Nasal-PAMPA: A novel non-cell-based high throughput screening assay for prediction of nasal drug permeability.

In nasal drug product development, screening studies are vital to select promising compounds or formulations. The Parallel Artificial Membrane Permeability Assay (PAMPA), a high throughput screening tool, has been applied to evaluate drug permeability across several barriers such as the skin or blood-brain barrier. Herein, a new nasal-PAMPA model was optimized to predict nasal permeability, using a biorelevant donor medium containing mucin. The apparent permeability (Papp) of 15 reference compounds was assessed in six different experimental conditions, and the most discriminating and predictive model was applied to a test drug (piroxicam) and mucoadhesive powder formulations loading the same drug. The model with 0.5% (w/v) mucin in the donor compartment and 2% (w/v) phosphatidylcholine in the lipid membrane accurately distinguished high and low permeable compounds. Additionally, it exhibited the highest correlation with permeation across human nasal epithelial cells, RPMI 2650 (R2 = 0.93). When applied to powder formulations, this model was sensitive to the presence of mucoadhesive excipients and the drug solid state. Overall, the nasal-PAMPA model was more rapid than cell-based assays, without requiring specialized training or equipment, showing to be a promising in vitro tool that can be applied in drug and formulation screening for nasal delivery.

Spray dried powders for nasal delivery: Process and formulation considerations.

Powders for nasal delivery have been recognized as advantageous dosage forms over liquids due to increased stability and residence time on nasal mucosa, with improved bioavailability. They can be manufactured by spray-drying, allowing the optimization of the particle properties that are critical to guarantee nasal deposition, as size and shape. It is also a scalable and flexible method already explored extensively in the pharmaceutical industry. However, it is important to understand how process parameters, particle physical properties and formulation considerations affect the product performance. Hence, this review aims to provide an overview of nasal powder formulation and processing through spray drying, with an emphasis on the variables that impact on performance. To this purpose, we describe the physical, biological and pharmacological phenomena prior to drug absorption as well as the most relevant powder properties. Formulation considerations including qualitative and quantitative composition are then reviewed, as well as manufacturing considerations including spray drying relevant parameters.

QbD approach to downstream processing of spray-dried amorphous solid dispersions - a case study.

In the current study, we demonstrate a structured approach to downstream process development for spray dried amorphous solid dispersions. Direct compression is generally not suitable due to typically poor flow of spray dried powders in tablets. Roller compaction (RC) is therefore the method of choice to enable spray dried dispersion downstream processing. Here, a structured experimental design of RC process parameters was used. The objective was to identify process conditions that lead to improved powder flow without compromising tablet robustness. Ten blends were compacted using different process parameters, and subsequently compressed into tablets. The impact of process parameters on granules and tablet properties was analyzed. We demonstrate that compaction force, gap and mesh aperture have major impact on RC outcomes. A combination of large gap and low force was identified as optimum combination of RC process parameters leading to powder flow improvement that could guarantee low tablet weight variation and at the same prevented loss of blend compressibility.

Cationic Surfactants: Self-Assembly, Structure-Activity Correlation and Their Biological Applications.

The development of biotechnological protocols based on cationic surfactants is a modern trend focusing on the fabrication of antimicrobial and bioimaging agents, supramolecular catalysts, stabilizers of nanoparticles, and especially drug and gene nanocarriers. The main emphasis given to the design of novel ecologically friendly and biocompatible cationic surfactants makes it possible to avoid the drawbacks of nanoformulations preventing their entry to clinical trials. To solve the problem of toxicity various ways are proposed, including the use of mixed composition with nontoxic nonionic surfactants and/or hydrotropic agents, design of amphiphilic compounds bearing natural or cleavable fragments. Essential advantages of cationic surfactants are the structural diversity of their head groups allowing of chemical modification and introduction of desirable moiety to answer the green chemistry criteria. The latter can be exemplified by the design of novel families of ecological friendly cleavable surfactants, with improved biodegradability, amphiphiles with natural fragments, and geminis with low aggregation threshold. Importantly, the development of amphiphilic nanocarriers for drug delivery allows understanding the correlation between the chemical structure of surfactants, their aggregation behavior, and their functional activity. This review focuses on several aspects related to the synthesis of innovative cationic surfactants and their broad biological applications including antimicrobial activity, solubilization of hydrophobic drugs, complexation with DNA, and catalytic effect toward important biochemical reaction.

Key production parameters for the development of solid lipid nanoparticles by high shear homogenization.

