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Open Access Publications from the University of California

Scholarly Works (15 results)

  • Thesis
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UC Santa Barbara Electronic Theses and Dissertations (2019)

In order to increase the efficacy of the cancer drug paclitaxel (PTX), scientists must develop more effective drug carriers that well solubilize PTX and achieve targeted delivery. Loading cytotoxic drugs like PTX into nanoparticles that are engineered to accumulate in cancer tissue by physical or chemical means is expected to increase local drug concentrations and minimize systemic side effects. Due to its hydrophobic nature, PTX loads into the actual membrane of lipid nanoparticles (LNPs)—closely associating with lipid tails—and has limited solubility in lipid membranes. In vitro cell viability experiments demonstrate a direct correlation between delivery efficacy and the duration of PTX solubility in LNPs. This study importantly reveals enhanced delivery when PTX is loaded below, rather than at, its drug-loading saturation level. Based on PTX loading into membranes, we expect and indeed observe that PTX solubility depends strongly on the types of lipids used in LNP formulations. Here we report the PTX-loading and delivery efficacy of a selection of lipids that enhance delivery of other therapeutics (hydrophilic drugs, nucleic acids) to see if they similarly improve PTX delivery: inverted cone-shaped lipids (DOPE, GMO), poly-unsaturated (18:2, 18:3) lipids, cationic lipids (DOTAP, MVL5), and polymer-conjugated lipids (2k and 5k PEG-lipids).

We developed a complementary approach using physical and cell biology characterization methods to elucidate structure-function relationships of LNP composition. DIC microscopy observations are used to generate kinetic phase diagrams to compare PTX solubility in different LNP formulations. Small-angle x-ray scattering (SAXS) shows that PTX-loading thins lipid membranes and determines the self-assembly structures of LNPs. The in vitro delivery efficacy of LNP formulations is determined by incubating PTX-loaded LNPs with PC3 (prostate) and M21 (melanoma) immortalized human cancer cell lines and measuring the resultant cell death. Fluorescent microscopy and flow cytometry provide qualitative and quantitative information about how LNPs interact with cells based on their composition. Finally, cryoTEM images offer important clues about how LNP structure and stability directly impact cell interactions and PTX delivery. In particular, we highlight how LNP vesicles transition to various disk, worm, and sphere micelles as the fraction of cationic or PEG-lipid increases. Using these methods, we have found several promising strategies to improve LNPs for PTX delivery. Studying fundamental LNP properties to determine composition-function relationships is a smarter strategy than empirical discovery because it identifies specific ways to thoughtfully design better drug delivery systems.

    Cover page: Designing lipid nanoparticles toward targeted drug delivery: Fundamental studies identify key compositional properties to improve formulations for the hydrophobic cancer drug paclitaxel
    • Article
    • Peer Reviewed
    UC Santa Barbara Previously Published Works (2021)
    Lipid carriers of hydrophobic paclitaxel (PTX) are used in clinical trials for cancer chemotherapy. Improving their loading capacity requires enhanced PTX solubilization. We compared the time-dependence of PTX membrane solubility as a function of PTX content in cationic liposomes (CLs) with lipid tails containing one (oleoyl; DOPC/DOTAP) or two (linoleoyl; DLinPC/newly synthesized DLinTAP) cis double bonds by using microscopy to generate kinetic phase diagrams. The DLin lipids displayed significantly increased PTX membrane solubility over DO lipids. Remarkably, 8 mol% PTX in DLinTAP/DLinPC CLs remained soluble for approximately as long as 3 mol% PTX (the solubility limit, which has been the focus of most previous studies and clinical trials) in DOTAP/DOPC CLs. The increase in solubility is likely caused by enhanced molecular affinity between lipid tails and PTX, rather than by the transition in membrane structure from bilayers to inverse cylindrical micelles observed with small-angle X-ray scattering. Importantly, the efficacy of PTX-loaded CLs against prostate cancer cells (their IC50 of PTX cytotoxicity) was unaffected by changing the lipid tails, and toxicity of the CL carrier was negligible. Moreover, efficacy was approximately doubled against melanoma cells for PTX-loaded DLinTAP/DLinPC over DOTAP/DOPC CLs. Our findings demonstrate the potential of chemical modifications of the lipid tails to increase the PTX membrane loading 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.
      Cover page: Paclitaxel loading in cationic liposome vectors is enhanced by replacement of oleoyl with linoleoyl tails with distinct lipid shapesCreative Commons 'BY-NC-ND' version 4.0 license
      • Article
      UC Santa Barbara Previously Published Works (2020)
      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 Δ 9 ; DOTAP/DOPC) or two (linoleoyl; C18:2 Δ 9 ; 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.
