Enhancing Solubility and Bioefficacy of Stilbenes by Liposomal Encapsulation-The Case of Macasiamenene F.

Stilbenes in food and medicinal plants have been described as potent antiphlogistic and antioxidant compounds, and therefore, they present an interesting potential for the development of dietary supplements. Among them, macasiamenene F (MF) has recently been shown to be an effective anti-inflammatory and cytoprotective agent that dampens peripheral and CNS inflammation in vitro. Nevertheless, this promising molecule, like other stilbenes and a large percentage of drugs under development, faces poor water solubility, which results in trickier in vivo administration and low bioavailability. With the aim of improving MF solubility and developing a form optimized for in vivo administration, eight types of conventional liposomal nanocarriers and one type of PEGylated liposomes were formulated and characterized. In order to select the appropriate form of MF encapsulation, the safety of MF liposomal formulations was evaluated on THP-1 and THP-1-XBlue-MD2-CD14 monocytes, BV-2 microglia, and primary cortical neurons in culture. Furthermore, the cellular uptake of liposomes and the effect of encapsulation on MF anti-inflammatory effectiveness were evaluated on THP-1-XBlue-MD2-CD14 monocytes and BV-2 microglia. MF (5 mol %) encapsulated in PEGylated liposomes with an average size of 160 nm and polydispersity index of 0.122 was stable, safe, and the most promising form of MF encapsulation keeping its cytoprotective and anti-inflammatory properties.

Development of 3D Printed Multi-Layered Orodispersible Films with Porous Structure Applicable as a Substrate for Inkjet Printing.

The direct tailoring of the size, composition, or number of layers belongs to the advantages of 3D printing employment in producing orodispersible films (ODFs) compared to the frequently utilized solvent casting method. This study aimed to produce porous ODFs as a substrate for medicated ink deposited by a 2D printer. The innovative semi-solid extrusion 3D printing method was employed to produce multilayered ODFs, where the bottom layer assures the mechanical properties. In contrast, the top layer provides a porous structure for ink entrapment. Hydroxypropyl methylcellulose and polyvinyl alcohol were utilized as film-forming polymers, glycerol as a plasticizer, and sodium starch glycolate as a disintegrant in the bottom matrix. Several porogen agents (Aeroperl® 300, Fujisil®, Syloid® 244 FP, Syloid® XDP 3050, Neusilin® S2, Neusilin® US2, and Neusilin® UFL2) acted as porosity enhancers in the two types of top layer. ODFs with satisfactory disintegration time were prepared. The correlation between the porogen content and the mechanical properties was proved. A porous ODF structure was detected in most samples and linked to the porogen content. SSE 3D printing represents a promising preparation method for the production of porous ODFs as substrates for subsequent drug deposition by 2D printing, avoiding the difficulties arising in casting or printing medicated ODFs directly.

Rational Design of Self-Emulsifying Pellet Formulation of Thymol: Technology Development Guided by Molecular-Level Structure Characterization and Ex Vivo Testing.

The growing need for processing natural lipophilic and often volatile substances such as thymol, a promising candidate for topical treatment of intestinal mucosa, led us to the utilization of solid-state nuclear magnetic resonance (ss-NMR) spectroscopy for the rational design of enteric pellets with a thymol self-emulsifying system (SES). The SES (triacylglycerol, Labrasol®, and propylene glycol) provided a stable o/w emulsion with particle size between 1 and 7 µm. The ex vivo experiment confirmed the SES mucosal permeation and thymol delivery to enterocytes. Pellets W90 (MCC, Neusilin®US2, chitosan) were prepared using distilled water (90 g) by the M1−M3 extrusion/spheronisation methods varying in steps number and/or cumulative time. The pellets (705−740 µm) showed mostly comparable properties­zero friability, low intraparticular porosity (0−0.71%), and relatively high density (1.43−1.45%). They exhibited similar thymol release for 6 h (burst effect in 15th min ca. 60%), but its content increased (30−39.6 mg/g) with a shorter process time. The M3-W90 fluid-bed coated pellets (Eudragit®L) prevented undesirable thymol release in stomach conditions (<10% for 3 h). A detailed, ss-NMR investigation revealed structural differences across samples prepared by M1−M3 methods concerning system stability and internal interactions. The suggested formulation and methodology are promising for other lipophilic volatiles in treating intestinal diseases.

Use of droplet-based microfluidic techniques in the preparation of microparticles.

