In vitro Models for Predicting Bioadhesion Fracture Strength to Ex Vivo Animal Buccal Tissue.

Commitment to the 3Rs principle (Replacement, Reduction, and Refinement) led to the development of a cell-based system to measure buccal bioadhesion in vitro and replace the use of porcine buccal and esophageal tissues (PBT and PET, respectively). Additionally, the aim is to bridge the gap in knowledge regarding the bioadhesion properties of PBT and PET. The in vitro models are based on the human buccal epithelial cell line-TR146 without ("Model I") or with ("Model II") 5% (w/v) mucous layer. The in vitro setup also provides a method to evaluate the bioadhesion between two soft materials. Standard bioadhesive hydrogels (alginate, chitosan, and gelatin) are used to test and compare the results from the in vitro models to the ex vivo tissues. The ex vivo and in vitro models show increased bioadhesion as the applied force and contact time increases. Furthermore, Model I exhibits bioadhesion values-of alginate, chitosan, and gelatin-comparable to those obtained with PBT. It is also found that contact time and applied force similarly affect PBT and PET bioadhesion, while PET exhibits greater values. In conclusion, Model I can replace PBT for measuring bioadhesion and be incorporated into the experimental design of bioadhesive DDS, thus minimizing animal tissue usage.

Algae-Based Nanoparticles for Oral Drug Delivery Systems.

Drug administration by oral delivery is the preferred route, regardless of some remaining challenges, such as short resident time and toxicity issues. One strategy to overcome these barriers is utilizing mucoadhesive vectors that can increase intestinal resident time and systemic uptake. In this study, biomimetic nanoparticles (NPs) were produced from 14 types of edible algae and evaluated for usage as oral DDSs by measuring their size, surface charge, morphology, encapsulation efficiency, mucoadhesion force, and cellular uptake into Caco-2 cells. The NPs composed of algal materials (aNPs) exhibited a spherical morphology with a size range of 126-606 nm and a surface charge of -9 to -38 mV. The mucoadhesive forces tested ex vivo against mice, pigs, and sheep intestines revealed significant variation between algae and animal models. Notably, Arthospira platensis (i.e., Spirulina) NPs (126 ± 2 nm, -38 ± 3 mV) consistently exhibited the highest mucoadhesive forces (up to 3127 ± 272 µN/mm²). Moreover, a correlation was found between high mucoadhesive force and high cellular uptake into Caco-2 cells, further supporting the potential of aNPs by indicating their ability to facilitate drug absorption into the human intestinal epithelium. The results presented herein serve as a proof of concept for the possibility of aNPs as oral drug delivery vehicles.

Biocoating-A Critical Step Governing the Oral Delivery of Polymeric Nanoparticles.

Decades of research into the topic of oral nanoparticle (NP) delivery has still not provided a clear consensus regarding which properties produce an effective oral drug delivery system. The surface properties-charge and bioadhesiveness-as well as in vitro and in vivo correlation seem to generate the greatest number of disagreements within the field. Herein, a mechanism underlying the in vivo behavior of NPs is proposed, which bridges the gaps between these disagreements. The mechanism relies on the idea of biocoating-the coating of NPs with mucus-which alters their surface properties, and ultimately their systemic uptake. Utilizing this mechanism, several coated NPs are tested in vitro, ex vivo, and in vivo, and biocoating is found to affect NPs size, zeta-potential, mucosal diffusion coefficient, the extent of aggregation, and in vivo/in vitro/ex vivo correlation. Based on these results, low molecular weight polylactic acid exhibits a 21-fold increase in mucosal diffusion coefficient after precoating as compared to uncoated particles, as well as 20% less aggregation, and about 30% uptake to the blood in vivo. These discoveries suggest that biocoating reduces negative NP charge which results in an enhanced mucosal diffusion rate, increased gastrointestinal retention time, and high systemic uptake.

Time-dependent mucoadhesion of conjugated bioadhesive polymers.

