Bulk Mixing Research Articles

Direct comparison between millifluidic and bulk-mixing platform in the synthesis of amorphous drug-polysaccharide nanoparticle complex

Amorphous drug-polysaccharide nanoparticle complex (or drug nanoplex) had emerged as an ideal supersaturating delivery system of poorly-soluble drugs attributed to its many attractive characteristics. Herein we presented for the first time direct comparison between two nanoplex synthesis platforms, i.e. millifluidics and bulk mixing, representing continuous and batch production modes, respectively. They were compared by the resultant nanoplex’s (1) physical characteristics (size, zeta potential, and payload), (2) preparation efficiency, (3) storage stability, (4) dissolution rate/supersaturation generation, and (5) production consistency. The effects of key variables in drug-polysaccharide complexation (pH, charge ratio) were investigated in both platforms. Perphenazine and dextran sulfate were used as the drug and polysaccharide models, respectively.The results showed that both platforms shared similar dependences on pH and charge ratio with similar optimal preparation conditions, where the pH was the governing variable through its influence on size and zeta potential, Nanoplexes having mostly similar characteristics (size ≈70–90nm, zeta potential ≈−50mV) were produced by both platforms, except for the payload where bulk mixing resulted in lower payload (65% versus 85%). The lower payload, however, resulted in its superior supersaturation generation. Nevertheless, millifluidics was favored attributed to its superior production consistency and scalability.

New insights into the role of selective and volumetric heating during microwave extraction: Investigation of the extraction of polyphenolic compounds from sea buckthorn leaves using microwave-assisted extraction and conventional solvent extraction

We report a direct comparison of microwave heating and conventional heating in solvent extraction by using exactly the same reaction conditions (including heating rate) in the extraction of polyphenols from dried sea buckthorn leaves. We have for the first time decoupled the effects of bulk heating rate and mixing regime from the fundamental microwave heating mechanism. We show that although microwave selective heating can increase the yield and quality of the polyphenols extracted, if the same bulk heating rate is applied there is no difference in treatment time and therefore theoretical energy requirements of the process. The first implication of these results for process intensification is that if microwave selective heating can be enhanced in scaled up processes through electromagnetic design, the extract yield and quality may be increased further. The second implication is that conventional extraction processes could be designed to provide the same heating rate and hence treatment time as microwave extraction, but any potential energy and space savings would have to be balanced against the increase in capital cost and complexity of the equipment. That said, the very small penetration depth of microwaves into ethanol/water solvent also poses design challenges in the scale up of microwave equipment.

Microfluidic-aided fabrication of nanoparticles blend based on chitosan for a transdermal multidrug delivery application

The aim of this work was to provide a microfluidic-aided fabrication of nanoparticles based on chitosan/poly(N-isopropylacrylamide-co-acrylic acid)/cellulose laurate [CS/P(NIPAAm-co-AAc/CL] blend for transdermal multidrug delivery applications. The scanning electron microscopy (SEM) and dynamic light scattering (DLS) results showed that the diameter sizes of samples were in the range from 200 to 300nm along with a narrow size distribution. Also, the CS-based nanoparticles containing tretinoin and clindamycin phosphate prepared using microfluidic technique exhibited a sustained control release of the drugs as well as minimum inhibitory and bactericidal concentrations compared to the samples fabricated via bulk mixing method. The thermal stability of the drugs loaded nanoparticles revealed a reduction in degradation for those fabricated by using microfluidic technique at 45°C for one month. Afterward, the in vivo assessments confirmed that by applying the microfluidically generated nanoparticles containing two drugs, a declined superficial reddening (erythema) and suitable transdermal permeation as well as residency were happened with respect to the those samples prepared via bulk mixing method and also the drugs solution alone. Finally, these CS-based nanoparticles showed sufficient potential used for transdermal multidrug delivery applications.