Microparticles are widely used in myriad fields such as pharmaceuticals, foods, cosmetics, and other industrial fields. Compared with traditional methods for synthesizing microparticles, microfluidic techniques provide very powerful platforms for creating highly controllable emulsion droplets as templates for fabricating uniform microparticles with advanced structures and functions. Microfluidic techniques can generate emulsion droplets with precisely controlled size, shape, and composition. A more precise preparation process brings an effective tool to control the release profile of the drug and introduces an easily accessible reproducibility. The paper gives information about basic droplet-based set-ups and examples of attainable microparticle types preparable by this method.

Use of droplet-based microfluidic techniques in the preparation of microparticles.

Microparticles are widely used in myriad fields such as pharmaceuticals, foods, cosmetics, and other industrial fields. Compared with traditional methods for synthesizing microparticles, microfluidic techniques provide very powerful platforms for creating highly controllable emulsion droplets as templates for fabricating uniform microparticles with advanced structures and functions. Microfluidic techniques can generate emulsion droplets with precisely controlled size, shape, and composition. A more precise preparation process brings an effective tool to control the release profile of the drug and introduces an easily accessible reproducibility. The paper gives information about basic droplet-based set-ups and examples of attainable microparticle types preparable by this method.

Hypromellose - A traditional pharmaceutical excipient with modern applications in oral and oromucosal drug delivery.

Hydroxypropylmethylcellulose (HPMC), also known as Hypromellose, is a traditional pharmaceutical excipient widely exploited in oral sustained drug release matrix systems. The choice of numerous viscosity grades and molecular weights available from different manufacturers provides a great variability in its physical-chemical properties and is a basis for its broad successful application in pharmaceutical research, development, and manufacturing. The excellent mucoadhesive properties of HPMC predetermine its use in oromucosal delivery systems including mucoadhesive tablets and films. HPMC also possesses desirable properties for formulating amorphous solid dispersions increasing the oral bioavailability of poorly soluble drugs. Printability and electrospinnability of HPMC are promising features for its application in 3D printed drug products and nanofiber-based drug delivery systems. Nanoparticle-based formulations are extensively explored as antigen and protein carriers for the formulation of oral vaccines, and oral delivery of biologicals including insulin, respectively. HPMC, being a traditional pharmaceutical excipient, has an irreplaceable role in the development of new pharmaceutical technologies, and new drug products leading to continuous manufacturing processes, and personalized medicine. This review firstly provides information on the physical-chemical properties of HPMC and a comprehensive overview of its application in traditional oral drug formulations. Secondly, this review focuses on the application of HPMC in modern pharmaceutical technologies including spray drying, hot-melt extrusion, 3D printing, nanoprecipitation and electrospinning leading to the formulation of printlets, nanoparticle-, microparticle-, and nanofiber-based delivery systems for oral and oromucosal application. Hypromellose is an excellent excipient for formulation of classical dosage forms and advanced drug delivery systems. New methods of hypromellose processing include spray draying, hot-melt extrusion, 3D printing, and electrospinning.

Targeting Human Thrombus by Liposomes Modified with Anti-Fibrin Protein Binders.

Development of tools for direct thrombus imaging represents a key step for diagnosis and treatment of stroke. Nanoliposomal carriers of contrast agents and thrombolytics can be functionalized to target blood thrombi by small protein binders with selectivity for fibrin domains uniquely formed on insoluble fibrin. We employed a highly complex combinatorial library derived from scaffold of 46 amino acid albumin-binding domain (ABD) of streptococcal protein G, and ribosome display, to identify variants recognizing fibrin cloth in human thrombus. We constructed a recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptide of the Bß chain fused to TolA protein. Ribosome display selection followed by large-scale Enzyme-Linked ImmunoSorbent Assay (ELISA) screening provided four protein variants preferentially binding to insoluble form of human fibrin. The most specific binder variant D7 was further modified by C-terminal FLAG/His-Tag or double His-tag for the attachment onto the surface of nanoliposomes via metallochelating bond. D7-His-nanoliposomes were tested using in vitro flow model of coronary artery and their binding to fibrin fibers was demonstrated by confocal and electron microscopy. Thus, we present here the concept of fibrin-targeted binders as a platform for functionalization of nanoliposomes in the development of advanced imaging tools and future theranostics.

Influence of formulation and process parameters on the properties of Cu2+/alginate particles prepared by external ionic gelation evaluated by principal component analysis.

