Progress in Topical and Transdermal Drug Delivery Research—Focus on Nanoformulations

Skin is the largest organ and a multifunctional interface between the body and its environment. It acts as a barrier against cold, heat, injuries, infections, chemicals, radiations or other exogeneous factors, and it is also known as the mirror of the soul. The skin is involved in body temperature regulation by the storage of fat and water. It is an interesting tissue in regard to the local and transdermal application of active ingredients for prevention or treatment of pathological conditions. Topical and transdermal delivery is an emerging route of drug and cosmetic administration. It is beneficial for avoiding side effects and rapid metabolism. Many pharmaceutical, technological and cosmetic innovations have been described and patented recently in the field. In this review, the main features of skin morphology and physiology are presented and are being followed by the description of classical and novel nanoparticulate dermal and transdermal drug formulations. The biophysical aspects of the penetration of drugs and cosmetics into or across the dermal barrier and their investigation in diffusion chambers, skin-on-a-chip devices, high-throughput measuring systems or with advanced analytical techniques are also shown. The current knowledge about mathematical modeling of skin penetration and the future perspectives are briefly discussed in the end, all also involving nanoparticulated systems

ex vivo–in vivo comparison of drug penetration analysis by confocal Raman microspectroscopy and tape stripping

When it comes to skin penetration analysis of a topically applied formulation, the number of suitable methods is limited, and they often lack in spatial resolution. In vivo studies are pivotal, especially in the approval of a new product, but high costs and ethical difficulties are limiting factors. For that reason, good ex vivo models for testing skin penetration are crucial. In this study, caffeine was used as a hydrophilic model drug, applied as a 2% (w/w) hydrogel, to compare different techniques for skin penetration analysis. Confocal Raman microspectroscopy (CRM) and tape stripping with subsequent HPLC analysis were used to quantify caffeine. Experiments were performed ex vivo and in vivo. Furthermore, the effect of 5% (w/w) 1,2-pentanediol on caffeine skin penetration was tested, to compare those methods regarding their effectiveness in detecting differences between both formulations

Curcumin Nanonization Using An Alternative Small-Scale Production Unit: Selection of Proper Stabilizer Applying Basic Physicochemical Consideration and Biological Activity Assessment of Nanocrystals

As the number of poorly soluble drugs is increasing, nanocrystals have become very interesting due to wide range of application possibilities. Curcuminwas used as a model active ingredient in this work. Even though it has many proven positive effects, due to its physicochemical issues, its possibilities have not been fully exploited. The goal of this work was to select optimal conditions for a top-down method for curcumin nanosuspension production, and to perform their comprehensive characterization applying complementary methodologies: dynamic light scattering, polarization and atomic force microscopy, thermal analysis, X-ray powder diffraction, antioxidant activity evaluation, release kinetics assessment, and screening of potential biological effects applying cell viability assays on normal human lung fibroblasts, human melanoma and human adenomacarcinoma cells. After 30 min of milling, nanosuspensions stabilized by polysorbate 80 and by its combinations with sucrose palmitate showed good stability, while curcumin crystal structure was unaltered. Obtained nanocrystals were well defined, with average diameter 120-170 nm and PDI of about 0.25, zeta potential was below -30 mV and pH~5 for all formulations. Nanodispersions exhibited high antioxidant potential and improved dissolution rate compared to the corresponding coarse dispersions. Although curcumin nanodispersions exhibited significant antiproliferative effect to each cancer cell line, the highest effect was towards adenocarcinoma cells

Monitoring Dermal Penetration and Permeation Kinetics of Topical Products; the Role of Raman Microspectroscopy

