Enhanced Loading of Paclitaxel in Cationic Liposomes by Replacement of Oleoyl with Linoleoyl Lipid Tails with Benefits in Cancer Therapeutics from In Vitro Studies

Lipid-based carriers of the hydrophobic drug paclitaxel (PTX) are used in clinical trials as next-generation agents for cancer chemotherapy. Improving the loading capacity of these carriers requires enhanced PTX solubilization. We compared the solubility of PTX in cationic liposomes (CLs) with lipid tails containing one (oleoyl; C18:1 Δ9; DOTAP/DOPC) or two (linoleoyl; C18:2 Δ9; DLinTAP/DLinPC) cis double bonds with newly synthesized cationic DLinTAP (2,3-dilinoleoyloxypropyltrimethylammonium methylsufate). We used differential-interference-contrast microscopy to directly observe PTX crystal formation and generate kinetic phase diagrams representing the time-dependence of PTX solubility as a function of PTX content in the membrane. Replacing tails bearing one cis double bond (DO lipids) with those bearing two (DLin lipids) significantly increased PTX membrane solubility in CLs. Remarkably, 8 mol% PTX in DLinTAP/DLinPC CLs remained soluble for approximately as long as 3 mol% PTX (the membrane solubility limit which has been the focus of most previous fundamental studies and clinical trials) in DOTAP/DOPC CLs. The large increase in solubility is likely caused by enhanced molecular affinity between lipid tails and PTX upon replacement of oleoyl by linoleoyl tails, rather than by the transition in membrane structure from lipid bilayers to inverse cylindrical micelles observed in small-angle X-ray scattering. Importantly, the efficacy of PTX-loaded CLs against human prostate cancer (PC3) cells from measurements of the IC50 of PTX cytotoxicity was unaffected by changing the lipid tails, and toxicity of the CL carrier alone was negligible. Moreover, efficacy was approximately doubled against human melanoma (M21) cells for PTX-loaded DLinTAP/DLinPC over DOTAP/DOPC CLs. The findings demonstrate the potential of chemical modifications of the lipid tails to increase the PTX membrane loading well over the typically used 3 mol% while maintaining (and in some cases even increasing) the efficacy of CLs. The increased PTX solubility will aid the development of liposomal PTX carriers that require significantly less lipid to deliver a given amount of PTX, reducing side effects and costs.


