of Cl− as compared with PF6− allows the interdigitated low temperature phase. Extra energy is needed in order to better organize this LC phase, as revealed by endothermic transitions observed upon (second) heating, which diminish in size and shift in position while stepping from 5Lau to 5Ste (Figure 1b). However, the noninterdigitated, compact, high-temperature phase becomes more stable due to the lower volume and higher mobility of the counterion. Thus, at high temperature the LC phases of 5Lau−5Ste are more thermodynamically stable than the corresponding LC phases generated with congeners 4. When the lipid assemblies are hydrated through a repeated freeze−thaw procedure, water penetrates the lipid bilayers, generating multilamellar vesicles (MLVs). Intrabilayer interactions are decoupled from interbilayer ones, allowing a better estimation of the impact of hydrophobic tail on the self-

assembling of these novel lipids (Figure 2). Data from Figure 2 proves that gel/liquid crystalline transition temperatures are not affected dramatically by the counterion used. Irrespective of anion, the transition temperature of MLVs increases with the elongation of the tail, as expected. However, the transition temperature difference between two lipid homologues increases considerably as compared with the thermotropic case. This behavior is attributed to the hydrophobic effect, which increases steeply with tail elongation, enhancing the intrabilayer cohesion of cationic amphiphiles. Tail unsaturation (Ste → Ole) causes a dramatic decrease in transition temperature due to a more disordered packing in the bilayer, similar to the thermotropic case. Comparing data from Figure 2a and Figure 2b also allows us to evaluate the effect of counterion on the stability of the self-assembly. The chaotropic hexafluorophosphate counterion is more hydrophobic than chloride and was shown to prefer to locate in the close vicinity of the pyridinium polar head, 2755

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Figure 4. The effect of cationic lipid/DNA charge ratio on the size (nm, panel a) and zeta potential (mV, panel b) of lipoplexes generated from three different formulation based on lipid 5 Ole: cationic lipid alone, or coformulated with cholesterol (C) or DOPE (D) at 1:1 molar ratio). All formulations are able to efficiently compact the nucleic acid, forming lipoplexes around 175 nm in diameter, as confirmed by TEM data (panel c, depicting 5 Ole/DNA 3/1 charge ratio lipoplexes, bar = 50 nm). Note the blackberry-like, compact shape of lipoplexes and their distinct internal lamellar structure (panel c, inset). The zeta potential measurements were confirmed by electrophoretic mobility experiments (panel d, depicting gel electrophoresis of lipoplexes generated from 5 Ole/D at various cationic lipid/DNA charge ratios, against starting (uncomplexed) DNA, and ladder DNA).

packing parameter P close to 1 for these lipids. The zeta potential data (Figure 3c), which monotonously decreases from 4Myr to 4Ste, is also suggests the strong self-assembling via PF6− bridging the cationic polar heads. The SUVs generated from 4Ste were unstable and coalesced shortly after preparation. The lipid congeners 5 had a more homogeneous behavior, being rather easy to formulate even in concentrated form. Taking into consideration that the two lipid series have very similar gel/liquid crystalline transition temperatures (Figure 2), this is probably due to better hydration properties of chloride counterion and to its different localization at the water/oil interface. Thus, Cl− ion is borderline kosmotropic and therefore is much better hydrated than PF6−. It is also known to be associated rather loosely with the polar head of charged amphiphiles.43 Hence, its residence in the Stern layer is reduced as compared with PF6−, allowing more flexibility of the individual lipid molecules. The electrostatic repusion between the pyridinium polar heads is greater for Cl− versus PF6− since it is less efficiently buffered by the counterion. The increased bilayer flexibility of chlorides 5 as compared with hexafluorophosphate congeners 4 allows higher bilayer curvatures and smaller, more flexible vesicles. Chlorides 5 are also better hydrated due to these counterion effects, and they can be formulated much more easily. The zeta potential of the liposomes generated from pure lipids decreases with the increase of chain length in both series 4 and 5 due to a stronger self-assembly and more rigid supra-molecular complexes that localize the counterions to a greater extent and efficiently shields the positive charges of the polar heads.43 Chain unsaturation diminishes this tight molecular packing and fluidifies the bilayers, hence the liposomes generated from lipids 4Ole and 5Ole have zeta potentials similar to shorterchain congeneres (Figure 3).