The study of human skin represents an important area of research and development in dermatology, toxicology, pharmacology and cosmetology, in order to assess the effects of exogenous agents, their interaction, their absorption mechanism, and/or their toxicity towards the different cutaneous structures. The processes can be parameterised by mathematical models of diffusion, of varying degrees of complexity, and are commonly measured by Franz cell diffusion, in vitro, and tape stripping, in vitro or in vivo, techniques which are recognised by regulatory bodies for commercialisation of dermally applied products. These techniques do not directly provide chemically specific measurement of the penetration and/or permeation of formulations in situ, however. Raman microspectroscopy provides a non-destructive, non-invasive and chemically specific methodology for in vitro, and in vivo investigations, in-situ, and can provide a powerful alternative to the current gold standard methods approved by regulatory bodies. This review provides an analysis of the current state of art of the field of monitoring dermal penetration and permeation kinetics of topical products, in vitro and in vivo, as well as the regulatory requirements of international guidelines governing them. It furthermore outlines developments in the analysis of skin using Raman microspectroscopy, towards the most recent demonstrations of quantitative monitoring of the penetration and permeation kinetics of topical products in situ, for in vitro and in vivo applications, before discussing the challenges and future perspectives of the field

Freeze-dried nanocrystal dispersion of novel deuterated pyrazoloquinolinone ligand (DK-I-56-1): Process parameters and lyoprotectant selection through the stability study

Recently, nanocrystal dispersions have been considered as a promising formulation strategy to improve the bioavailability of the deuterated pyrazoloquinolinone ligand DK-I-56-1 (7‑methoxy-2-(4‑methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one). In the current study, the freeze-drying process (formulation and process parameters) was investigated to improve the storage stability of the previously developed formulation. Different combinations of lyoprotectant (sucrose or trehalose) and bulking agent (mannitol) were varied while formulations were freeze-dried under two conditions (primary drying at -10 or -45 °C). The obtained lyophilizates were characterized in terms of particle size, solid state properties and morphology, while the interactions within the samples were analyzed by Fourier transform infrared spectroscopy. In the preliminary study, three formulations were selected based on the high redispersibility index values (around 95%). The temperature of primary drying had no significant effect on particle size, but stability during storage was impaired for samples dried at -10 °C. Samples dried at lower temperature were more homogeneous and remained stable for three months. It was found that the optimal ratio of sucrose or trehalose to mannitol was 3:2 at a total concentration of 10% to achieve the best stability (particle size < 1.0 μm, polydispersity index < 0.250). The amorphous state of lyoprotectants probably provided a high degree of interaction with nanocrystals, while the crystalline mannitol provided an elegant cake structure. Sucrose was superior to trehalose in maintaining particle size during freeze-drying, while trehalose was more effective in keeping particle size within limits during storage. In conclusion, results demonstrated that the appropriate combination of sucrose/trehalose and mannitol together with the appropriate selection of lyophilization process parameters could yield nanocrystals with satisfactory stability

Freeze-dried nanocrystal dispersion of novel deuterated pyrazoloquinolinone ligand (DK-I-56-1); process parameters and cryoprotectant selection through stability study

