Topical Nanoemulsions as Delivery Systems for Green Extracts of Pterocaulon balansae Aiming at the Treatment of Sporotrichosis.

Coumarins are benzopyrones found in several plant genera, including Pterocaulon (Asteraceae). These compounds represent an important source of new treatments, especially as antimicrobial and antifungal agents. In this study, two coumarin-rich extracts from Pterocaulon balansae using green technologies were obtained through aqueous maceration (AE) and supercritical fluid extraction (SFE). Such extracts were incorporated into nanoemulsions (NAE and NSFE) composed of a medium-chain triglyceride oil core stabilized by phospholipids. The nanoemulsions exhibited droplet sizes between 127 and 162 nm, pH above 5.0, and viscosity of approximately 1.0 cP, properties compatible with the topical route. The coumarins permeation/retention from formulations through ear porcine skin using Franz-type diffusion cells were evaluated. Whatever the extract, coumarins were distributed in skin layers, especially in the dermis in both intact and impaired (tape stripping) skin. In addition, a significant increase in coumarins that reached up to the receptor fluid was observed for impaired skin, with increases of approximately threefold for NAE and fourfold for NSFE. Finally, antifungal activity of nanoemulsions was evaluated according to minimum inhibitory concentrations, and the values were 250 µg/mL for all strains tested. The overall results demonstrated the feasibility of incorporating P. balansae extracts into nanoemulsions and showed a potential alternative for the treatment of sporotrichosis.

Regulatory Framework, Challenges, and Initial Strategic Planning for Advanced Therapy Products (PTAs) Development in Brazil.

Advanced Therapies are a class of innovative complex biological products used for therapeutic purposes, encompassing cell therapy, tissue engineering, and gene therapy products. These are promising therapeutic strategies for several complex diseases with low or non-existent therapeutic alternatives. The proper transposition of basic research in this area into medicinal products must comply with regulatory requirements. Here we review the main regulatory recommendations, emphasizing on the Brazilian regulation. The critical points are the manufacturing process, challenges in characterizing the product, development of non-clinical trials, lack of adequate animal models representative of the clinical situation, and absence of valid and measurable therapeutic endpoints. Based on that, we propose a framework for strategic planning of pre-clinical studies in this field. The detailed example involves producing a nonviral vector-based gene editing product, but the regulations and methods may be extrapolated for developing different types of advanced therapies.

Metastatic Melanoma: A Preclinical Model Standardization and Development of a Chitosan-Coated Nanoemulsion Containing Temozolomide to Treat Brain Metastasis.

Melanoma is the most aggressive type of skin cancer. Brain metastasis is the worst scenario in metastatic melanoma and the treatment options for these patients are limited. Temozolomide (TMZ) is a chemotherapy agent used to treat primary central nervous system tumors. Our objective was to develop chitosan-coated nanoemulsion containing temozolomide (CNE-TMZ) for nasal route administration to melanoma brain metastasis treatment. A preclinical model of metastatic brain melanoma was standardized, and the efficiency of the developed formulation was further determined in vitro and in vivo. The nanoemulsion was done by spontaneous emulsification method and the formulation was characterized by size, pH, polydispersity index, and zeta potential. Culture assessments to determine cell viability were done in the A375 human melanoma cell line. To determine the safety of formulation, healthy C57/BL6 mice were treated with a nanoemulsion without TMZ. The model in vivo used B16-F10 cells implanted by stereotaxic surgery in C57/BL6 mice brains. The results demonstrate that the preclinical model used showed to be useful to analyze the efficiency of new candidate drugs to treat melanoma brain metastasis. The chitosan-coated nanoemulsions with TMZ showed the expected physicochemical characteristics and demonstrated safety and efficacy, reducing around 70% the tumor size compared to control mice, and presenting a tendency in mitotic index reduction, becoming an interesting approach to treat melanoma brain metastasis.

Nasal administration of a temozolomide-loaded thermoresponsive nanoemulsion reduces tumor growth in a preclinical glioblastoma model.

