Cell-Based Targeting to Hepatocytes.

Different drug delivery strategies to the hepatocytes which can target the hepatocytes precisely.

1. Cell-Based Targeting to Hepatocytes By Umesh Chaudhary NIPERAPHD2023PE10 1

2. The Liver The liver is the largest, one of the most crucial and vital organ in the human body. It plays most important role in our body. Functions: Detoxification Nutrient Processing Bile Production Protein Synthesis Blood Sugar Regulation 2

3. Types of cells in the liver Liver consists of different types of cells: Parenchymal cells Hepatocytes 2. Non-parenchymal cells Kupffer cells Stellate cells Liver sinusoidal endothelial cells Hepatocytes are the major cell type of the liver Hepatocytes contribute to 80% of total mass. 3 Damania , A., Jain, E., & Kumar, A. (2013).  Advancements in in vitro hepatic models: application for drug screening and therapeutics. Hepatology International, 8(1), 23–38.

4. Cont … Böttger R, Pauli G, Chao PH, Al Fayez N, Hohenwarter L, Li SD. Lipid-based nanoparticle technologies for liver targeting. Adv Drug Deliv Rev. 2020;154-155:79-101. 4

5. Various Hepatocytic Disease Viral Hepatitis Hepatocellular carcinoma Malaria Autoimmune hepatitis Wilson's disease H epatocytes are targets for HCC, malaria and viral pathogens such as hepatitis. Specificity : Reduces off-target effects and systemic toxicity. Enhanced Efficacy: Higher drug concentrations delivered directly to the site of action. Reduced Dosage: Smaller doses may be effective, lowering costs and side effects. Why Target Hepatocytes? 5

6. Receptors Present on Hepatocytes Galactose and N- acetylgalactosamine Asialofetuin Lactose Lactosyl-norcantharidin Folic Acid Transferrin. (LDL), apolipoprotein B, apolipoprotein E High-density lipoprotein (HDL), apolipoprotein A-I Asialoglycoprotein Receptor (ASGPR) Folate Receptors Transferrin receptors LDL receptors Scavenger Receptor Class B Type I (SR-BI) Ligands Receptors Mishra N, Yadav NP, Rai VK, Sinha P, Yadav KS, Jain S, Arora S. Efficient hepatic delivery of drugs: novel strategies and their significance. Biomed Res Int. 2013;2013:382184. 6

7. Asialoglycoprotein Receptor (ASGPR): Predominantly found on the sinusoidal surfaces of hepatocytes. Mainly involved in the endocytosis and clearance of glycoproteins from the bloodstream. Binds specifically to glycoproteins that have exposed terminal galactose (GAL) or N- acetylgalactosamine (GalNAc) residues. 7

8. Targeting Strategies 8 Passive Targeting Active Targeting Via EPR effect Liver sinusoidal fenestrations Receptor-Mediated Targeting Antibody-Based Targeting L iver does not exhibit impermeable basal lamina. Smaller particles below 100 nm escapes interaction with KC and can achieve higher hepatocytes targeting H ydrophilic neutral or negatively charged nano carriers decreases the KC uptake and increases the hepatocytic uptake Hydrophilic coating recommended to decreased KC uptake with higher parenchymal uptake include dextran, phosphatidylinositol, poloxamers, polyvinylpyrrolidone, and cellulose derivatives

9. EPR Effect 9 https://doi.org/10.3389/fphar.2014.00077 The enhanced permeability and retention (EPR) effect is the mechanism by which high–molecular weight nontargeted drugs and prodrugs accumulate in tissues that offer increased vascular permeability, such as in sites of cancer.