In this work, we report the development and optimization of solid lipid nanoparticles (SLN) production by a simple, fast, and cost-effective high shear homogenization process. A screening of several solid lipids (Compritol 888 ATO, Precirol ATO 5, Cetyl Palmitate, Dynasan 118, Imwitor 900K, Stearic acid) has been carried out in combination with Poloxamer 188 as the selected surfactant, based on the mean particle size and polydispersity index. The improvement of the physical stability of the SLN dispersions was achieved by the use of a cationic lipid (cetyl trimethylammonium bromide) reaching zeta potential values above +60 mV. Combining the optimized speed and time of shear, monodispersed SLN (PdI < 0.25) under the nanometer range could be produced.

Surface-tailored anti-HER2/neu-solid lipid nanoparticles for site-specific targeting MCF-7 and BT-474 breast cancer cells.

CAB51, a compact antibody against human epithelial growth receptor 2 (HER2, ErbB2), has been linked to cationic Solid Lipid Nanoparticles (SLN) via streptavidin-biotin interaction and their targeting potential evaluated against breast cancer cells. The amount of streptavidin and biotinylated antibody was optimised by monitoring the mean complex size (intensity weighed average diameter), polydispersity index and immediate stability in phosphate buffer saline (PBS). The effect on MCF-7 and BT-474 cells was evaluated at concentrations of 0.01 mg/mL and 0.1 mg/mL (counted as solid lipid). Streptavidin adsorption onto SLN surface had no influence on cell viability. Linking the antibody showed a synergistic effect on cell viability at lowest concentration tested (0.01 mg/mL) which was lower than that observed after exposure to SLN alone or antibody alone. At the higher tested concentration (0.1 mg/mL), the observed toxicity was entirely governed by the inherent toxicity of the SLN themselves. Streptavidin adsorption had no effect on accumulation in cells, while the antibody-containing complexes showed clearly increased internalisation in both cell lines. In HER2/neu positive BT-474 higher internalisation was observed than in HER2/neu negative MCF-7.

Hansen solubility parameters (HSP) for prescreening formulation of solid lipid nanoparticles (SLN): in vitro testing of curcumin-loaded SLN in MCF-7 and BT-474 cell lines.

Curcumin, a phenolic compound from turmeric rhizome (Curcuma longa), has many interesting pharmacological effects, but shows very low aqueous solubility. Consequently, several drug delivery systems based on polymeric and lipid raw materials have been proposed to increase its bioavailability. Solid lipid nanoparticles (SLN), consisting of solid lipid matrix and a surfactant layer can load poorly water-soluble drugs, such as curcumin, deliver them at defined rates and enhance their intracellular uptake. In the present work, we demonstrate that, despite the drug's affinity to lipids frequently used in SLN production, the curcumin amount loaded in most SLN formulations may be too low to exhibit anticancer properties. The predictive curcumin solubility in solid lipids has been thoroughly analyzed by Hansen solubility parameters, in parallel with the lipid-screening solubility tests for a range of selected lipids. We identified the most suitable lipid materials for curcumin-loaded SLN, producing physicochemically stable particles with high encapsulation efficiency (>90%). Loading capacity of curcumin in SLN allowed preventing the cellular damage caused by cationic SLN on MCF-7 and BT-474 cells but was not sufficient to exhibit drug's anticancer properties. But curcumin-loaded SLN exhibited antioxidant properties, substantiating the conclusions that curcumin's effect in cancer cells is highly dose dependent.

Preclinical safety of solid lipid nanoparticles and nanostructured lipid carriers: Current evidence from in vitro and in vivo evaluation.

Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) were designed as exceptionally safe colloidal carriers for the delivery of poorly soluble drugs. SLN/NLC have the particularity of being composed of excipientsalready approved for use in medicines for human use, which offers a great advantage over any other nanoparticulate system developed from novel materials. Despite this fact, any use of excipients in new route of administration or in new dosage form requires evidence of safety. After 25 years of research on SLN and NLC, enough evidence on their preclinical safety has been published. In the present work, published data on in vitro and in vivo compatibility of SLN/NLC have been surveyed, in order to provide evidence of high biocompatibility distinguished by intended administration route. We also identified critical factors and possible weak points in SLN/NLC formulations, such as the effect of surfactants on the cell viability in vitro, which should be considered for further development.

Looking out for cancer stem cells' properties: the value-driving role of CD44 for personalized medicines.