The time-dependent bioadhesive performance of various polymers was evaluated using a texture analyzer apparatus and freshly excised rat small intestinal tissue. A series of novel bioadhesive polymers were prepared by conjugating L-phenylalanine, L-tyrosine, and L-DOPA to either a low molecular weight poly (butadiene-maleic anhydride) or a high molecular weight poly (ethylene-maleic anhydride). Bioadhesive force was characterized as a function of time relative to polycarbophil, a slightly cross-linked poly (acrylic acid)-derivative, revealing different fracture strengths and tensile work for each of the six backbone-side chain conjugations that were studied. While polycarbophil showed a rapid and significant loss of bioadhesion over the testing period, the newly developed synthetic polymers were able to maintain their bioadhesive performance over the course of 91 min with the overall magnitude of bioadhesion corresponding to the hydrogen bonding potential of the associated side chains. These results highlight the potential of these polymers for use in the development of more effective bioadhesive oral drug delivery systems.

Single Step Double-walled Nanoencapsulation (SSDN).

A quick fabrication method for making double-walled (DW) polymeric nanospheres is presented. The process uses sequential precipitation of two polymers. By choosing an appropriate solvent and non-solvent polymer pair, and engineering two sequential phase inversions which induces first precipitation of the core polymer followed by precipitation of the shell polymer, DW nanospheres can be created instantaneously. A series of DW formulations were prepared with various core and shell polymers, then characterized using laser diffraction particle sizing, scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectroscopy, and differential scanning calorimetry (DSC). Atomic force microscopy (AFM) imaging confirmed existence of a single core polymer coated with a second polymer. Insulin (3.3% loading) was used as a model drug to assess its release profile from core (PLGA) and shell (PBMAD) polymers and resulted with a tri-phase release profile in vitro for two months. Current approaches for producing DW nanoparticles (NPs) are limited by the complexity and time involved. Additional issues include aggregation and entrapment of multiple spheres and the undesired formation of heterogeneous coatings. Therefore, the technique presented here is advantageous because it can produce NPs with distinct, core-shell morphologies through a rapid, spontaneous, self-assembly process. This method not only produces DW NPs, but can also be used to encapsulate therapeutic drug. Furthermore, modification of this process to other core and shell polymers is feasible using the general guidelines provided in this paper.

Concise Review: Fabrication, Customization, and Application of Cell Mimicking Microparticles in Stem Cell Science.

Stem and non-stem cell behavior is heavily influenced by the surrounding microenvironment, which includes other cells, matrix, and potentially biomaterials. Researchers have been successful in developing scaffolds and encapsulation techniques to provide stem cells with mechanical, topographical, and chemical cues to selectively direct them toward a desired differentiation pathway. However, most of these systems fail to present truly physiological replications of the in vivo microenvironments that stem cells are typically exposed to in tissues. Thus, cell mimicking microparticles (CMMPs) have been developed to more accurately recapitulate the properties of surrounding cells while still offering ways to tailor what stimuli are presented. This nascent field holds the promise of reducing, or even eliminating, the need for live cells in select, regenerative medicine therapies, and diagnostic applications. Recent, CMMP-based studies show great promise for the technology, yet only reproduce a small subset of cellular characteristics from among those possible: size, morphology, topography, mechanical properties, surface molecules, and tailored chemical release to name the most prominent. This Review summarizes the strengths, weaknesses, and ideal applications of micro/nanoparticle fabrication and customization methods relevant to cell mimicking and provides an outlook on the future of this technology. Moving forward, researchers should seek to combine multiple techniques to yield CMMPs that replicate as many cellular characteristics as possible, with an emphasis on those that most strongly influence the desired therapeutic effects. The level of flexibility in customizing CMMP properties allows them to substitute for cells in a variety of regenerative medicine, drug delivery, and diagnostic systems. Stem Cells Translational Medicine 2018;7:232-240.

Amplified CPEs enhancement of chorioamnion membrane mass transport by encapsulation in nano-sized PLGA particles.

