A short review on the applicability and use of cubosomes as nanocarriers.

Abstract: Cubosomes are nanostructured lipid-based particles that have gained significant attention in the field of drug delivery and nanomedicine. These unique structures consist of a three-dimensional cubic lattice formed by the self-assembly of lipid molecules. The lipids used to construct cubosomes are typically nonionic surfactants, such as monoolein, which possess both hydrophilic and hydrophobic regions, allowing them to form stable, water-dispersible nanoparticles. One of the key advantages of cubosomes is their ability to encapsulate and deliver hydrophobic as well as hydrophilic drugs. The hydrophobic regions of the lipid bilayers provide an ideal environment for incorporating lipophilic drugs, while the hydrophilic regions can encapsulate water-soluble drugs. This versatility makes cubosomes suitable for delivering a wide range of therapeutic agents, including small molecules, proteins, peptides, and nucleic acids. The unique structure of cubosomes also offers stability and controlled release benefits. The lipid bilayers provide a protective barrier, shielding the encapsulated drugs from degradation and improving their stability. Moreover, the cubic lattice arrangement enables the modulation of drug release kinetics by varying the lipid composition and surface modifications. This allows for the development of sustained or triggered drug release systems, enhancing therapeutic efficacy and reducing side effects. Furthermore, cubosomes can be easily modified with targeting ligands or surface modifications to achieve site-specific drug delivery, enhancing therapeutic selectivity and reducing off-target effects. In conclusion, cubosomes offer a versatile and promising platform for the delivery of therapeutic agents. In this manuscript, we will highlight some of these applications.

Peg-Grafted Liposomes for L-Asparaginase Encapsulation.

L-asparaginase (ASNase) is an important biological drug used to treat Acute Lymphoblastic Leukemia (ALL). It catalyzes the hydrolysis of L-asparagine (Asn) in the bloodstream and, since ALL cells cannot synthesize Asn, protein synthesis is impaired leading to apoptosis. Despite its therapeutic importance, ASNase treatment is associated to side effects, mainly hypersensitivity and immunogenicity. Furthermore, degradation by plasma proteases and immunogenicity shortens the enzyme half-life. Encapsulation of ASNase in liposomes, nanostructures formed by the self-aggregation of phospholipids, is an attractive alternative to protect the enzyme from plasma proteases and enhance pharmacokinetics profile. In addition, PEGylation might prolong the in vivo circulation of liposomes owing to the spherical shielding conferred by the polyethylene (PEG) corona around the nanostructures. In this paper, ASNase was encapsulated in liposomal formulations composed by 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) containing or not different concentrations of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [methoxy (polyethylene glycol)-2000] (DSPE-PEG). Nanostructures of approximately 142-202 nm of diameter and polydispersity index (PDI) of 0.069 to 0.190 were obtained and the vesicular shape confirmed by Transmission Electron Microscopy (TEM and cryo-TEM). The encapsulation efficiency (%EE) varied from 10% to 16%. All formulations presented activity in contact with ASNase substrate, indicating the liposomes permeability to Asn and/or enzyme adsorption at the nanostructures' surface; the highest activity was observed for DMPC/DSPE-PEG 10%. Finally, we investigated the activity against the Molt 4 leukemic cell line and found a lower IC50 for the DMPC/DSPE-PEG 10% formulation in comparison to the free enzyme, indicating our system could provide in vivo activity while protecting the enzyme from immune system recognition and proteases degradation.

Thymus vulgaris essential oil + tobramycin within nanostructured archaeolipid carriers: A new approach against Pseudomonas aeruginosa biofilms.