Glioblastoma (GB) is the worst and most common primary brain tumor. Temozolomide (TMZ), an alkylating agent, is widely used for treating primary and recurrent high-grade gliomas. However, at least 50% of TMZ treated patients do not respond to TMZ and the development of chemoresistance is a major problem. Here, we designed a lipid nanoemulsion containing a thermoresponsive polymer (poloxamer 407) aiming to improve TMZ release into the brain via nasal delivery. Increasing amounts of poloxamer 407 were added to preformed nanoemulsions (250 nm-range) obtained by spontaneous emulsification. The influence of the polymer concentration (from 2.5% to 12.5%) and temperature on viscosity was clearly evidenced. Such effect was also noticed on the mucoadhesiveness of formulations, as well as TMZ release rate and retention/permeation through nasal porcine mucosa using Franz-type diffusion cells. From these results, a formulation containing 10% of poloxamer (NTMZ-P10) was selected for further experiments by nasal route. A significantly higher TMZ amount was observed in the brain of rats from NTMZ-P10 in comparison with controls. Finally, our results show that formulation reduced significantly tumor growth by three-fold: 103.88 ± 43.67 mm3 (for NTMZ-P10) and 303.28 ± 95.27 mm3 (control). Overall, these results suggest the potential of the thermoresponsive formulation, administered by the non-invasive nasal route, as a future effective glioblastoma treatment.

Oral Pharmacokinetics of Hydroxycinnamic Acids: An Updated Review.

Hydroxycinnamic acids (HCAs) such as caffeic acid (CA), chlorogenic acid (CGA), coumaric acid (COA) isomers, ferulic acid (FA) and rosmarinic acid (RA) are natural phenolic acids with widespread distribution in vegetal foods and well-documented pharmacological activities. However, the low bioavailability of HCAs impairs their administration by the oral route. The present review addresses new findings and important factors/obstacles for their oral administration, which were unexplored in the reviews published a decade ago concerning the bioavailability of phenolic acids. Based on this, the article aims to perform an updated review of the water solubility and gastrointestinal stability of HCAs, as well as describe their oral absorption, distribution, metabolism and excretion (ADME) processes by in vitro, ex vivo, in situ and in vivo methods.

Effect of Hydrogel Containing Achyrocline satureioides (Asteraceae) Extract-Loaded Nanoemulsions on Wound Healing Activity.

Achyrocline satureioides (Lam.) DC extract-loaded nanoemulsions have demonstrated potential for wound healing, with promising effects on keratinocyte proliferation. We carried out the first in vivo investigation of the wound healing activity of a hydrogel containing A. satureioides extract-loaded nanoemulsions. We prepared hydrogels by adding the gelling agent (Carbopol® Ultrez) to extract-loaded nanoemulsions (~250 nm in diameter) obtained by spontaneous emulsification. The final flavonoid content in formulation was close to 1 mg/mL, as estimated by ultra-fast liquid chromatography. Permeation/retention studies using porcine ear skin showed that flavonoids reached deeper layers of pig ear skin when it was damaged (up to 3.2 µg/cm² in the dermis), but did not reach the Franz-type diffusion cell receptor fluid. For healing activity, we performed a dorsal wound model using Wistar rats, evaluating the lesion size, anti-inflammatory markers, oxidative damage, and histology. We found that extract-loaded formulations promoted wound healing by increasing angiogenesis by ~20%, reducing inflammation (tumor necrosis factor α) by ~35%, decreasing lipid damage, and improving the re-epithelialization process in lesions. In addition, there was an increase in the number of blood vessels and hair follicles for wounds treated with the formulation compared with the controls. Our findings indicate that the proposed formulation could be promising in the search for better quality healing and tissue reconstruction.

Cyclodextrin-Based Delivery Systems and Hydroxycinnamic Acids: Interactions and Effects on Crucial Parameters Influencing Oral Bioavailability-A Review.