        Creative Commons 'BY-NC-ND' version 4.0 license
        • Article
        UC Santa Barbara Previously Published Works (2023)
        Lipids, and cationic lipids in particular, are of interest as delivery vectors for hydrophobic drugs such as the cancer therapeutic paclitaxel, and the structures of lipid assemblies affect their efficacy. We investigated the effect of incorporating the multivalent cationic lipid MVL5 (+5 e ) and poly(ethylene glycol)-lipids (PEG-lipids), alone and in combination, on the structure of fluid-phase lipid assemblies of the charge-neutral lipid 1,2-dioleoyl- sn -glycero-phosphocholine (DOPC). This allowed us to elucidate lipid–liposome structure correlations in sonicated formulations with high charge density, which are not accessible with univalent lipids such as the well-studied DOTAP (+1 e ). Cryogenic TEM allowed us to determine the structure of the lipid assemblies, revealing diverse combinations of vesicles and disc-shaped, worm-like, and spherical micelles. Remarkably, MVL5 forms an essentially pure phase of disc micelles at 50 mol% MVL5. At higher (75 mol%) content of MVL5, short and intermediate-length worm-like micellar rods were observed and, in ternary mixtures with PEG-lipid, longer and highly flexible worm-like micelles formed. Independent of their length, the worm-like micelles coexisted with spherical micelles. In stark contrast, DOTAP forms mixtures of vesicles, disc micelles and spherical micelles at all studied compositions, even when combined with PEG-lipids. The observed similarities and differences in the effects of charge (multivalent versus univalent) and high curvature (multivalent charge versus PEG-lipid) on assembly structure provide insights into parameters that control the size of fluid lipid nanodiscs, relevant for future applications.
          Creative Commons 'BY-NC-ND' version 4.0 license
          • Article
          UC Santa Barbara Previously Published Works (2023)
          Paclitaxel (PTX) is a hydrophobic small-molecule cancer drug that loads into the membrane (tail) region of lipid carriers such as liposomes and micelles. The development of improved lipid-based carriers of PTX is an important objective to generate chemotherapeutics with fewer side effects. The lipids 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE) and glyceryl monooleate (GMO) show propensity for fusion with other lipid membranes, which has led to their use in lipid vectors of nucleic acids. We hypothesized that DOPE and GMO could enhance PTX delivery to cells through a similar membrane fusion mechanism. As an important measure of drug carrier performance, we evaluated PTX solubility in cationic liposomes containing GMO or DOPE. Solubility was determined by time-dependent kinetic phase diagrams generated from direct observations of PTX crystal formation using differential-interference-contrast optical microscopy. Remarkably, PTX was much less soluble in these liposomes than in control cationic liposomes containing univalent cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl- sn -glycero-3-phosphatidylcholine (DOPC), which are not fusogenic. In particular, PTX was not substantially soluble in GMO-based cationic liposomes. The fusogenicity of DOPE and GMO is related to the negative spontaneous curvature of membranes containing these lipids, which drives formation of nonlamellar self-assembled phases (inverted hexagonal or gyroid cubic). We used synchrotron small-angle x-ray scattering to determine whether PTX solubility is governed by lipid membrane structure (condensed with DNA in pellet form) or by local intermolecular interactions. The results suggest that local intermolecular interactions are of greater importance and that the negative spontaneous curvature-inducing lipids DOPE and GMO are not suitable components of lipid carriers for PTX delivery regardless of carrier structure.
            Creative Commons 'BY-NC-ND' version 4.0 license
            • Article
            • Peer Reviewed
            UC Santa Barbara Previously Published Works (2023)
            Lipids, and cationic lipids in particular are of interest as delivery vectors for hydrophobic drugs such as the cancer therapeutic paclitaxel, and the structures of lipid assemblies affect their efficacy. We investigated the effect of incorporating the multivalent cationic lipid MVL5 (+5e) and poly(ethylene glycol)-lipids (PEG-lipids), alone and in combination, on the structure of fluid-phase lipid assemblies of the charge-neutral lipid 1,2-dioleoyl-sn-glycero-phosphocholine (DOPC). This allowed us to elucidate lipid-assembly structure correlations in sonicated formulations with high charge density, which are not accessible with univalent lipids such as the well-studied DOTAP (+1e). Cryogenic transmission electron microscopy (cryo-TEM) allowed us to determine the structure of the lipid assemblies, revealing diverse combinations of vesicles and disc-shaped, worm-like, and spherical micelles. Remarkably, MVL5 forms an essentially pure phase of disc micelles at 50 mol % MVL5. At a higher (75 mol %) content of MVL5, short- and intermediate-length worm-like micellar rods were observed, and in ternary mixtures with PEG-lipid, longer and highly flexible worm-like micelles formed. Independent of their length, the worm-like micelles coexisted with spherical micelles. In stark contrast, DOTAP forms mixtures of vesicles, disc micelles, and spherical micelles at all studied compositions, even when combined with PEG-lipids. The observed similarities and differences in the effects of charge (multivalent versus univalent) and high curvature (multivalent charge versus PEG-lipid) on the assembly structure provide insights into parameters that control the size of fluid lipid nanodiscs, relevant for future applications.
