Ovule siRNAs methylate protein-coding genes in trans.

Twenty-four-nucleotide (nt) small interfering RNAs (siRNAs) maintain asymmetric DNA methylation at thousands of euchromatic transposable elements in plant genomes in a process called RNA-directed DNA methylation (RdDM). RdDM is dispensable for growth and development in Arabidopsis thaliana, but is required for reproduction in other plants, such as Brassica rapa. The 24-nt siRNAs are abundant in maternal reproductive tissue, due largely to overwhelming expression from a few loci in the ovule and developing seed coat, termed siren loci. A recent study showed that 24-nt siRNAs produced in the anther tapetal tissue can methylate male meiocyte genes in trans. Here we show that in B. rapa, a similar process takes place in female tissue. siRNAs are produced from gene fragments embedded in some siren loci, and these siRNAs can trigger methylation in trans at related protein-coding genes. This trans-methylation is associated with silencing of some target genes and may be responsible for seed abortion in RdDM mutants. Furthermore, we demonstrate that a consensus sequence in at least two families of DNA transposons is associated with abundant siren expression, most likely through recruitment of CLASSY3, a putative chromatin remodeler. This research describes a mechanism whereby RdDM influences gene expression and sheds light on the role of RdDM during plant reproduction.

Abundant expression of maternal siRNAs is a conserved feature of seed development.

Small RNAs are abundant in plant reproductive tissues, especially 24-nucleotide (nt) small interfering RNAs (siRNAs). Most 24-nt siRNAs are dependent on RNA Pol IV and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and establish DNA methylation at thousands of genomic loci in a process called RNA-directed DNA methylation (RdDM). In Brassica rapa, RdDM is required in the maternal sporophyte for successful seed development. Here, we demonstrate that a small number of siRNA loci account for over 90% of siRNA expression during B. rapa seed development. These loci exhibit unique characteristics with regard to their copy number and association with genomic features, but they resemble canonical 24-nt siRNA loci in their dependence on RNA Pol IV/RDR2 and role in RdDM. These loci are expressed in ovules before fertilization and in the seed coat, embryo, and endosperm following fertilization. We observed a similar pattern of 24-nt siRNA expression in diverse angiosperms despite rapid sequence evolution at siren loci. In the endosperm, siren siRNAs show a marked maternal bias, and siren expression in maternal sporophytic tissues is required for siren siRNA accumulation. Together, these results demonstrate that seed development occurs under the influence of abundant maternal siRNAs that might be transported to, and function in, filial tissues.

Coarse-Grained Molecular Dynamics Simulations of Paclitaxel-Loaded Polymeric Micelles.

A continuous manufacturing technology based on coaxial turbulent jet in coflow was previously developed to produce paclitaxel-loaded polymeric micelles. Herein, coarse-grained molecular dynamics (CG-MD) simulations were implemented to better understand the effect of the material attributes (i.e., the drug-polymer ratio and the ethanol concentration) and process parameters (i.e., temperature) on the self-assembly process of polymeric micelles as well as to provide molecular details on micelle instability. An all-atom (AA) poly (ethylene glycol)-poly (lactic acid) (PEG-PLA) polymer model was developed as the reference for parameterizing a coarse-grained (CG) model, and the AA polymer model was further validated with experimental glass transition temperature (Tg). The model transferability was verified by comparing structural properties between the AA and CG models. The CG model was further validated with experimental data, including micelle particle size measurements and drug encapsulation efficiency. Furthermore, the encapsulation of paclitaxel into the polymeric micelles was included in the simulations, taking into consideration the interactions between the paclitaxel and the polymers. The results from various points of view demonstrated a strong dependence of the shape of the micelles on the drug encapsulation, with micelles transitioning from spherical to ellipsoidal structures with an increasing paclitaxel amount. Simulation data were also used to identify the critical aggregation number (i.e., the number of polymer and drug molecules required for transition from one shape to another). Improved micellar structural stability was found with a larger micellar size and less solvent accessibility. Lastly, an evaluation was performed on the micellar dissociation free energy using a steered molecular dynamics simulation over a range of temperatures and ethanol concentrations. These simulations revealed that at higher ethanol and temperature conditions, micelles become destabilized, resulting in greater paclitaxel release. The increased drug release was determined to originate from the solvation of the hydrophobic core, which promoted micellar swelling and an associated reduction in hydrophobic interactions, leading to a loosely packed micellar structure.