Hydroxycinnamic acids (HCAs) are a subclass of phenolic acids presenting caffeic acid (CA), chlorogenic acid (CGA), coumaric acid (COA) isomers, ferulic acid (FA), and rosmarinic acid (RA) as the major representants, being broadly distributed into vegetal species and showing a range of biological potentials. Due to the low oral bioavailability of the HCAs, the development of delivery systems to promote better administration by the oral route is demanding. Among the systems, cyclodextrin (CD)-based delivery systems emerge as an important technology to solve this issue. Regarding these aspects, in this review, CD-based delivery systems containing HCAs are displayed, described, and discussed concerning the degree of interaction and their effects on crucial parameters that affect the oral bioavailability of HCAs.

Gene editing strategies to treat lysosomal disorders: The example of mucopolysaccharidoses.

Lysosomal storage disorders are a group of progressive multisystemic hereditary diseases with a combined incidence of 1:4,800. Here we review the clinical and molecular characteristics of these diseases, with a special focus on Mucopolysaccharidoses, caused primarily by the lysosomal storage of glycosaminoglycans. Different gene editing techniques can be used to ameliorate their symptoms, using both viral and nonviral delivery methods. Whereas these are still being tested in animal models, early results of phase I/II clinical trials of gene therapy show how this technology may impact the future treatment of these diseases. Hurdles related to specific hard-to-reach organs, such as the central nervous system, heart, joints, and the eye must be tackled. Finally, the regulatory framework necessary to advance into clinical practice is also discussed.

The proteomic and particle composition of human platelet lysate for cell therapy products.

Following health agencies warning, the use of animal origin supplements should be avoided in biological products proposed as therapy in humans. Platelet lysate and several other growth factors sources are alternatives to replace fetal calf serum, the current gold standard in clinical-grade cell culture. However, the platelet supplement's content lacks data due to different production methods. The principle behind these products relays on the lysis of platelets that release several proteins, some of which are contained in heterogeneous granules and coordinate biological functions. This study aims to analyze the composition and reproducibility of a platelet lysate produced with a standardized method, by describing several batches' protein and particle content using proteomics and dynamic light scattering. Proteomics data revealed a diversified protein content, with some related to essential cellular processes such as proliferation, morphogenesis, differentiation, biosynthesis, adhesion, and metabolism. It also detected proteins responsible for activation and binding of transforming growth factor beta, hepatocyte growth factor, and insulin-like growth factor. Total protein, biochemical, and growth factors quantitative data showed consistent and reproducible values across batches. Novel data on two major particle populations is presented, with high dispersion level at 231 ± 96 d.nm and at 30 ± 8 d.nm, possibly being an important way of protein trafficking through the cellular microenvironment. This experimental and descriptive analysis aims to support the content definition and quality criteria of a cell supplement for clinical applications.

Curcumin and Quercetin-Loaded Lipid Nanocarriers: Development of Omega-3 Mucoadhesive Nanoemulsions for Intranasal Administration.

Curcumin (CUR) and quercetin (QU) are potential compounds for treatment of brain diseases such as neurodegenerative diseases (ND) because of their anti-inflammatory and antioxidant properties. However, low water solubility and poor bioavailability hinder their clinical use. In this context, nanotechnology arises as a strategy to overcome biopharmaceutical issues. In this work, we develop, characterize, compare, and optimize three different omega-3 (ω-3) fatty acids nanoemulsions (NEs) loaded with CUR and QU (negative, cationic, gelling) prepared by two different methods for administration by intranasal route (IN). The results showed that formulations prepared with the two proposed methods exhibited good stability and were able to incorporate a similar amount of CUR and QU. On the other side, differences in size, zeta potential, in vitro release kinetics, and permeation/retention test were observed. Considering the two preparation methods tested, high-pressure homogenization (HPH) shows advantages, and the CQ NE- obtained demonstrated potential for sustained release. Toxicity studies demonstrated that the formulations were not toxic for Caenorhabditis elegans. The developed ω-3 fatty acid NEs have shown a range of interesting properties for the treatment of brain diseases, since they have the potential to increase the nose-to-brain permeation of CUR and QU, enabling enhanced treatments efficiency.

