Development of an injectable alginate collagen hydrogel for cardiac delivery of extra cellular vesicles.pptx

Presentation of article. M.pharm. extra cellular vesicles, Hydrogel, Myocardial infraction, Alginate, collagen, Heart failure and especially myocardial infraction is the main cause of morbidity and mortality World wide, remaining major public health challenge each year. Novel therapeutic approaches based on tissue engineering and regenerative medicine are emerging as promising treatment to repair the injured myocardial and reduce the need for heart transplantation.

1. A LIPOSOME-MICELLE-HYBRID (LMH) ORAL DELIVERY SYSTEM FOR POORLY WATERSOLUBLE DRUGS: ENHANCING SOLUBILIZATION AND INTESTINAL TRANSPORT   GRADUATE SCHOOL OF PHARMACY DEPARTMENT OF PHARMACEUTICS PRESENTED BY   JIGNA KHASIYA M.PHARM SEM II GUIDED BY DR. D. M. PATEL ASSOCIATE PROFESSOR 1

2. Impact factor : 5.13 2

3. List of Contents : Introduction Rational Hypothesis Aim & Objectives Materials and Method Result Conclusion References 3

4. Introduction : Liposome-micelle-hybrid (LMH) : The Liposome-Micelle-Hybrid (LMH) is a drug delivery system that combines liposomes and micelles to create a hybrid structure with unique properties. Liposomes are spherical vesicles composed of lipid bilayers, while micelles are colloidal aggregates formed by surfactant molecules in a specific concentration range. In the LMH system, liposomes and micelles work synergistically to enhance the solubilization and oral absorption of poorly water-soluble drugs.  The liposomes provide a protective environment for the drugs, shielding them from degradation and improving their stability. On the other hand, micelles play a crucial role in solubilizing hydrophobic drugs by forming mixed micelles with lipids from the liposome bilayer.  4

5. Lovastatin : Lovastatin is a highly lipophilic Biopharmaceutical Classification System (BCS) class II drug (high permeability and low solubility) with limited aqueous solubility and low oral bioavailability. Lovastatin is a medication belonging to the statin class used to lower cholesterol levels. It is taken orally and may have side effects like muscle pain and liver toxicity. 5 Fig.1. Mechanism of action

6. Caco-2 cells : Caco-2 monolayer is a widely used in vitro cell culture model that mimics the human intestinal epithelium.  It consists of a monolayer of cells derived from a human colon carcinoma cell line called Caco-2.  Caco-2 cells differentiate and form a monolayer resembling the brush border epithelium of the small intestine.  They possess characteristic microvilli on the apical side, tight junctions between cells, and basolateral membranes. They express various drug transporters, such as P-glycoprotein (P- gp ), which plays a crucial role in efflux transport across the intestinal epithelium. 6 This Photo by Unknown author is licensed under CC BY .

7. Rational : Liposomes and micelles can be prone to stability issues, including aggregation, leakage, and drug precipitation.  Limited drug loading capacity, especially for hydrophobic drugs. Challenges in crossing biological barriers for targeted drug delivery. Achieving controlled and sustained drug release from liposomes and micelles can be challenging.  7 Figure : 2

8. Need for Liposome-Micelle-Hybrid (LMH) Formulation: Enhanced drug loading by combining liposomal and micellar drug solubilization and encapsulation. Improved stability to prevent leakage and maintain drug efficacy. Targeted delivery potential by incorporating ligands or antibodies on the LMH formulation. Controlled drug release for optimal therapeutic outcomes. Improved pharmacokinetics, including enhanced absorption and prolonged circulation time. 8

9. Aim :  To develop A liposome-micelle-hybrid (LMH) oral delivery system for poorly water soluble drugs: Enhancing solubilization and intestinal transport. 9

10. Hypothesis :  10 Micelle Liposome Liposome-micelle-hybrid (LMH)  Enhance Bioavailability Enhance solubilization and intestinal transport Figure : 3

11. Objectives : 11

12. Materials & Method : TPGS (d-α- tocopheryl polyethylene glycol 1000 succinate)  Phosphatidylcholine (PC)  Cholesterol (CHO)  Phosphate buffer solution (PBS)  Lovastatin Sodium Hydroxide  Sodium chloride Polyethylene glycol 400 12

13. Formulation and characterization : Formulation of LOV loaded micelles : 13 Fig. 4. S olvent evaporation method 

14. Formulation and characterization : Formulation of liposomes : 14 Fig. 5. T hin-film hydration method  Powdered Lipids and Lovastatin Drug Sodium Chloride solution Sonication (10 min) [Phosphatidylcholine (PC), cholesterol (CHO), 1,2-distearoyl-sn-glycero3-phospho-1′-rac-glycerol sodium-salt ( DSPGNa )] 