Introduction
Despite immense progress in treatment options and their effectiveness over recent decades, cancer remains a leading cause of death. Thus, there is an ongoing need for high-efficacy cancer chemotherapy with reduced side effects. Paclitaxel (PTX, Figure 1A,B) 1 is a potent and widely used (>$1 billion/yr) cancer drug for treating ovarian, breast, lung, pancreatic, and other cancers. 1-10 PTX inhibits mitosis by stabilizing microtubules, which are inherently dynamical in vivo, and subsequently activates apoptotic signaling pathways that lead to cell death. [1][2][3]5,[11][12][13][14] Because PTX is hydrophobic and poorly soluble in water, it has to be delivered by a carrier (vector). 15,16 However, the carrier employed in the prevalent PTX formulation Taxol®, 17 polyoxyethylated castor oil and ethanol, has been linked to severe hypersensitivity reactions requiring premedication. [18][19][20] Development of more efficient and safer PTX carriers has been an ongoing challenge for decades. [21][22][23][24][25][26][27] Albumin-bound PTX is an example of earlier success in carrier development and was approved by the FDA in 2005 (Abraxane®; a nontargeted nanoparticle formulation). This formulation appears to have fewer adverse reactions than Taxol and eliminates drug-carrier toxicity, but reports on whether it improves patient survival are mixed. 21,[28][29][30] Increasing the capacity (PTX loading) of the carrier is desirable because it means less carrier is required for a given PTX dose, reducing both cost and side effects stemming from the carrier.
Furthermore, developing PTX carriers with higher efficacy, i.e., lower IC50 of PTX cytotoxicity, also reduces drug-related side effects because less PTX is required to exert its cytotoxic effect.
Finally, based on its biochemical mechanism of action, PTX should be effective against most cancer cells. Therefore, development of novel, improved vectors for PTX , which, for example, may be able to deliver PTX to an expanded range of tissues, could also open treatment avenues against an expanded range of cancers. For example, Abraxane appears to be effective to treat metastatic melanoma, whereas Taxol is not. 31 Liposomes are highly versatile and widely studied carriers of hydrophilic as well as hydrophobic drugs in therapeutic applications, in particular for cancer. 21,[32][33][34][35][36][37][38][39][40][41][42][43][44][45][46] Most widely-known liposomal formulations, such as Doxil and Myocet, contain the cancer drug doxorubicin in the interior of the liposome (yellow oval in Figure 1C). This is not feasible for PTX, however. PTX is much more hydrophobic (logP = 3.96) than doxorubicin (logP = 1.3), which can even be administered directly, without a solubilizing agent. In addition, the doxorubicin formulations rely on design principles that do not translate to PTX, because PTX lacks the functional groups that permit doxorubicin to be loaded via pH-or ion-gradient loading methods (forming reversibly soluble crystals within the liposomal aqueous pocket). 47 Instead, hydrophobic drugs such as PTX are solubilized by and incorporated into the nonpolar (hydrocarbon chain) bilayer membrane of lipid-based carriers (red spheres in Figure 1C). 16,37,[48][49][50] Cationic liposomes (CLs; consisting of mixtures of cationic and neutral lipids) are particularly attractive as a lipid-based carrier for PTX because positively charged particles have been shown to passively target the tumor neovasculature, 38,40,43,[51][52][53][54][55][56] which has a greater negative charge than other tissues. 39,53 Moreover, CLs are particularly attractive as a lipid-based carrier for PTX because CLs are a prevalent nonviral vector (investigated as alternatives to engineered viruses) for the delivery of therapeutic nucleic acids (NAs, e.g., plasmid DNA or siRNA; electrostatically condensed with membranes with cationic headgroups). 34, This enables the use of CLs for combination therapies. The CL formulation EndoTAG (aka SB05), which served as a starting point for our investigations, has completed Phase II clinical trials and is currently in phase III trials. 26,52 EndoTAG consists of CLs of the univalent cationic lipid DOTAP (2,3dioleoyloxypropyltrimethylammonium chloride) and neutral DOPC (1,2-dioleoyl-sn-glycero-3phosphatidylcholine) loaded with PTX (50:47:3 molar ratio). 37,38,40,41,82,83 Because PTX is loaded in the bilayer of liposomes by hydrating a mixture of the lipid and PTX, the initial "loading efficiency" is 100%. However, if the amount of PTX in the membrane is larger than the membrane solubility limit, PTX precipitates out of the membrane and forms crystals over time. Once PTX has phase-separated into stable, water-insoluble crystals, the drug loses efficacy. 37,48,[84][85][86] Thus, it is crucial that PTX remains soluble in the membrane on timescales relevant for delivery. Relatively few studies have investigated the PTX solubility limit in different types of membranes, and not many common themes have emerged. 49,50,[87][88][89][90][91][92] Nearly all animal studies and clinical trials with liposome-PTX carriers have been conducted at 3 mol% PTX content, 37,38,41,82,83,93,94 the first reported membrane solubility limit of PTX. 95 Rarely, liposomal PTX formulations with higher membrane solubility have been reported, but they did not alter the structure of the lipid tails and did not provide a systematic approach to increase PTX loading. 89,96 The PTX membrane solubility strongly depends on lipid tail structure because the location of the drug within the bilayer implies that tails exhibiting favorable local packing interactions with PTX will suppress PTX self-association, nucleation, and crystal growth. PTX is quickly expelled from membranes consisting of chain-ordered saturated lipid tails or those that have a high concentration of cholesterol. 37,49,50,87,88 Lipids with chain-melted mono-unsaturated tails, on the other hand, are used in many of the lipid-based PTX carriers in development, such as EndoTAG®, LEP-ETU, and DHP107. 21,93 In this work, we instead focused on tails with multiple cis double bonds because these increase chain disorder and modify chain flexibility. We hypothesized that this increased chain disorder and altered flexibility would affect molecular affinity to PTX compared to tails with one cis double bond. Currently there are only a few instances of commercial therapeutics containing lipids with poly-unsaturated fatty acid tails. 32,97 A notable exception is DLin-MC3-DMA (Figure 2), the cationic lipid component of patisiran (Onpattro®), 98 which became the first FDA-approved siRNA therapeutic in 2018. We note, however, that the siRNA component interacts with the lipid's headgroup in the case of patisiran and thus, unlike in our system, the active ingredient has no direct interactions with the polyunsaturated tails. (As an aside, poly-unsaturated fatty acids have been studied as therapeutic entities in and of themselves for their anti-oxidant properties. 99,100 ) We pursued the development of CL carriers with a tail structure that improves solubility of PTX in their hydrophobic membrane. Such vectors require less lipid to deliver a given amount of PTX, reducing costs and side effects. Carriers with high solubility have also shown increased efficacy, 48 allowing administration of lower total doses of PTX. We obtained promising initial results of increased PTX membrane solubility using CLs prepared from DOTAP (oleoyl (C18:1) tails; see Figure 2) and DLinPC (linoleoyl (C18:2) tails; see Figure 2) (at a molar ratio of DOTAP/DLinPC/PTX=30/70-x/x). 101 Encouraged by these results, we synthesized the univalent cationic lipid DLinTAP from linoleic acid and used it to prepare PTX-loaded CLs containing lipids with exclusively C18:2 tails.
We used differential-interference-contrast (DIC) microscopy to directly observe PTX crystal formation and generate kinetic phase diagrams, characterizing the time-dependence of PTX solubility as a function of PTX content for CLs with lipid tails containing either one   Cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Invitrogen). Cells were cultured at 37°C in a humidified atmosphere with 5% CO 2 and split at a 1:5 ratio after reaching ≥80% confluency (every 48-72 hours) during maintenance.