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum.

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetium genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a 'near-complete' draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. The Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.

Factors Influencing the Immunogenicity and Immunotoxicity of Cyclic RGD Peptide-Modified Nanodrug Delivery Systems.

c(RGDyK)-modified liposomes have been shown to be immunogenic and potentially trigger acute systemic anaphylaxis upon repeated intravenous injection in both BALB/c nude mice and ICR mice. However, questions concerning the potential influence of mouse strains, immunization routes, drug carrier properties, and changes in c(RGDyK) itself on the immunogenicity and resultant immunotoxicity (anaphylaxis) of cyclic RGD peptide-modified nanodrug delivery systems remain unanswered. Here, these potential impact factors were investigated, aiming to better understand the immunological properties of cyclic RGD peptide-based nanodrug delivery systems and seek for solutions for this immunogenicity-associated issue. It was revealed that anaphylaxis caused by intravenous c(RGDyK)-modified drug delivery systems might be avoided by altering the preimmunization route (i.e., subcutaneous injection), introducing positively charged lipids into the liposomes and by using micelles or red blood cell membrane (RBC)-based drug delivery systems as the carrier. Different murine models showed different incidences of anaphylaxis following intravenous c(RGDyK)-liposome stimulation: anaphylaxis was not observed in both SD rats and BALB/c mice and was less frequent in C57BL/6 mice than that in ICR mice. In addition, enlarging the peptide ring of c(RGDyK) by introducing amino sequence serine-glycine-serine reduced the incidence of anaphylaxis post the repeated intravenous c(RGDyKSGS)-liposome stimulation. However, immunogenicity of cyclic RGD-modified drug carriers could not be reversed, although some reduction in IgG antibody production was observed when ICR mice were intravenously stimulated with c(RGDyK)-modified micelles, RBC membrane-based drug delivery systems and c(RGDyKSGS)-liposomes instead of c(RGDyK)-liposomes. This study provides a valuable reference for future application of cyclic RGD peptide-modified drug delivery systems.

Formulation and evaluation of itraconazole liposomes for Hedgehog pathway inhibition.

Itraconazole (ITZ) is an FDA-approved antifungal agent that has recently been explored for novel biological properties. In particular, ITZ was identified as a potent inhibitor of the hedgehog (Hh) pathway, a cell signalling pathway that has been linked to a variety of cancers and accounts for ∼25% of paediatric medulloblastoma (MB) cases. To date, there is not a targeted therapeutic option for paediatric MB, resulting in long-term side effects such as hormone deficiency, organ damage and secondary cancers. A primary obstacle for developing targeted therapy for brain ailments is the presence of the blood-brain barrier (BBB), which protects the brain from potentially harmful substances. Due to its size and hydrophobicity, ITZ does not penetrate the BBB. Alternatively, liposomes are being increasingly used within the clinic to increase drug bioavailability, target specificity and BBB permeability. With this in mind, we have successfully developed ITZ-containing liposomes with an optimal size for BBB penetration (<100 nm) and encapsulation efficiency (∼95%) by utilizing a continuous manufacturing approach-turbulent coaxial jet in co-flow. Our preliminary in vitro data demonstrate that these liposomes inhibit the Hh pathway, albeit at a reduced level in comparison to free ITZ. (196/250 words).

Maternal components of RNA-directed DNA methylation are required for seed development in Brassica rapa.