Pb‐isotope compositions of the Tasik Chini volcanic‐hosted massive sulfide deposit, Central Belt of Peninsular Malaysia: Implication for source region and tectonic setting

AbstractLead isotope data of sulfides and host volcanic rocks from the Bukit Botol and Bukit Ketaya deposits, the two representative deposits of the Tasik Chini volcanic‐hosted massive sulfide (VHMS) deposit, Central Belt of Peninsular Malaysia, are reported. Lead isotope compositions of the associated sulfide minerals and volcanic rocks from the Bukit Botol deposit exhibit homogeneous and less radiogenic values (206Pb/204Pb showing a range of composition from 18.14 to 18.20, 207Pb/204Pb between 15.52 and 15.59 and 208Pb/204Pb from 37.96 to 38.35). Similarly, the Pb isotopic compositions of the host volcanic rocks from the Bukit Ketaya deposit yielded a narrow range to those of the sulfide samples (206Pb/204Pb from 18.04 to 18.20, 207Pb/204Pb between 15.43 and 15.57 and 208Pb/204Pb of 37.96 to 38.30). The uniform Pb‐isotope compositions of the sulfides in the ore horizon and the host volcanic rocks from both deposits suggest a derivation from a similar source reservoir and mineralization processes. In the framework of the tectonic model for the Central Belt of Peninsular Malaysia, both deposits display a range of lead isotopic compositions originated from mixing of bulk crust/juvenile arc and minor mantle sources, which are typical for VHMS deposits in an island arc–back arc setting.

Local versus volume-integrated turbulence and mixing in breaking internal waves on slopes

Using direct numerical simulations (DNS), we explore local and volume-integrated measures of turbulence and mixing in breaking internal waves on slopes. We consider eight breaking wave cases with a range of normalized pycnocline thicknesses $k\unicode[STIX]{x1D6FF}$, where $k$ is the horizontal wavenumber and $\unicode[STIX]{x1D6FF}$ is the pycnocline thickness, but with similar incoming wave properties. The energetics of wave breaking is quantified in terms of local turbulent dissipation and irreversible mixing using the method of Scotti & White (J. Fluid Mech., vol. 740, 2014, pp. 114–135). Local turbulent mixing efficiencies are calculated using the irreversible flux Richardson number $R_{f}^{\ast }$ and are found to be a function of the turbulent Froude number $Fr_{k}$. Volume-integrated measures of the turbulent mixing efficiency during wave breaking are also made, and are found to be functions of $k\unicode[STIX]{x1D6FF}$. The bulk turbulent mixing efficiency ranges from 0.25 to 0.37 and is maximized when $k\unicode[STIX]{x1D6FF}\approx 1$. In order to connect local and bulk mixing efficiency measures, the variation in the bulk turbulent mixing efficiency with $k\unicode[STIX]{x1D6FF}$ is related to the turbulent Froude number at which the maximum total mixing occurs over the course of the breaking event, $Fr_{k}^{max}$. We find that physically, $Fr_{k}^{max}$ is controlled by the vertical length scale of billows at the interface during wave breaking.

Fluid flow fosters prebiotic chemistry

When it comes to understanding the origins of life, scientists have already determined that biochemical building blocks—amino acids, nucleobases, and sugars—can form under early-Earth-like conditions. But figuring out how those simple species combined in a dilute solution to form complex macromolecules has been harder. A new study suggests a key role for fluid dynamics in microscale pore networks within mineral structures at hydrothermal vents (Proc. Natl. Acad. Sci. USA 2017, DOI: 10.1073/pnas.1612924114). A team led by Victor M. Ugaz at Texas A&M University studied a model system of pore-mimicking cylindrical cells using computational and experimental methods. They found that thermal gradients characteristic of alkaline hydrothermal vent systems result in chaotic fluid flow. That fluid flow transports organic molecules from the bulk fluid to catalytically active pore surfaces, where the species may adsorb and react. Simultaneously, the chaotic flow also provides bulk mixing that prevents localized deple...

Comparison of the physical characteristics of monodisperse non-ionic surfactant vesicles (NISV) prepared using different manufacturing methods

Non-ionic surfactant vesicles (NISV) are synthetic membrane vesicles formed by self-assembly of a non-ionic surfactant, often in a mixture with cholesterol and a charged chemical species. Different methods can be used to manufacture NISV, with the majority of these requiring bulk mixing of two phases. This mixing process is time-consuming and leads to the preparation of large and highly dispersed vesicles, which affects the consistency of the final product and could hinder subsequent regulatory approval. In this study, we have compared the physical characteristics of NISV prepared using two conventional methods (thin-film hydration method and heating method) with a recently introduced microfluidic method. The resulting particles from these methods were assessed for their physical characteristics and in vitro cytotoxicity. Through microfluidics, nano-sized NISV were prepared in seconds, through rapid and controlled mixing of two miscible phases (lipids dissolved in alcohol and an aqueous medium) in a microchannel, without the need of a size reduction step, as required for the conventional methods. Stability studies over two months showed the particles were stable regardless of the method of preparation and there were no differences in terms of EC50 on A375 and A2780 cell lines. However, this work demonstrates the flexibility and ease of applying lab-on-chip microfluidics for the preparation of NISV that could be used to significantly improve formulation research and development, by enabling the rapid manufacture of a consistent end-product, under controlled conditions.

