Tadpole-Like Anisotropic Polymer/Lipid Janus Nanoparticles for Nose-to-Brain Drug Delivery: Importance of Geometry, Elasticity on Mucus-Penetration Ability.

Asymmetric geometry (aspect ratio >1), moderate stiffness (i.e., semielasticity), large surface area, and low mucoadhesion of nanoparticles are the main features to reach the brain by penetrating across the nasal mucosa. Herein, a new application has been presented for the use of multifunctional Janus nanoparticles (JNPs) with controllable geometry and size as a nose-to-brain (N2B) delivery system by changing proportions of Precirol ATO 5 and polycaprolactone compartments and other operating conditions. To bring to light the N2B application of JNPs, the results are presented in comparison with polymer and solid lipid nanoparticles, which are frequently used in the literature regarding their biopharmaceutical aspects: mucoadhesion and permeability through the nasal mucosa. The morphology and geometry of JPs were observed via cryogenic-temperature transmission electron microscopy images, and their particle sizes were verified by dynamic light scattering, atomic force microscopy, and scanning electron microscopy. Although all NPs showed penetration across the mucus barrier, the best increase in penetration was observed with asymmetric and semielastic JNPs, which have low interaction ability with the mucus layer. This study presents a new and promising field of application for a multifunctional system suitable for N2B delivery, potentially benefiting the treatment of brain tumors and other central nervous system diseases.

The nanostructure of polyelectrolyte complexes of QPDMAEMA-b-POEGMA copolymers and oppositely charged polyelectrolytes, and their stability in the presence of serum albumin.

Quaternized poly(2-(dimethylamino) ethyl methacrylate)-b-poly(oligo(ethyleneglycol) methyl ether methacrylate) (QPDMAEMA-b-POEGMA) is a copolymer of a positively charged block and a non-ionic hydrophilic block. The positively charged block, QPDMAEMA, electrostatically interacts with oppositely charged polymers, e.g., poly(acrylic acid) (PAA) and DNA, to form a complex. This complex is stable in aqueous solution due to the hydrophilic block, POEGMA, which provides colloidal stability and biocompatibility. Polyplexes can be used as non-viral vectors in gene therapy. Polyplexes are essential for delivering genetic materials into cells because they protect the genetic material from degradation before reaching the target cells, thus increasing the transfection efficiency. However, currently used polyplexes show a low transfection efficiency in vivo, probably because the polyplexes are exposed to blood proteins, such as serum albumin, which cause their dissociation. The main goal of this research is the morphology characterization of QPDMAEMA-b-POEGMA complexes with the sodium salt of polyacrylic acid (NaPAA), and with DNA by cryogenic transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS). These methods give qualitative and quantitative data about the morphology of the complexes. The morphology of the complexes was examined at different charge ratios (CRs). Complexes with NaPAA form core-corona spherical micelles and vesicular structures, whereas complexes with DNA form lamellar and hexagonal structures. The QPDMAEMA-b-POEGMA and DNA complexes were also examined after exposing them to bovine serum albumin (BSA). We found that BSA does not affect the complexes for seven days. This morphology characterization is essential for better design and formulation of vectors for gene therapy and polyelectrolyte complexes for biomedical applications.

Cubosomes for in vivo fluorescence lifetime imaging.

Herein we provided the first proof of principle for in vivo fluorescence optical imaging application using monoolein-based cubosomes in a healthy mouse animal model. This formulation, administered at a non-cytotoxic concentration, was capable of providing both exogenous contrast for NIR fluorescence imaging with very high efficiency and chemospecific information upon lifetime analysis. Time-resolved measurements of fluorescence after the intravenous injection of cubosomes revealed that the dye rapidly accumulated mainly in the liver, while lifetimes profiles obtained in vivo allowed for discriminating between free dye or dye embedded within the cubosome nanostructure after injection.

Cancer-cell-targeted theranostic cubosomes.