1. INTRODUCTION Nanocrystal dispersions are considered as the universal formulation strategy for brick dust substances. However, the stability of these systems to aggregation represents a big issue. To overcome this, nanocrystal dispersions are usually solidified by freeze-drying (lyophilization). During this process the risk of aggregation is considered to be high, due to ice formation and/or water loss. To prevent the aggregation, For the particle size preservation, therefore, it is necessary to add cryoprotectants/lyoprotectants, among which sugars are most commonly used. To ensure good structure of the cake, bulking agents are often included in formulations, as well [1,2], although in nanocrystalline dispersions the combination of cryoprotectants and bulking agents is not frequent nor much investigated. Nanocrystals of DK-I-56-1 (7‑methoxy‑2-(4‑methoxy‑d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one), patent protected pyrazoloquinolinone ligand, have been developed recently, and characterized in terms of physicochemical properties and pharmacokinetics after intraperitoneal administration in mice. These formulations were stable for three weeks [3]. Our aim in this study was to improve the stability by freeze-drying, and investigate the influence of different concentrations and physical form of cryoprotectants (sucrose, trehalose) and bulking agent (mannitol) as well as different primary drying conditions on the aggregation prevention. 2. MATERIALS AND METHODS 2.1. Materials DK-I-56-1 was synthesized at the Department of Chemistry and Biochemistry, University of Wisconsin—Milwaukee, USA. The following other materials were used: polysorbate 80, poloxamer 407, sucrose, mannitol (Sigma-Aldrich Laborchemikalien GmbH, Germany) and trehalose (Carl Roth GmbH, Germany). 2.2. Lyophilization Nanocrystal dispersions stabilized by polysorbate 80 and poloxamer 407 were prepared by wet ball milling [3]. After addition of mannitol (M), sucrose (S), or trehalose (T) alone or in combination samples were freeze- dried. Two processes were applied: (1) freezing at -80 °C (3 h), primary drying at -10 °C, 0.340 mbar, secondary drying at 25 °C (24 h) or (2) freezing at -50 °C (3 h), primary drying at -45 °C, 0.2 mbar (21 h), secondary drying at 20 °C (30 h). Samples were stored in crimped vials at 25 °C (lyophilization 1) or 2-8 ºC (lyophilization 2) for three months. 2.3. Physicochemical characterization Particle size (z-ave) was measured by Zetasizer Nano ZS (Malvern Instruments, UK) and Mastersizer (Malvern Mastersizer 2000 Malvern, UK). Redispersibility index (RDI) was calculated as z-ave (before)/z-ave (after) and expressed in percentages. Physical state of samples was determined by differential scanning calorimetry (DSC1; Mettler Toledo, Switzerland),powder X-ray diffraction (Rigaku Smartlab X-ray Diffractometer) and polarized light microscopy (PLM) (Carl Zeiss ApoTome Imager Z1 microscope Zeiss, Germany). 3. RESULTS AND DISCUSSION Right after preparation, nanocrystal dispersions were with submicron particle size around 160 nm, and PDI below 0.2, suggesting narrow size distribution. In the cryoprotectant screening phase, sucrose and/or mannitol were added in different concentrations. It was shown that 10% of the total stabilizer concentration was needed for the particle size preservation: the achieved RDI was above 95%, while cakes with sucrose alone or in combination with mannitol in ratio 1:1 or 3:2 were also with satisfied appearance (Figure 1). Lyophilization was conducted above or below the glass transition temperature of the maximally freeze-concentrated solution (Tg’) (around -39 ºC). When primary drying was performed at -10 °C, no aggregation was noticed right after lyophilization, but particle size increased significantly, lowering down the RDI to < 50%, after one month storage at 25 °C. This was confirmed by laser diffraction. In lyophilization 2, with primary drying at temperature below Tg’, trehalose was also used in the same concentration as sucrose and in combination with mannitol. Interestingly, in this process parameters setup, sucrose or trehalose alone did not prevent aggregation during freeze-drying. Particle size remained almost unchanged in formulation S+M 3+2 (RDI 95%) or slightly higher in T+M 3+2 (RDI 90%), after three months storage, suggesting it was most probably the optimal combination for the stabilization. Physical state analysis revealed that sucrose and mannitol in samples lyophilized by process 1 were in crystalline state, as well as sucrose when used alone in lyophilization 2. Trehalose, on the other hand was amorphous in all samples containing it. Amorphous state of lyoprotectants allows maximal hydrogen bonding due to higher molecule flexibility and availability of hydroxyl groups [3]. Surprisingly, mannitol as a substance with high crystallization tendency was with low crystallinity in lyophilizates. These observations were confirmed by PLM. It is possible that it formed interactions with sucrose or nanocrystal stabilizers [4]. 4. CONCLUSION Results from this study demonstrated freeze- drying as an important technique for the improvement of nanocrystals stability. However, the selection of cryoprotectant and bulking agent ratio beside process parameters (primary drying at -45 ºC) was crucial to get freeze-dried samples with good stability. Sucrose or trehalose in combination with mannitol (ratio 3+2) in total concentration 10% successfully hindered aggregation, thus prolonging the stability to 3 months at 2-8 ºC. 5. REFERENCES 1. Van Eerdenbrugh, B., et al. Top-down production of drug nanocrystals: nanosuspension stabilization, miniaturization and transformation into solid products. International journal of pharmaceutics, 2008. 364(1): 64-75. 2. Trenkenschuh, E., and Friess, W. Freeze-drying of nanoparticles: How to overcome colloidal instability by formulation and process optimization. European Journal of Pharmaceutics and Biopharmaceutics, 2021.165: 345-360. 3. Mitrović, J.R., et al. Overcoming the low oral bioavailability of deuterated pyrazoloquinolinone ligand DK-I-60-3 by nanonization: A knowledge-based approach. Pharmaceutics, 2021. 13(8): 1188. 4. Kumar, S., et al. Sugars as bulking agents to prevent nano-crystal aggregation during spray or freeze-drying. International journal of pharmaceutics, 2014. 471(1-2): 303-311. ACKNOWLEDGMENT This research was supported by the Science Fund of the Republic of Serbia, grant No. 7749108, project Neuroimmune aspects of mood, anxiety and cognitive effects of leads/drug candidates acting at GABAA and/or sigma-2 receptors: In vitro/in vivo delineation by nano- and hiPSC-based platforms-NanoCellEmoCog

