Comparison of two kinds of nanomedicine for targeted gene therapy: premodified or postmodified gene delivery systems

Abstract

Background

The applications of ligand-polyethylene glycol (PEG)-modified nanocarriers have now emerged, as well as recognized strategies to provide the vectors with active targeting properties. In this research, premodification and postmodification were compared using the same ligand, ie, a novel conjugated mannan-containing PEG and L-α-phosphatidylethanolamine (PE).

Methods

Premodified and postmodified solid lipid nanoparticles were prepared and the characteristics of the two kinds of vehicles were evaluated. The modified vectors were then administered intravenously to rats and the in vivo targeting behavior of the complexes was investigated in liver macrophages.

Results

By carefully formulating the carriers with an optimal ratio of mannan-containing PEG-PE, postmodified vehicles displayed more efficient gene expression in rat Kupffer cells both in vitro and in vivo.

Conclusion

Postmodified gene carriers are superior to premodified gene vectors, although the latter is also promising for targeted gene delivery. This discovery could guide our future research.

Keywords:

• gene therapy
• nanomedicine
• mannan
• polyethylene glycol
• L-α-phosphatidylethanolamine
• ligands
• premodification
• postmodification

Supplementary data

In vivo gene delivery (Figure 9)

Details of the experiments and data analysis are as follows: Cells were washed in cold phosphate-buffered saline three times and the fluorescence intensity of the cells was determined using a flow cytometer equipped with a 488 nm argon laser for excitation. For each sample, about 10,000 events were collected and fluorescence was detected. Signals were amplified in logarithmic mode for fluorescence to determine the positive events by a standard gating technique. The percentage of positive events was calculated as the events within the gate divided by the total number of events, excluding cell debris. The percentage of cells transfected with plasmid-enhanced green fluorescent protein appeared at the upper right and lower right quadrants of the diagram. The data for the quadrantal diagram of the in vivo study are as follows:

1. Naked plasmid-enhanced green fluorescent protein 24 hours post transfection

   Quad X mean Y mean Events%Total%Gated

   1. UL 6.8 18.3 5 0.04 74.01

   2. UR 107.6 78.6 31 0.26 1.18

   3. LL 2.4 8.5 11986 97.42 24.33

   4. LR 43.8 14.1 65 0.54 0.47

   Gene expression UR + LR = 1.65%

2. Naked plasmid-enhanced green fluorescent protein 48 hours post transfection

   Quad X mean Y mean Events%Total%Gated

   1. UL 6.8 18.3 5 0.04 69.27

   2. UR 107.6 78.6 31 0.26 2.03

   3. LL 2.4 8.5 11986 97.42 28.38

   4. LR 43.8 14.1 65 0.54 0.32

   Gene expression: UR + LR = 2.35%

3. Naked plasmid-enhanced green fluorescent protein 72 hours post transfection

   Quad X mean Y mean Events%Total%Gated

   1. UL 6.8 18.3 5 0.04 71.36

   2. UR 107.6 78.6 31 0.26 1.47

   3. LL 2.4 8.5 11986 97.42 26.39

   4. LR 43.8 14.1 65 0.54 0.75

   Gene expression: UR + LR = 2.22%

4. SLN/DNA 24 hours post transfection

   Quad X mean Y mean Events%Total%Gated

   1. UL 6.8 18.3 5 0.04 61.72

   2. UR 107.6 78.6 31 0.26 5.56

   3. LL 2.4 8.5 11986 97.42 27.86

   4. LR 43.8 14.1 65 0.54 4.86

   Gene expression: UR + LR = 10.42%

5. SLN/DNA 48 hours post transfection

   Quad X mean Y mean Events%Total%Gated

   1. UL 6.8 18.3 5 0.04 58.24

   2. UR 107.6 78.6 31 0.26 12.41

   3. LL 2.4 8.5 11986 97.42 23.79

   4. LR 43.8 14.1 65 0.54 5.52

   Gene expression: UR + LR = 17.93%

6. SLN/DNA 72 hours post transfection

   Quad X mean Y mean Events%Total%Gated

   1. UL 6.8 18.3 5 0.04 58.24

   2. UR 107.6 78.6 31 0.26 12.41

   3. LL 2.4 8.5 11986 97.42 23.79

   4. LR 43.8 14.1 65 0.54 5.52

   Gene expression: UR + LR = 17.93%

7. PEG-PE-SLN/DNA 24 hours post transfection

   Quad X mean Y mean Events%Total%Gated

   1. UL 6.8 18.3 5 0.04 70.06

   2. UR 107.6 78.6 31 0.26 6.40

   3. LL 2.4 8.5 11986 97.42 18.61

   4. LR 43.8 14.1 65 0.54 4.93

   Gene expression: UR + LR = 11.33%

8. PEG-PE-SLN/DNA 48 hours post transfection

   Quad X mean Y mean Events%Total%Gated

   1. UL 6.8 18.3 5 0.04 52.16

   2. UR 107.6 78.6 31 0.26 16.87

   3. LL 2.4 8.5 11986 97.42 25.30

   4. LR 43.8 14.1 65 0.54 5.65

   Gene expression: UR + LR = 22.52%

9. PEG-PE-SLN/DNA 72 hours post transfection

   Quad X mean Y mean Events%Total%Gated

   1. UL 6.8 18.3 5 0.04 49.35

   2. UR 107.6 78.6 31 0.26 19.08

   3. LL 2.4 8.5 11986 97.42 25.05

   4. LR 43.8 14.1 65 0.54 6.52

   Gene expression: UR + LR = 25.6%

10. A-MN-SLN/DNA 24 hours post transfection

    Quad X mean Y mean Events%Total%Gated

    1. UL 6.8 18.3 5 0.04 57.53

    2. UR 107.6 78.6 31 0.26 11.42

    3. LL 2.4 8.5 11986 97.42 22.82

    4. LR 43.8 14.1 65 0.54 8.23

    Gene expression: UR + LR = 19.65%

11. A-MN-SLN/DNA 48 hours post transfection

    Quad X mean Y mean Events%Total%Gated

    1. UL 6.8 18.3 5 0.04 46.36

    2. UR 107.6 78.6 31 0.26 22.48

    3. LL 2.4 8.5 11986 97.42 25.25

    4. LR 43.8 14.1 65 0.54 5.88

    Gene expression: UR + LR = 28.36%

12. A-MN-SLN/DNA 72 hours post transfection

    Quad X mean Y mean Events%Total%Gated

    1. UL 6.8 18.3 5 0.04 48.78

    2. UR 107.6 78.6 31 0.26 20.93

    3. LL 2.4 8.5 11986 97.42 20.77

    4. LR 43.8 14.1 65 0.54 9.52

    Gene expression: UR + LR = 30.45%

13. B-MN-SLN/DNA 24 hours post transfection

    Quad X mean Y mean Events%Total%Gated

    1. UL 6.8 18.3 5 0.04 49.74

    2. UR 107.6 78.6 31 0.26 17.78

    3. LL 2.4 8.5 11986 97.42 23.14

    4. LR 43.8 14.1 65 0.54 9.34

    Gene expression: UR + LR = 27.12%

14. B-MN-SLN/DNA 48 hours post transfection

    Quad X mean Y mean Events%Total%Gated

    1. UL 6.8 18.3 5 0.04 44.73

    2. UR 107.6 78.6 31 0.26 25.15

    3. LL 2.4 8.5 11986 97.42 17.21

    4. LR 43.8 14.1 65 0.54 12.88

    Gene expression: UR + LR = 38.03%

15. B-MN-SLN/DNA 72 hours post transfection

    Quad X mean Y mean Events%Total%Gated

    1. UL 6.8 18.3 5 0.04 37.26

    2. UR 107.6 78.6 31 0.26 32.67

    3. LL 2.4 8.5 11986 97.42 19.23

    4. LR 43.8 14.1 65 0.54 10.83

    Gene expression: UR + LR = 43.5%

Disclosure

The authors report no conflicts of interest in this work.