bridging the lipid molecules and neutralizing the repulsions of the positively charged polar heads.54 Lipid hexafluorophosphates were always much harder to fully hydrate than their chloride counterparts and only repeated freeze−thawing cycles on rather dilute (1 mg/mL) samples could achieve full hydration of these lipids. The strong self-assembling induced by the dialkoxyaryl hydrophobic anchor and the lipophilic PF6− counterion created difficulties in formulation of these lipids for biological testing (vide infra). Interestingly, for shorter alkyl tails such as 4Myr and 4Lau, the hexafluorophosphate counterions tend to form lipid clusters on cooling from the liquid crystalline fluid phase, broadening substantially the transition peak and revealing several subtransitions (attributed to metastable clusters) within the liquid crystalline/gel transition. Mention must be made that the decyl derivatives did not form a liquid crystalline phase in hydrated form, irrespective of the counterion used. For proper association with DNA, the cationic lipids must be formulated in single unilamellar vesicles (SUVs) in order to maximize the lipid area available for DNA condensation and to ensure the formation of lipid/DNA complexes as structurally homogeneous as possible. In consequence, all lipid MLVs were transformed into SUVs via sonication, using a sonication program previously optimized for this type of cationic lipids, at 65 °C (above the gel/liquid crystalline temperature of all lipids).38 DLS and zeta potential experiments were performed to evaluate the size of SUVs and their surface charge (Figure 3). Lipids 4, bearing the hexafluorophosphate counterion, were extremely difficult to formulate, even after repeated sonication cycles. Samples had to be diluted in order to increased the ultrasonic power received by a lipid unit. In fact, we were not able to generate stock solutions of liposomes from lipids 4 more concentrated than 0.1 mM. This is probably due to the clustering effect of the PF6− counterion in conjunction with this structural design that enhances self-assembling and confers a 2756

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Figure 5. Size (nm, panel a), zeta potential (mV, panel b), transfection efficiency (RLU/μg protein, NCI-H23 cell line, panel c), and cytotoxicity (% of control, NCI-H23 cell line, panel d) of lipoplexes generated from lipids 5 (alone, or coformulated with cholesterol (C) or DOPE (D) at 1:1 molar ratio) as compared with the standard transfection system DOTAP/C (1:1 molar ratio) as reference. The cationic lipid/DNA charge ratio was 3/1 in all cases.

Since the behavior of cationic lipids formulated alone was quite heterogeneous due to enhanced self-assembling induced by hydrophobic tails and counterions, we tried to homogenize their physicochemical and self-assembling properties by blending them with two neutral lipids, cholesterol and DOPE, at 1/1 molar ratio. These mixtures were hydrated using the standard freeze−thaw procedure, then sonicated to generate SUVs. Their sizes and zeta potentials are presented in

Figure 3. Data of Figure 3 suggests a limited success of this strategy: the size of liposomes obtained from hexafluorophosphate lipids 4 coformulated with cholesterol and DOPE is still generally bigger than the size of the corresponding liposomes generated from lipids 5 and the same colipids. DOPE was more effective than cholesterol in diminishing the cohesive effect of PF6− counterion, probably due to its bigger steric demands and higher fluidity. Equimolar blending with these colipids also 2757