The Impact of the Oil Phase Selection on Physicochemical Properties, Long-Term Stability, In Vitro Performance and Injectability of Curcumin-Loaded PEGylated Nanoemulsions

A nanotechnology-based approach to drug delivery presents one of the biggest trends in biomedical science that can provide increased active concentration, bioavailability, and safety compared to conventional drug-delivery systems. Nanoemulsions stand out amongst other nanocarriers for being biodegradable, biocompatible, and relatively easy to manufacture. For improved drug-delivery properties, longer circulation for the nanoemulsion droplets should be provided, to allow the active to reach the target site. One of the strategies used for this purpose is PEGylation. The aim of this research was assessing the impact of the oil phase selection, soybean or fish oil mixtures with medium chain triglycerides, on the physicochemical characteristics and injectability of curcumin-loaded PEGylated nanoemulsions. Electron paramagnetic resonance spectroscopy demonstrated the structural impact of the oil phase on the stabilizing layer of nanoemulsions, with a more pronounced stabilizing effect of curcumin observed in the fish oil nanoemulsion compared to the soybean oil one. The design of the experiment study, employed to simultaneously assess the impact of the oil phase, different PEGylated phospholipids and their concentrations, as well as the presence of curcumin, showed that not only the investigated factors alone, but also their interactions, had a significant influence on the critical quality attributes of the PEGylated nanoemulsions. Detailed physicochemical characterization of the NEs found all formulations were appropriate for parenteral administration and remained stable during two years of storage, with the preserved antioxidant activity demonstrated by DPPH and FRAP assays. In vitro release studies showed a more pronounced release of curcumin from the fish oil NEs compared to that from the soybean oil ones. The innovative in vitro injectability assessment, designed to mimic intravenous application, proved that all formulations tested in selected experimental setting could be employed in prospective in vivo studies. Overall, the current study shows the importance of oil phase selection when formulating PEGylated nanoemulsion

Melanoma Spheroids Grown Under Neural Crest Cell Conditions Are Highly Plastic Migratory/Invasive Tumor Cells Endowed with Immunomodulator Function

International audienceBACKGROUND: The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi-subpopulations of cancer cells some of which may possess stem cell-like properties. Although useful, the sphere-formation assay to identify stem cell-like or tumor initiating cell subpopulations in melanoma has been challenged, and it is unclear if this model can predict a functional phenotype associated with aggressive tumor cells. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic or advanced primary tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages and enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not enhanced self-renewal or tumorigenicity when compared to their adherent counterparts. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that spheroids display enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Our findings reveal a novel immune-modulator function of melanoma spheroids and suggest specific roles for spheroids in invasion and in evasion of antitumor immunity. CONCLUSION/SIGNIFICANCE: The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroids reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and suggested that aggressiveness and heterogeneity of melanoma tumors can be supported by subpopulations other than cancer stem cells. Therefore, it could be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability