Structure and biological activity of pathogen-like synthetic nanomedicines

Abstract

Here we characterize the structure, stability and intracellular mode of action of DermaVir nanomedicine that is under clinical development for the treatment of HIV/AIDS. This nanomedicine comprises pathogen-like pDNA/PEIm nanoparticles (NPs) having the structure and function resembling spherical viruses that naturally evolved to deliver nucleic acids to the cells. Atomic force microscopy demonstrated spherical 100 - 200 nm NPs with a smooth polymer surface protecting the pDNA in the core. Optical absorption determined both the NP structural stability and biological activity relevant to their ability to escape from the endosome and release the pDNA at the nucleus. Salt, pH and temperature influence nanomedicine shelf-life and intracellular stability. This approach facilitates the development of diverse polyplex nanomedicines where the delivered pDNA-expressed antigens induce immune responses to kill infected cells.

From the clinical editor: The authors investigated DermaVir nanomedicine comprised of pathogen-like pDNA/PEIm nanoparticles with structure and function resembling spherical viruses. DermaVir delivery of pDNA expresses antigens that induce immune responses to kill HIV infected cells.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

Figure 1. Investigation of the particle sizes of pDNA/PEIm NPs

a, AFM image of pDNA in the absence of PEIm on mica surface. The pDNA strands form a random mesh typical of polymer solutions of long overlapping chains. b, AFM images of DV2 on mica. The NPs have smooth interfaces. c, AFM image of DV1 on mica. Hair-like protrusions of presumably uncompactified DNA are clearly visible. d, Magnified AFM image of the DV1. The minimum pDNA width is 6nm indicating that this part of the pDNA is uncoated DNA. At other places the pDNA is thicker (about 10 nm) suggesting that PEIm coats the pDNA. e, Representative particle size distributions of DV1 and DV2 measured by DLS; ■–DV1 and ■–DV2.

Figure 1. Investigation of the particle sizes of pDNA/PEIm NPs

a, AFM image of pDNA in the absence of PEIm on mica surface. The pDNA strands form a random mesh typical of polymer solutions of long overlapping chains. b, AFM images of DV2 on mica. The NPs have smooth interfaces. c, AFM image of DV1 on mica. Hair-like protrusions of presumably uncompactified DNA are clearly visible. d, Magnified AFM image of the DV1. The minimum pDNA width is 6nm indicating that this part of the pDNA is uncoated DNA. At other places the pDNA is thicker (about 10 nm) suggesting that PEIm coats the pDNA. e, Representative particle size distributions of DV1 and DV2 measured by DLS; ■–DV1 and ■–DV2.

Figure 1. Investigation of the particle sizes of pDNA/PEIm NPs

a, AFM image of pDNA in the absence of PEIm on mica surface. The pDNA strands form a random mesh typical of polymer solutions of long overlapping chains. b, AFM images of DV2 on mica. The NPs have smooth interfaces. c, AFM image of DV1 on mica. Hair-like protrusions of presumably uncompactified DNA are clearly visible. d, Magnified AFM image of the DV1. The minimum pDNA width is 6nm indicating that this part of the pDNA is uncoated DNA. At other places the pDNA is thicker (about 10 nm) suggesting that PEIm coats the pDNA. e, Representative particle size distributions of DV1 and DV2 measured by DLS; ■–DV1 and ■–DV2.

Figure 1. Investigation of the particle sizes of pDNA/PEIm NPs

a, AFM image of pDNA in the absence of PEIm on mica surface. The pDNA strands form a random mesh typical of polymer solutions of long overlapping chains. b, AFM images of DV2 on mica. The NPs have smooth interfaces. c, AFM image of DV1 on mica. Hair-like protrusions of presumably uncompactified DNA are clearly visible. d, Magnified AFM image of the DV1. The minimum pDNA width is 6nm indicating that this part of the pDNA is uncoated DNA. At other places the pDNA is thicker (about 10 nm) suggesting that PEIm coats the pDNA. e, Representative particle size distributions of DV1 and DV2 measured by DLS; ■–DV1 and ■–DV2.

Figure 1. Investigation of the particle sizes of pDNA/PEIm NPs

a, AFM image of pDNA in the absence of PEIm on mica surface. The pDNA strands form a random mesh typical of polymer solutions of long overlapping chains. b, AFM images of DV2 on mica. The NPs have smooth interfaces. c, AFM image of DV1 on mica. Hair-like protrusions of presumably uncompactified DNA are clearly visible. d, Magnified AFM image of the DV1. The minimum pDNA width is 6nm indicating that this part of the pDNA is uncoated DNA. At other places the pDNA is thicker (about 10 nm) suggesting that PEIm coats the pDNA. e, Representative particle size distributions of DV1 and DV2 measured by DLS; ■–DV1 and ■–DV2.

Figure 2. Spectrophotometric and zeta potential investigation of pDNA/PEIm NPs (DV2)

a, , UV-VIS-spectra of pDNA/PEIm NP and its components; pDNA (○), PEIm (□), pDNA/PEIm NP (△). b, Titration of pDNA (10μg/ml) with PEIm by UV-spectrophotometry (■) and zeta potential analysis (○). N/P=0 sample contains only pDNA. c, UV-VIS-spectra of pDNA (○), pDNA/PEIm NP before SDS treatment (△) and pDNA/PEIm NP after SDS treatment (×). SDS absorbance was subtracted from the spectrum. d, Residual gel electrophoresis of the pDNA (lane #2) and the NP before (lane #3) and after (lane #4) SDS treatment.

Figure 2. Spectrophotometric and zeta potential investigation of pDNA/PEIm NPs (DV2)

a, , UV-VIS-spectra of pDNA/PEIm NP and its components; pDNA (○), PEIm (□), pDNA/PEIm NP (△). b, Titration of pDNA (10μg/ml) with PEIm by UV-spectrophotometry (■) and zeta potential analysis (○). N/P=0 sample contains only pDNA. c, UV-VIS-spectra of pDNA (○), pDNA/PEIm NP before SDS treatment (△) and pDNA/PEIm NP after SDS treatment (×). SDS absorbance was subtracted from the spectrum. d, Residual gel electrophoresis of the pDNA (lane #2) and the NP before (lane #3) and after (lane #4) SDS treatment.

Figure 2. Spectrophotometric and zeta potential investigation of pDNA/PEIm NPs (DV2)

a, , UV-VIS-spectra of pDNA/PEIm NP and its components; pDNA (○), PEIm (□), pDNA/PEIm NP (△). b, Titration of pDNA (10μg/ml) with PEIm by UV-spectrophotometry (■) and zeta potential analysis (○). N/P=0 sample contains only pDNA. c, UV-VIS-spectra of pDNA (○), pDNA/PEIm NP before SDS treatment (△) and pDNA/PEIm NP after SDS treatment (×). SDS absorbance was subtracted from the spectrum. d, Residual gel electrophoresis of the pDNA (lane #2) and the NP before (lane #3) and after (lane #4) SDS treatment.

Figure 2. Spectrophotometric and zeta potential investigation of pDNA/PEIm NPs (DV2)

a, , UV-VIS-spectra of pDNA/PEIm NP and its components; pDNA (○), PEIm (□), pDNA/PEIm NP (△). b, Titration of pDNA (10μg/ml) with PEIm by UV-spectrophotometry (■) and zeta potential analysis (○). N/P=0 sample contains only pDNA. c, UV-VIS-spectra of pDNA (○), pDNA/PEIm NP before SDS treatment (△) and pDNA/PEIm NP after SDS treatment (×). SDS absorbance was subtracted from the spectrum. d, Residual gel electrophoresis of the pDNA (lane #2) and the NP before (lane #3) and after (lane #4) SDS treatment.

Figure 3

Investigation of pDNA/PEIm NP stability. a, Hc of DV1 measured in different time points after NP preparation; (□) 4°C, (○) 25°C, (△) 37°C. b, Hc of DV2 measured in different time points after NP preparation; (□) 4°C, (○) 25°C, (△) 37°C. c, Hc of DV1 (□), DV2 (●) and control pDNA (×) measured in different time points after incubating at 37°C.

Figure 3

Investigation of pDNA/PEIm NP stability. a, Hc of DV1 measured in different time points after NP preparation; (□) 4°C, (○) 25°C, (△) 37°C. b, Hc of DV2 measured in different time points after NP preparation; (□) 4°C, (○) 25°C, (△) 37°C. c, Hc of DV1 (□), DV2 (●) and control pDNA (×) measured in different time points after incubating at 37°C.

Figure 3

Investigation of pDNA/PEIm NP stability. a, Hc of DV1 measured in different time points after NP preparation; (□) 4°C, (○) 25°C, (△) 37°C. b, Hc of DV2 measured in different time points after NP preparation; (□) 4°C, (○) 25°C, (△) 37°C. c, Hc of DV1 (□), DV2 (●) and control pDNA (×) measured in different time points after incubating at 37°C.

Figure 4. Effect of the ionic environment on the hyperchromicity of the pDNA/PEIm NPs

a, , Linear correlation between the pH of the solvent and the Hc% of pDNA/PEIm NPs. NP formulations were prepared using the same pDNA and PEIm at N/P ratio of 4 in different pH TEAM solutions. The pH of the DV1 and DV2 are marked with the arrows. (y=1.9403x+6.3354; r2=0.98) b, Inverse correlation between the NaCl concentration of pDNA solution and the measured Hc% of NP. Meq on the x axis stands for molar equivalents calculated on the phosphate concentration of pDNA (y=−5.1448x+29.761; r2=0.95).

Figure 4. Effect of the ionic environment on the hyperchromicity of the pDNA/PEIm NPs

a, , Linear correlation between the pH of the solvent and the Hc% of pDNA/PEIm NPs. NP formulations were prepared using the same pDNA and PEIm at N/P ratio of 4 in different pH TEAM solutions. The pH of the DV1 and DV2 are marked with the arrows. (y=1.9403x+6.3354; r2=0.98) b, Inverse correlation between the NaCl concentration of pDNA solution and the measured Hc% of NP. Meq on the x axis stands for molar equivalents calculated on the phosphate concentration of pDNA (y=−5.1448x+29.761; r2=0.95).

Figure 5. Effect of the hyperchromicity on gene expression

a, , Biological activity of DV1 and DV2. b Demonstration of the higher nuclease resistance of DV2 compared to DV1 formulation. Lanes: #1: marker; #2: DV1 digested with aspecific endonuclease and decomplexed; #3: DV2 digested with aspecific endonuclease and decomplexed; #4: pDNA digested with aspecific endonuclease; #5 control, untreated pDNA. c, Hypothetical model of pDNA nanomedicine uptake and antigen expression exemplified with the DermaVir HIV nanomedicine candidate. When Hc is optimal, the nanomedicine escapes from endosomal degradation, loosens in the cytosol and the pDNA reaches the nucleus, where the encoded several antigens are expressed. d, Complement activation of DV1, DV2, pDNA and PEIm. Positive control is Zymosan.

Figure 5. Effect of the hyperchromicity on gene expression

a, , Biological activity of DV1 and DV2. b Demonstration of the higher nuclease resistance of DV2 compared to DV1 formulation. Lanes: #1: marker; #2: DV1 digested with aspecific endonuclease and decomplexed; #3: DV2 digested with aspecific endonuclease and decomplexed; #4: pDNA digested with aspecific endonuclease; #5 control, untreated pDNA. c, Hypothetical model of pDNA nanomedicine uptake and antigen expression exemplified with the DermaVir HIV nanomedicine candidate. When Hc is optimal, the nanomedicine escapes from endosomal degradation, loosens in the cytosol and the pDNA reaches the nucleus, where the encoded several antigens are expressed. d, Complement activation of DV1, DV2, pDNA and PEIm. Positive control is Zymosan.

Figure 5. Effect of the hyperchromicity on gene expression

a, , Biological activity of DV1 and DV2. b Demonstration of the higher nuclease resistance of DV2 compared to DV1 formulation. Lanes: #1: marker; #2: DV1 digested with aspecific endonuclease and decomplexed; #3: DV2 digested with aspecific endonuclease and decomplexed; #4: pDNA digested with aspecific endonuclease; #5 control, untreated pDNA. c, Hypothetical model of pDNA nanomedicine uptake and antigen expression exemplified with the DermaVir HIV nanomedicine candidate. When Hc is optimal, the nanomedicine escapes from endosomal degradation, loosens in the cytosol and the pDNA reaches the nucleus, where the encoded several antigens are expressed. d, Complement activation of DV1, DV2, pDNA and PEIm. Positive control is Zymosan.

Figure 5. Effect of the hyperchromicity on gene expression

a, , Biological activity of DV1 and DV2. b Demonstration of the higher nuclease resistance of DV2 compared to DV1 formulation. Lanes: #1: marker; #2: DV1 digested with aspecific endonuclease and decomplexed; #3: DV2 digested with aspecific endonuclease and decomplexed; #4: pDNA digested with aspecific endonuclease; #5 control, untreated pDNA. c, Hypothetical model of pDNA nanomedicine uptake and antigen expression exemplified with the DermaVir HIV nanomedicine candidate. When Hc is optimal, the nanomedicine escapes from endosomal degradation, loosens in the cytosol and the pDNA reaches the nucleus, where the encoded several antigens are expressed. d, Complement activation of DV1, DV2, pDNA and PEIm. Positive control is Zymosan.