This work was devoted to the development of a new type of lipid-based (cubosome) theranostic nanoparticle able to simultaneously host camptothecin, a potent anticancer drug, and a squarain-based NIR-emitting fluorescent probe. Furthermore, to confer targeting abilities on these nanoparticles, they were dispersed using mixtures of Pluronic F108 and folate-conjugated Pluronic F108 in appropriate ratios. The physicochemical characterization, performed via SAXS, DLS, and cryo-TEM techniques, proved that aqueous dispersions of such cubosomes can be effectively prepared, while the photophysical characterization demonstrated that these nanoparticles may be used for in vivo imaging purposes. The superior ability of these innovative nanoparticles in targeting cancer cells was emphasized by investigating the lipid droplet alterations induced in HeLa cells upon exposure to targeted and nontargeted cubosomes.

Lipid Segregation in Membranes of Anionic Liposomes Adsorbed onto Polycationic Brushes.

Two-phased: Complexation of liposomes to spherical polycationic brushes induces lipid segregation in the liposomal membrane. The greater the initial anionic lipid content in the membrane, the more the electroneutral lipid dilutes the induced anionic clusters.

Nanostructure of complexes between cationic lipids and an oppositely charged polyelectrolyte.

The morphology of aqueous solutions of polyelectrolytes and oppositely charged lipids is the subject of extensive colloid science research, because of their application in industry and medicine, the latter especially for gene therapy. In this work, we show that complexes of two different cationic lipids with the polyelectrolyte sodium poly(acrylic acid), PAA, share similar morphology with the complexes of those lipids with nucleic acids, implying a broader and universal packing phenomenon. We characterized by direct-imaging cryogenic-temperature transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), and zeta (ζ)-potential two cationic lipids, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and bis(11-ferrocenylundecyl) dimethylammonium bromide (BFDMA), which are used in gene transfection, at equivalent lipid/polyelectrolyte charge ratio. Our results revealed that, for both types of complexes, onion-like multilamellar nanostructures formed, which exhibited similar morphology as in complexes of DNA or oligonucleotides (lipoplexes), based on the same lipids. Our findings suggest that the onion-like packing may be energetically favorable for a wide range of polyelectrolyte-liposome systems, from oligonucleotides and DNA to PAA.

Composition and properties of complexes between spherical polycationic brushes and anionic liposomes.

A spherical polycationic brush (SPB) is made by graft-polymerizing a cationic monomer onto the surface of a 100 nm polystyrene bead. It is possible to adsorb anionic liposomes (40-60 nm diameter) onto the SPBs while maintaining the liposome integrity. The liposomes were constructed with phosphatidyl choline (PC) admixed with 0.05-0.4 mol fraction of an dianionic lipid, cardiolipin (CL(2-)). As shown by electrophoretic mobility measurements, SPB-to-liposome complexation leads to a conversion from the initial positive charge of the copolymer to a negative charge. The higher the CL(2-) content of the liposomes, the lower the concentration needed for charge neutralization. Dynamic light scattering (DLS) revealed that multicomplex aggregates are formed with a maximum size at the SPB/liposome charge-equivalence point. Experiments with fluorescent-labeled liposomes show that at low CL(2-) content about 80 liposomes are adsorbed per SPB. As the mole fraction of CL(2-) increases from 0.05 to 0.4, fewer liposomes adsorb owing to electrostatic repulsion among neighboring liposomes. The effect of added NaCl also depends upon the CL(2-) content. With 0.05 mol fraction CL(2-), the SPB/liposome complex dissociates into its components at 0.15 M NaCl. With a mole fraction of >0.1, complexes fail to dissociate even at 1.2 M NaCl. Additional information about the SPB/liposome morphology was obtained from cryo-TEM. For example, cryo-TEM data confirm liposome integrity upon complexation, a behavior that contrasts with the liposome destruction as found with adsorption to many other types of surfaces.

The effect of intercalants on the host liposome.