Brain and visceral gene editing of mucopolysaccharidosis I mice by nasal delivery of the CRISPR/Cas9 system.

BACKGROUND: Mucopolysaccharidosis type I (MPS I) is an inherited disease caused by deficiency of the enzyme alpha-l-iduronidase (IDUA). MPS I affects several tissues, including the brain, leading to cognitive impairment in the severe form of the disease. Currently available treatments do not reach the brain. Therefore, in this study, we performed nasal administration (NA) of liposomal complexes carrying two plasmids encoding for the CRISPR/Cas9 system and for the IDUA gene targeting the ROSA26 locus, aiming at brain delivery in MPS I mice. METHODS: Liposomes were prepared by microfluidization, and the plasmids were complexed to the formulations by adsorption. Physicochemical characterization of the formulations and complexes, in vitro permeation, and mucoadhesion in porcine nasal mucosa (PNM) were assessed. We performed NA repeatedly for 30 days in young MPS I mice, which were euthanized at 6 months of age after performing behavioral tasks, and biochemical and molecular aspects were evaluated. RESULTS: Monodisperse mucoadhesive complexes around 110 nm, which are able to efficiently permeate the PNM. In animals, the treatment led to a modest increase in IDUA activity in the lung, heart, and brain areas, with reduction of glycosaminoglycan (GAG) levels in serum, urine, tissues, and brain cortex. Furthermore, treated mice showed improvement in behavioral tests, suggesting prevention of the cognitive damage. CONCLUSION: Nonviral gene editing performed through nasal route represents a potential therapeutic alternative for the somatic and neurologic symptoms of MPS I and possibly for other neurological disorders.

Achyrocline satureioides (Lam.) DC (Asteraceae) Extract-Loaded Nanoemulsions as a Promising Topical Wound Healing Delivery System: In Vitro Assessments in Human Keratinocytes (HaCaT) and HET-CAM Irritant Potential.

Achyrocline satureioides (Lam.) DC Asteraceae extracts (ASEs) have been investigated for the treatment of various skin disorders. This study reports the effects of ASE-loaded nanoemulsions (NEASE) on the cellular viability, death by necrosis, and migration of immortalized human keratinocytes (HaCaT cell line), as well as the irritant potential through the hen's egg chorioallantoic membrane test (HET-CAM). NEASE exhibited a polydispersity index above 0.12, with a droplet size of 300 nm, ζ-potential of -40 mV, and content of flavonoids close to 1 mg/mL. No cytotoxicity of the ASE was observed on HaCaT by MTT assay (up to 10 µg/mL). A significant increase of HaCaT viability was observed to NEASE (up to 5 µg/mL of flavonoids), compared to treatment with the ASE. The necrosis death evaluation demonstrated that only NEASE did not lead to cell death at all the tested concentrations. The scratch assay demonstrated that NEASE was able to increase the cell migration at low flavonoid concentrations. Finally, the HET-CAM test proved the non-irritative potential of NEASE. Overall, the results indicate the potential of the proposed formulations for topical use in wound healing, in view of their promising effects on proliferation and migration in keratinocytes, combined with an indication of the absence of cytotoxicity and non-irritating potential.

Optimization of Coumarins Extraction from Pterocaulon balansae by Box-Behnken Design and Anti-Trichomonas vaginalis Activity.