Chemical penetration enhancers (CPEs) have long been used for mass transport enhancement across membranes. Many CPEs are used in a solution or gel and could be a solvent. The use of CPEs is mainly limited due to their toxicity/irritation levels. This study presents the evaluation of encapsulated CPEs in nano-sized polymeric particles on the chorioamnion (CA) membrane mass transport. CPEs' mass encapsulated in nanoparticles was decreased by 10,000-fold. Interestingly, this approach resulted in a 6-fold increase in mass transport across the CA. This approach may also be used with other CPEs' base applications necessitating lower CPE concentration. Applying Ultrasound (US) has shown to increase the release rate of and also the mass transport across the CA membrane. It is proposed that encapsulated CPEs penetrate into the CA membrane thus prolonging their exposure, possibly extending their penetration into the CA membrane, while insonation also deepens their penetration into the CA membrane.

Ultrasound Effect on Cancerous versus Non-Cancerous Cells.

Previous studies have found that cancer cells whose metastatic potential is low are more vulnerable to mechanical stress-induced trauma to their cytoskeleton compared with benign cells. Because ultrasound induces mechanical stresses on cells and tissues, it is postulated that there may be a way to apply ultrasound to tumors to reduce their ability to metastasize. The difference between low-malignant-potential cancer cells and benign cells could be a result of their different responses to the mechanical stress insonation induced. This hypothesis was tested in vitro and in vivo. Low-malignant-potential cells were found to be more sensitive to insonation, resulting in a significantly higher mortality rate compared with that of benign cells, 89% versus 21%, respectively. This effect can be controlled by varying ultrasound parameters: intensity, duration, and duty cycle. Thus, the results presented in this study suggest the application of ultrasound to discriminate between benign and malignant cells.

Blotting from PhastGel to Membranes by Ultrasound.

Ultrasound based approach for enhanced protein blotting is proposed. Three minutes of ultrasound exposure (1 MHz, 2.5 W/cm(2)) was sufficient for a clear transfer of proteins from a polyacrylamide gel (PhastGel) to nitrocellulose or Nylon 66 Biotrans membrane. The proteins evaluated were prestained sodium dodecyl sulfate-polyacrylamide standards (18,500-106,000 Da) and 14C-labeled Rainbow protein molecular weight markers (14,300-200,000 Da).

Fetal membrane transport enhancement using ultrasound for drug delivery and noninvasive detection.

PURPOSE: The purpose of this research was to evaluate the effect of ultrasound on mass transport across fetal membrane for direct fetal drug delivery and sensing of the amniotic fluid in a noninvasive manner. METHODS: Post-delivery human fetal membranes (chorioamnion) were used for in vitro experiments, in which the effect of ultrasound on transport across fetal membrane of fluorescent model molecule (250 kDa) was evaluated. Ex vivo experiments were carried out on a whole rat amniotic sac. The model molecule or alpha-fetoprotein was injected into the amniotic sac through the placenta. Transport of these molecules across pre- and post-insonation of the amniotic sac was evaluated. The ultrasound enhancement's mechanism was also assessed. RESULTS: The greatest enhancement in mass transport (43-fold) in vitro was achieved for 5 min of insonation (20 kHz, 4.6 W/cm(2), 5 mm distance). Ex vivo results showed a rapid increase (23-fold) in mass transport of the model molecule and also for alphafetoprotein following 30 s of insonation (20 kHz, 4.6 W/cm(2), 5 mm distance). CONCLUSIONS: Mass transport across fetal membranes was enhanced post-insonation both in vitro and ex vivo in a reversible and transient manner. We suggest that exterior (to the amniotic sac) ultrasound-induced cavitation is the main mechanism of action.

The synergistic effect of ultrasound and chemical penetration enhancers on chorioamnion mass transport.