The expression of CD44 tags cells with stemness-associated properties (cancer initiating cells or cancer stem cells - CSC). This membrane glycoprotein with a cytoplasmic domain indirectly associated with the cellular cytoskeleton, has a crucial role in tumorigenesis. The CD44 receptor enables the cell to respond to changes in tumor microenvironment, promoting several signaling events related to tumor initiation, progression and fixation in distant host tissues. Although the contribution of this transmembrane protein in gene regulation remains unclear, its overexpression in adenocarcinomas, mostly supported by microRNA (miR)-mediated upregulation of target mRNA, is widely accepted. Herein, we gather the evidence that CD44 is one of the most predominant markers of malignant cells and may be found in diverse phenotypes associated with tumor progression. Additionally, CD44 tumor receptors were found to have different roles at a transcriptional level. Thus, innovative therapeutic strategies should rely heavily on its metastasis-promoting ability. Furthermore, the concept of selectively targeting cell sub-populations may be used to develop specific therapeutic and/or diagnostic systems. An approach based on targeting CD44⁺ cells might provide a strategy to design guided-therapeutic systems against multiple malignant cells including putative CSC.

Surface engineering of silica nanoparticles for oral insulin delivery: characterization and cell toxicity studies.

The present work aimed at studying the interaction between insulin and SiNP surfaced with mucoadhesive polymers (chitosan, sodium alginate or polyethylene glycol) and the evaluation of their biocompatibility with HepG2 and Caco-2 cell lines, which mimic in vivo the target of insulin-loaded nanoparticles upon oral administration. Thus, a systematic physicochemical study of the surface-modified insulin-silica nanoparticles (Ins-SiNP) using mucoadhesive polymers has been described. The surfacing of nanoparticle involved the coating of silica nanoparticles (SiNP) with different mucoadhesive polymers, to achieve high contact between the systems and the gut mucosa to enhance the oral insulin bioavailability. SiNP were prepared by a modified Stöber method at room temperature via hydrolysis and condensation of tetraethyl orthosilicate (TEOS). Interaction between insulin and nanoparticles was assessed by differential scanning calorimetry (DSC), X-ray and Fourier-transform infrared (FTIR) studies. The high efficiency of nanoparticles' coating resulted in more stable system. FTIR spectra of insulin-loaded nanoparticles showed amide absorption bands which are characteristic of α-helix content. In general, all developed nanoparticles demonstrated high biocompatible, at the tested concentrations (50-500 µg/mL), revealing no or low toxicity in the two human cancer cell lines (HepG2 and Caco-2). In conclusion, the developed insulin-loaded SiNP surfaced with mucoadhesive polymers demonstrated its added value for oral administration of proteins.

Cationic solid lipid nanoparticles interfere with the activity of antioxidant enzymes in hepatocellular carcinoma cells.

Solid lipid nanoparticles (SLN) are colloidal drug and/or gene carriers developed from solid lipids and surfactants that are considered safe. Cationic SLN, usually used for formulating poorly water-soluble drugs and for gene delivery purposes, as positively charged particles may attach to cellular surfaces and be internalized more easily than negatively charged SLN, but they can also cause damage. The main aim of this work was to test a set of cationic SLN and investigate its influence on the amount of reactive oxygen species (ROS), on antioxidant enzymes activities and on possible oxidative damage to membrane lipids in HepG2 cells. The Dichlorofluorescein assay revealed great increase in ROS presence after cell exposure to SLN. While the exposure to SLN increased the activities of superoxide dismutase and glutathione peroxidase it decreased glutathione reductase activity. Although no significant increase in thiobarbituric reactive species was found, a decrease in sulfhydryl groups was detected. These results indicate that cationic SLN caused oxidative stress in HepG2 cells, but under reported exposure conditions HepG2 cells could attenuate the stress and thus the damage to cellular components was minimal.

Nanotoxicology applied to solid lipid nanoparticles and nanostructured lipid carriers - a systematic review of in vitro data.

Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) were developed as alternative to other colloidal carriers. They were designed to overcome lipid nanoemulsions and liposomes in stability and ability to control the release of an encapsulated substance, and at the same time to be better tolerated than polymeric nanoparticles. Since the patenting of SLN discovery, large amount of data became available on the behaviour of these systems in vitro. SLN/NLC have many prerequisites to be a well tolerated carrier - the currently available data seem to confirm it, but there are also some contradictory results. In this review, we collected the available data from cytotoxicity, oxidative stress and hemocompatibility studies in vitro and analysed their outcomes. We also provide a summary of the available data in a form of reference table.