When phospholipids are vigorously dispersed in water, liposomes are formed. In the present study, we have explored the effect of intercalant concentration on various properties of unilamellar liposomes. Liposomes were sonically intercalated with vitamin E acetate (VitEAc) and hypericin (Hy) until no difference in light transmission was observed, which reflects the formation of liposomes of minimal diameter. Our studies indicate that the intercalant structure and concentration have an influence on the liposome diameter, which could be directly measured by cryogenic transmittance electronic microscopy. Thus, intercalated VitEAc substantially decreased the diameter of unilamellar dimyristoylphosphatidylcholine liposomes, whereas Hy did not. In addition, we followed peak intensities in the absorbance and fluorescence spectra of Hy as a function of intercalant concentration in the liposomal solution. Initially, the fluorescence intensity increased linearly with concentration; however, the curve then arched asymptotically, followed by a decrease in fluorescence at yet higher concentrations. Because the Hy monomer is the only species that emits fluorescence, we believe that the decrease of fluorescence intensity is the result of Hy aggregation.

Liposome fusion rates depend upon the conformation of polycation catalysts.

Cryo-TEM and NaCl-leakage experiments demonstrated that the cationic polymer polylysine induces fusion of anionic liposomes but that the cationic polymer poly(N-ethyl-4-vinylpyridinium bromide) (PEVP) does not, although both polymers bind strongly to the liposomes. The difference was traced to the thickness of the coatings at constant charge coverage. Polylysine is believed to form planar ß-sheets that are sufficiently thin to allow membrane fusion. In contrast, looping and disorganization among adsorbed PEVP molecules physically prevent fusion. A similar effect is likely to be applicable to important polycation-induced fusion of cell membranes.

Complexation of anionic liposomes with spherical polycationic brushes.

Spherical polycationic brushes, consisting of polystyrene particles with linear cationic macromolecules grafted onto their surfaces, were electrostatically complexed with small unilamellar anionic liposomes. Complexation was monitored using a multimethod approach that included laser electrophoresis, dynamic light scattering, fluorescence, cryogenic transmission electron microscopy, and conductivity. Liposomes adsorb onto the outer edges of the brushes rather than penetrate into their dense polycationic layer. The integrity of the liposomes remains unaltered when the liposomes reside on the polycationic brushes. The resulting complexes (roughly 40 liposomes per brush) do not dissociate into their components upon exposure to physiological solutions. The system is potentially useful in that liposomes are gathered into well-defined clusters with a high encapsulating potential. Multicomponent constructs can be easily prepared if polycationic brushes are allowed to bind to a mixture of liposomes that encapsulate different guests. This work provides an example of "systems chemistry" whereby as many as eight components, each with its own particular location and function (i.e., polystyrene core, polycationic graft, egg lecithin, cardiolipin, two fluorescent dyes, water, and buffer), collectively self-assemble.

Liposomes remain intact when complexed with polycationic brushes.

Anionic liposomes adsorb onto the surface of spherical polymer particles bearing grafted linear cationic macromolecules. The size, shape, and encapsulation ability of the liposomes remain unchanged upon adsorption, thus providing immobilized self-organizing containers that have potential applications in the biomedical field.

Cryo-Imaging of Hydrogels Supermolecular Structure.

Gelatin, derived from collagen, has both the mechanical properties required for tissue growth, as well the functional domains required for cell binding. In its natural state, gelatin derives its properties from a network of structured, intertwined, triple helical chains, which is stabilized by hydrogen bonds at temperatures below 37 °C. The mechanical properties of such a structure can be further controlled by additional enzymatic cross-linking. But, in contrast to simple polymer systems, the response to an imposed deformation is here determined by two competing factors: the establishment of the cross-linked mesh vs. the self-assembly of the fibrils into larger and stronger hierarchical structures. Therefore, properties deduced from the response to measurements such as rheology or swelling, are a combination of these two very different factors, hence a modeling is impossible unless more precise knowledge regarding the internal structure is available. The cryogenic-temperature scanning electron microscopy (cryo-SEM) was adopted to image the fully hydrated gelatin network in which distinct chain folding was observed at low densities, while cross-linked networks were observed at higher densities. Based on these images, a theoretical model which results in good agreement between the mesh sizes of both networks and their mechanical properties was developed.

