Towards analyzing the potential of exosomes to deliver microRNA therapeutics.

Exosome selectivity mechanisms underlying exosome-target cell interactions and the specific traits affecting their capability to communicate still remain unclear. Moreover, the capacity of exosomes to efficiently deliver their molecular cargos intracellularly needs precise investigation towards establishing functional exosome-based delivery platforms exploitable in the clinical practice. The current study focuses on: (a) exosome production from normal MRC-5 and Vero cells growing in culture, (b) physicochemical characterization by dynamic light scattering (DLS) and cryo-transmission electron microscopy; (c) cellular uptake studies of rhodamine-labeled exosomes in normal and cancer cells, providing to exosomes either "autologous" or "heterologous" cellular delivery environments; and (d) loading exogenous Alexa Fluor 488-labeled siRNA into exosomes for the assessment of their delivering capacity by immunofluorescence in a panel of recipient cells. The data obtained thus far indicate that MRC-5 and Vero exosomes, indeed exhibit an interesting delivering profile, as promising "bio-shuttles," being pharmacologically exploitable in the context of theranostic applications.

Influence of the freeze-drying process on the physicochemical and biological properties of pre-heated amphotericin B micellar systems.

The moderate heat treatment of amphotericin B (AmB) in its micellar form (M-AmB) results in superaggregates (H-AmB) that present a substantially lower toxicity and similar activity. The aim of this work was to evaluate the H-AmB behavior after a freeze-drying process. H-AmB and M-AmB micelles were evaluated before and after freeze-drying concerning their physicochemical and biological properties by spectrophotometry and activity/toxicity assay, respectively. Four concentrations of M-AmB and H-AmB were studied aiming to correlate their aggregation state and the respective biological behavior: 50 mg L(-1), 5 mg L(-1), 0.5 mg L(-1), and 0.05 mg L(-1). Then, potassium leakage and hemoglobin leakage from red blood cells were used to evaluate the acute and chronic toxicity, respectively. The efficacy of M-AmB and H-AmB formulations was assessed by potassium leakage from Candida albicans and by the broth microdilution method. After heating, in addition to an evident turbidity, a slight blueshift from 327 to 323 nm was also observed at the concentrations of 50 and 5 mg L(-1) for H-AmB. Additionally, an increase in the absorbance at 323 nm at the concentration of 0.5 mg L(-1) was detected. Concerning the toxicity, H-AmB caused significantly lower hemoglobin leakage than M-AmB. These results were observed for H-AmB before and after freeze-drying. However, there was no difference between H-AmB and M-AmB concerning their activity. Accordingly, the freeze-drying cycle did not show any influence on the behavior of heated formulations, highlighting the suitability of such a method to produce a new AmB product with a long shelf life and with both greater efficiency and less toxicity.

Are phytosomes a superior nanodelivery system for the antioxidant rutin?

Rutin, a strong antioxidant, has been implicated in the prevention of liver inflammation. However, low solubility and permeability through the gut wall limit development of rutin as a therapeutic agent for oral administration. Phytosomes are described as lipid nanocarriers with a complexation between the phospholipid headgroups and entrapped phytochemicals. The aim of this research was to compare the structure of rutin liposomes to rutin phytosomes. FT-IR, DSC and NMR were employed to investigate the presence of any molecular interactions between the formulation components. The FT-IR spectra showed that a new -OH bond had formed in the rutin phytosomes, suggesting the formation of a molecular complex. 31P NMR experiments revealed that the DPPC molecule is altered when formulated as liposomes but that these changes were greater for samples from the phytosome formulation. DSC data revealed that when rutin was added to DPPC there was a significant shift in the transition temperature of DPPC. Further, the shift was greater in the THF solvent used to produce phytosomes compared to CHCl3 used to produce liposomes. 1H NMR spectra of the phytosome samples indicated three additional peaks that were greater than in the liposome formulation. ROESY NMR spectra provided evidence supporting the interaction between rutin and DPPC in both liposomes and phytosomes. The apparent differences in molecular interaction between liposomes and phytosomes did not however impact rutin release in biorelevant media or during in vitro small intestinal lipolysis.

Influence of drug load and physical form of cinnarizine in new SNEDDS dosing regimens: in vivo and in vitro evaluations.

The aim of this work was to evaluate the influence of drug load and physical form of cinnarizine (CIN) in self-nanoemulsifying drug delivery systems (SNEDDS) on absorption in rats. Further, the predictivity of the dynamic in vitro lipolysis model was evaluated. The following dosing regimens were assessed: (1) CIN dissolved in SNEDDS at 80% of equilibrium solubility (Seq) (SNEDDS 80%); (2) supersaturated SNEDDS with CIN dissolved at 200% Seq (super-SNEDDS solution); (3) SNEDDS suspension with CIN added at 200% Seq (CIN partially dissolved and partially suspended) (super-SNEDDS suspension); (4) drug-free SNEDDS co-dosed with aqueous CIN suspension (Chasing principle), and (5) CIN aqueous suspension. The CIN dose was kept constant for all dosing regimens. Therefore, the super-SNEDDS solution and super-SNEDDS suspension contained 2.5-fold less SNEDDS pre-concentrate than SNEDDS 80% and the Chasing principle. In vivo, a higher AUC after dosing CIN in SNEDDS 80% and the Chasing principle was obtained when compared to the super-SNEDDS solution, super-SNEDDS suspension, and aqueous suspension. In vitro, a higher extent of CIN in the aqueous phase was observed for all SNEDDS-containing dosing regimens, compared to the aqueous suspension. Since the drug level in the aqueous phase is traditionally considered as the fraction available for absorption, a lack of in vitro-in vivo relation was observed. This study revealed that the physical form of CIN in the current SNEDDS does not affect CIN absorption and solubilization, whereas the drug load, or amount of co-dosed lipid, significantly influenced CIN bioavailability.