              Creative Commons 'BY-NC-ND' version 4.0 license
              • Article
              • Peer Reviewed
              UC Santa Barbara Previously Published Works (2023)
              Paclitaxel (PTX) is a hydrophobic small-molecule cancer drug that loads into the membrane (tail) region of lipid carriers such as liposomes and micelles. The development of improved lipid-based carriers of PTX is an important objective to generate chemotherapeutics with fewer side effects. The lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and glyceryl monooleate (GMO) show propensity for fusion with other lipid membranes, which has led to their use in lipid vectors of nucleic acids. We hypothesized that DOPE and GMO could enhance PTX delivery to cells through a similar membrane fusion mechanism. As an important measure of drug carrier performance, we evaluated PTX solubility in cationic liposomes containing GMO or DOPE. Solubility was determined by time-dependent kinetic phase diagrams generated from direct observations of PTX crystal formation using differential-interference-contrast optical microscopy. Remarkably, PTX was much less soluble in these liposomes than in control cationic liposomes containing univalent cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), which are not fusogenic. In particular, PTX was not substantially soluble in GMO-based cationic liposomes. The fusogenicity of DOPE and GMO is related to the negative spontaneous curvature of membranes containing these lipids, which drives formation of nonlamellar self-assembled phases (inverted hexagonal or gyroid cubic). To determine whether PTX solubility is governed by lipid membrane structure or by local intermolecular interactions, we used synchrotron small-angle X-ray scattering. To increase the signal/noise ratio, we used DNA to condense the lipid formulations into lipoplex pellets. The results suggest that local intermolecular interactions are of greater importance and that the negative spontaneous curvature-inducing lipids DOPE and GMO are not suitable components of liposomal carriers for PTX delivery.
                Creative Commons 'BY-NC-ND' version 4.0 license
                • Article
                • Peer Reviewed
                UC Santa Barbara Previously Published Works (2017)
                Lipid-based particles are used worldwide in clinical trials as carriers of hydrophobic paclitaxel (PTXL) for cancer chemotherapy, albeit with little improvement over the standard-of-care. Improving efficacy requires an understanding of intramembrane interactions between PTXL and lipids to enhance PTXL solubilization and suppress PTXL phase separation into crystals. We studied the solubility of PTXL in cationic liposomes (CLs) composed of positively charged 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP) and neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) as a function of PTXL membrane content and its relation to efficacy. Time-dependent kinetic phase diagrams were generated from observations of PTXL crystal formation by differential-interference-contrast microscopy. Furthermore, a new synchrotron small-angle x-ray scattering in situ methodology applied to DOTAP/DOPC/PTXL membranes condensed with DNA enabled us to detect the incorporation and time-dependent depletion of PTXL from membranes by measurements of variations in the membrane interlayer and DNA interaxial spacings. Our results revealed three regimes with distinct time scales for PTXL membrane solubility: hours for >3 mol% PTXL (low), days for ≈ 3 mol% PTXL (moderate), and ≥20 days for < 3 mol% PTXL (long-term). Cell viability experiments on human cancer cell lines using CLPTXL nanoparticles (NPs) in the distinct CLPTXL solubility regimes reveal an unexpected dependence of efficacy on PTXL content in NPs. Remarkably, formulations with lower PTXL content and thus higher stability show higher efficacy than those formulated at the membrane solubility limit of ≈3 mol% PTXL (which has been the focus of most previous physicochemical studies and clinical trials of PTXL-loaded CLs). Furthermore, an additional high-efficacy regime is seen on occasion for liposome compositions with PTXL ≥9 mol% applied to cells at short time scales (hours) after formation. At longer time scales (days), CLPTXL NPs with ≥3 mol% PTXL lose efficacy while formulations with 1-2 mol% PTXL maintain high efficacy. Our findings underscore the importance of understanding the relationship of the kinetic phase behavior and physicochemical properties of CLPTXL NPs to efficacy.