Small RNAs trigger repressive DNA methylation at thousands of transposable elements in a process called RNA-directed DNA methylation (RdDM). The molecular mechanism of RdDM is well characterized in Arabidopsis, yet the biological function remains unclear, as loss of RdDM in Arabidopsis causes no overt defects, even after generations of inbreeding. It is known that 24 nucleotide Pol IV-dependent siRNAs, the hallmark of RdDM, are abundant in flowers and developing seeds, indicating that RdDM might be important during reproduction. Here we show that, unlike Arabidopsis, mutations in the Pol IV-dependent small RNA pathway cause severe and specific reproductive defects in Brassica rapa. High rates of abortion occur when seeds have RdDM mutant mothers, but not when they have mutant fathers. Although abortion occurs after fertilization, RdDM function is required in maternal somatic tissue, not in the female gametophyte or the developing zygote, suggesting that siRNAs from the maternal soma might function in filial tissues. We propose that recently outbreeding species such as B. rapa are key to understanding the role of RdDM during plant reproduction.

Perspectives on Physicochemical and In Vitro Profiling of Ophthalmic Ointments.

Ophthalmic ointments are unique in that they combine features of topical drug delivery, the ophthalmic route and ointment (semisolid) formulations. Accordingly, these complex formulations are challenging to develop and evaluate and therefore it is critically important to understand their physicochemical properties as well as their in vitro drug release characteristics. Previous reports on the characterization of ophthalmic ointments are very limited. Although there are FDA guidance documents and USP monographs covering some aspects of semisolid formulations, there are no FDA guidance documents nor any USP monographs for ophthalmic ointments. This review summarizes the physicochemical and in vitro profiling methods that have been previously reported for ophthalmic ointments. Specifically, insight is provided into physicochemical characterization (rheological parameters, drug content and content uniformity, and particle size of the API in the finished ointments) as well as important considerations (membranes, release media, method comparison, release kinetics and discriminatory ability) in in vitro release testing (IVRT) method development for ophthalmic ointments. Graphical Abstract Summary of the physicochemcial profiling and in vitro drug release testing (IVRT) for ophthalmic ointments.

The banana (Musa acuminata) genome and the evolution of monocotyledonous plants.

Bananas (Musa spp.), including dessert and cooking types, are giant perennial monocotyledonous herbs of the order Zingiberales, a sister group to the well-studied Poales, which include cereals. Bananas are vital for food security in many tropical and subtropical countries and the most popular fruit in industrialized countries. The Musa domestication process started some 7,000 years ago in Southeast Asia. It involved hybridizations between diverse species and subspecies, fostered by human migrations, and selection of diploid and triploid seedless, parthenocarpic hybrids thereafter widely dispersed by vegetative propagation. Half of the current production relies on somaclones derived from a single triploid genotype (Cavendish). Pests and diseases have gradually become adapted, representing an imminent danger for global banana production. Here we describe the draft sequence of the 523-megabase genome of a Musa acuminata doubled-haploid genotype, providing a crucial stepping-stone for genetic improvement of banana. We detected three rounds of whole-genome duplications in the Musa lineage, independently of those previously described in the Poales lineage and the one we detected in the Arecales lineage. This first monocotyledon high-continuity whole-genome sequence reported outside Poales represents an essential bridge for comparative genome analysis in plants. As such, it clarifies commelinid-monocotyledon phylogenetic relationships, reveals Poaceae-specific features and has led to the discovery of conserved non-coding sequences predating monocotyledon-eudicotyledon divergence.

The most deeply conserved noncoding sequences in plants serve similar functions to those in vertebrates despite large differences in evolutionary rates.

In vertebrates, conserved noncoding elements (CNEs) are functionally constrained sequences that can show striking conservation over >400 million years of evolutionary distance and frequently are located megabases away from target developmental genes. Conserved noncoding sequences (CNSs) in plants are much shorter, and it has been difficult to detect conservation among distantly related genomes. In this article, we show not only that CNS sequences can be detected throughout the eudicot clade of flowering plants, but also that a subset of 37 CNSs can be found in all flowering plants (diverging ∼170 million years ago). These CNSs are functionally similar to vertebrate CNEs, being highly associated with transcription factor and development genes and enriched in transcription factor binding sites. Some of the most highly conserved sequences occur in genes encoding RNA binding proteins, particularly the RNA splicing-associated SR genes. Differences in sequence conservation between plants and animals are likely to reflect differences in the biology of the organisms, with plants being much more able to tolerate genomic deletions and whole-genome duplication events due, in part, to their far greater fecundity compared with vertebrates.