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structure of lipoplexes indicates the efficient internally balanced positive charge density in the pyridinium polar head of new cationic lipids and for the strong lamellar self-assembling of these cylindrical engineered amphiphiles. It confirms the translation of liquid crystalline assembling properties of these amphiphiles from liposomes (hydrated bilayers) to lipoplexes (DNA/lipid bilayer composite structures). Following the main findings of this DNA compaction set of experiments, lipids 5 formulated either alone, with cholesterol, or with DOPE at 1/1 molar ratio, together with DOTAP/C 1/ 1 reference transfection system, were associated with luciferase reporter plasmid DNA at 3/1 ± charge ratio and were assessed for transfection efficiency and associated cytotoxic effect on the NCI-H23 lung carcinoma cell line (Figure 5). Cells were incubated with lipoplexes for 48 h, after which they were lysed with Triton X, and the content of their cytoplasm was assesed for luciferase content, normalizing the results for total protein content. Reduced-serum media (Optimem) was used in all cases. A WST-1 cytotoxicity assay56 was done in parallel using identical transfection parameters. The lipoplex size and zeta potential are also presented (Figure 5a,b), for correlating the physicochemical characteristics of the lipoplexes with their biological properties. It can be observed that the size and zeta potential data from Figure 5a,b matches the results of previous experiment (Figure 4), confirming the ability of all members of this new class of amphiphiles to efficiently compact the reporter luciferase plasmid. Homogeneous lipoplex formulations, with sizes around 175−200 nm and zeta potentials around +40 mV, were generated from cationic lipids 5Dec-5Ole. Larger sizes were observed for formulations based on lipid 5Ste, which was more difficult to formulate due to its high Tc (Figure 2) that makes it stiffer than its congeners at temperatures used in lipoplex formulation and testing (25−37 °C). Cholesterol increases the stiffeness of the cationic lipids formulations, while DOPE had an opposite effect. Importantly, lack of flexibility of 5Lau-5Ste due to their enhanced self-assembling within the bilayer when formulated alone or with cholesterol translated into a low transfection efficiency. Only formulations containing the very fluid lipids 5Dec and 5Ole proved efficient, with decyl chains being more transfection-efficient than oleyl congeners when formulated alone or with cholesterol at 1/1 molar ratio. Lipoplexes based on 5Dec/C lipid formulation surpassed the efficiency of standard transfection system DOTAP/C, while lipoplexes generated from lipid 5Dec alone, 5Ole alone, and 5Ole/C displayed about the same transfection efficiency as DOTAP/C. However, the 5Dec-based formulations were significantly more cytotoxic than 5Ole-based ones and DOTAP/C, despite similar physicochemical characteristics (Figure 5). The leveling effect of DOPE (Tc < 0 °C) on bilayer fluidity at 37 °C and its known fusogenicity57 translated into a rather homogeneous transfection profile for lipids 5Lau5Ste, about half of DOTAP/C transfection efficiency. Cytotoxicity was very good, these formulations being practically nontoxic. Lipids 5Dec and 5Ole coformulated with DOPE were very transfection-efficient, both surpassing DOTAP/C, but the cytotoxic effect was again higher for 5Dec/DOPE as compared with DOTAP/C and 5Ole/DOPE. Lipoplexes generated from 5Ole/DOPE 1/1 were the most efficient, displaying higher transfection efficiency than DOTAP/C and having a lower cytotoxic effect than this standard transfection system, at similar physicochemical parameters (Figure 5). Lipoplexes generated from lipids 5Dec and 5Ole were the most efficient, with transfection efficiency/cytotoxicity ratios