Currently, the method of external ionic gelation for the preparation of alginate particles is successfully used not only in the field of pharmacy and medicine, but also especially in the field of biotechnology. Therefore, the preparation of alginate particles and their subsequent evaluation using principal component analysis was the key task of our experiment. To optimize this method, we focused on the evaluation of the effect of formulation (the polymer concentration, the hardening solution concentration) and process parameters (the outer diameter of the injection needle) on the properties of the resulting beads (yield, sphericity factor, equivalent diameter and swelling capacity at pH 6). Using multivariate data analysis, the major influence on the resulting properties of the prepared particles was confirmed only in sodium alginate concentration. Obtained results verified the reliable and safe potential of the external ionic gelation for preparation alginate-based particulate dosage forms.

Highly Soluble Drugs Directly Granulated by Water Dispersions of Insoluble Eudragit® Polymers as a Part of Hypromellose K100M Matrix Systems.

The aim of the present study was to investigate the suitability of insoluble Eudragit® water dispersions (NE, NM, RL, and RS) for direct high-shear granulation of very soluble levetiracetam in order to decrease its burst effect from HPMC K100M matrices. The process characteristics, ss-NMR analysis, in vitro dissolution behavior, drug release mechanism and kinetics, texture profile analysis of the gel layer, and PCA analysis were explored. An application of water dispersions directly on levetiracetam was feasible only in a multistep process. All prepared formulations exhibited a 12-hour sustained release profile characterized by a reduced burst effect in a concentration-dependent manner. No effect on swelling extent of HPMC K100M was observed in the presence of Eudragit®. Contrary, higher rigidity of formed gel layer was observed using combination of HPMC and Eudragit®. Not only the type and concentration of Eudragit®, but also the presence of the surfactant in water dispersions played a key role in the dissolution characteristics. The dissolution profile close to zero-order kinetic was achieved from the sample containing levetiracetam directly granulated by the water dispersion of Eudragit® NE (5% of solid polymer per tablet) with a relatively high amount of surfactant nonoxynol 100 (1.5%). The initial burst release of drug was reduced to 8.04% in 30 min (a 64.2% decrease) while the total amount of the released drug was retained (97.02%).

N-Oxy lipid-based click chemistry for orthogonal coupling of mannan onto nanoliposomes prepared by microfluidic mixing: Synthesis of lipids, characterisation of mannan-coated nanoliposomes and in vitro stimulation of dendritic cells.

New synthetic aminooxy lipid was designed and synthesized as a building block for the formulation of functionalised nanoliposomes (presenting onto the outer surface of aminooxy groups) by microfluidic mixing. Orthogonal binding of cellular mannan (Candida glabrata (CCY 26-20-1) onto the outer surface of functionalised nanoliposomes was modified by orthogonal binding of reducing termini of mannans to oxime lipids via a click chemistry reaction based on aminooxy coupling (oxime ligation). The aminooxy lipid was proved as a suitable active component for preparation of functionalised nanoliposomes by the microfluidic mixing method performed with the instrument NanoAssemblr™. This "on-chip technology" can be easily scaled-up. The structure of mannan-liposomes was visualized by transmission and scanning electron microscopy, including immunogold staining of recombinant mannan receptor bound onto mannosylated-liposomes. The observed structures are in a good correlation with data obtained by DLS, NTA, and TPRS methods. In vitro experiments on human and mouse dendritic cells demonstrate selective internalisation of fluorochrome-labelled mannan-liposomes and their ability to stimulate DC comparable to lipopolysaccharide. We describe a potentially new drug delivery platform for mannan receptor-targeted antimicrobial drugs as well as for immunotherapeutics. Furthermore, the platform based on mannans bound orthogonally onto the surface of nanoliposomes represents a self-adjuvanted carrier for construction of liposome-based recombinant vaccines for both systemic and mucosal routes of administration.

Multi-layered nanofibrous mucoadhesive films for buccal and sublingual administration of drug-delivery and vaccination nanoparticles - important step towards effective mucosal vaccines.

Nanofibre-based mucoadhesive films were invented for oromucosal administration of nanocarriers used for delivery of drugs and vaccines. The mucoadhesive film consists of an electrospun nanofibrous reservoir layer, a mucoadhesive film layer and a protective backing layer. The mucoadhesive layer is responsible for tight adhesion of the whole system to the oral mucosa after application. The electrospun nanofibrous reservoir layer is intended to act as a reservoir for polymeric and lipid-based nanoparticles, liposomes, virosomes, virus-like particles, dendrimers and the like, plus macromolecular drugs, antigens and/or allergens. The extremely large surface area of nanofibrous reservoir layers allows high levels of nanoparticle loading. Nanoparticles can either be reversibly adsorbed to the surface of nanofibres or they can be deposited in the pores between the nanofibres. After mucosal application, nanofibrous reservoir layers are intended to promote prolonged release of nanoparticles into the submucosal tissue. Reversible adsorption of model nanoparticles as well as sufficient mucoadhesive properties were demonstrated. This novel system appears appropriate for the use in oral mucosa, especially for sublingual and buccal tissues. To prove this concept, trans-/intramucosal and lymph-node delivery of PLGA-PEG nanoparticles was demonstrated in a porcine model. This system can mainly be used for sublingual immunization and the development of "printed vaccine technology".