Liposome Formation Using a Coaxial Turbulent Jet in Co-Flow.

PURPOSE: Liposomes are robust drug delivery systems that have been developed into FDA-approved drug products for several pharmaceutical indications. Direct control in producing liposomes of a particular particle size and particle size distribution is extremely important since liposome size may impact cellular uptake and biodistribution. METHODS: A device consisting of an injection-port was fabricated to form a coaxial turbulent jet in co-flow that produces liposomes via the ethanol injection method. By altering the injection-port dimensions and flow rates, a fluid flow profile (i.e., flow velocity ratio vs. Reynolds number) was plotted and associated with the polydispersity index of liposomes. RESULTS: Certain flow conditions produced unilamellar, monodispersed liposomes and the mean particle size was controllable from 25 up to >465 nm. The mean liposome size is highly dependent on the Reynolds number of the mixed ethanol/aqueous phase and independent of the flow velocity ratio. CONCLUSIONS: The significance of this work is that the Reynolds number is predictive of the liposome particle size, independent of the injection-port dimensions. In addition, a new model describing liposome formation is outlined. The significance of the model is that it relates fluid dynamic properties and lipid-molecule physical properties to the final liposome size.

Drug Distribution in Microspheres Enhances Their Anti-Inflammatory Properties in the Gottingen Minipig.

The foreign body reaction (FBR), one of the body's defense mechanisms against foreign materials, results in loss of implant biocompatibility. A popular strategy to prevent FBR is the constant release of dexamethasone in the tissue surrounding the implant. However, FBR prevention has not been sufficiently studied in large animal models, which offer a better representation of the human subcutaneous tissue physiology. Accordingly, a long-term strategy to prevent FBR to subcutaneous implants in a large animal model is necessary to translate the existing research for clinical applications. Here, a poly(lactic-co-glycolic) (PLGA) microsphere/poly(vinyl alcohol) (PVA) hydrogel composite coating for one-month prevention of FBR in Gottingen minipigs was developed. A modified PLGA microsphere formulation process is presented, that utilizes coprecipitation of dexamethasone and PLGA. Traditional methods result in heterogeneous distribution of large drug crystals in the microsphere matrix, which in turn results in low drug loading since the drug crystal size is close to that of the microspheres. The modified microsphere preparation method showed homogeneous distribution of dexamethasone, which in turn gave rise to increased drug loading, low burst release, and minimal lag phase. Elimination of the lag phase was dictated from previous work that compared FBR between rats and minipigs. The ability of the coatings to improve implant biocompatibility was successfully tested in vivo via histological examination of explanted tissue from the area surrounding the implants. The biocompatible coatings presented here are suitable for miniaturized implantable devices, such as biosensors, that require constant communication with the local microenvironment.

Formulation and performance of danazol nano-crystalline suspensions and spray dried powders.

PURPOSE: This study focuses on the formulation optimization, in vitro and in vivo performance of differently sized nano-crystalline liquid suspensions and spray-dried powders of a poorly soluble BCS class II compound i.e. Danazol. METHODS: A DoE approach was utilized to optimize stabilizer concentration and formulate danazol (BCS class II) nano-crystalline suspensions and dry powders via wet milling followed by spray drying. Solubility studies were performed to select best stabilizers. Particle size, PXRD, contact angle measurement and in vitro dissolution were utilized in characterization of the liquid and spray-dried powder formulations. RESULTS: The liquid nano-crystalline suspensions followed particle size-dependent dissolution rates i.e. faster dissolution for smaller crystals. The spray-dried nano-crystal powders did not show fast dissolution profiles compared to the liquid nano-crystalline suspension. The poor dissolution of the spray-dried powder correlated to its high LogP value (i.e. LogP 4.53) and poor wetting (or polar surface-area). In vivo bioavailability studies showed superior performance of the liquid nano-crystalline suspensions compared to other milled and un-milled formulations. CONCLUSION: Wet-milling and spray-drying optimization for danazol nano-crystalline suspension was performed. This study indicates that drug candidates with high LogP values and low polar surface area may not be suitable for formulation as dry nano-crystals.

