Liposomal carriers as possible therapeutic vesicles : a morpho-functional investigation

1. Liposomalcarriersaspossibletherapeuticvesicles: a morpho-functionalinvestigation • Michela Battistelli • Università degli Studi di Urbino Carlo Bo, Urbino, PU Therapeutic Nanoproducts: from Biology to Innovative Technology. June 19th - 20th, 2019

2. IRON DEFICIENCY • “a health-related condition in which iron availability is insufficient to meet the body’s needs and which can co-exist with or without anemia” • (Cappellini MD. et al. Am J Hematol2017;92:1068-78.) • Common and widespread both in industrialized and non industrialized country. • (Baltussen R. et al. J Nutr, 2004. Oct;134(10):2678-84. Camaschella C., Am J Hematol., 2015;2015:8-13) • It represents the only nutritional disorder, significantly prevalent in developed countries, affecting over 2 million people (about over 30% of the world population is anemic). • (Pasricha, SR. et al. Blood 2013; 121: 2607– 17) • Iron is an essential element that is needed to form hemoglobin, an oxygen carrying protein inside red blood cells. • (Musallam KM. et al. Curr Med Res Opin. 2018 Jan;34(1):81-93) • It is essential for proper cellular function i. e. energy metabolism, cell signaling, gene expression, and the regulation of cell growth and differentiation. • (Coffey R and Ganz T. J Biol Chem. 2017 Aug 4;292(31):12727-12734)

3. The term Anemia denotes a reduction in the oxygen-carrying capacity of blood. • This is a condition in whichbloodhas a lowernumberofcirculating redbloodcellsor a decrease in the hemoglobin concentration. • Iron deficiency results from depletion of iron stores and occurs when iron absorption is insufficient compared to the metabolic iron demands. - The ratio between the iron absorbed and the amount ingested is typically low, but may range from 5% to 35% depending on circumstances and type of iron. A number of dietary factors influence iron absorption.

4. Young children and womenwho are pregnant or in postpartum are more frequently and severelyaffected by irondeficiency anemia, due to high irondemandsduringgrowth and pregnancy.

5. Significant slowdown in psychomotor and neurological development in new-borns. • Low cognitive function in preschool children. • Low cognitive function and learning disabilities in school age children. • During the adulthood iron deficiency is associated with low productivity, both manually and mentally. • Mental retardation and maternal and perinatal mortality are most serious results. • 20% of maternal death, it is enough to change dietary habits, but it is necessary to use iron dietary supplements.

6. Treatment of irondeficency • Preventfurtheriron loss, and provideironsupplementation, bothtocorrectanaemia and replenish body store • Oralironsupplementation • Limits of iron supplementation: low absorption, metallic flavour, gastrointestinal disturbances, • nausea, constipation or diarrhea and oxidative stress. • Intravenoussupplementation

7. NANOCARRIERS Nanocontainers can be used for iron transport; they represent a new technology that modifies pharmacokinetics and reduces toxicity. Nanotechnology is a new approach that in recent years has made excellent progress and has been used for many industrial applications, allowing significant advances in the biomedical field as well.

8. In particular, in the biomedical area, nanotechnology is intensively studied to improve the results obtainable through diagnosis and therapy of the most common pathologies. Liposomes are sphericalvesicles with an aqueous volume enterelyenclosed by a double phospholipidlayer membrane

9. The use of liposomes as carriers is significant for pharmacologically active substances that have a low therapeutic index (as some anticancer, antibiotics, etc.), because they make it possible to reduce the concentration of the drugs and improve the bioavailability, with reduction of side effects. Negative stainingmake a contrast with the outside, if the membrane isintact and leaves the innercompartmentunstained. (Guescini M. et al. Int J Mol Sci. 2017 Nov 22;18(11); Battistelli M and Falcieri E, BiologySubmitted 2019)

10. - Exosomes, whose diameters range from 40 to 120 nm. - Microvesicles assuming a diameter that goes from 100 to 500nm. - Dying cells release 500 nm–2 μm vesicular apoptotic bodies. While micro-vesicles can operate as ‘safe containers’ mediating inter-cellular communication, apoptotic bodies appear after the disassembly of an apoptotic cell into subcellular fragments. The formation of ApoBDs is an important process deriving from the apoptotic death of the cell, which is considered a peculiarity of apoptosis.

11. Liposomes are vesicular structures with a double lipid membrane and an internal empty space with an aqueous phase. Liposomes appear very important in the biomedical field as a delivery system, pharmaceutically active substances. • Biofer (Ironsulfate and Vitamin C in liposomes, patented in 1994) iswidelyused in foodindustry, as additive for milk fortification and dairyproducts • (Boccio and other, 1995; Zubillaga and other, 1996; Gotelli and other, 1996; Boccio and other, 1996; Boccio and other, 1997a,b; Boccio and other, 1998). • Biofershelf life isoneyear and the storage temperature isbetween +8 and +15°C. • Lifervitis a newliposomewith a prolongedshelf life with 5 nutrients: Ironsulfate, Vitamin C, Folic Acid, Vitamin B6 and Vitamin B12 • (Lysionek and other, 1998; Uicich and other, 1999; Boccio and other, 2000; Lysionek and other, 2000; Lysionek and other, 2001 a, b).

12. OUR EXPERIENCE………………. • Membrane stabilityduring the dryingprocess • liquid / gel form “native” liposomes. • isotonicsolution and hypotonicsolution (distilled water). • Negative staining • 2) Liposomeinternalization • Humanmyelomonocyticcellline U937 wasgrown in RPMI 1640, supplementedwith 10% heat-inactivatedfetal bovine serum, 2 mm glutamine, 1% antibiotics and wasmaintained at 37 °C in humidified air with 5% CO2 • Conventional electron microscopy

13. Vesiclesdimensiondecreasing from native form to the dryingform in isotonicsolution. Dehydrationreduces the fusion beetween the liposomes, and probablystabilizes the individualvesicles.

14. Vesiclesdimensiondecreasing from native form to the dryingform in hypotonicsolution.

15. Bioferappearsdiffuselylocalizedoutside the cell and near plasma membrane, forming electron dense clusters. It can be internalized from the intercellularspace.

16. Lifervitappearsdiffuselylocalizedoutside the cell and near plasma membrane, forming electron dense clusters. It can be internalized from the intercellularspace.

19. CONCLUSION • Liposomesmaintainmorphologicalstability in bothliquid and driedform. • Biofer and Lifervithave a goodpenetrationinto U937 cells. • Biofer and Lifervit do not induce cellulardamage. - Thisstudyconfirmed the Biofergoodbioactvity and alsodemonstrate the optimalinternalizationcapacityofLifervitto penetrate inside cells. • Furtherstudywill beperform on othercelltypes to understandliposomeinternalization. • …………..Theirbehaviorduring and aftertechnicalproceduresconfirmstheirpotential use in irondeficiency-correlateddisorders

20. AKNOWLEDGEMENTS Prof. Elisabetta Falcieri Dott. Sabrina Burattini Dott. Sara Salucci Prof. Michele Guescini Dr. Thomas De Paoli Dr. Bruno Riccardi LipoTech s.a., Buenos Aires Argentina