Trichomonas vaginalis causes trichomoniasis, a nonviral sexually transmitted infection with a high prevalence worldwide. Oral metronidazole is the drug of choice for the treatment of this disease, although high levels of T. vaginalis resistance to this agent are well documented in the literature. This study describes the anti-T. vaginalis activity of an optimized coumarin-rich extract from Pterocaulon balansae. Optimization was performed to maximize extraction of total coumarins by means of a 3-level Box-Behnken design, evaluating the effect of three factors: extraction time, plant : solvent ratio, and ethanol concentration. Optimum conditions were found to be 5 h extraction time and a plant : solvent ratio of 1% (w/v) and 60% (v/v) ethanol, which resulted in approximately 30 mg of total coumarins/g of dry plant. The coumarin-enriched extract exhibited a minimum inhibitory concentration of 30 µg/mL and an IC50 of 3.2 µg/mL against T. vaginalis, a low cytotoxicity, and a high selectivity index (18 for vaginal epithelial cells and 16 for erythrocytes). The coumarins permeation/retention profile through porcine vaginal mucosa was evaluated in Franz-type diffusion cells. After 8 h of kinetics, coumarins were detected in the tissue (4.93 µg/g) without detecting them in the receptor compartment. A significant increase of coumarins in the mucosa layers (8.18 µg/g) and receptor compartment (0.26 µg/g) was detected when a T. vaginalis suspension (2 × 105 trophozoites/mL) was previously added onto the mucosa. No alterations were visualized in the stratified squamous non-keratinized epithelium of the porcine vaginal mucosa after contact with the extract. Overall, these results suggest that the P. balansae coumarin-rich extract may have potential as a treatment for trichomoniasis.

Chitosan-coated rosmarinic acid nanoemulsion nasal administration protects against LPS-induced memory deficit, neuroinflammation, and oxidative stress in Wistar rats.

Rosmarinic acid (RA) lipid-nanotechnology-based delivery systems associate with mucoadhesive biopolymers for nasal administration has arisen as a new promising neuroprotective therapy for neurodegenerative disorders (ND). We have previously demonstrated the glioprotective effect of chitosan-coated RA nanoemulsions (RA CNE) against lipopolysaccharide (LPS)-induced damage in rat astrocyte primary culture. Here, we further investigate the protective effect of RA CNE nasal administration on LPS-induced memory deficit, neuroinflammation, and oxidative stress in Wistar rats, since these in vivo studies were crucial to understand the impact of developed delivery systems in the RA neuroprotective effects. The animals were treated through nasal route with RA CNE (2 mg·mL-1), free RA (2 mg·mL-1), blank CNE, and saline (control and LPS groups) administrations (n.a., 100 µL per nostril) twice a day (7 a.m./7 p.m.) for six days. On the sixth day, the animals received the last treatments and LPS was intraperitoneally (i.p.) administrated (250 µg·kg-1). Overall results, proved for the first time that the RA CNE nasal administration elicits a neuroprotective effect against LPS-induced damage, which was associated with increased 1.6 times recognition index, decreased 5.0 and 1.9 times in GFAP+ cell count and CD11b expression, respectively, as well as increased 1.7 times SH in cerebellum and decreased 3.9 times TBARS levels in cerebral cortex in comparison with LPS group. RA CNE treatment also facilitates RA bioavailability in the brain, confirmed by RA quantification. Free RA also demonstrates a protective effect in some studied parameters, although no RA was quantified in the brain.

Hydrogels containing soybean isoflavone aglycones-rich fraction-loaded nanoemulsions for wound healing treatment - in vitro and in vivo studies.

Soybean isoflavone aglycones have been investigated as potential wound healing compounds for topical application. The aim of this study was to evaluate the wound healing properties of a soybean isoflavone aglycones-rich fraction (IAF) when incorporated into lipid nanoemulsions dispersed in acrylic-acid hydrogels. Formulations exhibited a mean droplet size in the sub 200 nm range, negative ζ-potential (-60 mV), and displayed non-Newtonian pseudoplastic behavior. The addition of a gelling agent decreased the IAF release from formulations and improved the retention of these compounds in intact porcine ear skin when compared with a control propylene glycol solution. No IAF were detected in receptor fluid of Franz-type diffusion cells. However, increasing amounts of IAF were noticed in both skin layers and the receptor fluid when the tissue was partially injured (tape stripping), or when the epidermis was completely removed. In vitro studies showed that IAF elicits an increased proliferation and migration of keratinocytes (HaCaT cell line). Subsequently, the healing effect of the formulations was evaluated in a model of dorsal wounds in rats, by assessing the size of the lesions, histology, inflammatory markers, and antioxidant activity. Overall findings demonstrated the potential of IAF-loaded formulations to promote wound healing by increasing angiogenesis by ∼200 %, reducing the lipid oxidation (TBARS) by ∼52 % and the inflammation (TNFα) by ∼35 %, while increasing re-epithelialization by ∼500 %, visualized by the epithelium thickness.

