Purpose: Immune cells are very rigid and almost impossible to transfect them. Many approaches were reported to transfect immune cells like dendritic cell published elsewhere [1-4]. Repeated lys-ala-leu-ala peptide oriented DNA-lipid nanoparticles induced boom transgene expression and antigen presentation in BMDC (Bone Marrow Derived Dendritic Cell) [1, 2, 5]. Still the mechanism of repeated lys-ala-leu-ala peptide in gene expression is not clear enough though some researchers indicated to its helical property. However, alfa-helix property of protein-peptide is a very common phenomena for their secondary structure. Here a ternary complex, TCM was prepared in order to evaluate Lys-ala-leu-ala peptide in transgene expression from immune cell like DC.
Methods: TCM was prepared using CpG free DNA containing luciferase gene and protamine (N/P ratio of 1.5) followed by stearyl Lys-Ala-Leu-Ala peptide as post modification. The nano particle was prepared with a size of 147 nm as well as electrical potential of +32 mV, while measured in a zeta sizer Nano. The nanoparticles were transfected using bone marrow derived dendritic cells and luciferase activity was measured by a luminometer. HAT activity was measured using HAT assay kit as per the guidance of manufacturer protocol. A negative control of anacardic acid and octa arginine were used, where two positive controls of H3 and H4 were also used accordingly. Fluorescence was measured by an ELISA reader.
Two G-Protein couple receptor blockers like cetirizine and ranitidine were used to check G-protein couple dependency in terms of gene expression. Transgene expression shutdown was checked by luciferase activity.
Repeated lys-ala-leu-ala peptide was also used to prepare NBD-DOPE-KALA liposome containing sulfo-rhodamine. The labeled liposomes were purified by ultracentrifugation. The labeled liposome was transfected into the dendritic cell and examine under a Fluoview confocal microscope. The nucleuses of the cells were stained with the Hoechst 33342 Dye (blue fluorescent dye to stain dsDNA) just prior washing the cells. The cells were also treated with H1 blocker, cetirizine 0.9mM for one hour prior transfection of labeled nanoparticles.
Results: TCM has got a transgene expression of luciferase in dendritic cell more than 105 (RLU /mg protein) magnitude in comparison to that of a protamine complex (protamine core). Protamine core of DNA has got no gene expression at all (Figure 1a). Later the repeated lys-ala-leu-ala peptide (KALA) was evaluated for HAT activity and found the similarity effect of both H3 and H4, where an octa arginine peptide showed no remarkable similarity (Figure 1b).
Generally speaking, HAT caused histone acetylation, which is directly involved with transcriptional activation for gene expression and associated with euchromatin accumulation. Euchromatin accumulation favors transcriptional activation because of binding transcriptional factors to the regulatory sites on DNA in euchromatin, which is less densely compact. Moreover, the KALA peptide showed structural similarity with a vasoactive peptide hormone like secretin, which is G-protein couple receptor bound hormone. Both are of 27 amino acids peptides, where arginine-leucine rich in secretin hormone, and Lysine-leucine rich in KALA peptide (Figure 2), respectively.
When we used two G-protein receptor inhibitors like H1 and H2 blockers (cetirizine, ranitidine, respectively) (Figure 2), we found no gene expression at all both in luciferase activity and also in confocal microscopic view (Figure 3).
Collectively our data suggest that KALA has got the evidence of vasoactive potentiality here in terms of transgene expression in BMDC. However, further molecular studies should be appreciated for exploration of the studies, especially condensation/decondensation status of DNA in euchromatin zone . No conflict of interest is declared so far.
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2. Miura, Shaheen, Akita et al. Nucleic Acids Res. 2015; 43(3):1317-31.
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4. Un K. et al. Mol Pharm. 2011; 8(2):543-54.
5. Shaheen et al. PharmacologyOnline 2018, 1:1, 209-230.
6. Shaheen et al. Nucleic Acids Research, 2011, 39(7).
Md. Jashim Uddin– Associate Professor (Research), Department of Biochemistry, Vanderbilt University
M. Mustafezur Rahman– Head, Department of Pharmacy, Daffodil International University, Dhaka
A. K. Azad– Assistant Professor, Department of Pharmacy, Daffodil International University
M. M. Islam– Lecturer, Department of Pharmacy, Daffodil International University
M. Sarowar Hossain– Assistant Professor, Department of Pharmacy, Daffodil International University