reduced the impact of the counterion on the lipid assemblies since the colipid efficiently buffers the polar head electrostatic repulsions. Hence, the zeta potential values of the blended liposomes were generally higher than those of liposomes generated from pure lipids. Physicochemical properties of DOTAP/C (1/1 molar ratio) are presented as reference. Mention must be made that when the molar fraction of colipid in the formulations of cationic amphiphiles 4 and 5 was increased to 0.66 (molar ratio of cationic lipid/colipid of 1/2) the size and polydispersity of the liposomes increased dramatically, probably due to a packing parameter >1 for the lipid/colipid assembly. A very large polydispersity of the liposomes was also observed when cationic lipids 4 and 5 were formulated with DOPE or cholesterol at a molar ratio of cationic lipid/colipid of 2/1 (data not shown). Formulations based on hexafluorophosphates 4 were, again, very hard to formulate. We atribute this behavior to clusterization of cationic amphiphiles in the bilayer due to their strong self-assembling, enhanced at low colipid molar fraction by cohesive effect of PF6− counterion (vide supra). Based on these experimental observations, considering the equivalence of the two counterions in terms of transfection efficiency38 and the perfect biocompatibility of the Cl− counterion, we have decided to continue our biological study only with the lipid chlorides 5. 3.2. Lipoplex Optimization and Transfection Experiments. Optimization of cationic lipid/DNA ± charge ratio for Cl− lipoplexes was performed on 5Ole-based formulations (lipid alone or coformulated with cholesterol (C) or DOPE (D) at 1:1 molar ratio) since this lipid is the most fluid one (see nanoDSC experiment, Figure 2). The gWiz plasmid encoding the firefly luciferase (from Aldevron) was used in all cases for reliable translation of results to future transfection experiments. The ± charge ratio was varied in between 1/1 and 8/1, measuring the size and the zeta potential of the resulting lipoplexes (Figure 4). All formulations were able to efficiently condense the luciferase plasmid DNA reporter (6.7 kDa) to very small lipoplexes (∼175 nm in diameter), at cationic lipid/ DNA charge ratios of 2/1 to 3/1. Formulation containing colipids have displayed a faster dynamic, with 5Ole/C being able to fully condense the plasmid at 2/1 ± charge ratio, thus confirming previous findings37−40 for this type of pyridinium cationic lipids. In the case of formulations 5Ole/D and 5Ole alone full compaction (constant size) and constant (positive) zeta potential (around +40 mV) was achieved at 3/1 charge ratio (Figure 4a,b). Compaction was confirmed by TEM measurements (Figure 4c, for 5Ole formulated alone) and by gel electrophoresis experiments (exemplified for 5Ole/C, Figure 4c), which also confirmed the size/zeta potential measurements. The fact that cationic lipid 5Ole can efficiently compact alone DNA plamids, without having the positively charge diluted by colipids, constitutes an important finding. This is an unique feature displayed by many heterocyclic amphiphiles with soft, delocalized, cationic charge,37−40 that allows simplification of formulation process and enhancement of reproducibility of the nucleic acid compaction process, yielding more homogeneous lipoplexes (see TEM picture, Figure 4c). Structure of lipoplexes was determined by TEM to be quasi-spherical, blackberry-like. The internal lamellar, onion-like, structure with the nucleic acid sandwiched between cationic lipid bilayers12,55 can be clearly seen upon higher magnification of lipoplexes generated from 5Ole (Figure 4c, main and inset). This periodical internal 2758

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experiment due to much shorter exposure time of cells to the transfection system. The lipoplexes generated from lipid 5Ole were also more efficient that the corresponding SPYRIT-13based lipoplexes, which reached about 2/3 of DOTAP/C transfection efficiency.38 Interestingly, all formulations based on 5Ole were almost equipotent, reaching the previous transfection level of lipoplexes 5Ole formulated alone, and about half the transfection efficiency of 5Ole/C and 5Ole/DOPE. In contrast, lipoplexes based on lipid 5Dec were being almost completely devoid of transfection efficiency. These results are revealing different kinetics of transfection for formulations based on cationic lipids 5Ole and 5Dec. Lipid 5Ole is a fastacting compound, able to deliver DNA quickly without significant cytotoxicity, due to its optimum molecular parameters, flexibility (vide supra) and charge density, while 5Dec is significantly slower, probably efficient (at longer contact times) through significant temporal perturbation of internal and external membranes of the target cells, a process that can explain the higher cytotoxicity associated with its use. Overall, this experiment proved the impact of hydrophobic chain length toward the overall shape and conformational mobility of the molecule, and the importance of fine-tuning this parameter for efficient gene delivery. Lipofectamine was found to be more toxic, confirming literature data. Taking into consideration the good results obtained with 5Dec- and 5Ole-lipoplexes in transfecting the NCI-H23 lung malignancy at long contact times, we undertook a comprehensive screening of these formulations on breast (MCF-7), prostate (PC-3 and DU-145), lung (A549), and colon (Caco-2) carcinoma cell lines in similar conditions. Standard transfection systems DOTAP/C and Lipofectamine were also included in the screening for comparison (Figure 7). Data from Figure 7 revealed a strong dependence of transfection efficiency on the cell line type, as expected. It is a common belief in the field of cancer gene delivery that different tumor types will require different transfection systems, best adapted to overcome specific local delivery barriers and tumor metabolic particularities. An overview of the multicell line transfection experiment reveals the very good transfection profile of 5Ole-based formulations, which surpassed the similar formulations based on congener 5Dec. Lipoplexes derived from cationic lipid 5Ole also proved generally superior to standard transfection systems DOTAP/C and Lipofectamine, irrespective of cell line tested, confirming the previous findings. Most efficient was the formulation 5Ole/C, with a constantly good efficiency across cell lines tested, sometimes more than an order of magnitude higher than DOTAP/C or Lipofectamine. A very good transfection profile was displayed by 5Ole when coformulated with DOPE, but the efficiency of 5Ole/D lipoplexes was proved to be more cell line-dependent than 5Ole/C. Formulation 5Ole/D was about 5 times more efficient than standard transfection systems on breast cancer MCF-7 cell line and prostate carcinoma DU-145 (a relatively hard to transfect cell line), and displayed a similar transfection efficiency as compared with these referentials on prostate carcinoma PC-3, and lung cancer A549 cell lines. Remarkably, lipid 5Ole was able to transfect PC-3 and colon cancer Caco-2 cell lines with significant efficiency, reaching the transfection level of the standard transfection systems (Figure 7). By contrast, lipoplexes derived from lipid 5Dec were less efficient than those based on 5Ole congener, and their efficiency was highly dependent on the cell line. Formulation