Influence of pH Modulation on Dynamic Behavior of Gel Layer and Release of Weakly Basic Drug from HPMC/Wax Matrices, Controlled by Acidic Modifiers Evaluated by Multivariate Data Analysis.

The solubility of weakly basic drugs in passage through gastrointestinal tract leads to their pH-dependent release from extended release formulations and to lower drug absorption and bioavailability. The aim of this study was to modulate the micro-environmental pH of hypromellose/montanglycol wax matrices and to observe its influence on the release of weakly basic drug verapamil hydrochloride (VH) with a pH-dependent solubility with respect to gel layer formation and its dynamics. For this study, malic and succinic acids differing in their solubility and pKa were selected as pH modifiers. The dissolution studies were performed by the method of changing pH. Within the same conditions, pH, thickness, and penetration force of the gel layer were measured as well. From the PCA sub-model, it is evident that a higher acid concentration ensured lower gel pH and conditions for higher drug solubility, thus creating larger gel layer with smaller rigidity, resulting in higher VH release during the dissolution test. Incorporation of stronger and more soluble malic acid (100 mg/tablet) created the most acidic and the thickest gel layer through which a total of 74% of VH was released. Despite having lower strength and solubility, matrices containing succinic acid (100 mg/tablet) released a comparable 71% of VH in a manner close to zero-order kinetics. The thinner and less rigid gel layers of the succinic acid matrices allowed an even slightly faster VH release at pH 6.8 than from matrices containing malic acid. Thus acid solubility is more parametrically significant than acid pKa for drug release at pH 6.8.

Influence of different formulations and process parameters during the preparation of drug-loaded PLGA microspheres evaluated by multivariate data analysis.

The main objective of this study was to evaluate the influence of the formulation and process parameters on PLGA microparticles containing a practically insoluble model drug (ibuprofen) prepared by the o/w solvent evaporation method. Multivariate data analysis was used. The effects of altered stirring speed of a mechanical stirrer (600, 1000 rpm), emulsifier concentrations (PVA concentration 0.1 %, 1 %) and solvent selection (dichloromethane, ethyl acetate) on microparticle characteristics (encapsulation efficiency, drug loading, burst effect) were observed. It was found that with increased stirring speed, the PVA concentration or the use of ethyl acetate had a significantly negative effect on encapsulation efficiency. In addition, ethyl acetate had an adverse effect on the burst effect, while increased stirring speed had the opposite effect. Drug load was not affected by any particular variable, but rather by the interactions of evaluated variables.

The effect of acid pH modifiers on the release characteristics of weakly basic drug from hydrophlilic-lipophilic matrices.

The solubility of weakly basic drugs within passage though GI tract leads to pH-dependent or even incomplete release of these drugs from extended release formulations and consequently to lower drug absorption and bioavailability. The aim of the study was to prepare and evaluate hydrophilic-lipophilic (hypromellose-montanglycol wax) matrix tablets ensuring the pH-independent delivery of the weakly basic drug verapamil-hydrochloride by an incorporation of three organic acidifiers (citric, fumaric, and itaconic acids) differing in their concentrations, pK a, and solubility. The dissolution studies were performed by the method of changing pH values, which better corresponded to the real conditions in the GI tract (2 h at pH 1.2 and then 10 h at pH 6.8). Within the same conditions, pH of matrix microenvironment was measured. To determine relationships between the above mentioned properties of acidifiers and the monitored effects (the amount of released drug and surface pH of gel layer in selected time intervals-360 and 480 min), the full factorial design method and partial least squares PLS-2 regression were used. The incorporation of the tested pH modifiers significantly increased the drug release rate from matrices. PLS-components explained 75% and 73% variation in the X- and Y-data, respectively. The obtained results indicated that the main crucial points (p < 0.01) were the concentration and strength of acidifier incorporated into the matrix. Contrary, the acid solubility surprisingly did not influence the selected effects except for the surface pH of gel layer in time 480 min.