In our previous study we proposed the use of chemical penetration enhancers for noninvasive detection of fetus abnormalities that can also be utilized for direct fetal drug delivery. In an attempt to further increase the mass transport rate across the amniotic membrane, thus shortening the procedure and improving the applicability of the proposed procedure, the effect and mechanism of combining ultrasound exposure with chemical penetration enhancers' application were assessed. The combined effect was evaluated in vitro on post-delivery human amniotic membrane and ex vivo on rat's whole amniotic sac. Ultrasound effect has been assessed by dye experiments using a customized image analysis program. Additional insights of ultrasound effect's mechanism on biological membranes are presented. Previously we have determined that chemical penetration enhancers affect the fetal membranes via two mechanisms termed as 'extractors' and 'fluidizers'. In this study, we found that combining ultrasound with a 'fluidizer' CPE (e.g. bupivacaine) results in a synergistic enhancement (90-fold) of fetal membrane's mass transport, while combining ultrasound with 'extractors' (e.g. ethanol and NMP) results in an antagonistic effect. The combined procedure is faster and gain greater accuracy than the applications of sole chemical penetration enhancers.

Quaternized starch-based carrier for siRNA delivery: from cellular uptake to gene silencing.

RNAi therapeutics is a powerful tool for treating diseases by sequence-specific targeting of genes using siRNA. Since its discovery, the need for a safe and efficient delivery system for siRNA has increased. Here, we have developed and characterized a delivery platform for siRNA based on the natural polysaccharide starch in an attempt to address unresolved delivery challenges of RNAi. Modified potato starch (Q-starch) was successfully obtained by substitution with quaternary reagent, providing Q-starch with cationic properties. The results indicate that Q-starch was able to bind siRNA by self-assembly formation of complexes. For efficient and potent gene silencing we monitored the physical characteristics of the formed nanoparticles at increasing N/P molar ratios. The minimum ratio for complete entrapment of siRNA was 2. The resulting complexes, which were characterized by a small diameter (~30 nm) and positive surface charge, were able to protect siRNA from enzymatic degradation. Q-starch/siRNA complexes efficiently induced P-glycoprotein (P-gp) gene silencing in the human ovarian adenocarcinoma cell line, NCI-ADR/Res (NAR), over expressing the targeted gene and presenting low toxicity. Additionally, Q-starch-based complexes showed high cellular uptake during a 24-hour study, which also suggested that intracellular siRNA delivery barriers governed the kinetics of siRNA transfection. In this study, we have devised a promising siRNA delivery vector based on a starch derivative for efficient and safe RNAi application.

A novel approach for noninvasive drug delivery and sensing through the amniotic sac.

Current invasive prenatal tests (amniocentesis and chorionic villus sampling) are known for their risk to the fetus. In the last decade, the use and awareness of these prenatal tests have increased, resulting in growing demand for a safe, non-invasive, and accurate prenatal test. Chemical penetration enhancers (CPEs) have long been used to increase transport phenomena across skin and other membranes (e.g., tympanic membrane). The amniotic sac membrane is called the chorioamnion (CA) membrane and serves as the physical barrier between the fetus and the mother. In this research, the effect of CPEs on human CA mass transport was evaluated both in vitro and ex vivo. The results show that the tested CPEs exhibit an enhancing effect on CA mass transport. Based on the permeability results, two mechanisms of action were suggested: "extractors" and "fluidizers". Fourier transform infrared (FTIR) and rapid colorimetric screening measurements supported the mechanisms, based on which, more potent compounds were designed and tested for their enhancing effect. The enhancing mass transport effect of CPEs on CA membrane may be used both for sampling of cell-free DNA and for noninvasively administering drugs and other biological agents to the amniotic sac.

Ultrasound mediated transdermal drug delivery.

Transdermal drug delivery offers an attractive alternative to the conventional drug delivery methods of oral administration and injections. However, the stratum corneum serves as a barrier that limits the penetration of substances to the skin. Application of ultrasound (US) irradiation to the skin increases its permeability (sonophoresis) and enables the delivery of various substances into and through the skin. This review presents the main findings in the field of sonophoresis in transdermal drug delivery as well as transdermal monitoring and the mathematical models associated with this field. Particular attention is paid to the proposed enhancement mechanisms and future trends in the fields of cutaneous vaccination and gene therapy.