Lactosylated lipid calcium phosphate-based nanoparticles: A promising approach for efficient DNA delivery to hepatocytes

Document Type : Original Article


1 Department of Medical Biotechnology and Nanotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

2 Department of Pharmaceutical Nanotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

3 Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

4 Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

5 Targeted Drug Delivery Research Center, Mashhad University of Medical Sciences, Mashhad, Iran



Objective(s): For safe and effective gene therapy, the ability to deliver the therapeutic nucleic acid to the target sites is crucial. In this study, lactosylated lipid phosphate calcium nanoparticles (lac-LCP) were developed for targeted delivery of pDNA to the hepatocyte cells. The lac-LCP formulation contained lactose-modified cholesterol (CHL), a ligand that binds to the asialoglycoprotein receptor (ASGR) expressed on hepatocytes, and polyethyleneimine (PEI) in the core.  
Materials and Methods: Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) were used to monitor the chemical modification, and the physicochemical properties of NPs were studied using dynamic light scattering (DLS) and transmission electron microscopy (TEM). To evaluate transfection efficiency, cellular uptake and GFP expression were assessed using fluorescence microscopy and flow cytometry. 
Results: The results revealed that lactose-targeted particles (lac-LCP) had a significant increase in cellular uptake by hepatocytes. The inclusion of a low molecular weight PEI (1.8 KDa) with a low PEI/pDNA ratio of 1 in the core of LCP, elicited high degrees of GFP protein expression (by 5 and 6-fold), which exhibited significantly higher efficiency than PEI 1.8 KDa and Lipofectamine. 
Conclusion: The successful functionalization and nuclear delivery of LCP NPs described here indicate its promise as an efficient delivery vector to hepatocyte nuclei. 