Monoacyl phosphatidylcholine inhibits the formation of lipid multilamellar structures during in vitro lipolysis of self-emulsifying drug delivery systems.

The colloidal structures formed during lipolysis of self-emulsifying drug delivery systems (SEDDS) might affect the solubilisation and possibly the absorption of drugs. The aim of the current study is to elucidate the structures formed during the in vitro lipolysis of four SEDDS containing medium-chain glycerides and caprylocaproyl polyoxyl-8 glycerides (Labrasol), with or without monoacyl phosphatidylcholine (MAPC). In situ synchrotron small-angle X-ray scattering (SAXS) was combined with ex situ cryogenic transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS) to elucidate the generated structures. The SAXS scattering curves obtained during the lipolysis of MAPC-free SEDDS containing 43-60% w/w Labrasol displayed a lamellar phase peak at q=2.13nm-1 that increased with Labrasol concentration, suggesting the presence of multilamellar structures (MLS) with a d-spacing of 2.95nm. However, SEDDS containing 20-30% w/w MAPC did not form MLS during the lipolysis. The cryo-TEM and DLS studies showed that MAPC-free SEDDS formed coarse emulsions while MAPC-containing SEDDS formed nanoemulsions during the dispersion in digestion medium. From the first minute and during the entire lipolysis process, SEDDS both with and without MAPC generated uni-, bi-, and oligo-lamellar vesicles. The lipolysis kinetics in the first minutes of the four SEDDS correlated with an increased intensity of the SAXS curves and the rapid transformation from lipid droplets to vesicles observed by cryo-TEM. In conclusion, the study elucidates the structures formed during in vitro lipolysis of SEDDS and the inhibitory effect of MAPC on the formation of MLS.

In vitro and in vivo performance of monoacyl phospholipid-based self-emulsifying drug delivery systems.

This study investigates the effect of monoacyl phospholipid incorporation on the in vitro and in vivo performance of self-emulsifying drug delivery systems (SEDDS). Monoacyl phosphatidylcholine (Lipoid S LPC 80 (LPC)) was incorporated into four different fenofibrate (FF)-loaded long-chain SEDDS to investigate the impact of LPC on the emulsion droplet size, extent of digestion, colloidal structure evolution and drug precipitation during in vitro lipolysis simulating human conditions and drug bioavailability in a rat model. The four investigated SEDDS containing long-chain glycerides, polyoxyl 35 castor oil or polyoxyl 8 caprylocaproyl glycerides with or without LPC. In situ synchrotron small/wide-angle X-ray scattering (SAXS/WAXS) was used to simultaneously real-time monitor the kinetics of lamellar phase structure development and FF crystalline precipitation. Adding LPC increased the particle size and polydispersity of the dispersed SEDDS. The two LPC-free SEDDS generated lamellar phase structures (Lα) with d-spacing=4.76nm during digestion. Incorporating LPC into these systems inhibited the formation of lamellar phase structures. The amount of precipitated crystalline FF from the four SEDDS was similar during the first 15min but differed during the last 45min of in vitro digestion. The kinetics of colloidal structure development and FF precipitation was related to the digestion kinetics. The in vivo bioavailability data showed no significant differences between the four SEDDS, which correlates with the in vitro FF precipitation during the first 15min of lipolysis. Thus, the presence of LPC, different emulsion droplet sizes and concentration of lamellar phase structures observed in vitro did not correlate with the FF absorption in rats. The study suggests that later time points of the in vitro lipolysis overestimated FF precipitation in rats because of the high enzyme activity, the lack of gastric and absorption steps, and the low bile salts and phospholipid concentrations of the in vitro model.

How can micelle systems be rebuilt by a heating process?

The aim of this work was to evaluate how an aqueous micellar system containing Amphotericin B (AmB) and sodium deoxycholate (DOC) can be rebuilt after heating treatment. Also, a review of the literature on the physicochemical and biological properties of this new system was conducted. Heated (AmB-DOC-H) and unheated (AmB-DOC) micelles were then diluted at four different concentrations (50 mg · L(-1), 5 mg · L(-1), 0.5 mg · L(-1), and 0.05 mg · L(-1)) to perform physicochemical studies and a pharmacotoxicity assay, in which two cell models were used for the in vitro experiments: red blood cells (RBC) from human donors and Candida parapsilosis (Cp). While potassium (K(+)) and hemoglobin leakage from RBC were the parameters used to evaluate acute and chronic toxicity, respectively, the efficacy of AmB-DOC and AmB-DOC-H were assessed by K(+) leakage and cell survival rate from Cp. The spectral study revealed a slight change in the AmB-DOC aggregate peak from 327 nm to 323 nm, which is the peak for AmB-DOC-H. Although AmB-DOC and AmB-DOC-H exhibited different behavior for hemoglobin leakage, AmB-DOC produced higher leakage than AmB-DOC-H at high concentrations (from 5 mg · L(-1)). For K(+) leakage, both AmB-DOC and AmB-DOC-H showed a similar profile for both cell models, RBC and Cp (P < 0.05). AmB-DOC-H and AmB-DOC also revealed a similar profile of activity against Cp with an equivalent survival rate. In short, AmB-DOC-H showed much less toxicity than AmB-DOC, but remained as active as AmB-DOC against fungal cells. The results highlight the importance of this new procedure as a simple, inexpensive, and safe way to produce a new kind of micelle system for the treatment of systemic fungal infections.