Comet assay reveals no genotoxicity risk of cationic solid lipid nanoparticles.

Cationic solid lipid nanoparticles (cSLN) are colloidal carriers for genes or drugs, particularly lipophilic drugs. Several reports exist on their high efficiency, but only a few studies report the effect of cSLNs on living cells. In the present work, internalization, cell viability (alamar blue assay) and genotoxic potential (alkaline comet assay) of three cSLN formulations (A-C) were evaluated in HepG2 and Caco-2 cells. cSLN showed an average hydrodynamic diameter (z-ave) of 141-222 nm, zeta-potential of 55.0-72.5 mV and polidispersity indices (PdI) of 0.336-0.421. Dispersion in physiological buffers increased z-ave and PdI. 0.01 mg ml(-1) cSLN unaffected cell viability, but 1.0 mg ml(-1) significantly decreased it, being cSLN-C (Compritol-based) the most toxic and HepG2 the most affected. DNA damage was not significantly increased by 0.1 mg ml(-1) cSLN but damage was observed at 1.0 mg ml(-1) cSLN-C. Thus, no genotoxicity is to be expected at concentrations that do not reduce cell viability.

Trehalose is not a universal solution for solid lipid nanoparticles freeze-drying.

OBJECTIVE: To prepare stable and easy to handle formulation of solid lipid nanoparticles (SLNs) by freeze-drying with or without cryoprotectants, as appropriate. MATERIALS AND METHODS: SLNs were freeze-dried without cryoprotectants or with cryoprotectants in quantities selected by freeze-thaw test (sucrose, glucose) or literature search (trehalose, maltose). Appearance, re-dispersability and size distribution of re-dispersed samples were evaluated. RESULTS: SLN could be freeze-dried using 10% sucrose, trehalose or maltose. Trehalose was effective in protecting one of presented formulations that was already very stable on its own; its efficiency in protecting other two formulations was limited. DISCUSSION: Our results are in line with various reports of successful freeze-drying of SLN, but considering the stability of original dispersions, no improvement was achieved. CONCLUSION: We confirmed that trehalose is among the most suitable cryoprotectant for SLN, however it did not improve shelf-life of the most stable formulation.

Chain-length dependence of insertion, desorption, and translocation of a homologous series of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled aliphatic amines in membranes.

We present a complete characterization of the kinetics of interaction between the homologous series of fluorescent fatty amines with the fluorescent moiety 7-nitrobenz-2-oxa-1,3-diazol-4-yl covalently bound to the amine group, NBD-C(n) (n = 8-16), and a lipid bilayer in the liquid disordered phase. The insertion into and the desorption from the lipid bilayer, as well as the rate of translocation across the two bilayer leaflets, has been measured at different temperatures, allowing an estimation of the thermodynamic parameters in the formation of the transition state. This is the first report on the complete characterization of the kinetics of the interaction of a large series of structurally homologous amphiphiles. In a recent paper from this research group, the equilibrium interaction of NBD-C(n) (n = 4-10) with POPC bilayers and serum albumin was reported. This information allows the calculation of the equilibrium distribution of the amphiphiles among the aqueous phase, serum proteins, and biomembranes. The data presented in this manuscript complement its characterization with information on the kinetics of the interactions, making possible the quantitative evaluation of their pharmacokinetics. The rate of translocation is shown to decrease with increasing alkyl chain length up to n = 12, becoming relatively insensitive to further increases in n. The Gibbs free energy variation associated with the rate of desorption from the lipid bilayer increased linearly with n, with ΔΔG(‡o) = 3.4 ± 0.5 kJ mol(-1) per methylene group. It was also found that the process of insertion in the lipid bilayer is not diffusion-limited, although it is close to this limit for the smaller amphiphiles in the homologous series at high temperatures.

The effects of cholesterol and ß-sitosterol on the structure of saturated diacylphosphatidylcholine bilayers.

The structures of DMPC and DPPC bilayers in unilamellar liposomes, in the presence of 33.3 mol% cholesterol or the plant sterol ß-sitosterol, have been studied by small-angle neutron scattering. The bilayer thickness d(L) increases in a similar way for both sterols. The repeat distance in multilamellar liposomes, as determined by small-angle X-ray diffraction, is larger in the presence of ß-sitosterol than in the presence of cholesterol. We observe that each sterol modifies the interlamellar water layer differently, cholesterol reducing its thickness more efficiently than ß-sitosterol, and conclude that cholesterol suppresses bilayer undulations more effectively than ß-sitosterol.

