Head on Comparison of Self- and Nano-assemblies of Gamma Peptide Nucleic Acid Amphiphiles

Abstract

Peptide nucleic acids (PNAs) are nucleic acid analogs with superior hybridization properties and enzymatic stability than deoxyribonucleic acid (DNA). In addition to gene targeting applications, PNAs have garnered significant attention as bio-polymer due to the Watson-Crick -based molecular recognition and flexibility of synthesis. Here, we engineered PNA amphiphiles using chemically modified gamma PNA (8 mer in length) containing hydrophilic diethylene glycol units at the gamma position and covalently conjugated lauric acid (C12) as a hydrophobic moiety. Gamma PNA (γPNA) amphiphiles self-assemble into spherical vesicles. Further, we formulate nano-assemblies using the amphiphilic γPNA as a polymer via ethanol injection-based protocols. We perform comprehensive head-on comparison of the physicochemical and cellular uptake properties of PNA derived self- and nano-assemblies. Small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) analysis reveal ellipsoidal morphology of γPNA nano-assemblies that results in superior cellular delivery compate to the spherical self-assembly. Next, we compare the functional activities of γPNA self-and nano-assemblies in lymphoma cells via multiple endpoints, including gene expression, cell viability, and apoptosis-based assays. Overall, we establish that γPNA amphiphile is a functionally active bio-polymer to formulate nano-assemblies for a wide range of biomedical applications.

Keywords: PNA amphiphiles; gamma peptide nucleic acids (γPNAs); micro-RNA-155; nano-assemblies; self-assembly.

Figure 1.

(A) Chemical structures of DNA, PNA, MiniPEGγPNA (^MPγPNA) monomers and lauric acid (LA). (B) The sequence of oncomiR-155 target and sequence of PNAs used in the study. The designed PNAs were complementary to the seed region of oncomiR-155 (underlined segment). γPNA-155 was synthesized using MiniPEG γPNA monomers and PNA-155 was synthesized using regular PNA monomers. Lauric acid was appended to N terminus or 5’ end and one lysine (K) was appended to the C terminus or 3’ end of both the PNAs. 5-Carboxytetramethylrhodamine (TAMRA) dye was appended to the C terminus or 3’ end of both the PNAs for visualization purposes. OOO is 11-amino-3,6,9-Trioxaundecanoic Acid linker.

Figure 2.

(A) The hydrodynamic diameter, (B) polydispersity index (PDI), and (C) surface charge of γPNA-155, γPNA-155 nano-assemblies (NA), PNA-155 and PNA-155 NA. The results are presented as mean ± SD (n=3). The 10x dilution of γPNA-155 NA and PNA-155 NA was used for characterization. γPNA-155 and PNA-155 dilutions were prepared at the same concentration as the 10x dilutions of their respective NAs. (D) Transmission electron microscopy (TEM) images of γPNA-155, γPNA-155 NA, PNA-155, and PNA-155 NA.

Figure 3.

The SANS patterns of (A) γPNA-155 and (B) γPNA-155 NA. (C) The SAXS pattern of γPNA-155 NA. (D) The cross section of the oblate ellipsoid shape of γPNA-155 NA. (E) The fitting results of γPNA-155 (SANS) and γPNA-155 NA (SANS and SAXS). The lighter and darker dashed lines correspond to the contributions of the larger and smaller morphologies, respectively.

Figure 4.

(A) Confocal microscopy images of live HeLa cells indicating uptake of γPNA-155 and γPNA-155 nano-assemblies (NA) at different PNA equivalent dose after 24 hours of treatment. The scale bar represents 80 pixels. Blue indicates nucleus and red indicates TAMRA. (B) The flow cytometry histograms showing uptake of γPNA-155 and γPNA-155 NA. (C) The quantification γPNA-155 and γPNA-155 NA containing HeLa cells at indicated doses via flow cytometry. The results are presented as mean ±SD (n=3) and t-test was used for statistical analysis. (D) Confocal microscopy images of live U2932 cells after 24 hours of treatment with γPNA-155 and γPNA-155 NA at different PNA equivalent doses. The scale bar represents 80 pixels. Blue represents nucleus and red is TAMRA. (E) Flow cytometry histograms depicting the uptake of γPNA-155 and γPNA-155 NA in U2932 cells after 24 hours of treatment. (F) The quantitative representation of γPNA-155 and γPNA-155 NA containing U2932 cells. The results are represented as mean±SD (n=3) and t-test was used for statistical analysis. **p<0.01, ***p<0.001.

Figure 5.

(A) The quantitative representation of γPNA-155 and γPNA-155 nano-assemblies (NA) uptake in HeLa cells at 37°C and 4°C after 2 hour of incubation. The results are represented as mean±SD (n=3) and t-test was used for statistical analysis. ****p<0.0001. (B) Graph representing the change in cellular transport of γPNA-155 and γPNA-155 NA in HeLa cells via flow cytometry analysis after inhibition of endocytic pathways using genistein, chlorpromazine, and amiloride which inhibits caveolae-mediated, clathrin-mediated, and macropinocytosis cellular transport respectively. The results are represented as mean±SD (n=3) and t-test was used for statistical analysis. **p<0.01, ***p<0.001, ****p<0.0001. (C) The fluorescent images of HeLa cells showing uptake of γPNA-155 and γPNA-155 NA after small molecule (chlorpromazine, genistein, amiloride) mediated inhibition of endocytic pathways and 2 hour treatment duration. Scale bar represents 80 pixels. Blue indicates nucleus and red is TAMRA.

Figure 6.

(A) The graph representing cellular viability of peripheral blood mononuclear cells (PBMC) after 48-hour treatment with γPNA-155 and γPNA-155 nano-assemblies (NA). Untreated cells were used as control. Results are presented as mean±SD (n=5). (B) The fold change in U2932 miR-155 levels after 48-hour treatment with γPNA-155 and γPNA-155 NA at indicated doses. The results are presented as mean±SD (n=3) and t-test was used for statistical analysis. *p<0.05, **p<0.01, ****p<0.0001. (C) The fold change in levels of downstream targets of miR-155 in U2932 cells after 48-hour treatment with γPNA-155 and γPNA-155 NA at 0.5 uM dose. The results are presented as mean±SD (n=3) and t-test was used for statistical analysis. *p<0.05, **p<0.01. (D) The change in percentage of live U2932 cells after treatment with γPNA-155 and γPNA-155 NA at 0.5 uM PNA equivalent dose. The results are presented as mean±SD (n=3) and t-test was used for statistical analysis. *p<0.05, ****p<0.0001.