Chemical and mechanical modulation of polymeric micelle assembly.

Recently, polymeric micelles self-assembled from amphiphilic polymers have been studied for various industrial and biomedical applications. This nanoparticle self-assembly typically occurs in a solvent-exchange process. In this process, the quality of the resulting particles is uncontrollably mediated by polymeric solubility and mixing conditions. Here, we hypothesized that improving the solubility of an amphiphilic polymer in an organic solvent via chemical modification while controlling the mixing rate of organic and aqueous phases would enhance control over particle morphology and size. We examined this hypothesis by synthesizing a poly(2-hydroxyethyl)aspartamide (PHEA) grafted with controlled numbers of octadecyl (C18) chains and oligovaline groups (termed "oligovaline-PHEA-C18"). The mixing rate of DMF and water was controlled either by microfluidic mixing of laminar DMF and water flows or through turbulent bulk mixing. Interestingly, oligovaline-PHEA-C18 exhibited an increased solubility in DMF compared with PHEA-C18, as demonstrated by an increase of mixing energy. In addition, increasing the mixing rate between water and DMF using the microfluidic mixer resulted in a decrease of the diameter of the resulting polymeric micelles, as compared with the particles formed from a bulk mixing process. Overall, these findings will expand the parameter space available to control particle self-assembly while also serving to improve existing nanoparticle processing techniques.

Modeling of Gas-Steel-Slag Three-Phase Flow in Ladle Metallurgy: Part I. Physical Modeling

To obtain a better understanding the gas-steel-slag three-phase flow in ladle metallurgy with eccentric gas bubbling, both the single-plug-stirred and dual-plug-stirred water model systems were employed. The plume Froude number derived from the buoyancy of the bubble plume was used to characterize the plume two-phase flow. The elctrical conductivity measurement technique was applied to measure the mixing time. A video technique was used to monitor the slag eye and the open software called ImageJ was taken to quantify the slag eye area. Some experiments were carried out to determine the location of the probe in the ladle where the measured mixing times can be interpreted as the bulk mixing times. The eccentric gas injection in the ladle bottom can improve the mixing efficiency in the ladle. Shorter mixing times can be achieved by injecting gas through two porous plugs, located diametrically opposite at mid-bath radius position (α=180°). A critical gas flow rate is proposed based on the formation of slag eye. The mixing time will decreases sharply at the condition of slag eye formation and collapse alternately. The critical gas flow rate increases with increasing the slag layer thickness and decreasing the porous plug angles. Four fators effect on the slag eye area were investigated: the gas flow rate, slag layer thickness, porous plug locations and angles. A semi-empirical model was developed based on the experimental data of the present work to describe the slag eye area as a function of the heights of the two liquids and the gas flow rate. The present correlation for slag eye area was reviewed against many previous different liquid-liquid systems.

Microfluidic Platform for the Continuous Production and Characterization of Multilamellar Vesicles: A Synchrotron Small-Angle X-ray Scattering (SAXS) Study.

A microfluidic platform combined with synchrotron small-angle X-ray scattering (SAXS) was used for monitoring the continuous production of multilamellar vesicles (MLVs). Their production was fast and started to evolve within less than 0.43 s of contact between the lipids and the aqueous phase. To obtain nanoparticles with a narrow size distribution, it was important to use a modified hydrodynamic flow focusing (HFF) microfluidic device with narrower microchannels than those normally used for SAXS experiments. Monodispersed MLVs as small as 160 nm in size, with a polydispersity index (PDI) of approximately 0.15 were achieved. The nanoparticles produced were smaller and had a narrower size distribution than those obtained via conventional bulk mixing methods. This microfluidic platform therefore has a great potential for the continuous production of monodispersed NPs.

