The intranasal administration of nanostructured lipid carriers (NLC) has been suggested as a promising strategy to improve the fast treatment of epilepsy. This route allows drug passage directly from the nose to the brain, avoiding the need of bypassing the blood brain barrier. In addition, the quality-by-design (QbD) approach is a useful tool for the optimization of manufacturing variables, resulting in effective and safe pharmaceutical formulations. Herein, the quality target profile product (QTPP) and critical quality attributes (CQAs) are identified and a risk assessment analysis is conducted to qualitatively detect the most critical material attributes (CMAs) and critical process parameters (CPPs). The aim of this work was to use the QbD approach to optimize a NLC formulation for the nose-to-brain delivery of diazepam, improving the emergency therapy of epilepsy. The studies began with the screening of excipients and the assessment of lipid-drug compatibility. The central composite design was used to evaluate the effects of CMAs (ratio of solid and liquid lipids and amount of emulsifiers) on the CQAs of the NLC formulation (particle size, polydispersity index (PDI), zeta potential (ZP) and encapsulation efficiency (EE)). The results showed that the most adequate ratios of lipids and emulsifiers were 6.65:2.85 and 4.2:0.3 (%, w/w), with values of 84.92 nm, 0.18, -18.20 mV and 95.48% for particle size, PDI, ZP and EE, respectively. This formulation was selected for further studies related to the optimization of CPPs.

Triple-negative breast cancer (TNBC) is a highly aggressive type of cancer with limited therapeutic options. However, this type of cancer cells have shown overexpression of folate receptors, which bind with folic acid (FA) with high affinity. This feature can be used for therapeutic targeting in combination with nanocarriers, such as liposomes. In order to further examine the potential of increased efficacy by targeting the folate receptor, we prepared folate conjugated liposomes (DSPC/Chol/PEG/DSPE-PEG-FA) and loaded them with doxorubicin (DOX), an anticancer drug. For this, we first synthesized and verified the conjugate between FA and PEG-lipid (FA-PEG-lipid). After that, liposomes were prepared with thin film hydration method followed by probe sonication. Three different types of targeted liposomes were evaluated having concentrations of FA-PEG-Lipid in their membranes (0.1 mol%, 0.5 mol% and 1mol%). The various formulations were evaluated for their uptake (FITC-dextran encapsulating liposomes) and for their cytotoxicity (DOX-loaded liposomes) using three different cancer cell lines, MDA-MB-231 (epithelial human breast cancer cells), 4T1 (murine mammary carcinoma cells) and MCF7 (Human breast cancer cells); the two first are TNBC cells the third is not. Cellular uptake results proved that increasing amounts of FA on the surface of liposomes results in enhance uptake by TNBC cells. Furthermore, the antitumor activity of liposomal-DOX was significantly increase by folate conjugation on their surface, especially in the case of the TNBC cancer cells.

The resveratrol, due to its antioxidant property, has the potential to be successfully applied in the prevention and the treatment of neurological disorders (Parkinson’s and Alzheimer’s disease). Nevertheless, its traditional administration (intravenous, oral) and bioavailability are limited by its physical-chemical characteristics (solubility, chemical instability, sensitivity to heat, UV-light and pH). The ‘nose-to-brain’ application, as an alternate administration route, represents a way to reach the brain without the limitations of the blood-brain barrier, while the use of nano-sized drug delivery systems, like the liposomes, can overcome the developmental and therapeutic issues of the formulations. This research paper shows the application of Risk Assessment, the key element of the Quality by Design mindset, in the development of a liposomal resveratrol-containing formulation with brain target and nasal administration.

The study intends to demonstrate the definition of the quality target product profile, the selection of the critical factors, and the application of the RA to get a detailed view on the critical parts of the development process.

On these terms, the factors with the most significant impact on the product quality among the critical material attributes (phospholipids, API content, cholesterol ratio, surface modification), furthermore the production process (temperature, oxidation and light protection) were identified; subsequently, an RA-based liposome preparation process was described.

The formulation procedures of ‘nose-to-brain’ liposomal systems loaded with drugs with many limiting factors meet several risks; however, the adaption of the QbD tools helps to focus on the aimed final product quality and achieve effective experimental designs.