surpasing DOTAP/C. These are the lipids who were able to induce the biggest local bilayer anisotropy due to their high fluidity generated via short or unsaturated alkoxy hydrocarbonate chains, and to their cylindrical yet flexible shape. Since the physicochemical parameters of their corresponding lipoplexes were almost identical, the difference in transfection efficiency and associated cytotoxic effect can be justified only considering different transfection mechanisms for the two lipids. To shed some light in this direction we have tested the same lipoplexes of lipids 5Dec and 5Ole, DOTAP/C, and of the additional standard (polycationic) transfection system Lipofectamine, on the same NCI-H23 lung cancer cell line, but under different transfection conditions. In the new experiment cells were exposed to lipoplex formulations for only 2 h, instead of 48 h as previously done. After this preincubation time, the lipoplex formulations were removed, cells were washed with PBS, and have subsequently received regular serum-containing media. At 48 h post-transfection cells were harvested and were assessed for luciferase amount expressed, normalizing the results for protein content (Figure 6a). A WST-1 cytotoxicity assay was done in parallel, in similar conditions (Figure 6b). The results of this transient transfection experiment are shown in Figure 6. It can be observed that lipoplexes generated from lipid 5Ole were again more efficient than DOTAP and Lipofectamine, although the transfection level for all formulations was inferior to the level reached in the previous

Figure 6. Transfection efficiency (RLU/μg protein, NCI-H23 cell line, panel a) and associated cytotoxicity (% of control, NCI-H23 cell line, panel b) for transient transfection with lipoplexes generated from lipids 5 Dec and 5 Ole (alone, or coformulated with cholesterol (C) or DOPE (D) at 1:1 molar ratio), as compared with the standard transfection systems DOTAP/C (1:1 molar ratio) and Lipofectamine as references. Note that all SPYRIT-13 ester-linked cationic lipids displayed transfection activities inferior to DOTAP/C.38. 2759

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and other factors, which can interact with lipoplexes and interfere with the transfection process.18,58,59 These interactions are responsible for lipoplex premature inactivation in vivo and account in part for the in vitro-in vivo translational problemes encountered by many groups. Therefore, we have decided to test the transfection efficiency of 5Dec and 5Ole-based cationic lipid formulations, together with standard transfection systems DOTAP/C and Lipofectamine, on NCI-H23 lung cancer cell line at different serum concentrations. We have used transfection conditions similar to experiments depicted in Figures 5 and 7, the only variable parameter being the percent of serum in the culture media, adjusted between 0% and 40% to mimic the conditions encountered by lipoplexes during in vivo transfections (Figure 8). An examination of data from Figure 8 reveals that standard transfection systems DOTAP/C and Lipofectamine, although very efficient in the absence of serum, are inactivated fast by increased levels of serum in the transfection media, probably due to the instability of the lipoplex structure in the presence of serum proteins and other constitutive elements. A similar behavior is displayed by lipoplexes generated from lipid 5Ole alone, which probably has a very high charge density despite the soft charge on individual polar head. However, when the charge density was reduced through coformulation with cholesterol or with DOPE the resistance to serum action was increased dramatically. Thus, 5Ole/C and especially 5Ole/D displayed excellent transfection profiles in the presence of increasing levels of serum, while the standard transfection systems were practically devoid of any significant biological effect. A similar trend was observed for formulation 5Dec/D, with a slightly diminished transfection efficiency, thus validating our new cationic lipid design with enhanced self-assembling properties. Interestingly, lipoplexes generated from cationic lipid 5Dec alone were inefficient under the conditions tested, while 5Dec/C formulation had a curious transfection profile, its efficiency linearly increasing with the concentration of serum in transfection media. The low cytotoxicity and excellent transfection properties of the lipoplexes generated from 5Ole/DOPE lipid mixture in the presence of serum prompted us to test the ability of these lipoplexes to deliver DNA into primary neuronal cells, which are known to be extremely difficult to transfect and highly sensitive to various toxic materials. We hypothesized that a lipoplex based on cationic lipids having a dopamine-like backbone will be recognized and bound by dopamine receptors on the surface of dopaminergic neurons, followed by internalization of the lipoplex and the transfection of target neuron. The internalization and transfection events will be favored by the efficient membrane destabilization/poration properties of the pyridinium polar head. Primary cocultures of neurons and glial cells from Sprague− Dawley rat neonates (1−2 days old) were made on glass coverslips following established experimental protocols.60 In order to test the potential selectivity of the transfection agent on dopaminergic neurons, we harvested neurons and glial cells from nucleus accumbens (rich in dopaminergic neurons) and from the prefrontal cortex, which contains a mixed population of neurons. After 3 days of culture ex-vivo the neurons were transfected with lipoplexes obtained from 5Ole/DOPE (1/1 molar ratio) and gWiz GFP plasmid expressing the green fluorescent protein, generated using a cationic lipid/DNA ± charge ratio of 3 as in the previous experiments. The cell cultures were incubated with lipoplexes diluted with Neurobasal-A media containing 10% serum for 30 min, after which

