key: cord-0307387-tk4y78ls authors: Simón-Gracia, Lorena; Scodeller, Pablo; Fisher, William S.; Sidorenko, Valeria; Steffes, Victoria M.; Ewert, Kai K.; Safinya, Cyrus R.; Teesalu, Tambet title: Paclitaxel-loaded PEGylated Cationic Lipid Nanodiscs and Small Liposomes with Protruding Brush Conformation PEG-chains Show Tumor Penetration and Proapoptotic Caspase-3 Activation date: 2022-03-29 journal: bioRxiv DOI: 10.1101/2022.03.28.486128 sha: 09b838ffdb0c2fded2952f2e270fc1ebb08bf65d doc_id: 307387 cord_uid: tk4y78ls Novel approaches are required to address the urgent need to develop lipid-based carriers of paclitaxel (PTX) and other hydrophobic drugs for cancer chemotherapy. Carriers based on cationic liposomes (CLs) (e.g. EndoTAG-1®) have shown promise in animal models and advanced to late-stage clinical trials. We recently found that the addition of cone-shaped poly(ethylene glycol)-lipid (PEG-lipid) to PTX-loaded CLs (CLsPTX) drives a transition to a phase of sterically stabilized, higher curvature (smaller) lipid nanoparticles consisting of coexisting PEGylated CLsPTX and PTX-containing nanodiscs (bicelles) (nanodiscsPTX). At PEG coverages in the brush regime, these PEGylated CLsPTX and nanodiscsPTX showed significantly improved delivery and cytotoxicity to human cancer cells in vitro. Here, we investigated the PTX loading and stability of the CLsPTX and nanodiscsPTX at high concentration and assessed their circulation half-life and biodistribution in vivo. At higher content of PEG-lipid (5 and 10 mol% of a PEG-lipid with PEG molecular weight 5000 g/mol), the mixture of PEGylated CLsPTX and nanodiscsPTX formed almost exclusively particles of sub-micron size and incorporated up to 2.5 mol% PTX without crystallization for at least 20 h. Remarkably, compared to lipid vector preparations containing 2 and 5 mol% PEG5K-lipid (with the PEG chains in the mushroom regime), those at 10 mol% with PEG chains in the extended brush conformation showed significantly more tumor penetration and increased proapoptotic caspase-3 activation in 4T1 breast cancer lesions modeled in immunocompetent mice. Chemotherapy continues to be a common and effective treatment option for cancer. However, a number of the drugs used in chemotherapy have poor water solubility and therefore must be formulated with a vector (carriers) to enable their use. A prime example of this is the potent cytotoxic drug paclitaxel (PTX) [1] [2] [3] [4] . PTX is highly hydrophobic with very low water solubility, and it is ineffective in its insoluble crystalline form. Nonetheless, PTX is one of the most commonly used anti-cancer drugs, with multibillion dollar sales of PTX-based medicines each year 5-11 . Currently, the most prominent formulations of PTX are Taxol® (where PTX is solubilized using nonionic Kolliphor EL surfactant) and Abraxane® (where PTX is formulated in albumin nanoparticles) 5,12-14 . Serious side effects, both due to the vector and the indiscriminate delivery of PTX throughout the body, are common and dose-limiting for these PTX formulations [15] [16] [17] . Major efforts are therefore underway to develop improved PTX vectors for cancer chemotherapy with reduced side effects: novel carrier materials to avoid the hypersensitivity reactions associated with the use of Kolliphor EL (formerly Cremophor EL, polyoxyethylated castor oil), carriers with higher efficacy to allow the administration of lower total doses of PTX, and platforms for delivery of PTX that improve targeting to malignant tissues 18-27 . Liposomes are promising and versatile carriers (vectors) of drugs in therapeutic applications due to their biocompatibility and ability to sequester a wide range of molecules. These payloads include hydrophilic drugs and nucleic acids (NAs, e.g., DNA, siRNA and mRNA) in addition to hydrophobic drugs 5, [28] [29] [30] [31] [32] [33] [34] [35] [36] . A prominent example of a liposomal carrier of a hydrophobic drug is Doxil®, 37 the formulation