Foreign Body Reaction to Subcutaneous Implants.

Subcutaneously implanted materials trigger the host's innate immune system, resulting in the foreign body reaction. This reaction consists of protein adsorption on the implant surface, inflammatory cell infiltration, macrophage fusion into foreign body giant cells, fibroblast activation and ultimately fibrous encapsulation. This series of events may affect the function of subcutaneous implants, such as inhibition of drug diffusion from long-acting drug delivery depots and medical device failure. The foreign body reaction is a complex phenomenon and is not yet fully understood; ongoing research studies aim to elucidate the cellular and molecular dynamics involved. Recent studies have revealed information about the specific role of macrophages and their differential activation towards pro- and anti-inflammatory states, as well as species differences in the timing of collagen deposition and fibrosis. Understanding of the diverse processes involved in the foreign body reaction has led to multiple approaches towards its negation. Delivery of tissue response modifiers, such as corticosteroids, NSAIDs, antifibrotic agents, and siRNAs, has been used to prevent or minimize fibrosis. Of these, delivery of dexamethasone throughout the implantation period is the most common method to prevent inflammation and fibrosis. More recent approaches employ surface modifications to minimize protein adsorption to 'ultra-low' levels and reduce fibrosis. However, the diverse nature of the processes involved in the foreign body reaction favor the use of corticosteroids due to their wide spectrum action compared to other approaches. To date, combination approaches, such as hydrophilic coatings that reduce protein adsorption combined with delivery of dexamethasone are the most effective.

Freeze-anneal-thaw cycling of unilamellar liposomes: effect on encapsulation efficiency.

PURPOSE: Freeze-thaw cycling is an important processing step in the preparation of liposomes that leads to the encapsulation of drug molecules. There is considerable variability in the number of freeze-thaw cycles reported in the literature. This work is designed to aid in liposomal formulation design by gaining an insight into the drug encapsulation process and an understanding of liposome stabilization during various thawing conditions. METHODS: The effects of different thawing temperatures, as well as "annealing" at subzero temperatures on a liposome formulation, are reported here. RESULTS: Two freeze-anneal-thaw (FANNT) cycles (freezing to -196°C, annealing at -1.4°C for ~30 min, thawing at 65°C) resulted in the maximum predicted encapsulation efficiency without causing any significant change in particle size or zeta potential. Annealing at -22°C was shown to be destabilizing due to limited hydration of the liposomes in the frozen state. CONCLUSIONS: It was shown that two important processes are occurring during the FANNT cycling that affect liposome encapsulation efficiency. The first is drug diffusion in the frozen state and the second is fusion/destabilization of the liposomes. This is the first report on the annealing of liposomes and understanding the mechanism of drug encapsulation using the freeze-thaw cycling method.

Microspheres prepared with PLGA blends for delivery of dexamethasone for implantable medical devices.