BACKGROUND: Pseudomonas aeruginosa biofilms in the respiratory tract of patients with an excessive inflammatory context are difficult to eradicate. New medicines that simultaneously target biofilms and inflammation should be developed. HYPOTHESIS: Co-delivery of Thymus vulgaris essential oil (EOT) and tobramycin (TB) by nanostructured archaeolipids carriers (NAC) could support nebulization as well as improve EOT and TB antioxidant, anti-inflammatory and antibiofilm activity. METHODS: NAC(EOT+TB) were prepared by loading EOT and TB in NAC having a compritol and miglyol core, covered with a shell of archaeolipids, extracted from the hyperhalophylic archaebacteria Halorubrum tebenquichense, and Tween 80. NAC(EOT+TB) were structurally characterized, including DSC thermograms, Raman spectra, TB release profile, EOT volatilization and in vitro antioxidant activity. In addition, stability upon nebulization, autoclaving and storage were assessed. The antibiofilm activity on P. aeruginosa PAO1 established biofilm of NAC(EOT+TB) and the cytotoxicity on human lung epithelial cells and macrophage were determined, as well as intracellular reactive oxygen species (ROS) production and cytokines release on LPS stimulated cells. RESULTS: NAC(EOT+TB) showed a size of 197 ± 16 nm with PdI of 0.3 ± 0.1 and ζ Potential of -38 ± 3 mV. Structural characterization suggested that EOT was trapped in the compritol-miglyol core and TB was distributed between the surface of nanoparticles and free in solution. NAC(EOT+TB) displayed a dual release profile of TB, a delayed release of EOT and improved EOTs in vitro antioxidant activity. While NAC(EOT+TB) preserved its structural features after nebulization, autoclaving and 18 months of storage, carriers without archaeolipids gelled at room temperature and showed a significant increase of size after the same storage time. Below cytotoxic concentration, NAC(EOT+TB) decreased bacteria viability and enhanced the disruption of established PAO1 biofilms compared to free TB and EOT. Also, the strong entrapment of EOT in NAC(EOT+TB) delayed its volatilization, decreased intracellular ROS production and maintained its anti-inflammatory activity in LPS stimulated cells. CONCLUSION: Combination of EOT + TB within NAC(EOT+TB) result in a stable and nebulizable formulation that enhanced the antioxidant and anti-biofilm activity of free ingredients, improved their ability to decrease intracellular ROS and provided anti-inflammatory activity, at non-cytotoxic concentrations on eukaryotic cells.

Structural, thermodynamic and functional studies of human 71 kDa heat shock cognate protein (HSPA8/hHsc70).

Human 71 kDa heat shock cognate protein (HSPA8, also known as Hsc70, Hsp70-8, Hsc71, Hsp71 or Hsp73) is a constitutively expressed chaperone that is critical for cell proteostasis. In the cytosol, HSPA8 plays a pivotal role in folding and refolding, facilitates protein trafficking across membranes and targets proteins for degradation, among other functions. Here, we report an in solution study of recombinant HSPA8 (rHSPA8) using a variety of biophysical and biochemical approaches. rHSPA8 shares several structural and functional similarities with others human Hsp70s. It has two domains with different stabilities and interacts with adenosine nucleotides with dissociation constants in the low micromolar range, which were higher in the presence of Mg2+. rHSPA8 showed lower ATPase activity than its homolog HSPA5/hGrp78/hBiP, but it was 4-fold greater than that of recombinant HSPA1A/hHsp70-1A, with which it is 86% identical. Small angle X-ray scattering indicated that rHSPA8 behaved as an elongated monomeric protein in solution with dimensions similar to those observed for HSPA1A. In addition, rHSPA8 showed structural flexibility between its compacted and extended conformations. The data also indicated that HSPA8 has capacity in preventing the aggregation of model client proteins. The present study expands the understanding of the structure and activity of this chaperone and aligns with the idea that human homologous Hsp70s have divergent functions.

X-Ray Characterization of Pharmaceutical and Cosmetic Lipidic Nanoparticles for Cutaneous Application.

Starting from the second half of the 1900s, the advent of nanotechnology in medicine has provoked a profound revolution in this area; at present, nanomedicine delivered a remarkably large set of research and clinically useful tools as diagnostic devices, contrast agents, analytical tools, physical therapy applications, and drugdelivery vehicles. Concerning nanoformulations for drug delivery, they are constituted by nanoparticles with dimensions lower than 1 µm, usually characterized by improved pharmacokinetics, taking advantage of specific targeting, and reduced side effects. The contributors to the present chapter are reviewing a range of papers related to the structural characterization of nanoformulations by X-ray diffraction techniques. The whole of the considered papers underlines the essential role that biophysical techniques have acquired as an essential prerequisite to understanding stability, bioavailability, and lipid, biopolymer, and drug organization in nanoformulations.

Polymeric micelles of pluronic F127 reduce hemolytic potential of amphiphilic drugs.