Arsonoliposomes (ARSL) constitute a particular class of liposomes that incorporate arsonolipids (ARS) in their membranes. ARSL realize selective toxicity to cancer cells, thus being an important tool in the treatment of cancer. Folic Acid (FA) is widely used in targeted drug delivery, due to its high affinity for the folate receptors that are overexpressed in cancer cell membranes. The aim of our studies was to develop novel TNBC-targeted ARSL, by incorporating folic acid-conjugated PEG-lipid in their membrane and loading them with anticancer drug doxorubicin (DOX). ARSL incorporating 0.1 mol% of FA-PEG-lipid (folate-PEG-DSPE) were prepared and loaded with DOX, using the active loading protocol. They were characterized for their size distribution, zeta potential and drug entrapment efficiency (%). Their cytotoxic activity towards TNBC cell lines, particularly MDA-MB-231 (epithelial human breast cancer cells) and 4T1 (murine mammary carcinoma cells) was evaluated by the MTT-assay. Additionally MCF7 (Human breast cancer cells), and HEK-293 (Human embryonic kidney cells) were evaluated as control cells. The first results demonstrated enhanced toxicity of this novel type of ARSL towards cancer cells, which is particularly interesting and deserves further exploitation.

Abstract:

Osteosarcoma (OS) is a rare, aggressive bone tumor that impacts mostly children and young adults. Despite numerous therapeutic efforts, OS still presents low patient survival rate, high metastasis, and relapse occurrence¹. To surpass that, polymeric micelles have been researched for the targeted co-delivery of genetic material and drugs.

In this work, mixed polymeric micelles with cationic properties containing polyethyleneimine (PEI), Pluronics® F68 and P123 were prepared. For this purpose, Pluronic^® F68 was activated by the addition of diacrylate groups in the two hydroxyl groups at the ends of the Pluronic chain and conjugated with PEI. The prepared nanosystem was structurally characterized by FTIR and NMR spectroscopy and the morphology was assessed by TEM. Following this, Pluronic^® P123 was incorporated in the formulation in a ratio of 2:1 regarding the concentration of Pluronic^® F68-PEI and Pluronic^® P123. Particle size, polydispersity index (PDI) and zeta potential were assessed by Dynamic and Electrophoretic Light Scattering, respectively. The morphology was evaluated by TEM.

Pluronic^® F68 diacrylate conjugated with PEI was synthesized successfully as confirmed by FTIR and NMR analysis. It was possible to observe small-sized micelles (around 153 nm) with a PDI of 0.346 and zeta potential of 12.59 mV with irregular spherical shape, a darker core and a lighter shell. The incorporation of Pluronic^® P123 in the formulation lowered particle size (119.6 nm) and resulted in spherical micelles. However, zeta potential decreased (7.3 mV) due the presence of Pluronic^® P123, but remained positive.

A mixed polymeric micelle composed of Pluronics^® F68 and P123 complexed with PEI was developed and characterized, allowing to achieve a stable, small-sized nanosystem, characteristics that suggest a capability to surpass multidrug resistance and perform active targeting. Further incorporation of miRNA145 in the outer cationic shell will be tested for the capacity to inhibit OS metastasis and proliferation.

Key-words: mixed polymeric micelles; polyethyleneimine; Pluronic® F68; Pluronic®P123; Osteosarcoma

References:

¹ Melim, C.; Jarak, I.; Veiga, F.; Figueiras, A. The potential of micelleplexes as a therapeutic strategy for osteosarcoma disease. 3 Biotech (2020), 10(4), 147. DOI: 10.1007/s13205-020-2142-5

Funding:

This work received financial support from National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência) through the project UID/QUI/50006/2013, co-financed by European Union (FEDER under the Partnership Agreement PT2020). It was also supported by the grant FCT PTDC/BTM-MAT/30255/2017 (POCI-01- 0145-FEDER-030255) from the Portuguese Foundation for Science and Technology (FCT) and the European Community Fund (FEDER) through the COMPETE2020 program.

