This study reports high-efficiency delivery of nucleic acids to eukaryotic cells using MEND particles containing polycation-condensed nucleic acids encapsulated in an R8-DOPE lipid envelope. The R8 peptide was shown to be the most efficient oligoarginine.29
MEND particles are non-cytotoxic and achieve transfection efficiencies as high as adenovirus. The high efficiency of MEND particles is attributed at least in part to the fact that the R8 modification promotes cellular uptake by macropinocytosis, which improved intracellular trafficking towards more efficient gene expression. Thus, MEND-mediated gene delivery provides a novel and efficient mechanism for non-viral transfection of eukaryotic cells, which may be useful for clinical applications including gene therapy.
R8-MEND particles can be prepared with or without fusogenic lipids such as DOPE/CHEMS; however, in experiments described here, fusogenic lipids are required to enhance cytosolic delivery for optimal transfection efficiency. The lipid envelope of MEND particles can also be modified with other functional devices such as polyethyleneglycol, a targeting ligand or a NLS. Thus, MEND can be readily adapted to specific biological systems to achieve desired biological effects.
Recent studies show that TAT–Cre fusion proteins as well as R8 and TAT peptides enter cells primarily via macropinocytosis.30–32
These results are consistent with our observation that R8-MEND particles enter the cell via macropinocytosis. In addition, consistent with reports suggesting that macropinosomes fuse with lysosomes at a low rate in non-phagocytic cells,30,33
liposomes modified with a high surface density of the R8 peptide only partially colocalize with lysosomes.11
Because of these properties, the particles are resistant to lysosomal degradation, and nuclear delivery and subsequent gene expression are enhanced. However, complexes formed between plasmid DNA (pDNA) and R8 or STR-R8 peptides are internalized via classical endocytosis and become trapped in endosomes.12,34
This suggests that the topology of R8 on a particle surface has a significant effect on the mechanism by which the particle enters the cell. Similar observations were made previously regarding the impact of the topology of the fusogenic peptide GALA on endosomal escape.7
Thus, the topology and content of a lipid envelope significantly influence both particle uptake and the function of devices embedded in the envelope. Although the diameters of MENDs1-3 were somewhat different (Supplementary Table 1
online), it is suggested that this difference has no significant effect on the uptake pathway of the particles. We have previously reported that 5% R8-liposomes of around 100 nm was taken up by macropinocytosis.11
Here, we confirmed that the larger R8-MEND3 particles (330 nm) are taken up mainly by the same pathway (). Even much smaller arginine-rich peptides and peptide-fusion proteins were taken up by macropinocytosis.30,32
This suggests that the size difference of different MENDs does not affect the uptake pathway. In addition, the uptake via macropinocytosis involves the formation of large macropinosomes (>1 μm), which allows the internalization of small as well as larger MENDs. This is clearly different form the classical clathrin-mediated endocytosis (involves endosomes with a size limit of around 150 nm),2
which may not explain the uptake of MEND3 particles. Regarding the transfection efficiency of different MENDs, we believe that the superiority of MEND3 is mainly because of the enhanced fusiogenic ability of the DOPE/CHEMS combination11
rather than a size effect. However, the possibility that larger particles may sediment more on the cell surface,35
thus allowing more internalization, cannot be completely excluded.
The ultimate goal of our studies is to develop an efficient non-viral delivery system that can be used for in vivo
gene therapy. Here, the potential of MEND particles for in vivo
gene therapy was tested by applying MEND particles carrying a LacZ
reporter gene or a gene encoding constitutively active BMPR1a to the dorsal skin of 4-week-old ICR mice. Four-week-old mice were chosen for this experiment because most hair follicles are at anagen phase in this strain of animals, as judged by histology, AP activity and proliferation ability (Supplementary Figure 3
online). For LacZ
delivery, efficient LacZ
delivery and expression were observed in hair follicles of MEND3-LacZ
-treated animals after 2 weeks (~24% β-galactosidase-positive hair follicles for MEND-mediated delivery vs 0.2% β-galactosidase-positive hair follicles for Lipofectamine-mediated delivery). These data demonstrate that R8-MEND particles have significant potential for efficient in vivo
gene therapy and suggest that R8-MEND particles may be superior to Lipofectamine particles for in vivo
delivery of LacZ
to mouse skin. Although the reasons for superior results with MEND particles are not exactly known, they may include the relatively small diameter of MEND particles and the resistance of MEND particles to lysosomal degradation. Consistent with previous studies by Domashenko et al
transfection of LacZ
was poor when MEND3 particles were applied to 8-week-old mice (data not shown), because hair follicles are in late catagen to telogen phase in 8–week-old mice (Supplementary Figure 3
In vivo experiments with MEND3 particles carrying the caBmpr1a gene also gave encouraging results. Thus, delivery of constitutively active BMPR1a resulted in a prolonged anagen phase and extended cycling in treated hair follicles. This conclusion is based on the observations that (i) hair follicles were deeper in the subcutis space in treated mice than in control mice, (ii) a fraction of hair follicles in treated mice were in late anagen or early catagen phase, whereas hair follicles in control mice were in late catagen or telogen phase as judged by AP activity and (iii) proliferating cells were detected 2 weeks after treatment in hair follicles exposed to MEND3-caBmpr1a but not in control hair follicles. Additional studies are necessary to understand fully the molecular consequences of expression of caBmpr1a and subsequent activation of BMP signaling in treated skin. However, it is striking that a single application of MEND3-caBmpr1a had such significant biological effects in vivo.
In summary, this study describes a non-viral gene delivery system called MEND that is based on compacted DNA coated in an R8-DOPE lipid envelope. MEND particles mediate highly efficient non-cytotoxic delivery of nucleic acids in vitro and in vivo. To our knowledge, this is the first virus-like core-shell structure, which can control the topology of functional devices to maximize gene expression. We propose that MEND particles be considered a prototype nanomachine, and we encourage continued research to develop this system for efficient and safe gene therapy.