Figure 7. Transfection efficiency (RLU/μg protein) of lipoplexes generated from lipids 5 Dec and 5 Ole (alone, or coformulated with cholesterol (C) or DOPE (D) at 1:1 molar ratio) as compared with the standard transfection system DOTAP/C (1:1 molar ratio) and Lipofectamine as references, on MCF7 (a), PC-3 (b), DU 145 (c), A459 (d), and Caco-2 (e) cancer cell lines.

5Dec/D was the most transfection-competent, with significant efficiency on colon cancer Caco-2 cell line. Interestingly, on prostate cancer DU-145 cell line, a relatively hard to transfect malignancy, all formulations derived from 5Dec were again equipotent, reaching the level of DOTAP/C and Lipofectamine. Overall, data from Figure 7 confirmed that lipids 5Dec and 5Ole have very different mechanisms of action, with 5Ole being overall superior to both its congener 5Dec and to the two standard transfection systems tested. Work is in progress to shed more light toward these issues. An important delivery barrier against synthetic transfection systems in vivo is constituted by the presence of serum proteins 2760

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Figure 8. Transfection efficiency (RLU/μg protein, NCI-H23 cell line) of lipoplexes generated from lipids 5 Dec (a) and 5 Ole (b) formulated alone, or coformulated with cholesterol (C) or DOPE (D) at 1:1 molar ratio, as compared with the standard transfection systems DOTAP/C (1:1 molar ratio) and Lipofectamine as references, in the presence of media without serum (0%) or with increased serum concentrations (5%, 10%, 20% and 40%).

the transfection mixture was removed and replaced with FBScontaining media. After 48h incubation time at 37 °C the cells were retrieved from incubator, washed with Hank’s balanced salt solution (HBSS) and loaded with Fura-2 a.m. calcium indicator.60−62 The coverslips with cells were mounted on an organ bath and the cells were imaged for presence of GFP and basal cytoplasmic calcium with a Nikon Eclipse Ti fluorescence microscope (Figure 9). As seen from Figure 9, our novel transfection system proved efficient for transfecting neurons from both nucleus accumbens and prefrontal cortex, while leaving glial cells unaltered. The viability of both neurons and glial cells was tested posttransfection using the metabolism of calcium-binder Fura-2. Thus the membrane-permeant precursor Fura-2 a.m. enters all cells and it is metabolized to Fura-2, in which form it can bind cytoplasmatic calcium. Only viable cells can metabolize Fura-2 a.m. to Fura-2, and the presence of Fura-2 complex with Ca2+ constitutes direct evidence that the cells are functional. In order to validate the selectivity of transfection system on cells that do not present dopamine receptors, we have performed identical transfection experiments on rat cultured myometrial smooth muscle cells. Imaging results showed lack of transfection on these particular cells. Calcium imaging