Feasibility of lipid nanoparticles for ocular delivery of anti-inflammatory drugs.

Due to the multiple barriers imposed by the eye against the penetration of drugs, the ocular delivery and targeting are considered difficult to achieve. A major challenge in ocular drug therapy is to improve the poor bioavailability of topically applied ophthalmic drugs by overcoming the severe constraints imposed by the eye on drug absorption. One of the promising strategies nowadays is the use of colloidal carrier systems characterized by a submicron-meter size. Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) represent promising alternatives to conventional and very popular ocular carrier systems, such as the nanoemulsions, liposomes, and polymeric nanoparticles. Nevertheless, taking into account the characteristics of the eye, morphometrical properties of the colloidal systems (e.g., average particle size and polydispersion) may represent a limiting factor for topical application without induced corneal irritation, being responsible for the selected system. This review article focuses on the application of lipid nanoparticles (SLN, NLC) as carriers for both non-steroidal and steroidal anti-inflammatory drugs for the treatment of ocular inflammatory disorders. Major benefits, as well as shortcomings, of ocular inflammation conditions are described, in particular upon management of inflammation induced by ocular surgery (e.g., cataracts, refractive surgery). Particular emphasis is given to the clinical choices currently available, while examining the most recent drugs that have been approved.

Formulating fluticasone propionate in novel PEG-containing nanostructured lipid carriers (PEG-NLC).

The aim of this study was to develop nanostructured lipid carriers (NLC) for topical delivery of fluticasone propionate (FP) with the aim to further improve the safety profile and decrease the adverse-side effects commonly reported in topical corticotherapy. NLC are colloidal drug-carriers consisting of a blend of a solid lipid and a small amount of liquid lipid since these carriers have proved to be effective in epidermal targeting in particular of glucocorticoids. NLC consisting of glyceryl palmito-stearate, and PEG-containing medium chain triglycerides mixture, stabilised by polysorbate 80 and soybean phosphatidylcholine were prepared. A mean particle size between 380 and 408 nm and entrapment efficacy of 95% were obtained for FP-loaded NLC. The crystallinity and polymorphic phase behaviour of FP-free and FP-loaded NLC were examined by differential scanning calorimetry and wide angle X-ray diffraction. Results revealed a low-crystalline structure and confirmed the incorporation of FP into the particles. The suitability of PEG-containing liquid lipids to form the lipid matrix of NLC was also confirmed.

Chapter 6 - Solid lipid nanoparticle formulations pharmacokinetic and biopharmaceutical aspects in drug delivery.

Solid lipid nanoparticles (SLNs) have emerged as important tools to modify the release profile for a large number of drugs including protein and peptide molecules. SLNs are produced from biocompatible and biodegradable lipid materials, making them a promising therapeutic strategy for drug targeting and delivery, and surmounting the inherent limitations of regulation acceptance. Due to their versatility in loading both lipophilic and hydrophilic molecules in the solid lipid matrix, SLNs depict the ability to prolong, extend or sustain the release profile of the loaded molecules, therefore reducing the repeated administration, and increasing the therapeutic value of a certain treatment. Additional advantages include reduction of drug toxicity and increase of drug bioavailability. To develop SLN formulations for drug targeting and delivery, a basic pharmacokinetic understanding of drug distribution is of major relevance, as well as the biopharmaceutical aspects of the administration route. This chapter provides a fundamental understanding of the pharmacokinetic properties of SLNs, which influence both biopharmaceutical and clinical profiles of the loaded molecules.

Nanostructured lipid carrier-based hydrogel formulations for drug delivery: a comprehensive review.

The scientific literature today provides several systems that can deliver active pharmaceutical ingredients (APIs) across the skin. These include reservoir matrices, matrix diffusion-controlled devices, multiple polymer devices and multilayer matrix assemblies. Among these, nanostructured lipid carriers (NLC) have emerged as novel systems composed of physiological lipid materials suitable for topical, dermal and transdermal administration. This review focuses on the design characteristics, production and composition of semi-solid formulations containing NLC as API carriers. One of the useful semi-solid systems are hydrogels, which can be used as vehicles to provide appropriate consistency for NLC formulations to be applied onto the skin. In the present review recent developments in the field are highlighted, including examples of APIs successfully entrapped within NLC now amenable for delivery via the skin. Further innovations in NLC composition and formulation, as well as in semi-solid hydrogel assemblies, are likely to expand the number of APIs available for topical, dermal and transdermal delivery.