Background: Topical application of flavonoids has recently received increased attention, however their use is limited due to a low aqueous solubility and related low in vivo absorption. Unsaturated long-chain monoglycerides emulsified in water, such as monoolein, lead to aqueous nanostructured dispersions of complex lyotropic liquid crystalline phases (lamellar, hexagonal, and cubic structure) able to carry lipophilic molecules^1-2. To potentially solve solubility problems of these molecules, and possibly to facilitate a topical application, the use of monoolein aqueous dispersions (MAD) was investigated on two model flavonoids, namely quercetin (QT) and rutin (RU).

Experimental: MAD were produced by emulsifying monoolein in water in the presence of sodium cholate used at two different concentrations, namely 0.15% and 0.25% with respect to the total weight of the formulation¹. In addition increasing concentrations of flavonoids were tested. MAD were then characterized in terms of size, morphology and drug content by mean of PCS, SDFFS, cryo-TEM and UV spectroscopy. Moreover in vitro studies concerning the drug release modalities, the cytotoxicity and the antioxidant activity of the produced MAD were also conducted.

Results and Discussion: MAD size was found around 250 nm. Cryo-TEM showed that the content of sodium cholate influences the morphological aspect of the MAD. Indeed MAD015 appear as a mixture of vesicles and cubic structures, whilst MAD025 are mainly characterized by unilamellar vesicular structures. Concerning drug content it was found that MAD025 increased at least 80-fold QT solubility, while the same was not found for RU. In addition, in vitro Franz cell experiments showed that the MAD are suitable for cutaneous application while experiments on antioxidant activity demonstrated the two-fold power of QT as compared to RU.

Conclusions: MAD can be potentially proposed for the delivery of the antioxidant molecule QT, whilst many studies have to be performed for finding a way to deliver RU.

References:

1. Esposito et al. Eur J Pharm Biopharm 80:306-312,2012.
2. Esposito et al. Mater Sci Eng C 33:4923–34,2013.

Acknowledgements: This study was funded by Unife FIR2018

The main objective of this work is to develop a high-throughput electrospinning method for production of hybrid (organic-inorganic) and bioactive scaffolds needed for bone regeneration. The work involved the comparison between the low-throughput and high-throughput systems and their feasibility towards bone regeneration application. For this reason, variety of formulations starting from the low-throughput system have been tested. The approach has been gradually improved to the high-throughput system. Then the bioactive scaffolds were developed using the w/o emulsion electrospinning. Emulsions were tested for the stability and systematically adjusted to define the most suitable formulation. Adjustments were done by different concentrations of hydrophilic and hydrophobic non-ionic surfactants as well as water phase components including polyvinyl alcohol (PVA). After setting up emulsion electrospinning process electrospun samples have been characterized by different methods including: scanning electron microscopy (SEM), confocal laser scanning microscopy, Fourier-transform infrared spectroscopy (FTIR). Based on the characterization results selected samples doped with different fluorophores have been additionally checked for the release tests. Our result suggest that the release of the bioactive molecules can be controlled by addition of the optimized ratio of polymers to the water phase. Moreover, metabolic activity tests have been performed demonstrating its potential use for cell proliferation.

Nowadays, an electrospinning process is one of the most promising technique to produce nanofibers. The popularity of this method is still significant, due to its simplicity and relatively low cost. Moreover, many electrospun compounds can be used in numerous applications in different areas, for instance medical and pharmaceutical fields, textile engineering, environmental remediation. However, a design of an efficient electrospinning system remains challenging, due to high number of parameters strongly affecting fibers properties and productivity of their formation.

Lately, a great attention is given to development of needleless configurations, characterized by multi-jets system, thanks to their enhanced output. Indeed, a surface of spinneret, where the liquid jet formation is initiated, plays crucial role to improve the process. The electric field profile of the spinning electrode directly influences the size and uniformity of the produced fibers and should be strongly considered in further development. Our study presents a comprehensive theoretical analysis of the most effective needleless electrospinning configurations and compares their electrostatic features. Our extensive analysis was prepared in COMSOL Multiphysics software, known as a great tool to describe in details physical phenomena and it can strongly help to select the proper electrospinning set up, depending on desired results.