10. Targeting Strategies 10 Passive Targeting Active Targeting Via EPR effect Liver sinusoidal fenestrations Receptor-Mediated Targeting Antibody-Based Targeting L iver does not exhibit impermeable basal lamina. Smaller particles below 100 nm escapes interaction with KC and can achieve higher hepatocytes targeting H ydrophilic neutral or negatively charged nano carriers decreases the KC uptake and increases the hepatocytic uptake Hydrophilic coating recommended to decreased KC uptake with higher parenchymal uptake include dextran, phosphatidylinositol, poloxamers, polyvinylpyrrolidone, and cellulose derivatives

11. Böttger R, Pauli G, Chao PH, Al Fayez N, Hohenwarter L, Li SD. Lipid-based nanoparticle technologies for liver targeting. Adv Drug Deliv Rev. 2020;154-155:79-101. 11

12. Research Evidences 12

13. 13 Dipali and co- workers developed lamivudine loaded NPs with PLGA functionalized with maleimide group and then conjugated L-HSA to target the hepatocytes. The confocal laser scanning microscopy was used to study the qualitative cell uptake behavior of the unconjugated (Mal-PLGA NP) and the conjugated NP (LHSA-Mal-PLGA NP). The internalization of both the NPs was monitored by loading them with Rhodamine B dye giving red fluorescence. HepG2 cells were used for the study

14. Method of preparation 14

15. Qualitative cell uptake study 15

16. 16 Giridharan and co-workers developed transferrin-targeted core-shell nanomedicine formed by encapsulating two drugs, doxorubicin and sorafenib against liver cancer.

17. Method of Preparation 17 Prepared by Freeze Thaw/ coacervation method (EDC) 3-ethylcarbodiimide hydrochloride T he expression of TfR in HepG2 cells was studied using flow cytometry . Following that, Th ey have studied intracellular uptake of the core-shell nanoparticles in cancer cells. APC- Allophycocyanin.

18. Transferrin receptor expression and cellular uptake 18 iron-loaded transferrin

19. 19 T he expression of TfR in HepG2 cells was studied using flow cytometry . A1 shows unstained cells (control). ~76% of the malignant cells were found to express TfR before iron chelation as noticed from Figure A2. After creating an iron deficient microenvironment by treating the cells with 100 μM Deferoxamine(DFO), the number of the TfR -expressing cells increased to ~89% ( Figure A3). Figure A4 shows that upon addition of iron-loaded transferrin ( holotransferrin ), the number was reduced to ~42%. Following that, Th ey have studied intracellular uptake of the core-shell nanoparticles in cancer cells. B1 shows that untargeted particles are taken-up by only ~70% of HepG2 cells. Upon conjugating the nanoparticles with transferrin, the uptake was effectively increased to ~83% as shown in Figure 3 , B2. After creating iron-deficient microenvironment, the uptake was further increased to ~94% ( Figure B3) due to the increase in the number of TfRexpressing cells. Upon addition of iron-loaded transferrin, the uptake was dropped to ~69% due to the saturation of TfR expression in these cells ( Figure B4).

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21. Method of preparation Haohao Wang and co-workers prepared FA PEG CTr albumin nanoparticles (FA-PEG- CTr -NPs) for targeting hepatocellular carcinoma (HCC). CTr (a lupine derivative with the strongest activity against HCC ). In vitro targeted uptake of nanoparticle were studied on the HepG2 and BEL-7402 cell line 21

22. In vitro targeted uptake of nanoparticle 22

23. Abbreviation Human hepatocellular carcinoma (HepG2) cells Confocal Laser Scanning Microscope (CLSM) FITC - (fluorescein isothiocyanate) DAPI- (4′,6-diamidino-2-phenylindole) APC- Allophycocyanin. (DCM) dichloromethane. Side Scatter (SSC) Forward Scatter (FSC) (EDC) 3-ethylcarbodiimide hydrochloride 23

24. Reference Böttger R, Pauli G, Chao PH, Al Fayez N, Hohenwarter L, Li SD. Lipid-based nanoparticle technologies for liver targeting. Adv Drug Deliv Rev. 2020;154-155:79-101. Mishra N, Yadav NP, Rai VK, Sinha P, Yadav KS, Jain S, Arora S. Efficient hepatic delivery of drugs: novel strategies and their significance. Biomed Res Int. 2013;2013:382184. Damania , A., Jain, E., & Kumar, A. (2013).  Advancements in in vitro hepatic models: application for drug screening and therapeutics. Hepatology International, 8(1), 23–38. https://doi.org/10.3389/fphar.2014.00077 https://doi.org/10.1016/j.jddst.2018.06.006 https://doi.org/10.1016/j.nano.2014.05.011 https://doi.org/10.1016/j.biopha.2023.115485 24