of another commonly used chemotherapeutic agent (doxorubicin). Doxil allows increased doxorubicin dosing because it reduces the cardiotoxicity associated with the administration of the free drug. However, the specific chemical properties of doxorubicin enable loading of the liposomes by a process that is not transferable to most other drugs including PTX. Cationic liposomes (CLs; consisting of mixtures of cationic (or ionizable) and neutral lipids) in particular are prevalent nonviral vectors for the delivery of therapeutic NAs as recently demonstrated with the mRNA vaccines against COVID-19 38-41 . However, they are also suitable carriers for hydrophobic drugs. A cationic lipid-based carrier of PTX that has advanced to phase III clinical trials is EndoTAG ® -1, which consists of DOTAP/DOPC CLs with 3 mol% PTX 19,25,35,42 (DOTAP, N-[2,3-dioleoyloxy-1-propyl]trimethylammonium chloride, is a univalent cationic lipid; DOPC, 1,2-dioleoyl-sn-glycerophosphocholine, is a naturally occurring neutral phospholipid.) EndoTAG-1 has been shown to target tumor endothelial cells in solid tumors via nonspecific electrostatic interactions with cell surface anionic sulfated proteoglycans 36, [42] [43] [44] [45] [46] . Unlike NAs and hydrophilic drugs (as well as doxorubicin nanocrystals in Doxil), which are packed in the aqueous medium between membranes, PTX is solubilized within the hydrophobic region of the membrane of lipid-based carriers. The properties of lipid-based vectors of PTX can be tuned and improved in a variety of ways. By chemically modifying the constituent lipids, in particular the tails which interact with and solubilize PTX, the solubility of PTX and thus the drug loading can be increased markedly 47 beyond the 3 mol% solubility "limit" 48 of the PTX content that has been a limitation for most efficacy studies and clinical trials to date 19, 25, 35, 42, 44 . Lipid carriers also offer the advantage of enabling future targeted delivery in vivo (e.g. via cyclic homing peptides attached to poly(ethylene glycol)-lipids (PEG-lipids) coating the CL vectors 49, 50 ). In addition, modification of the lipids and the lipid composition of CL vectors can drive structural transitions 29,51-55 , including the spontaneous formation of PEGylated micelles (discs, cylinders and spheroids observed in cryogenic TEM) coexisting with vesicles. 56, 57 For example, a recent publication reported that PEGylation of CL-based vectors of PTX via the addition The breakup of large liposomes into nanodiscs and very small vesicles strongly increases the solution entropy. However, an enthalpic elastic cost is incurred by the creation of membrane areas with high curvature (i.e., the surfaces of spherical or cylindrical micelles and the edges of discs). In the case of the discs seen in Figure 1 , this enthalpy cost is lowered to below the overall gain in solution entropy by a No PEG-lipid 10 mol% PEG-lipid segregation of cone-shaped PEG-lipids (with spontaneous curvature C 0 >0, Figure 2 ) into the highcurvature edges of the discs which stabilizes them. (C 0 , the spontaneous (i.e., preferred) curvature of the membrane, is determined by the "shape" of the constituting lipid molecules, which is set by the size of the area of the lipid headgroup relative to the size of the area of the tails 58-61 [ Figure 2 ].) Without such lipid segregation or lateral phase separation within the membrane (which is associated with its own cost in entropy for the segregating lipids), the increase in membrane curvature stemming from the addition of cone-shaped lipids such as PEG-lipids or highly charged lipids results in a reduction of the size of the lipid assemblies. The size (radius) of the assemblies will be of order of 1/C 0 , because the membrane curvature, C, of lipid assemblies (as measured in experiments) is typically maintained close to C 0 to avoid an elastic energy penalty [62] [63] [64] . Importantly, the PEGylated lipid vectors consisting of