PURPOSE: To develop and characterize microspheres using poly (lactic-co-glycolic acid) (PLGA) blends (PLGA5050 (25 KD) and PLGA6535 (70 KD)) for dexamethasone delivery to prevent foreign body response to implantable biosensors. METHODS: A single emulsion based oil/water solvent evaporation/extraction method was used to prepare microspheres. RESULTS: All the microspheres prepared exhibited the typical triphasic release profile, but with different initial burst release, lag phase and zero order release rates. The burst release was reduced when the two PLGA were mixed at a molecular level, whereas increase in burst release was observed when phase separation occurred. Microspheres prepared using PLGA blends had significantly shorter lag phase. The activation energy (Ea) of dexamethasone release from microspheres was similar to the Ea value of PLGA degradation. The release kinetics were significantly enhanced under accelerated conditions (45 and 53°C) without altering the release mechanism of the post-burst phase. A rank order correlation between accelerated and "real-time" release kinetics was observed. CONCLUSIONS: Polymer blends of PLGA can produce microspheres with reduced lag time. The accelerated release testing conditions investigated can discriminate the formulations and predict "real-time" release. Such accelerated release testing can be used as a rapid screening method to facilitate formulation development.

Impact of manufacturing variables on product performance of contraceptive levonorgestrel intrauterine systems.

The development of Levonorgestrel Intrauterine Systems (LNG-IUSs) stands as a formidable challenge due to their intricate design and reliance on specialized manufacturing methods. Pharmaceutical manufacturers face a labyrinth of process variables that demand precise identification and comprehension to establish a robust product design to ensure consistent performance. The current manuscript navigates through this complexity, describing a small-scale processing method for LNG-IUSs via addition and condensation curing processes, as well as investigating the influence of key manufacturing variables on LNG-IUS product performance. Different mixing speeds and time exhibited distinct impact on drug content uniformity within the IUS drug-polymer reservoirs. Surprisingly, no variation in drug release rates were observed. Curing temperature and time were the critical processing parameters of IUSs which were dependent on the polymer type (polydimethylsiloxane, PDMS) and drug loading. At lower curing temperatures, crosslinking in PDMS remained relatively unaffected, irrespective of drug loading. By contrast, elevating curing temperatures resulted in a drastic reduction in PDMS crosslinking densities at higher drug loading. This was attributed to increased drug volume fraction within the matrix, impeding optimal prepolymer chain mobility and rearrangement which is crucial for complete crosslinking. Interestingly, rapid curing led to increased PDMS crystallinity, thereby retarding drug release rates while concurrently compromising mechanical properties. PDMS curing chemistry, such as condensation cure (no filler) and addition cure (cured at room temperature), did not affect drug release rates of the LNG-IUSs. In the condensation cure-based LNG-IUS, the formulations prepared without filler had higher drug release rates than those containing silica or diatomaceous earth fillers. Overall, the present study unravels the intricate interplay between PDMS characteristics, processing variables, and product performance, offering fundamental insights into product design and manufacturing of brand and generic LNG-IUS products.

Tailoring drug release from long-acting contraceptive levonorgestrel intrauterine systems.

The wide array of polydimethylsiloxane (PDMS) variants available on the market, coupled with the intricate combination of additives in silicone polymers, and the incomplete understanding of drug release behavior make formulation development of levonorgestrel intrauterine systems (LNG-IUSs) formidable. Accordingly, the objectives of this work were to investigate the impact of excipients on formulation attributes and in vitro performance of LNG-IUSs, elucidate drug release mechanisms, and thereby improve product understanding. LNG-IUSs with a wide range of additives and fillers were prepared, and in vitro drug release testing was conducted for up to 12 months. Incorporating various additives and/or fillers (silica, silicone resins, silicone oil, PEG, etc.) altered the crystallization kinetics of the crosslinked polymer, the viscosity, and the microstructure. In addition, drug-excipient interactions can occur. Interestingly, additives which increased matrix hydrophobicity and hindered PDMS crystallization facilitated dissolution and permeation of the lipophilic LNG. The influence of additives and lubricants on the mechanical properties of LNG-IUSs were also evaluated. PDMS chemical substitution and molecular weight were deemed to be most critical polymer attributes to the in vitro performance of LNG-IUSs. Drugs with varying physicochemical characteristics were used to prepare IUSs, modeling of the release kinetics was performed, and correlations between release properties and the various physicochemical attributes of the model drugs were established. Strong correlations between first order release rate constants and both drug solubility and Log P underpin the partition and diffusion-based release mechanisms in LNG-IUSs. This is the first comprehensive report to provide a mechanistic understanding of material-property-performance relationships for IUSs. This work offers an evidence-based approach to rational excipient selection and tailoring of drug release to achieve target daily release rates in vivo. The novel insights gained through this research could be helpful for supporting development of brand and generic IUS products as well as their regulatory assessment.