High-Throughput Fabrication of Nanocomplexes Using 3D-Printed Micromixers

3D printing allows a rapid and inexpensive manufacturing of custom made and prototype devices. Micromixers are used for rapid and controlled production of nanoparticles intended for therapeutic delivery. In this study, we demonstrate the fabrication of micromixers using computational design and 3D printing, which enable a continuous and industrial scale production of nanocomplexes formed by electrostatic complexation, using the polymers poly(diallyldimethylammonium chloride) and poly(sodium 4-styrenesulfonate). Several parameters including polymer concentration, flow rate, and flow ratio were systematically varied and their effect on the properties of nanocomplexes was studied and compared with nanocomplexes prepared by bulk mixing. Particles fabricated using this cost effective device were equally small and homogenous but more consistent and controllable in size compared with those prepared manually via bulk mixing. Moreover, each micromixer could process more than 2 liters per hour with unaffected performance and the setup could easily be scaled-up by aligning several micromixers in parallel. This demonstrates that 3D printing can be used to prepare disposable high-throughput micromixers for production of therapeutic nanoparticles.

Millifluidic synthesis of amorphous drug-polysaccharide nanoparticle complex with tunable size intended for supersaturating drug delivery applications

The conventional bulk mixing method to prepare amorphous drug-polysaccharide nanoparticle complex (or drug nanoplex in short) has a major drawback in the lack of size control for the nanoplex produced, hence limiting its potential applications as a supersaturating drug delivery system for bioavailability enhancement of poorly soluble drugs. For this reason, we developed a continuous millifluidic synthesis platform of the drug nanoplex exhibiting high size tunability using curcumin (CUR) and chitosan (CHI) as the models for drug and polysaccharides, respectively. The nanoplex size tunability was achieved by controlling the residence time of the CUR and CHI solutions in the millifluidic reactor, where their slow diffusive mixing at the liquid-liquid interface resulted in a well-regulated nanoplex growth as a function of the residence time. The effects of the preparation pH, molecular weight of CHI, millifluidic tube diameter, and flowrate on the nanoplex size tunability were investigated from which the optimal preparation condition was determined. At the optimal condition, the CUR nanoplex was roughly ≈115nm in size with zeta potential of ≈15mV and ≈72% (w/w) CUR payload. The millifluidic synthesis also maintained the high CUR utilization rate (≈80%) exhibited by the bulk mixing method. Most importantly, the ability to produce significantly smaller nanoplex (sixfold smaller) via millifluidics led to the generation of higher (≈8.5× of CUR saturation solubility) and prolonged (≈8h) supersaturation level. These results bode well for the bioavailability enhancement potential of the drug nanoplex.

Plasma kinetic effects on interfacial mix

Mixing at interfaces in dense plasma media is a problem central to inertial confinement fusion and high energy density laboratory experiments. In this work, collisional particle-in-cell simulations are used to explore kinetic effects arising during the mixing of unmagnetized plasma media. Comparisons are made to the results of recent analytical theory in the small Knudsen number limit and while the bulk mixing properties of interfaces are in general agreement, some differences arise. In particular, “super-diffusive” behavior, large diffusion velocity, and large Knudsen number are observed in the low density regions of the species mixing fronts during the early evolution of a sharp interface prior to the transition to a slow diffusive process in the small-Knudsen-number limit predicted by analytical theory. A center-of-mass velocity profile develops as a result of the diffusion process and conservation of momentum.

Single-step microfluidic synthesis of transferrin-conjugated lipid nanoparticles for siRNA delivery

Microfluidic systems can accelerate clinical translation of nanoparticles due to their ability to generate nanoparticles in a well-controlled and reproducible manner. In this study, a single-step process based on microfluidic focusing (MF) was employed to synthesize transferrin-conjugated lipid nanoparticles (Tf-LNPs) and the method was compared with a multi-steps bulk mixing (BM) method. The results indicate that this single-step MF process enables rapid and efficient synthesis of Tf-LNPs, which were named Tf-LNPs-MF. Tf-LNPs-MF was shown to have a smaller size and more uniform structures compared to LNPs produced by multi-steps BM method (Tf-LNPs-BM). Furthermore, efficient cellular uptake of Tf-LNPs-MF in vitro as well as greater tumor inhibition in vivo proved that Tf-LNPs-MF had higher siRNA delivery efficiency in vitro and in vivo. Taken together, this single-step microfluidic synthesis significantly simplified the Tf-LNPs production and improved their drug delivery properties and may serve as a valuable tool for developing new cancer therapies.

