Organelle-selective gene delivery and expression in the chloroplast in planta using chitosan-complexed single-walled carbon nanotube carriers
In one aspect, a composition can include an organelle, and a nanoparticle having a zeta potential of less than −10 mV or greater than 10 mV contained within the organelle. In a preferred embodiment, the organelle can be a chloroplast and the nanoparticle can be a single-walled carbon nanotube associated with a strongly anionic or strongly cationic polymer.
1 . A composition, comprising:
a chitosan-complexed single-walled carbon nanotube complexed with a gene cassette for delivery to a chloroplast, wherein the chitosan-complexed single-walled carbon nanotube comprises chitosan is covalently bonded to the single-walled carbon nanotube;
wherein the gene cassette comprises plasmid DNA;
wherein the gene cassette and the chitosan-complexed single-walled carbon nanotube are present in a ratio of 1:3 to 1:10 w/w of gene cassette to chitosan-complexed single-walled carbon nanotube; and
wherein the chitosan-complexed single-walled carbon nanotube complexed with the gene cassette has a zeta potential in the range of −35 to −30 mV or in the range of 30 to 35 mV.
2 . The composition of claim 1 , wherein the chitosan is deacetylated.
3 . The composition of claim 1 , wherein the polysaccharide chitosan is pegylated.
4 . The composition of claim 1 , wherein the gene cassette includes a zinc finger nuclease, a TALEN vector or a CRISPR/Cas vector.
5 . A method of delivering genetic material to a plant chloroplast comprising:
contacting a plant with the composition of claim 1 .
6 . The method of claim 5 , wherein the plant is a watercress plant, a tobacco plant, a spinach plant or isolated Arabidopsis thaliana mesophyll protoplast.
7 . The method of claim 5 , further comprising releasing the genetic material in the interior of a chloroplast.
8 . The composition of claim 1 , wherein a threshold zeta potential is determined by Equation 1 :
ξ
*
=
±
(
ε
M
+
ε
W
ε
W
)
(
d
a
)
e
κ
(
d
-
a
)
(
Γ
-
a
γ
0
+
4
a
Δ
Δ
H
ρ
n
)
L
2
a
ε
0
ε
M
(
1
-
ε
M
ε
W
)
(
Equation
1
)
wherein:
ε M , a relative permittivity of a double lipid bilayers of the chloroplast, is about 2.2;
ε W , a relative permittivity of a medium of the chloroplast, is about 80;
ε 0 , a dielectric permittivity of vacuum, is about 8.854×10 −12 F/m;
“d” is an effective charge radius of the composition;
“a” is a radius of the composition;
κ −1 is a Debye-Huckle screening length of the composition;
Γ, a pore line tension of the chloroplast, is about 10 −12 N;
γ 0 , a resting membrane tension of the chloroplast, is about 0.6 mN/m;
ΔΔH, a change in free energy due to the lipid of the chloroplast binding on the composition, is about 0.05 k b T;
ρ n , a lipid density on the composition, is about 10 18 ; and
L, a thickness of the double lipid bilayers of the chloroplast, is about 1.1×10 −10 m,
wherein the radius of the composition is in the range of 104-190 nm.
9 . The composition of claim 1 , wherein the zeta potential is about −30 m V or about 30 m V.
10 . The composition of claim 1 , wherein the gene cassette and the chitosan-complexed single-walled carbon nanotube are present in a ratio of 1:3 to 1:6 w/w of gene cassette to chitosan-complexed single-walled carbon nanotube.
11 . A composition, comprising:
a chitosan-complexed single-walled carbon nanotube complexed with a gene cassette for delivery to a chloroplast in a plant,
wherein the chitosan-complexed single-walled carbon nanotube comprises chitosan is covalently bonded to the single-walled carbon nanotube;
wherein the gene cassette comprises plasmid DNA;
wherein the gene cassette and the chitosan-complexed single-walled carbon nanotube are present in a ratio of 1:3 to 1:10 w/w of gene cassette to chitosan-complexed single-walled carbon nanotube; and
wherein the chitosan-complexed single-walled carbon nanotube complexed with the gene cassette has a zeta potential in the range of −35 to −30 mV or in the range of 30 to 35 mV; and
wherein the plant is a watercress plant, a tobacco plant, a spinach plant or isolated Arabidopsis thaliana mesophyll protoplast.
12 . The composition of claim 1 , wherein the chloroplast is in a plant, and the composition is in contact with the plant.
13 . The composition of claim 11 , wherein the composition is in contact with the plant.