One of the main toxicities associated to intravenous administration of amphiphilic drugs is pronounced hemolytic activity. To overcome this limitation, we investigated the anti-hemolytic properties of polymeric micelles of Pluronics, triblock copolymers of poly(ethylene oxide) and poly(propylene oxide). We studied the encapsulation of the amphiphilic compound miltefosine (HePC) into polymeric micelles of Pluronics F108, F68, F127, L44, and L64. In vitro hemolysis indicated that, among the five copolymers studied, only F127 completely inhibited hemolytic effect of HePC at 50 µg/mL, this effect was also observed for other two amphiphilic molecules (cetyltrimethylammonium bromide and cethylpyridinium chloride). To better understand this interaction, we analyzed the HC50 (concentration causing 50% of hemolysis) for HePC free and loaded into F127 micelles. Copolymer concentration influenced the hemolytic profile of encapsulated HePC; for F127 the HC50 increased relative to free HePC (40 µg/mL) up to 184, 441, 736 and 964 µg/mL, for 1, 3, 6 and 9% F127, respectively. Interestingly, a linear relationship was found between HC50-HePC and F127 concentration. At 3% of F127, it is possible to load up to 300 µg/mL of HePC with no hemolytic effect. By achieving this level of hemolysis protection, a promising application is on the view, bringing the parenteral use of HePC and other amphiphilic drugs. Additionally, small-angle X-ray scattering (SAXS) was used to asses structural information on the interactions between HePC and F127 micelles.

Nanostructures for protein drug delivery.

Use of nanoscale devices as carriers for drugs and imaging agents has been extensively investigated and successful examples can already be found in therapy. In parallel, recombinant DNA technology together with molecular biology has opened up numerous possibilities for the large-scale production of many proteins of pharmaceutical interest, reflecting in the exponentially growing number of drugs of biotechnological origin. When we consider protein drugs, however, there are specific criteria to take into account to select adequate nanostructured systems as drug carriers. In this review, we highlight the main features, advantages, drawbacks and recent developments of nanostructures for protein encapsulation, such as nanoemulsions, liposomes, polymersomes, single-protein nanocapsules and hydrogel nanoparticles. We also discuss the importance of nanoparticle stabilization, as well as future opportunities and challenges in nanostructures for protein drug delivery.

How does growth hormone releasing hexapeptide self-assemble in nanotubes?

Growth hormone releasing peptide, GHRP-6, a hexapeptide (His-(D-Trp)-Ala-Trp-(D-Phe)-Lys-NH2, MW = 872.44 Da) that belongs to a class of synthetic growth hormone secretagogues, can stimulate growth hormone secretion from somatotrophs in several species including humans. In the present study, we demonstrate that GHRP-6 dispersed in aqueous solution, at pH 7.0, room temperature of 22 °C, is able to form long nanotubes, which is evidenced by combining small angle X-ray scattering (SAXS), transmission electron microscopy and molecular dynamics simulation results. Such nanotubes possess inner and outer cross-sections equal to 6.7(2) nm and 13.4(5) nm, respectively. The mechanism of peptide self-assembly was determined by molecular dynamics simulations revealing that the peptides self-assemble like amphiphilic molecules in aqueous solution in a partially interdigitated structure. In this case, the position of the positively charged amino terminus is located at the peptide-water interface, whereas the neutral NH2-capped carboxy terminus remains buried at the hydrophobic core. In contrast, the long side chain of Lys-6 stretches out of the hydrophobic core positioning its positive charge near the cylinder surface. The peptide configuration in the nanotube wall comes from the interplay between the hydrophobic interactions of the aromatic side chains of GHRP-6 and the electrostatic repulsion of its cationic charges. On increasing the peptide concentration, the long nanotubes self-arrange in solution displaying a bi-dimensional hexagonal-like packing in the SAXS curves, with a center-to-center distance of ∼15 nm. Further, we also show that the nanostructure formed in solution is quite stable and is preserved following transfer to a solid support.

How does the ethoxylated grafting of polyelectrolytes affect the self-assembly of polyanion-cationic surfactant complex salts?

A cationic surfactant and different anionic copolymers randomly grafted with side chains of ethylene oxide were used to prepare stoichiometric complex salts. Variations in the length or proportion of side chains were shown to be responsible for affecting the surfactant phase behavior in water, resulting in the observation of a number of structures characterized by small angle X-ray scattering measurements, including a hierarchical micellar system and different liquid-crystalline phases. Additionally, although aqueous mixtures of stoichiometric complex salts usually phase separate, the presence of a sufficiently high weight fraction of ethylene oxide side chains can enhance the solubility of the complex salt aggregates in water over a wide range of concentration. Moreover, a dispersion of an isotropic concentrated solution of complex salts is formed at higher temperatures in a reversible process. In summary, this study proves the importance of the polyion structure for tuning the properties of systems of complex salts.