Amphotericin B (AmB) is a high-molecular weight poorly soluble drug with antiparasitic activity (1, 2), which possesses a very low oral and topical bioavailability (1, 2). Transferosomes (TFs), which are ultradeformable vesicles consisting on lipids, an edge activator and a low amount of ethanol (<10%)(3), could be a suitable formulation to deliver AmB through the skin (4, 5).

TFs were obtained by thin film hydration method. A 3-level Box-Behnken design of experiments was used to optimise the formulation. Optimised AmB-TFs were characterised in terms of particle size, zeta-potential, drug loading and morphology. The permeability of the AmB-TFs was tested in vitro using Franz cells across different membranes (6) but also in vivo in CD-1 mice. The toxicological profile of the formulation was tested by performing histological studies of the mouse skin after a 6 h exposure and haemolytic studies using human RBCs (7). In vitro and in vivo antiparasitic efficacy was also assessed (8-10) in different species of Leishmania spp.

The optimised formulation according to the Box-Behnken design consisted of 14:86 w:w edge activator : lipids and a drug loading of 0.086%. The formulation exhibited a good physicochemical stability for 6 months under desiccated conditions. AmB-TFs illustrated a flux of 41.18 ± 1.39 µg/cm²/h across Strat-M® membrane and the diffusion fitted well the Korsmeyer-Peppas model. Upon histological evaluation minor swelling indicative of inflammation or epithelial hyperplasia were appreciated. Haemolytic studies demonstrated that AmB-TFs possessed a 10-fold higher HC₅₀ compared to equivalent AmB concentrations dissolved in DMSO. AmB-TFs showed excellent in vitro activity against against L. amazonensis and L. brazilensis promastigotes was in the micromolar range with a selectivity index above 5 in both cases. In vivo studies demonstrated a good permeation of the drug after topical application on healthy mouse skin which increased over exposure time and was accumulated within the epidermis and dermis. The amount of AmB recovered from the epidermis was well above the IC₅₀ and likely to be efficacious in eliminating microorganisms form the skin. This was confirmed by in vivo efficacy studies in which parasite load was decreased in a 98.24 ± 1.54%.

AmB-TFs enabled the permeation of AmB after topical administration that allowed therapeutically relevant amounts to be uptaken and accumulated within the dermis where parasites accumulate. The low toxicity of the formulation and minimal if any changes in the skin morphology allows for safe and effective non-invasive formulations for the treatment of these fungal and parasitic infections.

Self-emulsifying systems are becoming increasingly popular for oral delivery of poorly soluble drugs. They are divided into subgroups according to their components; Type IV formulations are prepared using only surfactants and/or co-surfactants. The aim of the study was to increase the solubility of Tadalafil, a weakly basic BCS Class II drug, by preparing Type IV formulations and using porous adsorbents. In this study, Labrasol, Kolliphor PS 20, Kolliphor PS 60, Kolliphor PS 80, Kolliphor CS 12, Kolliphor CS 20, Kolliphor HS 15, Kolliphor EL, Kolliphor ELP, Kolliphor PEG400, Gelucire 44/14 and Gelucire 48/16 were used as surfactants; Transcutol was used as a co-surfactant. The Kolliphor PS 80, Kolliphor EL, Kolliphor HS 15 formulations were prepared with a ratio of 2:1 with Transcutol to form droplet sizes less than 50 nm and PDI values below 0.2. The formulations sensitivity to heat change and pH change was analysed by performing stability tests. Stable formulations of Tadalafil were obtained using the formulations were prepared using Transcutol and Kolliphor PS 80 or Kolliphor EL. Solidification of the optimum Type IV formulations were made using Fluorite, Neusilin US2, Neusilin FL2, Syloid 3050 and Syloid 3150. Dissolution studies of the prepared Solid Type IV (S-Type IV) formulations were first performed in 0.1 N HCl, and the dissolution of the optimum S-Type IV formulations were examined in pH 4.5 and pH 6.8. It was determined that the S-Type IV formulations prepared with Neusilin US2 and Neusilin UFL2 (2:1) had provided a dissolution of over 80% at the end of the 1^st minute. The results indicated the potential bioavailability improvement of S-Type IV self-emulsifying delivery system for a BCS class II drug, Tadalafil due to great enhancement in its dissolution rate.