Purpose: Phytosterols are plant sterols with structural resemblance to cholesterol. United States Food and Drug Administration (FDA) and Health Canada have approved phytosterols as a cholesterol-lowering agent due to their ability to significantly reduce low-density lipoprotein cholesterol (LDL-C) in the range of 7-12%. However, phytosterols (lipophilic) have the potential to impart higher efficacy in the reduction of LDL-C if formulated in a delivery system that increases its bioavailability. In this work, we aim to develop and characterize a liposomal formulation containing phytosterols and tocopherols; the aim is to enhance cholesterol-lowering ability of phytosterols. We also aim to test the efficacy of brassicasterol (phytosterol unique to canola seeds) which has not yet been approved by the FDA. To prevent oxidation of phytosterols during formulation development and storage, tocopherols (vitamin E) are also added as antioxidants.

Methods: Liposomes containing phytosterols and tocopherols were prepared using phosphatidylcholine as the lipid carrier and formulated using three different approaches -i) thin layer hydration homogenization, ii) thin layer hydration ultra-sonication, and iii) Mozafari method. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for quantifying liposomal phytosterols and tocopherols.

Results: Liposomal vesicles prepared via homogenization and ultrasonication methods were significantly lower in size (<200 nm) in comparison to those produced by the Mozafari method (>200 nm). All three methods showed comparable zeta potential values (-9 to -14 mV), which is adequate for the physical stability of the vesicles. A new validated LC-MS/MS method with a total run time of seven minutes was applied to quantify four phytosterols (brassicasterol, campesterol, stigmasterol, and β-sitosterol) and three tocopherols (alpha, gamma, and delta) simultaneously. The run time of seven minutes is the shortest among reported methods to date. The liposomal formulation prepared by all three methods showed entrapment efficiency >89% for both phytosterols and tocopherols.

Conclusion: Liposomes containing phytosterols and tocopherols were successfully developed and characterized with the aim of enhancing the efficacy of phytosterols. In vivo studies will be conducted using hamster animal model to compare the efficacy of liposomal phytosterols to marketed phytosterols containing products.

Multicomponent crystallisation is a widely studied technique in pharmaceutical chemistry to enhance physical properties of API’s without chemically modifying the drug moiety itself. Such crystal systems can display superior properties such as solubility, stability and bioavailability and as such many API’s are formulated as salts with several co-crystal preparations also approved for use. Methods to produce multicomponent crystals are varied with solution crystallisation being the predominant method. Crystal morphologies also influence an API’s properties with needle shaped crystals dissolving slower and possess poor flow properties compared to a more equant block shape.

In this study, we develop a method for the production of multicomponent crystals via cosublimation. Samples are sublimed on a laboratory scale from both ends of standard 15 x 160 mm test tubes sealed under vacuum with two heaters were used to equalize the sublimation rates of the components. We have shown that a range of multicomponent pharmaceutical crystals can be prepared where the components have quite different sublimation rates and that for the first time, tailor made additives can be used to obtain unprecedented morphology control of gas phase crystal growth. Dramatic changes were observed in morphology from the gas phase in sharp contrast to morphology changes in solution which produce much more modest effects. Salt formation was observed to occur during gas phase crystallisations in accordance with the pKa rule of 3 and modelling studies were carried out to understand the nature of proton transfer in these crystals in the absence of a solvent. In addition, we have shown that in addition to binary systems, ternary crystals can also be obtained via this technique.

Uterine fibroids (UF) occupies the 2nd place in the structure of gynecological pathology and is often the cause of infertility. Precise ultrasound localization makes the disease a perfect target for suicidal gene therapy in situ. However, excessive extracellular matrix (ECM) of UF represents a formidable barrier for gene delivery by means of nanoparticles. Coating with polyanions can provide colloidal stability of the polyplexes and resistance to ECM. We developed αvβ3-integrin-targeted peptide-based carrier and anionic peptide coating for DNA delivery into αvβ3-expressing cells, including primary leiomyoma cells (PLC).

Arginine-histidine-rich peptide carriers conjugated with cycloRGD ligand were synthesized. The physicochemical properties of DNA-polyplexes with anionic coating were tested. The specificity of the DNA delivery was demonstrated in competitive uptake and transfection experiments in pancreatic carcinoma cells (PANC-1). Suicidal gene therapy with HSV1-TK gene with subsequent ganciclovir treatment was held for PLC obtained after myomectomy.

DNA condensation analysis shows that the addition of anionic peptides does not lead to the destruction of the nucleopeptide complexes. Moreover, negatively charged coating allowed successful transfection in the presence of serum. AlamarBlue and TrypanBlue exclusion assays of PLC showed a decline of proliferative activity among cells transfected with HSV1-TK gene carrying complexes in comparison with the control LacZ gene transfected cells.

The study shows that the utilization of peptide carriers modified with RGD-ligand and negatively charged peptides is a promising approach for the development of targeted DNA delivery systems. Developed carriers demonstrated high specificity and transfection efficacy of leiomyoma cells with subsequent successful suicidal gene therapy.

Introduction: Glial cell line-derived neurotrophic factor (GDNF) is a well-establish therapeutic agent for Parkinson’s disease (PD) ¹. Its therapeutic potential, being a glycosylated protein, will largely depend on the possibility of administering it with a glycosylation pattern similar to the one present in the native protein ². Moreover, although several approaches to deliver this protein to the brain have been described ³, a promising strategy would be the use of nanoparticles (NPs) containing GDNF in the dopamine-depleted brain areas. Therefore, the aim of this work was to develop and characterize biodegradable NPs loaded with recombinant GDNF produced in mammalian cells for brain tissue engineering.

Methods: GDNF was produced by our group ⁴. GDNF purity was visualized by SDS-PAGE followed by Coomasie Blue (CB) staining. PC12 cell-based bioassay was used to assess its bioactivity. GDNF was encapsulated in polymeric NPs by multiple emulsion solvent evaporation method using Total Recirculation One Machine System (TROMS). Dynamic light scattering was used to measure the mean particle diameter and polydispersity index (PDI). Encapsulation efficiency was quantified by ELISA. NPs morphology was characterized by scanning electron microscopy (SEM). GDNF in vitro release from NPs disrupted with DMSO was determined by ELISA.

Results: GDNF was highly pure. The NPs showed a diameter of 405.5 ± 2.9 nm and a PDI of 0.08 ± 0.03. The encapsulation efficiency was 61.6%. SEM analysis showed spherical particles. GDNF released within the first 24 hours was 19.10 ± 3.5%, followed by a phase of sustained-release with 50.6 ± 3.1% of hGDNF being released within 28 days. PC12 treatment with the hGDNF released from NPs induced outgrowth of neurites in these cells indicating that hGDNF remains bioactive after its nanoencapsulation.

Conclusions: GDNF-NPs has been successfully prepared using TROMS, showing a high protein loading and a sustained release. The developed nanosystem has great potential for brain tissue engineering applications.

Acknowledgements

1. Torres thanks the Spanish Ministry of Education (Programa FPU (FPU17/01212)) and Government of Navarra (2019_66_NAB9). E. Garbayo is supported by a “Ramon y Cajal Fellowship (RYC2018-025897-I).

References

1. E. Garbayo et al, Effective GDNF brain delivery using microspheres-A promising strategy for Parkinson’s disease, J. Control. Release. 135 (2009) .
2. R.A. Barker et al, GDNF and Parkinson’s Disease: Where Next? A Summary from a Recent Workshop, J. Parkinsons. Dis. (2020) .
3. P.V. Torres-Ortega et al, Micro- and nanotechnology approaches to improve Parkinson’s disease therapy, J. Control. Release. 295 (2019).
4. E. Ansorena et al. A simple and efficient method for the production of human glycosylated glial cell line-derived neurotrophic factor using a Semliki Forest virus expression system. Int J Pharm. (2013).

Introduction: Neuroblastoma is the most frequent pediatric extracranial solid tumor being responsible for 8-10% of all childhood malignancies. Patient outcome is still a major issue due to neuroblastoma’s heterogeneity and complex tumor biology.^1,2 Nanotechnology has been widely studied in cancer treatment with the aim of improving the therapeutic index of chemotherapeutic drugs.³ Among them, etoposide is a podophyllotoxin derivative given to neuroblastoma patients that often presents acute and late toxicities.⁴ In this study we have designed and characterized etoposide-loaded lipid nanoparticles (ETP-LN) with the aim of improving therapeutics in neuroblastoma management.

Methods: ETP-LN were prepared by the hot homogenization followed by ultrasonication method which does not involve the use of any organic solvent.⁵ Nanoparticles physiochemical properties were characterized (size, homogeneity, surface charge) by dynamic light scattering and drug loading was evaluated by UV-vis and UHPLC-UV methods. Cell viability studies (MTS assay) were performed in multiple human neuroblastoma cell lines: SK-N-BE (2), SK-N-BE(2)C and NGP-A (MYCN-amplified cells) and SH-SY5Y, CHLA-90, NBL-S and SK-N-SH (MCYN-non-amplified cells).