15. Formulation and characterization : Formulation of liposome-micelle-hybrids : 15 Liposomes were prepared by a thin-film hydration method The thin drug-lipid film was hydrated with the pre-formed LOV loaded micelle Sonication / Ultracentrifugation LOV was loaded into LMH Figure : 6 Two control formulations were obtained i.e. LMHIN : a blank lipid bilayer and LOV-loaded micellar inner core and  LMHEX: a LOV-loaded lipid bilayer and an aqueous inner core containing blank micelles

16. Result of Particle diameter and zeta potential : 16 Nanocarriers Particle size (nm) Polydispersity index (PDI) Zeta potential(mV) Drug loading (% w/w) Encapsulation efficiency (%) TPGS Micelles Blank 13.0 ± 1 0.25± 0.05 -1.41 ± 1.9 Loaded 11.0 ± 0.2 0.13 ± 0.02 -1.26± 1.5 2.05 ± 0.08 72± 19 PC/CHO/ DSPG Liposomes Blank 107 ± 4 0.229 ± 0.01 -33.0± 8.9 Loaded 94.0 ± 2 0.264± 0.01 -42.3± 1.1 5.04 ± 0.29 92 ± 4 LMH  149 ± 2 0.264± 0.01 -46.3± 1.9 5.58± 0.03 79 ± 5 Table 1.  Characterization of TPGS micelles, liposomes and LMH (mean ± SD, n = 3) in the presence or absence of LOV loading

17. Morphology : 17 1.The sample is transferred to a metal mesh and excess material removed. 2 . The sample forms a thin film across the holes in the mesh when it is shot into ethane at about –174  °C. 3. The water vitrifies around the sample, which then is cooled by liquid nitrogen during the measurements in the electron microscope. Fig. 7. Cryo-transmission electron microscopy (Cryo-TEM) 

18. Result of Cryo-TEM : 18 Fig. 8. Cryo-TEM images of LOV-loaded (a) DSPG/PC/CHO liposomes and (b) LMH

19. Lovastatin in vitro release study : 18 Fig. 9. D ialysis membrane diffusion technique  The dialysis bag was soaked for 2 h Filled it with 10mL of sample Dipped in a beaker containing the release medium (90 mL) under magnetic stirring (100 rpm)  Two release media were investigated, PBS (pH 7.4) - PEG-400 (0.5%) and PBS (pH 7.4)-PEG-400 (0.5%) plus 20% ethanol, (at 37 °C)

20. Result of  LOV release studies : 20 Fig. 10. LOV release from (a) micelles and LMHIN (blank lipid bilayer + loaded aqueous inner core) for 24 h (inset graphs) and up to 10 d with (PBS + PEG-400 (0.5%), (b) liposomes and LMHEX (loaded lipid bilayer + blank aqueous inner core) at 37 °C in dialysis bag (mean ± SD, n = 3) 

21. Result of  LOV release studies : 21 Fig. 11. LOV release from micelles, liposomes and LMH (lipid bilayer and aqueous inner core) in release media (PBSwith0.2% of tween 80)and20%ethanol at 37 °C in dialysis bag (mean ± SD, n = 3). A similar LOV release profile was observed from micelles and liposomes, with approximately 95% LOV release within 48 h from both nanocarriers. In comparison, retarded release was observed from LMH, with 48% LOV release within 48 h demonstrating a controlled release profile from the hybrid system (Fig.11).  Here, LOV loaded in micelles within the liposome core has provided controlled drug release. This retarded release from LMH compared to the individual nanocarriers, provides further evidence that micelles have been encapsulated within the liposomes. ​

22. Result of Caco-2 cell monolayer permeability studies : 22 Fig. 12. (a) Effect of 100 µM LOV encapsulated in micelles, liposomes, LMH and free LOV on the TEER in Caco-2 cell monolayers (21d old) in HBSS over the 2 h treatment period. Control without treatment (cells and buffer only).    (b) The A → B and B → A fluxes of LOV and LOV loaded nanocarriers (100 µM) were determined at pH 7.4 as a function of time (2 h at 37 °C).

23. Result of  Cellular transport, intracellular uptake and accumulation : 23 Fig. 13. (a) LOV transported across Caco-2 monolayers after 8 h of incubation in the A → B direction at 37 °C in the presence of micelles, liposomes and LMH  (b) Caco-2 cell uptake of LOV and LOV loaded nanocarriers.

24. Conclusion : 24

25. References : Bilquis Romana a,c , Md. Musfizur Hassana, 1 , Fabio Sonvicob, 2 , Gabriela Garrastazu Pereira b 3 “A liposome-micelle-hybrid (LMH) oral delivery system for poorly water soluble drugs: Enhancing solubilization and intestinal transport” https://doi.org/10.1016/j.ejpb.2020.07.022 25

26. Thank you 26