Cubosome formulations stabilized by a dansyl-conjugated block copolymer for possible nanomedicine applications.

We present here an innovative, fluorescent, monoolein-based cubosome dispersion. Rather than embedded within the monoolein palisade, the fluorescent imaging agent, namely dansyl, was conjugated to the terminal ethylene oxide moieties of the block copolymer Pluronic F108. We discuss the physicochemical and photophysical properties of this fluorescent Pluronic and of a cubosome formulation stabilized by a mixture of dansyl-conjugated and non-conjugated Pluronic, also including an anticancer drug (quercetin). Furthermore, we performed biocompatibility tests against HeLa cells to assess internalization and cytotoxicity features of this nanoparticles aqueous dispersion. Cryo-TEM, SAXS, and DLS analysis, proved the bicontinuous cubic inner nanostructure and the morphology of this fluorescent cubosome dispersion, while photophysical measurements and biocompatibility results basically validate their potential use for theranostic nanomedicine applications.

Capacious and programmable multi-liposomal carriers.

Spherical polycationic brushes (SPBs) were synthesized by grafting polycationic chains onto 100 nm polystyrene particles. These particles were exposed to unilamellar egg-lecithin (EL) liposomes with a mean diameter of 40 nm that had been rendered anionic via the presence of 10 molar% of phosphatidylserine (PS(1-)). The liposomes also contained 30 mole% of a morpholinocyclohexanol-based lipid (MOCH) that undergoes a conformational flip when the pH is decreased from 7.0 to 5.0. Mixtures of SPBs and liposomes at pH 7 gave an electrostatically-driven complex possessing, on average, about 40 liposomes for each SPB particle. It was found that the bound liposomes rapidly release much of their contents when the pH is reduced from 7.0 to 5.0 owing mostly to a MOCH conformational change that creates defects in the bilayer membrane. The drop in pH does not, however, induce a separation of the liposomes from the SPBs. Around 50-60% of the liposome contents escape before, it is reasoned, lateral and transmembrane motion of the membrane components heals the defects and prevents further release. Remarkably, the liposomes complexed with SPB release their cargo much faster than the identical but non-complexed liposomes.

DOTAP (and other cationic lipids): chemistry, biophysics, and transfection.

Cationic lipid-mediated nucleic acid and protein delivery is becoming increasingly popular for in vitro and in vivo applications. While the chemistry of cationic lipid carriers is moving very rapidly, and more and more sophisticated molecules are being developed, it is worthwhile to look back to understand what has been achieved in the field of cationic lipids and why in some cases delivery based on cationic lipids works and in other cases it does not. For this purpose, DOTAP is one of the best candidates; it is the most widely used cationic lipid, it is relatively cheap, and it is efficient in both in vitro and in vivo applications. The vast amount of data that have accumulated on DOTAP and related molecules could provide invaluable clues to biophysical, structural, and biological mechanisms of transfection by cationic lipids. While many issues of cationic lipid transfection still remain unclear, this review will attempt to address mainly the following issues: (1) interplay of physicochemical parameters of DOTAP formulations; (2) impact of physicochemical parameters on transfection (lipofection) efficiency by cationic reagents, in vitro and in vivo; (3) structure-activity relationships of cationic lipid formulations in cell culture and in the living organism. In addition, in vivo applications of cationic lipids are reviewed, and the problems of local versus systemic administration of lipoplexes are discussed.

Active oxygen chemistry within the liposomal bilayer. Part III: Locating Vitamin E, ubiquinol and ubiquinone and their derivatives in the lipid bilayer.

We have previously shown that the location and orientation of compounds intercalated within the lipid bilayer can be qualitatively determined using an NMR chemical shift-polarity correlation. We describe herein the results of our application of this method to analogs of Vitamin E, ubiquinol and ubiquinone. The results indicate that tocopherol--and presumably the corresponding tocopheroxyl radical--reside adjacent to the interface, and can, therefore, abstract a hydrogen atom from ascorbic acid. On the other hand, the decaprenyl substituted ubiquinol and ubiquinone lie substantially deeper within the lipid membrane. Yet, contrary to the prevailing literature, their location is far from being the same. Ubiquinone-10 is situated above the long-chain fatty acid "slab". Ubiquinol-10 dwells well within the lipid slab, presumably out of "striking range" of Vitamin C. Nevertheless, ubiquinol can act as an antioxidant by reducing C- or O-centered lipid radicals or by recycling the lipid-resident tocopheroxyl radical.