Results: Optimized ETP-LN showed a mean particle diameter of 105 ± 3.29 nm with a PDI value of 0.19±0.01, indicating homogenous particle distribution. The surface charge presented a negative zeta potential value of -19.9 ± 4.24 mV. UHPLC-UV showed better reproducibility than the UV-vis method. Nevertheless, both techniques indicated a similar drug loading (4.26 ± 0.18 µg/mg and 4.58 ± 0.60 µg/mg, respectively), corresponding to an encapsulation efficiency of 86.8 + 4.16 %. Preliminary in vitro cell-based cytotoxicity assay revealed that ETP-LN displayed similar IC₅₀ values in the studied cell lines when compared to the free drug, indicating that the encapsulation process did not affect etoposide’s antitumor efficacy.

Conclusions: ETP-LN were successfully developed and characterized. Further experiments in vitro and in vivo will be performed to evaluate the therapeutic potential of ETP-LN in neuroblastoma.

References:

1. Pastor, E. R. & Mousa, S. A. Current management of neuroblastoma and future direction. Crit. Rev. Oncol. Hematol. 138, 38–43 (2019).
2. Park, J. R., Eggert, A. & Caron, H. Neuroblastoma: Biology, Prognosis, and Treatment. Hematology/Oncology Clinics of North America 24, 65–86 (2010).
3. Rodríguez-Nogales, C., Gonzaíez-Ferna, Y., Aldaz, A., Couvreur, P. & Blanco-Prieto, M. J. Nanomedicines for Pediatric Cancers. (2018).
4. Relling, M. V. et al. Etoposide and antimetabolite pharmacology in patients who develop secondary acute myeloid leukemia. Leukemia 12, 346–352 (1998).
5. González-Fernández, Y., Brown, H. K., Patiño-García, A., Heymann, D. & Blanco-Prieto, M. J. Oral administration of edelfosine encapsulated lipid nanoparticles causes regression of lung metastases in pre-clinical models of osteosarcoma. Cancer Lett. 430, 193–200 (2018).

Introduction: Cannabidiol (CBD), the main non-psychotropic cannabinoid, has emerged as a potential therapeutic agent for the treatment of several disorders including cancer, neurodegenerative disorders and pain among others. However, its low aqueous solubility hinders the development of effective parenteral formulations [1]. The use of polymeric microparticles as CBD carriers could resolve this challenge and allows to obtain an extended CBD release after a single administration [2]. Among all the available polymers, poly(lactic-co-glycolic acid) (PLGA), FDA approved for various medical applications, is one the most used. Ionizing radiation has been proposed as an effective sterilizing method for PLGA microparticles, which is essential for their parenteral administration [3].

The aim of this work was to evaluate the effect of gamma sterilization on the characteristics of CBD loaded microparticles.

Methods: Microparticles with a CBD:PLGA ratio of 10:100 (10-Mps) and 20: 100 (20-Mps) were prepared by solvent evaporation technique, using PLGA-RG-502 as polymer, and sterilized by gamma irradiation at a dose of 25 kGy. All formulations were then characterized by DLS, SEM and DSC. CBD content and CBD release were also evaluated by HPLC.

Results: No differences in particle morphology and particle size were detected between sterile and non-sterile formulations. All microparticles exhibited a spherical shape, a smooth surface, and an average particle size around 25 µm. DSC analysis showed the absence of the CBD melting peak in sterile and non-sterile CBD microparticles, indicating that it is dissolved or molecularly dispersed within the polymeric matrix and that no crystallization processes occurred during sterilization. However, a reduction on PLGA glass transition was appreciated in both 10-Mps and 20-Mps sterile formulations compared with their non-sterile counterparts. A significant lower CBD content was also detected in sterile microparticles, observing a CBD degradation during sterilization of 13.75% and 10.28% in 10-Mps and 20-Mps respectively. Finally, a faster CBD release was appreciated in sterile microparticles compared with their counterparts, due to the faster PLGA degradation in sterilized microparticles.

Conclusions: Due to the CBD degradation during sterilization process and the acceleration of the release of this drug from PLGA microparticles, gamma irradiation is not an adequate method to sterilize CBD-PLGA-microparticles.

References:

[1]. Fraguas et.al. Drugs. 2018; 78(16):1665-1703.

[2]. Fraguas et.al. Int J Pharm.202; 574:118916

[3]. De Oliveira et.al. Mater Sci Eng C Mater Biol Appl. 2017; 80:438-448.