                  Cover page: 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.
                  • Article
                  • Peer Reviewed
                  UC Santa Barbara Previously Published Works (2017)
                  Lipid-based particles are used worldwide in clinical trials as carriers of hydrophobic paclitaxel (PTXL) for cancer chemotherapy, albeit with little improvement over the standard-of-care. Improving efficacy requires an understanding of intramembrane interactions between PTXL and lipids to enhance PTXL solubilization and suppress PTXL phase separation into crystals. We studied the solubility of PTXL in cationic liposomes (CLs) composed of positively charged 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP) and neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) as a function of PTXL membrane content and its relation to efficacy. Time-dependent kinetic phase diagrams were generated from observations of PTXL crystal formation by differential-interference-contrast microscopy. Furthermore, a new synchrotron small-angle x-ray scattering in situ methodology applied to DOTAP/DOPC/PTXL membranes condensed with DNA enabled us to detect the incorporation and time-dependent depletion of PTXL from membranes by measurements of variations in the membrane interlayer and DNA interaxial spacings. Our results revealed three regimes with distinct time scales for PTXL membrane solubility: hours for >3 mol% PTXL (low), days for ≈ 3 mol% PTXL (moderate), and ≥20 days for < 3 mol% PTXL (long-term). Cell viability experiments on human cancer cell lines using CLPTXL nanoparticles (NPs) in the distinct CLPTXL solubility regimes reveal an unexpected dependence of efficacy on PTXL content in NPs. Remarkably, formulations with lower PTXL content and thus higher stability show higher efficacy than those formulated at the membrane solubility limit of ≈3 mol% PTXL (which has been the focus of most previous physicochemical studies and clinical trials of PTXL-loaded CLs). Furthermore, an additional high-efficacy regime is seen on occasion for liposome compositions with PTXL ≥9 mol% applied to cells at short time scales (hours) after formation. At longer time scales (days), CLPTXL NPs with ≥3 mol% PTXL lose efficacy while formulations with 1-2 mol% PTXL maintain high efficacy. Our findings underscore the importance of understanding the relationship of the kinetic phase behavior and physicochemical properties of CLPTXL NPs to efficacy.
                    Cover page: 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 studiesCreative Commons 'BY-NC-ND' version 4.0 license
                    • Article
                    • Peer Reviewed
                    UC Davis Previously Published Works (2020)
                    Poly(ethylene glycol) (PEG) is a polymer used widely in drug delivery to create "stealth" nanoparticles (NPs); PEG coatings suppress NP detection and clearance by the immune system and beneficially increase NP circulation time in vivo. However, NP PEGylation typically obstructs cell attachment and uptake in vitro compared to the uncoated equivalent. Here, we report on a cationic liposome (CL) NP system loaded with the hydrophobic cancer drug paclitaxel (PTX) in which PEGylation (i.e., PEG-CLPTX NPs) unexpectedly enhances, rather than diminishes, delivery efficacy and cytotoxicity to human cancer cells. This highly unexpected enhancement occurs even when the PEG-chains coating the NP are in the transition regime between the mushroom and brush conformations. Cryogenic transmission electron microscopy (TEM) of PEG-CLPTX NPs shows that PEG causes the proliferation of a mixture of sterically stabilized nanometer-scale vesicles and anisotropic micelles (e.g., bicelles). Remarkably, the onset of bicelles at sub-monolayer concentrations of the PEG coat has to our knowledge not been previously reported; it was previously thought that PEG-lipid in this composition regime was incorporated into vesicles but did not alter their shape. Confocal microscopy and flow cytometry reveal significantly greater PTX cell uptake from stabilized PEG-CLPTX NPs (vesicles and bicelles) in contrast to bare CLPTX NPs, which can aggregate in cell medium. This underscores the ability of steric stabilization to facilitate NP entry into cells via distinct size-dependent endocytic pathways, some of which cannot transport large NP aggregates into cells. This study highlights the value of understanding how PEGylation alters NP shape and structure, and thus NP efficacy, to design next-generation stealth drug carriers that integrate active cell-targeting strategies into NPs for in vivo delivery.
                      Cover page: PEGylation of Paclitaxel-Loaded Cationic Liposomes Drives Steric Stabilization of Bicelles and Vesicles thereby Enhancing Delivery and Cytotoxicity to Human Cancer Cells
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