Focused ion beam-scanning electron microscopy provides novel insights of drug delivery phenomena.

Scanning electron microscopy (SEM) has long been a standard tool for morphological analyses, providing sub micrometer resolution of pharmaceutical formulations. However, analysis of internal morphologies of such formulations can often be biased due to the introduction of artifacts that originate from sample preparation. A recent advancement in SEM, is the focused ion beam scanning electron microscopy (FIB-SEM). This technique uses a focused ion beam (FIB) to remove material with nanometer precision, to provide virtually sample-independent access to sub-surface structures. The FIB can be combined with SEM imaging capabilities within the same instrumentation. As a powerful analytical tool, electron microscopy and FIB-milling are performed sequentially to produce high-resolution 3D models of structural peculiarities of diverse drug delivery systems or their behavior in a biological environment, i.e. intracellular or -tissue distribution. This review paper briefly describes the technical background of the method, outlines a wide array of potential uses within the drug delivery field, and focuses on intracellular transport where high-resolution images are an essential tool for mechanistical insights.

Itraconazole Nanosuspensions via Dual Centrifugation Media Milling: Impact of Formulation and Process Parameters on Particle Size and Solid-State Conversion as Well as Storage Stability.

Nanocrystal suspensions proved to be a potent enabling principle for biopharmaceutics classification system class II drugs with dissolution limited bioavailability. In the example of itraconazole (ITZ) as a model drug combined with electrosteric stabilization using hydroxypropyl cellulose (HPC-SL), sodium dodecyl sulfate (SDS) and polysorbate 80 (PS80), the impacts of formulation and process parameters of a dual centrifugal mill on material attributes such as particle size, zeta potential, particle morphology, storage stability and especially solid-state characteristics were evaluated. A minimal concentration of 0.9% (w/w) HPC-SL, 0.14% (w/w) SDS and 0.07% (w/w) PS80 was necessary for sufficient nanoparticle stabilization. Despite the minor effect of PS80, its presence was beneficial for electrosteric stabilization. Choosing lower stabilizer concentrations resulted in a pronounced increase in particle size due to agglomeration, which was confirmed by SEM imaging and a decrease in zeta potential in combination with an amorphization of the particles. Milling temperature had no significant impact on the particle size, whereas milling speed and the size of the milling beads used were found to have a strong impact on the critical material attributes such as particle size and polydispersity index. The smallest particle sizes could be obtained by using the smallest milling bead size. However, the smallest obtainable particle size could only be achieved by using two-fold stabilizer concentrations, as smaller particles exhibit a larger specific surface area.

Improving Transungual Permeation Study Design by Increased Bovine Hoof Membrane Thickness and Subsequent Infection.

Ungual formulations are regularly tested using human nails or animal surrogates in Franz diffusion cell experiments. Membranes sometimes less than 100 µm thick are used, disregarding the higher physiological thickness of human nails and possible fungal infection. In this study, bovine hoof membranes, healthy or infected with Trichophyton rubrum, underwent different imaging techniques highlighting that continuous pores traversed the entire membrane and infection resulted in fungal growth, both superficial, as well as in the membrane's matrix. These membrane characteristics resulted in substantial differences in the permeation of the antifungal model substance bifonazole, depending on the dosage forms. Increasing the thickness of healthy membranes from 100 µm to 400 µm disproportionally reduced the permeated amount of bifonazole from the liquid and semisolid forms and allowed for a more pronounced assessment of the effects by excipients, such as urea as the permeation enhancer. Similarly, an infection of 400-µm membranes drastically increased the permeated amount. Therefore, the thickness and infection statuses of the membranes in the permeation experiments were essential for a differential readout, and standardized formulation-dependent experimental setups would be highly beneficial.

In vitro and in vivo assessment of hydroxypropyl cellulose as functional additive for enabling formulations containing itraconazole.

Using polymers as additives to formulate ternary amorphous solid dispersions (ASDs) has successfully been established to increase the bioavailability of poorly soluble drugs, when one polymer is not able to provide both, stabilizing the drug in the matrix and the supersaturated solution. Therefore, we investigated the influence of low-viscosity hydroxypropyl cellulose (HPC) polymers as an additive in HPMC based ternary ASD formulations made by hot-melt extrusion (HME) on the bioavailability of itraconazole (ITZ). The partitioning potential of the different HPC grades was screened in biphasic supersaturation assays. Solid-state analytics were performed using differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD). The addition of HPCs, especially HPC-UL, resulted in a superior partitioned amount of ITZ in biphasic supersaturation assays. Moreover, the approach in using HPCs as an additive in HPMC based ASDs led to an increase in partitioned ITZ compared to Sporanox® in biorelevant biphasic dissolution studies. The results from the biphasic dissolution experiments correlated well with the in vivo studies, which revealed the highest oral bioavailability for the ternary ASD comprising HPC-UL and HPMC.