A stability-indicating ultra-fast liquid chromatography method for the assay of the main flavonoids of Achyrocline satureioides (Marcela) in porcine skin layers and nanoemulsions.

INTRODUCTION: Achyrocline satureioides (marcela or macela) is a plant widely used in folk medicine in South America. Recently, there has been increasing interest for the development of skin care products containing A. satureoides extracts, due to its well-documented antioxidant, antiherpetic, and wound healing properties. OBJECTIVES: The present study aimed to develop and validate a yet unexplored stability-indicating and robust ultra-fast liquid chromatography (UFLC) method for the simultaneous quantification of the main flavonoids of A. satureioides in extracts, nanoemulsions, and porcine skin layers. MATERIAL AND METHODS: The chromatographic separation of flavonoids quercetin, luteolin, and 3-O-methylquercetin was performed on a Luna C18 analytical column (100 mm × 2.0 mm i.d.; particle size 2.5 µm) using isocratic elution with methanol/phosphoric acid 1% (48:52 v/v) with a flow rate of 0.3 mL/min at 40°C. RESULTS: The method was found to be specific, linear (R > 0.998), precise, accurate, and robust for all flavonoids assayed in A. satureioides extract, nanoemulsions, and porcine ear skin. A low matrix effect was noted for all complex matrices. The stability-indicating UFLC method was evaluated by submitting isolated flavonoids, a mixture of standards, and A. satureioides extract to acidic, alkaline, oxidative, UV-A/UV-C light, and thermal stress conditions. No peaks were found co-eluting with the flavonoids of interest in all matrices. The robustness of the method was confirmed using Plackett-Burman experimental design. CONCLUSION: The short run time (8 min) and reliability of the method could be useful for the determination of A. satureioides flavonoids in topical product development since extracts of this medicinal plant have been used to treat various skin disorders.

Glioprotective Effect of Chitosan-Coated Rosmarinic Acid Nanoemulsions Against Lipopolysaccharide-Induced Inflammation and Oxidative Stress in Rat Astrocyte Primary Cultures.

Rosmarinic acid (RA) is a natural polyphenolic compound with a well-documented neuroprotective effect mainly associated with its anti-inflammatory and antioxidant activities. Recently, our research group developed and optimized chitosan-coated RA nanoemulsions (RA CNE) intended to be used for nasal delivery as a new potential neuroprotective therapy. In this sense, the present study aimed to evaluate the protective and/or therapeutic potential of RA CNE in inflammation/oxidative stress induced by LPS (1 µg mL-1) in rat astrocyte primary cultures. In summary, pre-treatment with RA CNE before exposure to LPS (protective protocol) reduced significantly the LPS-induced alterations in astrocyte cell viability, proliferation, and cell death by necrosis, which was not observed in therapeutic protocol. RA CNE protective protocol also enhanced anti-oxidative status by ~ 50% by decreasing oxygen reactive species production and nitric oxide levels and preventing total thiol content decrease. Finally, our results demonstrate the protective effect of RA CNE in migratory activation and GFAP expression of reactive astrocytes. Overall, our findings indicate for the first time the RA CNE glioprotective potential, associated with an increase in cell viability and proliferation, a preventive effect on cellular death by necrosis, migratory ability and hypertrophic reactive astrocytes, and the reparation of astrocyte redox state.

Compatibility study of rosmarinic acid with excipients used in pharmaceutical solid dosage forms using thermal and non-thermal techniques.