Changes induced in the properties of dielectric silicone elastomers by the incorporation of transition metal complexes

Three new, cheap, and easily obtained complexes of triethanolamine with Cu(II), Ni(II), and Co(II) were incorporated in a high-molecular-weight polydimethylsiloxane-α,ω-diol (number-average molecular weight = 440,000 g mol−1) together with silica nanoparticles. While silica has a well-known mechanical reinforcing role, adding the metal complexes with high dielectric permittivity aims to increase values for this characteristic of the prepared silicone composites in order to be useful for electromechanical applications. A simple procedure was employed for the incorporation of fillers by mechanical mixing in bulk without a solvent. The composites were processed as films and stabilized by curing initiated with 2,4-dichlorobenzoyl peroxide at high temperature (180°C) and pressure. The cross-linking yield was estimated on the basis of swelling in chloroform, while the cross-linking density was calculated from differential scanning calorimetry traces. The filler’s effect on mechanical and dielectric properties was assessed by appropriate measurements (tensile testing and dielectric spectroscopy). Changes in dielectric behavior as a result of the repeated deformation by stretching were also highlighted. Electromechanical sensitivity and ultimate tensile toughness values were estimated on the basis of these results as primary indices on suitability of these materials in transducers. The dielectric strength of the composite films was also evaluated by electrical breakdown field measurements.

Direct measurement of the propagation velocity of defects using coherent X-rays

The properties of artificially grown thin films are often strongly affected by the dynamic relationships between surface growth processes and subsurface structure. Coherent mixing of X-ray signals promises to provide an approach to better understand such processes. Here, we demonstrate the continuously variable mixing of surface and bulk scattering signals during real-time studies of sputter deposition of a-Si and a-WSi2 films by controlling the X-ray penetration and escape depths in coherent grazing-incidence small-angle X-ray scattering. Under conditions where the X-ray signal comes from both the growth surface and the thin film bulk, oscillations in temporal correlations arise from coherent interference between scattering from stationary bulk features and from the advancing surface. We also observe evidence that elongated bulk features propagate upwards at the same velocity as the surface. Furthermore, a highly surface-sensitive mode is demonstrated that can access the surface dynamics independently of the subsurface structure. Defects affect materials’ properties. A method is now presented for studying dynamic processes during the growth of thin films — specifically, the evolution of defects — based on the coherent mixing of bulk and surface X-ray scattering signals.

Tracking along-arc sediment inputs to the Aleutian arc using thallium isotopes

Sediment transport from the subducted slab to the mantle wedge is an important process in understanding the chemical and physical conditions of arc magma generation. The Aleutian arc offers an excellent opportunity to study sediment transport processes because the subducted sediment flux varies systematically along strike (Kelemen et al., 2003) and many lavas exhibit unambiguous signatures of sediment addition to the sub-arc mantle (Morris et al., 1990). However, the exact sediment contribution to Aleutian lavas and how these sediments are transported from the slab to the surface are still debated. Thallium (Tl) isotope ratios have great potential to distinguish sediment fluxes in subduction zones because pelagic sediments and low-temperature altered oceanic crust are highly enriched in Tl and display heavy and light Tl isotope compositions, respectively, compared with the upper mantle and continental crust.Here, we investigate the Tl isotope composition of lavas covering almost the entire Aleutian arc a well as sediments outboard of both the eastern (DSDP Sites 178 and 183) and central (ODP Hole 886C) portions of the arc. Sediment Tl isotope compositions change systematically from lighter in the Eastern to heavier in the Central Aleutians reflecting a larger proportion of pelagic sediments when distal from the North American continent. Lavas in the Eastern and Central Aleutians mirror this systematic change to heavier Tl isotope compositions to the west, which shows that the subducted sediment composition is directly translated to the arc east of Kanaga Island. Moreover, quantitative mixing models of Tl and Pb, Sr and Nd isotopes reveal that bulk sediment transfer of ∼0.6–1.0% by weight in the Eastern Aleutians and ∼0.2–0.6% by weight in the Central Aleutians can account for all four isotope systems. Bulk mixing models, however, require that fractionation of trace element ratios like Ce/Pb, Cs/Tl, and Sr/Nd in the Central and Eastern Aleutians occurs after the sediment component was mixed with the mantle wedge. Models of Sr and Nd isotopes that involve sediment melting require either high degrees of sediment melting (>50%), in which case trace element ratios like Ce/Pb, Cs/Tl, and Sr/Nd of Aleutian lavas need to be produced after mixing with the mantle, or significant fluid additions from the underlying oceanic crust with Sr and Nd isotope compositions indistinguishable from the mantle wedge as well as high Sr/Nd ratios similar to that of low (<20%) degree sediment melts.Thallium isotope data from Western Aleutian lavas exhibit compositions slightly lighter than the upper mantle, which implies a negligible sediment flux at this location and probably involvement of low-temperature altered oceanic crust in the generation of these lavas. In general, the lightest Tl isotope compositions are observed for the highest Sr/Y ratios and most unradiogenic Sr and Pb isotope compositions, which is broadly consistent with derivation of these lavas via melting of eclogitized altered oceanic crust.