Background: The encapsulation of Mesemchymal Stem Cells (MSCs) into a hydrogel provides a promising future in diverse biomedical applications. MSCs provide a regenerative wound healing microenvironment due to their capacity to produce and secrete growth factors and cytokines that enhance granulation tissue formation, angiogenesis and reduce inflammation, which result in accelerated wound closure^1-2. Hydrogels are three-dimensional hydrophilic polymeric able to retain large amounts of water or biological fluids. Hydrogels possess similar structure to the extracellular matrix (ECM) and they are used in regenerative medicine due to their biocompatibility and their capability to act as a growth medium³. Hyaluronic acid (HA) is a non-sulfated glycosaminoglycan; it is also the main element of ECM. For this reason and have been investigated due to their biocompatibility, biodegradability and hydrophilic character. HA also lacks gelation abilities, thus it is used in combination with natural gelling agents such as alginate (A)⁴.

Purpose: The aim of this work was the development and in vitro characterization of an alginate-hyaluronic acid based hydrogel for wound healing applications.

Methods: A-HA pre-gel solution was elaborated by dissolution of both autoclaved polymers in deionized water at concentrations of 2% and 1% (w/v), respectively. Then, 1 × 10⁶ cells/mL was added on the pre-gel solutions and mixed until complete homogenization. Finally, 1% of 100 mM CaCl₂ was added to obtain the hydrogel. Many important properties for the design of a hydrogel, such as swelling, degradation and porosity were studied. Swelling rate was assessed by a gravimetric method in phosphate buffer saline (PBS) at pH 7.4 and 37 ºC. The degradation was calculated by incubating fresh hydrogels under the same conditions. The porosity analysis was carried out by immersion of the dry hydrogels in PBS pH 7.4 at room temperature.

Results and Discussion: The swelling ratio value was around 1500%. The swelling behavior in wound healing can promote the transportation of nutrients and provide mechanical resilience to the delivery systems and the biological site of action. Degradation rate about 100% was reached in 300 min. The degradation of the hydrogel should be progressive and takes place simultaneously with the restoration of the new tissue. The calculation of the porosity resulted in values of 52%. It is an important characteristic of hydrogel because this highly porous structure allows and promotes nutrient transport, and facilitates cell proliferation and differentiation, which involves cell migrations.

Conclusions: These results confirm that this hydrogel present optimal characteristics to promote cell adhesion, proliferation and differentiation, thus it could be proposed as a suitable vehicle for cell delivery in tissue regeneration.

References:

¹Murphy K.C., Whitehead J., Zhou D., Ho S.S., Leach J.K. (2017). Engineering fibrin hydrogels to promote the wound healing potential of mesenchymal stem cell spheroids. Acta Biomater, 64, 176-186.

²Hu M.S., Borrelli M.R., Lorenz H.P., Longaker M.T., Wan D.C. (2018). Mesenchymal Stromal Cells and Cutaneous Wound Healing: A Comprehensive Review of the Background, Role, and Therapeutic Potential. Stem Cells International. Article ID 6901983.

³Tsou Y.H., Khoneisser J., Huang P.C., Xu X. (2016). Hydrogel as a bioactive material to regulate stem cell fate. Bioactive Materials, 1, 39-55.

⁴Antich, C., de Vicente, J., Jiménez, G., Chocarro, C., Carrillo, E., Montañez, E., Marchal, J. A. (2020). Bio-inspired hydrogel composed of hyaluronic acid and alginate as a potential bioink for 3D bioprinting of articular cartilage engineering constructs. Acta Biomaterialia, 106, 114-123.

Impaired fracture healing impacts patients’ quality of life and imposes a financial burden on healthcare services. Up to 10% of bone fractures result in delayed/non-union fractures, for which new treatments are urgently required. However, systemic delivery of bone anabolic molecules is often sub-optimal and can lead to significant side effects. In this study, we developed ultrasound (US) responsive nano-sized vehicles in the form of perfluorocarbon nanodroplets (NDs), as a means of targeting delivery of drugs to localised tissues. We tested the hypothesis that NDs could stably encapsulate BIO (GSK-3β inhibitor), which could then be released upon US stimulation to activate Wnt signalling and induce ossification.