Liposome Preparation
Suspensions of sonicated and unsonicated liposomes at a total molar concentration (lipid + PTX) of 1 mM for cell viability experiments, 5 mM for DIC microscopy, and 30 mM for smallangle X-ray scattering measurements were prepared as described previously. 48

DIC Microscopy (PTX Solubility and Kinetic Phase Diagrams)
Samples were prepared and assayed as described previously, 48

Small-Angle X-Ray Scattering
Samples for X-ray scattering were prepared by combining and vortexing 50 μL of a 30 mM aqueous liposome suspension with DNA solution (3.5 mg/mL in water) at a cationic lipid to DNA charge ratio of 1 in a 500 μL centrifuge tube. Following centrifugation in a table-top centrifuge at 5,000 rpm, the resulting pellets were transferred to quartz capillaries (Hilgenberg) with the help of excess supernatant. The capillaries were then centrifuged in a capillary rotor in a Universal 320R centrifuge (Hettich) at 10,000 g and 25 °C for 30 min. After centrifugation, the capillaries were sealed with a fast-curing epoxy glue.
SAXS measurements were carried out at the Stanford Synchrotron Radiation Laboratory, beamline 4-2, at 9 keV (λ=1.3776 Å) with an Si(111) monochromator. Scattering data was measured by a 2D area detector (MarUSA) with a sample to detector distance of ≈3.5 m (calibrated with silver behenate). The X-ray beam size on the sample was 150 μm in the vertical and 200 μm in the horizontal directions. Scattering data is reported as azimuthally averaged scattering intensity in q-space.

Cell Viability Assays
Cells were plated at a density of 5,000 cells/well in 96-well plates and incubated overnight.
Suspensions of sonicated liposomes were diluted in DMEM to reach the desired PTX concentration. The culture medium was removed from the wells by aspiration with a pipette, taking care not to aspirate any cells, and a total of 100 μL of the liposome suspension in DMEM was added to each well. Cells were incubated for 24 h before the liposome suspension was removed by manual pipetting and replaced with cell culture medium. After incubation for 48 h, the cell viability was measured using the CellTiter 96® AQueous-One Soution Cell Proliferation Assay (Promega). The assay solution was diluted 6-fold with DMEM, and a total of 120 μL of this solution was added to each well. After 1 h of incubation, absorbance was measured at 490 nm using a plate reader (Tecan M220). Each data point shown comprises four identically treated wells and is normalized to the absorbance obtained for untreated cells.
Liposomes with different PTX contents were added to cells such that the resulting PTX concentration per well was identical for each data point, independent of the formulation. Because of this, the lipid concentration at each data point varies between formulations with different PTX contents. 48 To determine the IC50 values, we used the solver add-in for Microsoft Excel to perform a nonlinear least squares fit of the cell viability data to the equation y=A+B/(1+[x/C] D ).
Here, y is the measured (normalized) cell viability, x is the total PTX concentration, A is the minimum cell viability, (A+B) is the maximum cell viability (B is the range of y), C is the IC50 (the concentration of PTX at which cell viability is halfway between the maximum and minimum values, i.e. where y=A+B/2), and D is the "slope factor" of the curve (indicating how steeply the viability declines). The minimum and range of cell viabilities (A and B) was given by the data, while C and D were used as fitting parameters. To be able to prepare CLs with tails that exclusively bear two cis double bonds (derived from linoleic acid, C18:2 Δ 9 ), we synthesized DLinTAP as shown in Scheme 1. The full details of the synthesis, which used a route analogous to that reported for DOTAP, 102 are reported in the supplementary material.

PTX Membrane Solubility
Upon hydration of films prepared from a mixture of cationic lipid (DLinTAP or DOTAP), neutral lipid (DLinPC or DOPC) and PTX, PTX-loaded CLs formed spontaneously. These CLs were studied directly (unsonicated CLs; uni-and multi-lamellar with a broad distribution of larger sizes and an average diameter of ≈800 nm) or after sonication (small unilamellar CLs with diameter < 200 nm). As mentioned in the Introduction, the initial "loading efficiency" is 100%, but, depending on the composition, PTX may phase separate and crystallize over time, decreasing the PTX loading of the CLs and reducing the amount of PTX that is effective against cancer cells.

DIC Microscopy
To compare the solubility of PTX in CLs prepared from DLin-and DO-lipids, we used differential interference contrast (DIC) microscopy. 48 Starting 2 h after sample hydration, samples were observed at regular time intervals to check for PTX crystals as evidence of phase separation. Figure 3 shows DIC micrographs illustrating the variety of size and shape in the  Figure 3B to Figure 3E). Aggregates of PTX crystals were common at contents above 6 mol%, including the feather-like crystals observed at 9 mol% ( Figure 3F). The aspect ratio of PTX crystals from DLinTAP/DLinPC CLs was typically much smaller than that of crystals formed from DOTAP-containing CLs (compare Figure 3A,D to Figure 3B,C,E). The overall smaller size and larger number of PTX crystals from DLinTAP/DLinPC CLs suggests that there are fewer nucleation and growth sites in DOTAP-based samples. Only at low loadings (PTX content <2 mol% and <3 mol% in sonicated and unsonicated samples, respectively) and long incubation times was PTX more soluble in DOTAP/DOPC CLs than in DLinTAP/DLinPC CLs. This may be due to oxidation of the DLin tails, because we took no special precautions to exclude oxygen from the small sample volumes during the repeated withdrawing of aliquots for DIC microscopy over time.

Small-Angle X-Ray Scattering
To investigate the effect of tail saturation on the structure of CL membranes, we used synchrotron small-angle X-ray scattering (SAXS) to determine the structure of CLs prepared A B from mixtures of DOTAP with either DOPC or DLinPC. To enhance the signal-to-noise ratio, we condensed the CLs with DNA. This has been shown to result in CL-DNA complexes where the equilibrium self-assembled structure of the membrane within the complex is determined by the spontaneous curvature (C 0 ) of the lipid self-assembly. 34,[103][104][105] The spontaneous curvature is, in turn, determined by the average shape of the lipid molecules. 106 DOPC/DOTAP CLs mixed with DNA form the lamellar (L α C ) phase because both DOPC and DOTAP have a cylindrical shape with C 0 ≈0 (see Figure 6). 103 We expected that DLinPC could be capable of forming the inverse hexagonal (H II ) nonbilayer structure due to the increase in unsaturation from one to two cis double bonds in the lipid tails. The two cis double bonds in DLinPC induce kinks in the lipid tails that can not readily be offset by gauche conformations in the single bonds, leading to the tails taking up a bigger lateral area compared to the headgroup area ( Figure 6). This results in an inverted-cone molecular shape as depicted in Figure 6, corresponding to negative spontaneous curvature (C 0 ˂ 0). A previous study on soy PC (a lipid mixture largely composed of DLinPC) with x-ray scattering and cryogenic TEM supports this hypothesis. 107    Figure S1 in the supplementary material, which shows the kinetic phase diagram for PTX solubility in CLs containing 70 mol% DOPE, DOTAP and PTX.
DNA complexes of these membranes form the H II C phase (see Figure S2 in the supplementary material) because the headgroup of DOPE (phosphoethanolamine) is smaller than that of DOPC(phosphocholine). Therefore, improved solubility of PTX in DLinTAP/DLinPC membranes is not due to their preference for the HIIC phase but rather the different interactions of DLin tails with PTX.  To facilitate comparison of the DLinTAP/DLinPC and DOTAP/DOPC formulations, Figure   7C plots their IC50 for cytotoxicity of PTX against PC3 cells as a function of their PTX content.

Cytotoxicity
The IC50 values of DLinTAP/DLinPC and DOTAP/DOPC CLs at each PTX content are very similar, demonstrating that cytotoxic efficacy is unaffected by the change in tail structure. The efficacy against PC3 cells increased (i.e., IC50 decreased) with increasing PTX content in formulations with both DO-and DLin-lipids, while previous studies had found an increase in the IC50 with PTX content for DO-lipid-based CLs. 48 A possible explanation for this is that, even at high PTX content, the PTX remained solubilized in the membranes long enough to exert its cytotoxic effects, allowing successful PTX delivery by metastable CLs. If such PTX-loaded CLs are not used immediately after preparation, however, PTX crystallizes and their efficacy drops. 48 We also investigated the efficacy of PTX-loaded DLinTAP/DLinPC CLs against a metastatic melanoma (M21) cell line, using the same range of CL formulations and PTX contents as for the PC3 cell line ( Figure 8A). Compared to PC3 cells, the viability of M21 cells decreased more gradually, with most of the drop in viability occurring between 10 and 65 nM PTX, depending on the specific formulation. This is consistent with prior investigations using PTX-loaded DOTAP/DOPC CLs. 48 We again assessed DLinTAP/DLinPC lipid toxicity using a CL-only control.
DLinTAP/DLinPC CLs without PTX caused >10% drop in cell viability only at or above lipid concentrations equivalent to those present when 75 nM PTX is delivered by CLs loaded with 2 mol% PTX ( Figure 8A, blue line). In contrast, the IC50 for the same CLs with PTX is 17.6 nM PTX, about four times lower. It is therefore unlikely that lipid toxicity contributed significantly to the measured IC50 values for PTX-loaded CLs at any PTX content, given that the lipid/PTX ratio for the formulations decreased more than the IC50 increased (see below). . The decrease in cell viability with PTX concentration is less steep than in part A, suggesting a lower efficacy. Previous work has shown that CLs alone do not contribute to cytotoxicity at the employed concentrations. 48 C) Plot of IC50 values for PTX cytotoxicity against M21 cells for the CL formulations based on lipids with linoleoyl ("DLin", red line) and oleoyl ("DO", black line) tails. Each IC50 was determined by fitting the corresponding cell viability curve (from parts A and B) as described in the Methods section.

Figure 8. Cytotoxicity of PTX-loaded CLs against M21 cells. A) Viability of M21 cells (relative to untreated cells) as a function of PTX concentration for cells treated with
Importantly, the efficacy of the DLinTAP/DLinPC/PTX formulations was about twofold higher (their IC50 values were two-fold lower) than that of the corresponding DOTAP/DOPC/PTX formulations. This effect amplifies the benefits that replacing oleoyl with linoleoyl tails cells brings by to increasing PTX membrane solubility (Figure 4). In contrast to PC3 cells, the IC50 increases (efficacy decreases) with increasing PTX content for both DOTAP/DOPC/PTX and DLinTAP/DLinPC/PTX formulations. This effect is less pronounced for the DLinTAP/DLinPC/PTX formulations, and the efficacy of the DLinTAP/DLinPC/PTX formulation at 9 mol% PTX is lower than that of the formulation at 2 mol% PTX.

M21 cells
Viability of M21 cells as a function of delivered PTX concentration for the positive control, DOTAP/DOPC CLs loaded with 2 to 9 mol% PTX, is shown in Figure 8B. The decrease in cell viability with PTX concentration is very gradual and slower than that for PTX-loaded DLinTAP/DLinPC CLs ( Figure 8A), suggesting that DLinTAP/DLinPC CLs induced cytotoxicity more effectively than DOTAP/DOPC CLs when used against M21 cells. According to literature data, DOTAP/DOPC CLs without PTX are not cytotoxic at concentrations well above those used in this experiment. 48 For a more quantitative comparison, Figure 8C  We expect that this work will motivate future studies using chemical modifications of lipid structure as well as computational modeling to further explore how altering lipid tails affects their molecular affinity to PTX. This, when combined with kinetic phase diagrams as presented here, should lead to a comprehensive understanding of how lipid shape and tail structure correlates to PTX membrane solubility, paving the way to improved cancer therapeutics.

Supplementary Material Contents
Detailed methods for the synthesis of DLinTAP; kinetic phase diagram and SAXS profile of DNA complexes of PTX-loaded CLs containing 70 mol% DOPE; tabulated cytotoxicity data; tables with the values for the IC50 and the slope factor of PTX cytotoxicity against PC3 and M21 cells.

Data Availability
The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.