Hybrid composites of xanthan and magnetic nanoparticles for cellular uptake.

We describe a fast and simple method to prepare composite films of magnetite nanoparticles and xanthan networks. The particles are distributed close to hybrid film surface, generating a coercivity of 27 ± 2 Oe at 300 K. The proliferation of fibroblast cells on the hybrid composites was successful, particularly when an external magnetic field was applied.

LPS-protein aggregation influences protein partitioning in aqueous two-phase micellar systems.

Lipopolysaccharide endotoxins (LPS) are the most common pyrogenic substances in recombinant peptides and proteins purified from Gram-negative bacteria, such as Escherichia coli. In this respect, aqueous two-phase micellar systems (ATPMS) have already proven to be a good strategy to purify recombinant proteins of pharmaceutical interest and remove high LPS concentrations. In this paper, we review our recent experimental work in protein partitioning in Triton X-114 ATPMS altogether with some new results and show that LPS-protein aggregation can influence both protein and LPS partitioning. Green fluorescent protein (GFPuv) was employed as a model protein. The ATPMS technology proved to be effective for high loads of LPS removal into the micelle-rich phase (%REM(LPS) > 98 %) while GFPuv partitioned preferentially to the micelle-poor phase (K GFP(uv) < 1.00) due to the excluded-volume interactions. However, theoretically predicted protein partition coefficient values were compared with experimentally obtained ones, and good agreement was found only in the absence of LPS. Dynamic light scattering measurements showed that protein-LPS interactions were taking place and influenced the partitioning process. We believe that this phenomenon should be considered in LPS removal employing any kind of aqueous two-phase system. Nonetheless, ATPMS can still be considered as an efficient strategy for high loads of LPS removal, but being aware that the excluded-volume partitioning theory available might overestimate partition coefficient values due to the presence of protein-LPS aggregation.

Conformational stability of peanut agglutinin using small angle X-ray scattering.

In this work, quaternary conformational studies of peanut agglutinin (PNA) have been carried out using small-angle X-ray scattering (SAXS). PNA was submitted to three different conditions: pH variation (2.5, 4.0, 7.4 and 9.0), guanidine hydrochloride presence (0.5-2M) at each pH value, and temperature ranging from 25 to 60°C. All experiments were performed in the absence and presence of T-antigen to evaluate its influence on the lectin stability. At room temperature and pH 4.0, 7.4 and 9.0, the SAXS curves are consistent with the PNA scattering in its crystallographic native homotetrameric structure, with monomers in a jelly roll fold, associated by non-covalent bonds resulting in an open structure. At pH 2.5, the results indicate that PNA tends to dissociate into smaller sub-units, as dimers and monomers, followed by a self-assembling into larger aggregates. Furthermore, the conformational stability under thermal denaturation follows the pH sequence 7.4>9.0>4.0>2.5. Such results are consistent with the conformational behavior found upon GndHCl influence. The presence of T-antigen does not affect the protein quaternary structure in all studied systems within the SAXS resolution.

Unraveling the Na,K-ATPase alpha(4) subunit assembling induced by large amounts of C(12)E(8) by means of small-angle X-ray scattering.

In the current work, we studied the effect of the nonionic detergent dodecyloctaethyleneglycol, C(12)E(8), on the structure and oligomeric form of the Na,K-ATPase membrane enzyme (sodium-potassium pump) in aqueous suspension, by means of small-angle X-ray scattering (SAXS). Samples composed of 2 mg/mL of Na,K-ATPase, extracted from rabbit kidney medulla, in the presence of a small amount of C(12)E(8) (0.005 mg/mL) and in larger concentrations ranging from 2.7 to 27 mg/mL did not present catalytic activity. Under this condition, an oligomerization of the alpha subunits is expected. SAXS data were analyzed by means of a global fitting procedure supposing that the scattering is due to two independent contributions: one coming from the enzyme and the other one from C(12)E(8) micelles. In the small detergent content (0.005 mg/mL), the SAXS results evidenced that Na,K-ATPase is associated into aggregates larger than (alphabeta)(2) form. When 2.7 mg/mL of C(12)E(8) is added, the data analysis revealed the presence of alpha(4) aggregates in the solution and some free micelles. Increasing the detergent amount up to 27 mg/mL does not disturb the alpha(4) aggregate: just more micelles of the same size and shape are proportionally formed in solution. We believe that our results shed light on a better understanding of how nonionic detergents induce subunit dissociation and reassembling to minimize the exposure of hydrophobic residues to the aqueous solvent.