Towards in vitro - In vivo correlation models for in situ forming drug implants.

In vitro-In vivo correlation (IVIVC) is a main focus of the pharmaceutical industry, academia and the regulatory sectors, as this is an effective modelling tool to predict drug product in vivo performance based on in vitro release data and serve as a surrogate for bioequivalence studies, significantly reducing the need for clinical studies. Till now, IVIVCs have not been successfully developed for in situ forming implants due to the significantly different in vitro and in vivo drug release profiles that are typically achieved for these dosage forms. This is not unexpected considering the unique complexity of the drug release mechanisms of these products. Using risperidone in situ forming implants as a model, the current work focuses on: 1) identification of critical attributes of in vitro release testing methods that may contribute to differences in in vitro and in vivo drug release from in situ forming implants; and 2) optimization of the in vitro release method, with the aim of developing Level A IVIVCs for risperidone implants. Dissolution methods based on a novel Teflon shape controlling adapter along with a water non-dissolvable glass fiber membrane (GF/F) instead of a water dissolvable PVA film (named as GF/F-Teflon adapter and PVA-Teflon adapter, respectively), and an in-house fabricated Glass slide adapter were used to investigate the impact of: the surface-to-volume ratio, water uptake ratio, phase separation rate (measured by NMP release in 24 h post injection in vitro or in vivo), and mechanical pressure on the drug release patterns. The surface-to-volume ratio and water uptake were shown to be more critical in vitro release testing method attributes compared to the phase separation rate and mechanical pressure. The Glass slide adapter-based dissolution method, which allowed for the formation of depots with bio-mimicking surface-to-volume ratios and sufficient water uptake, has the ability to generate bio-relevant degradation profiles as well as in vitro release profiles for risperidone implants. For the first time, a Level A IVIVC (rabbit model) has been successfully developed for in situ forming implants. Release data for implant formulations with slightly different PLGA molecular weights (MWs) were used to develop the IVIVC. The predictability of the model passed external validation using the reference listed drug (RLD), Perseris®. IVIVC could not be developed when formulations with different PLGA molar ratios of lactic acid to glycolic acid (L/G) were included. The present work provides a comprehensive understanding of the impact of the testing method attributes on drug release from in situ forming implants, which is a valuable practice for level A IVIVC development.

Manufacturing process of liposomal Formation: A coarse-grained molecular dynamics simulation.

A method of producing liposomes has been previously developed using a continuous manufacturing technology that involves a co-axial turbulent jet in co-flow. In this study, coarse-grained molecular dynamics (CG-MD) simulations were used to gain a deeper understanding of how the self-assembly process of liposomes is affected by the material attributes (such as the concentration of ethanol) and the process parameters (such as temperature), while also providing detailed information on a nano-scale molecular level. Specifically, the CG-MD simulations yield a comprehensive internal view of the structure and formation mechanisms of liposomes containing DPPC, DPPG, and cholesterol molecules. The importance of this work is that structural details on the molecular level are proposed, and such detail is not possible to obtain through experimental studies alone. The assessment of structural properties, including the area per lipid, diffusion coefficient, and order parameters, indicated that a thicker bilayer was observed at higher ethanol concentrations, while a thinner bilayer was present at higher temperatures. These conditions led to more water penetrating the interior of the bilayer and an unstable structure, as indicated by a larger contact area between lipids and water, and a higher coefficient of lipid lateral diffusion. However, stable liposomes were found through these evaluations at lower ethanol concentrations and/or lower process temperatures. Furthermore, the CG-MD model was further compared and validated with experimental and computational data including liposomal bilayer thickness and area per lipid measurements.