Main Subjects

1. Barros SA, Gollob JA. Safety profile of RNAi nanomedicines. Adv Drug Deliv Rev 2012; 64:1730-1737.
2. Seth S, Johns R, Templin MV. Delivery and biodistribution of siRNA for cancer therapy: Challenges and future prospects. Ther Deliv 2012; 3:245-261.
3. Goswami R, Subramanian G, Silayeva L, Newkirk I, Doctor D, Chawla K, et al. Gene therapy leaves a vicious cycle. Front Oncol 2019; 9:297-321.
4. Shahryari A, Saghaeian Jazi M, Mohammadi S, Razavi Nikoo H, Nazari Z, Hosseini ES, et al. Development and clinical translation of approved gene therapy products for genetic disorders. Front Genet 2019; 10:1-25.
5. Zu H, Gao D. Non-viral vectors in gene therapy: Recent development, challenges, and prospects. AAPS J 2021; 23:1-12.
6. Chung S, Lee CM, Zhang M. Advances in nanoparticle-based mRNA delivery for liver cancer and liver-associated infectious diseases. Nanoscale Horiz 2022; 8:10-28.
7. Witzigmann D, Kulkarni JA, Leung J, Chen S, Cullis PR, van der Meel R. Lipid nanoparticle technology for therapeutic gene regulation in the liver. Adv Drug Deliv Rev 2020; 159:344-363.
8. Ahmed M, Narain R. Carbohydrate-based materials for targeted delivery of drugs and genes to the liver. Nanomedicine (Lond) 2015; 10:2263-2288.
9. Snoeys J, Lievens J, Wisse E, Jacobs F, Duimel H, Collen D, et al. Species differences in transgene DNA uptake in hepatocytes after adenoviral transfer correlate with the size of endothelial fenestrae. Gene Ther 2007; 14:604-612.
10. Crispe IN. Hepatic T cells and liver tolerance. Nat Rev Immunol 2003; 3:51-62.
11. Huang KW, Lai YT, Chern GJ, Huang SF, Tsai CL, Sung YC, et al. Galactose derivative-modified nanoparticles for efficient siRNA delivery to hepatocellular carcinoma. Biomacromolecules 2018; 19:2330-2339.
12. Zhou X, Zhang M, Yung B, Li H, Zhou C, Lee LJ, et al. Lactosylated liposomes for targeted delivery of doxorubicin to hepatocellular carcinoma. Int J Nanomedicine 2012; 7:5465-5474.
13. Zhang M, Zhou X, Wang B, Yung BC, Lee LJ, Ghoshal K, et al. Lactosylated gramicidin-based lipid nanoparticles (Lac-GLN) for targeted delivery of anti-miR-155 to hepatocellular carcinoma. J Control Release 2013; 168:251-261.
14. Nasr M, Kira AY, Saber S, Essa EA, El-Gizawy SA. Telmisartan- loaded lactosylated chitosan nanoparticles as a liver specific delivery system: synthesis, optimization and targeting efficiency. AAPS PharmSciTech 2023; 24:144-159.
15. Hu Y, Haynes MT, Wang Y, Liu F, Huang L. A highly efficient synthetic vector: nonhydrodynamic delivery of DNA to hepatocyte nuclei in vivo. ACS Nano 2013; 7:5376-5384.
16. Yao J, Fan Y, Li Y, Huang L. Strategies on the nuclear-targeted delivery of genes. J Drug Target 2013; 21:926-939.
17. Malaekeh-Nikouei B, Gholami L, Asghari F, Askarian S, Barzegar S, Rezaee M, et al. Viral vector mimicking and nucleus targeted nanoparticles based on dexamethasone polyethylenimine nanoliposomes: Preparation and evaluation of transfection efficiency. Colloids Surf B Biointerfaces 2018; 165:252-261.
18. Sabin J, Alatorre-Meda M, Miñones J Jr, Domínguez-Arca V, Prieto G. New insights on the mechanism of polyethylenimine transfection and their implications on gene therapy and DNA vaccines. Colloids Surf B Biointerfaces 2022; 210:112219.
19. Sikor M, Sabin J, Keyvanloo A, Schneider MF, Thewalt JL, Bailey AE, et al. Interaction of a charged polymer with zwitterionic lipid vesicles. Langmuir 2010; 26:4095-4102.
20. Shi J, Chou B, Choi JL, Ta AL, Pun SH. Investigation of polyethylenimine/DNA polyplex transfection to cultured cells using radiolabeling and subcellular fractionation methods. Mol Pharm 2013; 10:2145-2156.
21. Casper J, Schenk SH, Parhizkar E, Detampel P, Dehshahri A, Huwyler J. Polyethylenimine (PEI) in gene therapy: Current status and clinical applications. J Control Release 2023; 362:667-691.
22. Wu Y, Gu W, Xu ZP. Enhanced combination cancer therapy using lipid-calcium carbonate/phosphate nanoparticles as a targeted delivery platform. Nanomedicine (Lond) 2019; 14:77-92.
23. Li J, Yang Y, Huang L. Calcium phosphate nanoparticles with an asymmetric lipid bilayer coating for siRNA delivery to the tumor. J Control Release 2012; 158:108-114.
24. Tang J, Li L, Howard CB, Mahler SM, Huang L, Xu ZP. Preparation of optimized lipid-coated calcium phosphate nanoparticles for enhanced in vitro gene delivery to breast cancer cells. J Mater Chem B 2015; 3:6805-6812.
25. Schaffer DV, Fidelman NA, Dan N, Lauffenburger DA. Vector unpacking as a potential barrier for receptor-mediated polyplex gene delivery. Biotechnol Bioeng 2000; 67:598-606.
26. Maestro S, Weber ND, Zabaleta N, Aldabe R, Gonzalez-Aseguinolaza G. Novel vectors and approaches for gene therapy in liver diseases. JHEP Rep 2021; 3:100300.
27. Kawakami S, Hashida M. Glycosylation-mediated targeting of carriers. J Control Release 2014; 190:542-555.
28. D’Souza AA, Devarajan PV. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications. J Control Release 2015; 203:126-139.
29. Li J, Chen YC, Tseng YC, Mozumdar S, Huang L. Biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery. J Control Release 2010; 142:416-421.
30. Wilson G. Effect of reductive lactosamination on the hepatic uptake of bovine pancreatic ribonuclease A dimer. J Biol Chem 1978; 253:2070-2072.
31. Lundquist JJ, Toone EJ. The cluster glycoside effect. Chem Rev 2002; 102:555-578.
32. Ma P, Liu S, Huang Y, Chen X, Zhang L, Jing X. Lactose mediated liver-targeting effect observed by ex vivo imaging technology. Biomater 2010; 31:2646-2654.
33. Wang X, Qi Y, Liu L, Ganbold T, Baigude H, Han J. Preparation and cell activities of lactosylated curdlan-triornithine nanoparticles for enhanced DNA/siRNA delivery in hepatoma cells. Carbohydr Polym 2019; 225:115252.
34. Pathak PO, Nagarsenker MS, Barhate CR, Padhye SG, Dhawan VV, Bhattacharyya D, et al. Cholesterol anchored arabinogalactan for asialoglycoprotein receptor targeting: synthesis, characterization, and proof of concept of hepatospecific delivery. Carbohydr Res 2015; 408:33-43.
35. Li H, Cui Y, Liu J, Bian S, Liang J, Fan Y, et al. Reduction breakable cholesteryl pullulan nanoparticles for targeted hepatocellular carcinoma chemotherapy. J Mater Chem B 2014; 2:3500-3510.
36. Bodnar M, Hartmann JF, Borbely J. Preparation and characterization of chitosan-based nanoparticles. Biomacromolecules 2005; 6:2521-2527.
37. Jerzykiewicz J, Czogalla A. Polyethyleneimine-based lipopolyplexes as carriers in anticancer gene therapies. Materials (Basel) 2021; 15:179.
38. Ewe A, Schaper A, Barnert S, Schubert R, Temme A, Bakowsky U, et al. Storage stability of optimal liposome-polyethylenimine complexes (lipopolyplexes) for DNA or siRNA delivery. Acta Biomater 2014; 10:2663-2673.
39. Lampela P, Soininen P, Urtti A, Männistö PT, Raasmaja A. Synergism in gene delivery by small PEIs and three different nonviral vectors. Int J Pharm 2004; 270:175-184.
40. Kunath K, von Harpe A, Fischer D, Petersen H, Bickel U, Voigt K, et al. Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: Comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine. J Control Release 2003; 89:113-125.
41. Kazemi Oskuee R, Dabbaghi M, Gholami L, Taheri-Bojd S, Balali-Mood M, Mousavi SH, et al. Investigating the influence of polyplex size on toxicity properties of polyethylenimine mediated gene delivery. Life Sci 2018; 197:101-108.