Figure 9. Images of cocultured cells (neurons and glial cells) from rat nucleus accumbens (top images) and prefrontal rat cortex (bottom images) 48 h post-transfection with 5Ole/DOPE_GFP. Left: DIC image; center: GFP fluorescence image (488 nm excitation, 540 nm emission); right: basal calcium fluorescence image (ratio 340 nm/380 nm excitation and 520 nm emission). Only neurons were transfected with GFP, showing neuronal selectivity of the transfection agent. All neurons and glial cells are viable post-transfection, showing Fura-2 calcium binder postloading.60,62 No significant cytotoxicity was observed under these transfection conditions.

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showed that cells were viable post-transfection, proving the reduced toxicity of the novel transfection system (Figure 10).

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ABBREVIATION LIST: Chol, C: cholesterol; DIC: differential interference contrast; DLS: dynamic light scattering; DOPE, D: dioleoylphosphatidylethanolamine; DOTAP: dioleoyloxytrimethylammonium propane; DOTMA: N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride; DSC: differential scanning calorimetry; GFP: green fluorescent protein; HBSS: Hank’s balanced salt solution; LC: liquid crystalline; MLV: multilamellar vesicle; SAXS: small angle X-ray scattering; SUV: single unilamellar vesicle; TEM: transmission electron microscopy; TOPM: thermal optical polarized microscopy; TXS: turbo X-ray source

4. CONCLUSIONS The antagonistic processes of DNA compaction/release required for efficient gene delivery can be managed efficiently by using pyridinium cationic lipids, which possess a soft positive charge that is optimally balanced for nucleic acid delivery. Conjugation of the pyridinium polar head with a novel dialkoxyphenyl structural moiety via an ethyl linker was shown to enhance self-assembly while maintaining a cylindrical, yet flexible, shape to the cationic amphiphiles − all design elements proved efficient in the past by us and by others. The supra-molecular assemblies generated with the novel pyridinium amphiphiles were characterized in bulk and in solution via a combination of techniques including DSC, nanoDSC, SAXS, and TOPM. Fine tuning of the self-assembling process at the level of counterion, hydrophobic chain length, and colipid produced synthetic transfection formulations with optimum charge density and elastic moduli for efficient DNA delivery in a variety of malignant cells in conditions mimicking in vivo environment. The most efficient formulation was able to transfect neurons from primary neuronal/glial cells cocultures, proving selective for neurons. Experiments are underway to fully assess the mechanism of cellular internalization and to optimize these synthetic gene delivery systems for in vivo neuronal reprogramming.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Detailed procedures for the synthesis of pyridinium cationic lipids, complete characterization of the novel amphiphiles, liposome and lipoplex preparation and characterization, procedures for transfection and cytotoxicity experiments, the procedure for transfection experiments in the presence of variable amounts of serum, neuronal culture, transfection, and viability imaging. This material is available free of charge via the Internet at http://pubs.acs.org.

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ACKNOWLEDGMENTS

Financial support from the NSF (CHE-0923077), Temple University Provost’s Office, and Temple University School of Pharmacy Dean’s Office is gratefully acknowledged. The authors are also grateful to Dr. Hongwen Zhou for his help with TEM experiments, to Dr. Brian E. Jones from Bruker AXS Inc. for performing the SAXS experiments, and to Mr. Guy Barbagelata (Bruker AXS), for his entire help and availability.

Figure 10. Images of rat myometrial smooth muscle cell culture 48 h post-transfection with 5Ole/DOPE_GFP. Left: DIC image; center: GFP fluorescence image (488 nm excitation, 540 nm emission); right: basal calcium fluorescence image (ratio 340 nm/380 nm excitation and 520 nm emission. No transfected cells were observed, proving cell type selectivity of the transfection system. All cells were viable posttransfection, showing Fura-2 metabolism postloading.60,61.

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AUTHOR INFORMATION

Corresponding Author

*Address: Department of Pharmaceutical Sciences, Temple University School of Pharmacy, 3307 N Broad Street, Philadelphia, PA-19140; Tel: 215-707-1749; Fax: 215-7075620; E-mail: [email protected]. Notes

The authors declare no competing financial interest. 2762

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