Rosmarinic acid (RA) is a phenolic compound that presents well-documented anti-inflammatory, antioxidant and antitumor activities, and based on its pharmacological potential and poor bioavailability, several solid dosage forms have been developed to RA delivery. Therefore, in literature, there are no reports about RA compatibility with excipients. In this regard, the aim of the present study was to evaluate, for the first time, the compatibility of RA with excipients commonly used in solid dosage forms at a 1:1 (RA:excipient) ratio using differential scanning calorimetry (DSC), thermogravimetry (TG), Fourier-transform infrared (FTIR), solid-state nuclear magnetic resonance (ssNMR), and isothermal stress testing (IST) coupled with liquid chromatography (LC). The excipients selected were hydroxypropyl methylcellulose (HPMC), microcrystalline cellulose (MCC), lactose monohydrate (LAC), polyvinylpyrrolidone (PVP), talc (TALC), croscarmellose sodium (CCS), and magnesium stearate (MgSTE). According to DSC results, physical interactions were found between RA and HPMC, LAC, CCS, and MgSTE. The TG analyses confirmed the physical interactions and suggested chemical incompatibility. FTIR revealed physical interaction of RA with TALC and MgSTE and the ssNMR confirmed the physical interaction showed by FTIR and excluded the presence of chemical incompatibility. By IST, the greatest loss of RA content was found to CCS and MgSTE (>15%), demonstrating chemical incompatibilities with RA. High temperatures used in DSC and TG analyses could be responsible for incompatibilities in binary mixtures (BMs) with HPMC and LAC, while temperature above 25 °C and presence of water were factors that promote incompatibilities in BMs with CCS and MgSTE. Overall results demonstrate that RA was compatible with MCC and PVP.

Recent Patents Concerning the use of Nanotechnology-based Delivery Systems as Skin Penetration Enhancers.

Nanotechnology-based delivery systems have been considered a promising approach for topical application, considering their characteristics of penetration into/across the skin. The present review aimed to evaluate the recent international scenario of patents concerning the use of nanotechnology- based delivery systems as skin penetration enhancers. A survey of recent patent documents was conducted by using the Espacenet patent database including the terms "skin" in the title and "promot* or enhanc* and penetrat* or absorp* or permeat*" and "nano*" with the truncation symbol (*) in the abstract of documents. A total of 110 patents were published from 2008 to 2018, with 94 technologies being considered. The results demonstrated an increase in innovations concerning nanotechnologybased delivery systems as skin penetration enhancers in recent years. Most patent applicants are from China (60.6%) and Korea (21.3%), and companies (68%) were the most prominent owners. The majority of patent applications (76%) were intended for cosmetic purposes; the types of products and nanostructures were also investigated. Overall results demonstrated the increased interest around the world in patenting products involving skin permeation promotion and nanotechnology for pharmaceutical and, mainly, for cosmetics purposes.

HPLC-UV method for temozolomide determination in complex biological matrices: Application for in vitro, ex vivo and in vivo studies.

A high-performance liquid chromatography method for temozolomide (TMZ) determination in complex biological matrices was developed and validated for application in in vitro, ex vivo and in vivo studies of new nanotechnology-based systems for TMZ nasal delivery. The method was able to quantify TMZ in nanoemulsions, following cellular uptake, in the porcine nasal mucosa and in mouse plasma and brain. Analyses were performed on a C18 column at 35°C, under UV detection at 330 nm. The mobile phase was methanol-acetic acid 0.5% (30:70, v/v), eluted at an isocratic flow rate of 1.1 mL/min. The method was found to be specific, precise, accurate, robust and linear (0.05 to 5 µg/mL) for TMZ determination in all matrices. No interference of TMZ degradation products was found under various stress conditions such as acidic, alkaline, oxidative, light and thermal exposure, demonstrating stability. The method was applied for the quantification of TMZ in different matrices, i.e. the efficiency of nanoemulsions in vitro in increasing TMZ cellular uptake, ex vivo TMZ permeation and retention in the porcine nasal mucosa tissue, and for in vivo TMZ quantification in mouse brain following intranasal nanoemulsion administration compared with free TMZ.