coexisting PEGylated CLs PTX and PTX-containing nanodiscs (nanodiscs PTX ), with PEG chains in the brush state, showed a significant enhancement of cellular uptake and cytotoxicity (i.e., efficacy) against human cancer cells in vitro compared to formulations similar to the benchmark EndoTAG-1, with cell viability decreasing in a PEG concentrationdependent manner 57 . This suggests that the size, stabilization, and possibly shape of the PEGylated lipid vector promotes cell uptake and thereby PTX delivery through different size-dependent endocytic pathways. The finding that incorporating PEG-lipids yields CL carriers of PTX with stable micellar structures and high efficacy in vitro invited development of functionalized liposomes for in vivo delivery applications. Thus, we selected a series of PEGylated CL vectors of PTX for studies of biodistribution and cytotoxic activity in vivo to further assess their potential for therapeutic applications. We Samples for cryogenic TEM were prepared and imaged as described previously 57 . For PTX solubility experiments, 2 μL each of cationic lipid formulations containing 10 mol% PEG5Klipid and 2, 2.5, or 3 mol% PTX were placed on glass microscope slides and covered by coverslips secured by parafilm cutouts. All remaining samples were incubated for 20 h at room temperature before 2 μL were again taken for imaging. For lipid solubility experiments, 2 μL each of samples of formulations with 2, 5, and 10 mol% PEG5K-lipid were placed on glass microscope slides and again covered by coverslips secured by parafilm cutouts. All slides were imaged at 40× magnification on an inverted Ti2-E (Nikon) microscope. ImageJ. Two to six random areas per tumor were chosen and this was repeated in three tumor-bearing mice for each group. The statistical analyses were performed with Statistica 8 software (StatSoft, USA) using the one-way ANOVA and Fisher LSD tests. As mentioned in the introduction, it is essential for the efficacy of PTX-loaded CLs that PTX remains soluble in the membrane. We previously found that the membrane solubility of PTX is slightly lower in PEGylated CLs than in bare CLs 48, 57 . To determine the optimal PTX content for the CLs employed in the current study, we assessed the solubility of PTX in CLs containing 10 mol% PEG5K-lipid, the highest PEG-lipid content used. The formation of therapeutically inert PTX crystals, due to PTX self-association in the membrane and subsequent phase separation, is an indicator of PTX insolubility 48, 65 . To measure PTX solubility in PEG-CL membranes, we monitored PTX crystal formation with DIC microscopy 48, 57 . Representative DIC micrographs of sonicated CLs containing 10 mol% PEG5K-lipid and 2 to 3 mol% PTX are displayed in Figure 3 . For studies of therapeutic efficacy, PTX-loaded CLs must be prepared at total lipid concentrations of 50 mg/mL or greater to achieve sufficiently high PTX dosages 66, 67 . We thus prepared sonicated CL To assess the potential of PEGylated cationic liposome vectors containing PTX for applications in vivo, we first studied the effect of differential PEGylation (at 2, 5, or 10 mol% of PEG5K-lipid) on the blood nanodiscs PTX ) showed a 2-fold higher signal of cleaved caspase-3 ( Figure 6C ), although this difference did not reach statistical significance, possibly due to the low PTX dose administered and the short time point used to observe the effect of PTX. Importantly, the signal for cleaved caspase-3 was observed in areas overlapping or adjacent to the fluorescent signal of the lipid formulation with 10 mol% PEG5Klipid ( Figure 6A ), suggesting that this formulation was able to efficiently release PTX and trigger cell apoptosis. Plant antitumor agents. VI. Isolation and structure of taxol Paclitaxel-Loaded Polymersomes for Enhanced Intraperitoneal Chemotherapy Teesalu, T. iRGD peptide conjugation potentiates intraperitoneal tumor delivery of paclitaxel with polymersomes Cationic liposome-nucleic acid complexes