NDs (~ 280nm) were prepared from phospholipids and liquid perfluorocarbon and their stability and drug loading was studied by NTA (Nano Tracking Analysis) and HPLC. ND cytotoxicity was assessed in patient derived, bone marrow stromal cells (BMSCs) with Alamar Blue (24h), and in vitro bioactivity of BIO-NDs was evaluated in a 3T3 Wnt-pathway reporter cell line with luciferase readout. To investigate the acoustic behaviour of NDs, 2% agarose (LM) containing NDs was injected into a bespoke bone fracture model (Sawbones) of various geometries and stimulated by US (1MHz, 5% duty cycle, 1MPa, 30s), allowing simultaneous capture of optical images and acoustic emissions. Femoral bone hole defects (1-2mm) were made in WT-MF1 mice (age: 8-12wks) and DiR-labelled NDs (100µL, 10⁹NDs/mL, i.v.) were injected post-fracture to determine biodistribution by IVIS imaging.

NDs were stable (4 and 37°C) and retained >90% BIO until US was applied, which caused ~100% release. ND exposure up to a concentration of 10⁹NDs/mL showed no cytotoxicity (24h). BIO-loaded NDs induced Wnt pathway activation in a dose dependent manner. Biodistribution of DiR-NDs in a femoral bone hole defect model in mice demonstrated increased localisation at the fracture site (~2-fold relative to that found in healthy mice or contralateral femurs at 48h).

Background: In recent years, a growing concern about resistance to anti-infective agents has emerged. One of the most common microbial agents is Candida albicans. Under certain conditions, C. albicans can cause infections of skin and mucosal tissues. Nystatin (Nys) is a broad spectrum antifungal, which is indicated for the treatment of mucosal infections caused by Candida ssp such as patients under radiological treatment. Nys is a photosensitive drug and very poorly soluble in aqueous media. Therefore, microencapsulation can be the solution for its limiting factors^1-2.

Purpose: The aim of this work was to design, develop and characterize two types of microparticles as appropriate nystatin delivery systems for topical use: alginate microparticles (AM) and chitosan coated alginate microparticles (CCM).

Methods: The formulation of the microparticles was based on the emulsification/internal gelation methodology with modification³. First, a W/O emulsion was elaborated. Sodium alginate aqueous solution, CaCo₃ and Nys were the ingredients of the internal phase, and vegetable oil the external phase⁴. The resulting microparticles were characterized in terms of particle size, percentage yield (PY), loading capacity (LD), encapsulation efficiency (EE) and mucoadhesion ability.

Results and Discussion: Microparticles ranged from 51.21 µm for AM to 57.20 µm for CCM. The PY values were 83.26% and for 79.67% AM and CCM, respectively. The LD values for the inside/surface were 6.78%/0.40% for AM and 4.87%/0.91% for CCM. The values of EE for inside and surface were 81.12%/12.07% for AM and 85.08%/9.19% for CCM. CCM was the system that exhibited the best mucoadhesive properties.

Conclusions: The ability of these systems to adhere mucous membranes has great appeal for the treatment of localized infections. Thus these microparticulate systems could be proposed as a suitable vehicle for this kind of mucosal infections being an alternative therapy.

Introduction

Cyclodextrins (CDs) have a wide range of applications in different field of drug formulation due to their complexation ability which could improve the solubility, stability, safety and bioavailability of drug molecules. The application of natural and chemically modified CDs could present inclusion complexes by different technological methods. CDs are able to extend the function of pharmaceutical additives therefore their application become effective and valuable tool for development of drug delivery systems during different administration routes. Our research interest focused on the development of preparation protocols using cyclodextrins combined with other additives to produce suitable formulations (to reach local or systemic effect) to get effective therapies in different diseases.

Materials and methods

Niflumic acid, Levodopa, and Ciprofloxacin were used as a model active ingredients for preparation of samples could be used by per os, intranasal and pulmonary application. α-, β-, HPβCD and different kind of polymer as stabilizer (PVA, PVP) were also applied. Samples were produced by kneading, solvent evaporation, co-grinding and spray drying technologies. The particle size distribution and morphology were determined with laser diffraction and scanning electron microscopy. The surface adhesion was analyzed using contact angle system. Physico-chemical properties analyzed with differential scanning calorimetry and X-ray powder diffraction. In vitro drug release was carried out by modified paddle method in different media. Aerodynamic properties have been tested in vitro using Cascade Impactor. A modified horizontal diffusion cell was used by the in vitro penetration test.

Results and discussion

The presentation will introduce case studies our applied material- and technological parameters during the preformulation of samples containing model drugs, CD and other additives. Per os, intranasal and pulmonar formulation aspects of prepared systems will be also summarized focusing on structural, micrometric and in vitro investigations.

Acknowledgement: This work was supported by the Ministry of Human Capacities, Hungary grant (20391- 3/2018/FEKUSTRAT) and 2.2.1-15-2016-00007 Project