Physicochemical, cytotoxic, and dermal release features of a novel cationic liposome nanocarrier.

A novel cationic liposome nanocarrier, having interesting performance in topical drug delivery, is here presented and evaluated for its features. Two penetration enhancers, namely monoolein and lauroylcholine chloride, are combined to rapidly formulate (15 min) a cationic liposome nanostructure endowed of excellent stability (>6 months) and skin penetration ability, along with low short-term cytotoxicity, as evaluated via the MTT test. Cytotoxicity tests and lipid droplet analysis give a strong indication that monoolein and lauroylcholine synergistically endanger long-term cells viability. The physicochemical features, investigated through SAXS, DLS, and cryo-TEM techniques, reveal that the nanostructure is retained after loading with diclofenac in its acid (hydrophobic) form. The drug release performances are studied using intact newborn pig skin. Analysis of the different skin strata proves that the drug mainly accumulates into the viable epidermis with almost no deposition into the derma. Indeed, the flux of the drug across the skin is exceptionally low, with only 1% release after 24 h. These results validate the use of this novel formulation for topical drug release when the delivery to the systemic circulation should be avoided.

Nanoparticles from lipid-based liquid crystals: emulsifier influence on morphology and cytotoxicity.

Here, monoolein-based nanoparticles (NPs), obtained through fragmentation of bulk liquid crystalline phases, and stabilized by two different emulsifiers, namely, Pluronic F127 (PF127) and lauroylcholine chloride (LCh), are investigated for structural features and for short-term in vitro cytotoxicity. Depending on the emulsifiers, different morphologies of the lipid NPs (cubosomes and liposomes) are obtained, as demonstrated by cryo-TEM images. Although NPs offer many advantages in medical applications and various chemicals used for their preparation are under investigation, so far there are no standardized procedures to evaluate cell biocompatibility. Two different protocols to evaluate the impact of these lipid NPs on biological systems are presented. Results show that nanoparticles stabilized by PF127 (cubosomes) display a relevant toxicity toward different cell lines, whereas those stabilized by LCh (liposomes) affect cell viability at a much lesser extent.

Delivery of antisense oligodeoxyribonucleotide lipopolyplex nanoparticles assembled by microfluidic hydrodynamic focusing.

A multi-inlet microfluidic hydrodynamic focusing (MF) system to prepare lipopolyplex (LP) containing Bcl-2 antisense deoxyoligonucleotide (ODN) was developed and evaluated. The lipopolyplex nanoparticles consist of ODN:protamine:lipids (1:0.3:12.5wt/wt ratio) and the lipids included DC-Chol:egg PC:PEG-DSPE (40:58:2mol/mol%). Using K562 human erythroleukemia cells, which contain an abundance of Bcl-2 and overexpression of transferrin receptors (TfR), and G3139 (oblimerson sodium or Genasense(TM)) as a model cell line and drug, respectively, the Bcl-2 down-regulation at the mRNA and protein levels as well as cellular uptake and apoptosis was compared between the conventional bulk mixing (BM) method and the MF method. The lipopolyplex size and surface charge were characterized by dynamic light scattering (DLS) and zeta potential (zeta) measurement, respectively, while the ODN encapsulation efficiency was determined by gel electrophoresis. Cryogenic transmission electron microscopy (Cryo-TEM) was used to determine the morphology of LPs. Our results demonstrated that MF produced LP nanoparticles had similar structures but smaller size and size distribution compared to BM LP nanoparticles. MF LP nanoparticles had higher level of Bcl-2 antisense uptake and showed more efficient down-regulation of Bcl-2 protein level than BM LP nanoparticles.