Effects of carrier agents on powder properties, stability of carotenoids, and encapsulation efficiency of goldenberry (Physalis peruviana L.) powder produced by co-current spray drying.

Maltodextrin, modified starch, inulin, alginate, gum arabic, and combinations thereof were used as carrier agents for spray drying of carotenoid-rich goldenberry (Physalis peruviana L.) juice and compared to cellobiose as an alternative carrier. Powders were analyzed with respect to particle size and morphology, yield, moisture content, cold water solubility, suspension stability, hygroscopicity, carotenoid encapsulation efficiency, and carotenoid retention during storage. A high initial carotenoid concentration after spray drying, a high encapsulation efficiency of 77.2%, and a slow carotenoid degradation kinetics favored the high carotenoid content of the cellobiose powder at the end of the storage. Cellobiose might protect the carotenoids from degradation processes by light exposure, high temperature, and oxygen due to a tighter particle crust and larger particle sizes. Therefore, cellobiose may be considered a potential carrier agent for the encapsulation of carotenoid-rich fruit juices.

Cell membrane fusing liposomes for cytoplasmic delivery in brain endothelial cells.

Direct cytoplasmic delivery is essential for susceptible molecules as proteins and some nucleic acids to improve their therapeutic efficacy in cells. Using liposomes for their delivery proved challenging due to known uptake by endocytosis followed by partial or complete lysosomal breakdown. Thus, "fusogenic" liposomes (FL) composed of the neutral lipid dioleoylphosphatidylethanolamine (DOPE) combined with the cationic lipid 1, 2-dioleoyl-3-trimethylammoniumpropane (DOTAP) were tested in different ratios for their cell membrane fusion ability and their cytoplasmic delivery was compared to "pH-sensitive" liposomes in murine brain endothelial cells (bEnd.3). They were loaded with cargos of different molecular sizes (calcein/ enhanced green fluorescent-protein (EGFP)/ EGFP coding plasmid) and their intracellular delivery was quantitatively and qualitatively analyzed. FL composed of equimolar ratios of DOPE and DOTAP showed the most efficient cytoplasmic delivery of all cargos by fusing with the cell membranes within the first 15 min of addition. Their EGFP plasmid delivery to cells was quantified to be 58.2 ±â€¯9.5 % of the total EGFP load and calcein delivery was measured in buffer to be 64.1 ±â€¯4.0 % of the total calcein load, and reduced in blood to 26.1 ±â€¯0.6 %. Thus our tested FL allowed a fast and abundant cytoplasmic delivery of cargos independent of their molecular sizes while avoiding endocytosis, although they also underwent fast fusion with erythrocytes. Seemingly, these carriers could be used as a powerful delivery tool for in-vitro purposes.

Nanosphere-shaped ammonio methacrylate copolymers: converting a pharmaceutical inactive ingredient to efficient therapeutics for experimental colitis.

Inflammatory bowel disease (IBD) refers to progressive inflammatory disorders that impair the gastrointestinal tract's structure and function. Given their selective accumulation in inflamed tissues, nanoparticles are promising drug delivery systems for IBD treatment. The hypothesis here was that drug-free nanoscaled cationic ammonio methacrylate copolymers (AMCNP) may have a beneficial therapeutic effect in murine TNBS-induced colitis. Type A and B AMCNP (RLNP and RSNP, respectively) were prepared and characterized in vitro, and were rectally administered in two concentrations (5 and 25 mg ml-1) for the treatment of two grades of murine experimental colitis. The impact of the nanoparticles upon the inflammatory markers, circulating LPS, intestinal permeability and colonic leukocyte populations was examined. Both RLNP and RSNP led to a significant mitigation of mild to moderate experimental colitis, as evident from the substantial reduction of myeloperoxidase (MPO) and alkaline phosphatase (AP) activities (more than two-fold, P < 0.05) and various pro-inflammatory cytokine concentrations (TNF-α, IL-1ß, IL-6, IL-12). The best therapeutic efficiency was observed when the particles were used at 5 mg ml-1, while the more cationic RLNP performed superior. When used against a severe grade of colitis, RLNP (5 mg ml-1) resulted in a significant decrease of tissue MPO and TNF-α. It was found that treatment with AMCNP resulted in significant intestinal immune cell depletion, intestinal barrier function improvement, and 1.5-2.5 times reduction of the systemic endotoxin concentration. These findings highlighted the fact that nanoscaling endows the cationic amphiphilic AMCs unique therapeutic properties, which help mitigate murine experimental colitis in the absence of any drug load. The results also provided a glimpse of possible underlying mechanisms through which nanoscaled AMCs might have exerted their therapeutic effect within this context.