Time scales of crystal mixing in magma mushes

AbstractMagma mixing is widely recognized as a means of producing compositional diversity and preconditioning magmas for eruption. However, the processes and associated time scales that produce the commonly observed expressions of magma mixing are poorly understood, especially under crystal‐rich conditions. Here we introduce and exemplify a parameterized method to predict the characteristic mixing time of crystals in a crystal‐rich magma mush that is subject to open‐system reintrusion events. Our approach includes novel numerical simulations that resolve multiphase particle‐fluid interactions. It also quantifies the crystal mixing by calculating both the local and system‐wide progressive loss of the spatial correlation of individual crystals throughout the mixing region. Both inertial and viscous time scales for bulk mixing are introduced. Estimated mixing times are compared to natural examples and the time for basaltic mush systems to become well mixed can be on the order of 10 days.

Microfluidic Assembly of pDNA/Cationic Liposome Lipoplexes with High pDNA Loading for Gene Delivery.

Microfluidics offers unique characteristics to control the mixing of liquids under laminar flow. Its use for the assembly of lipoplexes represents an attractive alternative for the translation of gene delivery studies into clinical trials on a sufficient throughput scale. Here, it was shown that the microfluidic assembly of pDNA/cationic liposome (CL) lipoplexes allows the formation of nanocarriers with enhanced transfection efficiencies compared with the conventional bulk-mixing (BM) process under high pDNA loading conditions. Lipoplexes generated by microfluidic devices exhibit smaller and more homogeneous structures at a molar charge ratio (R±) of 1.5, representing the ratio of lipid to pDNA content. Using an optimized model to fit small-angle X-ray scattering (SAXS) curves, it was observed that large amounts of pDNA induces the formation of aggregates with a higher number of stacked bilayers (N ∼ 5) when the BM process was used, whereas microfluidic lipoplexes presented smaller structures with a lower number of stacked bilayers (N ∼ 2.5). In vitro studies further confirmed that microfluidic lipoplexes achieved higher in vitro transfection efficiencies in prostate cancer cells at R ± 1.5, employing a reduced amount of cationic lipid. The correlation of mesoscopic characteristics with in vitro performance provides insights for the elucidation of the colloidal arrangement and biological behavior of pDNA/CL lipoplexes obtained by different processes, highlighting the feasibility of applying microfluidics to gene delivery.

Cell-Penetrating Peptides as Carriers for Oral Delivery of Biopharmaceuticals.

Oral delivery of biopharmaceuticals, for example peptides and proteins, constitutes a great challenge in drug delivery due to their low chemical stability and poor permeation across the intestinal mucosa, to a large extent limiting the mode of administration to injections, which is not favouring patient compliance. Nevertheless, cell-penetrating peptides (CPPs) have shown promising potential as carriers to overcome the epithelium, and this minireview highlights recent knowledge gained within the field of CPP-mediated transepithelial delivery of therapeutic peptides and proteins from the intestine. Two approaches may be pursued: co-administration of the carrier and therapeutic peptide in the form of complexes obtained by simple bulk mixing, or administration of covalent conjugates demanding more advanced production methodologies. These formulation approaches have their pros and cons, and which is to be preferred depends on the physicochemical properties of both the specific CPP and the specific cargo. In addition to the physical epithelial barrier, a metabolic barrier must be overcome in order to obtain CPP-mediated delivery of a cargo drug from the intestine, and a number of strategies have been employed to delay enzymatic degradation of the CPP. The mechanisms by which CPPs translocate across membranes are not fully understood, but possibly involve endocytosis as well as direct translocation, and the CPP-mediated transepithelial delivery of cargo drugs thus likely involves similar mechanisms for the initial membrane interaction and translocation. However, the mechanisms responsible for transcytosis of the cargo drug, if taken up by an endocytic mechanism, or direct translocation across the epithelium are so far not known.