for gene delivery and gene silencing Cationic Liposomes as Vectors for Nucleic Acid and Hydrophobic Drug Therapeutics Recent advances with liposomes as pharmaceutical carriers Liposomal drug delivery systems: From concept to clinical applications Lipid nanoparticle technology for therapeutic gene regulation in the liver Lipid Nanoparticle Technology for Clinical Translation of siRNA Therapeutics Advances and Challenges of Liposome Assisted Drug Delivery Liposomal paclitaxel formulations Fighting cancer: From the bench to bedside using second generation cationic liposomal therapeutics Doxil® -The first FDA-approved nano-drug: Lessons learned Phase I/II study of COVID-19 RNA vaccine BNT162b1 in adults Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine An mRNA Vaccine against SARS-CoV-2 -Preliminary Report Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine Vascular and pharmacokinetic effects of EndoTAG-1 in patients with advanced cancer and liver metastasis Neovascular targeting chemotherapy: Encapsulation of paclitaxel in cationic liposomes impairs functional tumor microvasculature Tumor-selective vessel occlusions by platelets after vascular targeting chemotherapy using paclitaxel encapsulated in cationic liposomes Paclitaxel Encapsulated in Cationic Liposomes Diminishes Tumor Angiogenesis and Melanoma Growth in a "Humanized" SCID Mouse Model Neovascular Targeting Therapy: Paclitaxel Encapsulated in Cationic Liposomes Improves Antitumoral Efficacy Paclitaxel loading in cationic liposome vectors is enhanced by replacement of oleoyl with linoleoyl tails with distinct lipid shapes Distinct Solubility and Cytotoxicity Regimes of Paclitaxel-Loaded Cationic Liposomes at Low and High Drug Content Revealed by Kinetic Phase Behavior and Cancer Cell Viability Studies Synthesis of linear and cyclic peptide-PEG-lipids for stabilization and targeting of cationic liposome-DNA complexes Competition of charge-mediated and specific binding by peptidetagged cationic liposome-DNA nanoparticles in vitro and in vivo An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery A columnar phase of dendritic lipid-based cationic liposome-DNA complexes for gene delivery: Hexagonally ordered cylindrical micelles embedded in a DNA honeycomb lattice Highly Efficient Gene Silencing Activity of siRNA Embedded in a Nanostructured Gyroid Cubic Lipid Matrix Liquid crystalline phases of dendritic lipid-DNA self-assemblies: Lamellar, hexagonal, and DNA bundles Cationic Liposomes as Spatial Organizers of Nucleic Acids in One, Two, and Three Dimensions: Liquid Crystal Phases with Applications in Delivery and Bionanotechnology Patterned Threadlike Micelles and DNA-Tethered Nanoparticles: A Structural Study of PEGylated Cationic Liposome-DNA Assemblies PEGylation of Paclitaxel-Loaded Cationic Liposomes Drives Steric Stabilization of Bicelles and Vesicles thereby Enhancing Delivery and Cytotoxicity to Human Cancer Cells Theory of self-assembly of hydrocarbon amphiphiles into micelles and bilayers Theory of self-assembly of lipid bilayers and vesicles Intermolecular and Surface Forces Structure of the inverted hexagonal (HII) phase, and non-lamellar phase transitions of lipids Elastic Properties of Lipid Bilayers: Theory and Possible Experiments Structure and Dynamics of Membranes Statistical thermodynamics of surfaces, interfaces, and membranes Taxol-Lipid Interactions: Taxol-Dependent Effects on the Physical Properties of Model Membranes Vascular targeting by EndoTAG™-1 enhances therapeutic efficacy of conventional chemotherapy in lung and pancreatic cancer Cationic lipid complexed camptothecin (EndoTAG®-2) improves antitumoral efficacy by tumor vascular targeting Shape Effect of Glyco-Nanoparticles on Macrophage Cellular Uptake and Immune Response This research study was supported by the National Institutes of Health under Award R01GM130769 (CRS,KKE,WF, mechanistic studies on developing lipid nanoparticles for drug delivery), by the