We designed “chain-reactive burst” system as follows.
Each liposome contains triggers and drugs inside, and aptamers for the trigger on its surface. When liposomes are destroyed, new triggers and drugs are released. To achieve liposomal burst by outside triggers, we propose the following two approaches.
i) Bending approach
ii) Flower micelle approach
First, we considered a theory to disrupt liposomes by a trigger DNA signal through calculation. If a liposome is destroyed, its size becomes smaller. We estimated the free energy gap between the two liposomal states: a large liposome and a small one. And discuss which size of liposomes is more stable.
Please see the details (Go to Calculation).
Fig.1 Process of bending approach
Our bending approach consists of the following four steps.
1.Cholesterol-conjugated DNA strands (in the rest of this document, referred to as “aptamers”) attach to the surfaces of liposomes.
2. Then, DNA origami complementary to the aptamer is added as triggers.
3. Triggers bind to the surfaces of liposomes and give a load on the membrane.
4. Due to the load by triggers, liposomes are destroyed.
a) Mechanism of bending membranesTo destroy liposomes, we focused on the mechanism the living things use to bend cell membranes. We consider that if we could make use of the mechanism of bending membranes (destabilizing membranes), it would lead to the collapse of membranes. The following three mechanisms have been proposed as of now (Membrane-bending proteins)
The mechanism A is that amphipathic molecules are inserted into the cell membrane and the bending is caused. The inner hydrophobic part of the lipid bilayer has a strong adhesive power for the two leaflets. Thus, once the amphipathic molecules are inserted into one leaflet of the membrane and expand it, the other leaflet bends according to it, making its surface area smallest.
The mechanism B is that the molecule attached to the membrane becomes a rigid scaffold and distort the membrane under itself, or stabilize the already bended membrane.
The mechanism C is that lipid molecules are clustered in one leaflet of the membrane and the inequality of lipid quantity makes the membrane bend.
Most membrane bending proteins combine the above three mechanisms.
In addition, a theory that protein crowding causes the bending of cell membranes ( Membrane bending by protein- protein crowding) has recently been suggested. This mechanism is that the collision of membrane proteins produces lateral pressure on membranes and distorts them.
Due to the above reasons, the efficient design for destabilizing membranes is the structures that :
b) Rigid scaffoldsTo make rigid scaffolds, we took note of DNA origami, because DNA origami is a method for making rigid structures of any shape. Moreover, we adopted a 2D structure to make the surface area largest.
We also designed rectangle and triangle to make the pressure of the collision highest.
Fig.3 Rectangle origami and triangle origami
We suppose that rectangle and triangle structures are most effective for the following reasons.
Rectangle is expected to work as one scaffold in itself; triangle (the most efficient figure that covers a sphere) structures, to gather and work as one big rigid scaffold.
The design of our rectangular DNA origami is as below.
Fig.4 Rectangular origami
We used caDNAno2 for our DNA origami design.
The DNA origami has a rectangle shape of 67.6nm (26 helixes) by 127 nm (374 bases).
We cut out a smaller rectangle of 10 helixes by 161 bases at one edge of this origami, so that we could distinguish the two sides during AFM (Atomic Force Microscope) observation.
Besides, to destabilize the membrane by inserting this origami, we designed 141 staples at the center of the origami to hybridize with aptamers (These aptamers give our origami amphipathicity), and enabled it to insert into the membrane.
To sum up, the aptamer not only connects DNA origami and liposomes but also inserts into the membrane and destabilizes it.
Fig.6 Unstable liposome
ii)Flower micelle approach
Fig.7 Process of flower micelle approach
There is a method called flower micelles for collapsing liposomes.
In this method, we cover the surfaces of liposomes with many copolymer rings. The rings can be distorted by heating, place some stress on the liposomes, and collapse them.
We tried to collapse liposomes by applying this mechanism of flower micelles.
1. First, we mix aptamers (the same strands as used in i) Bending approach), loop strands, and liposomes.
Each of the loop strands is designed to have two complementary parts to aptamers at its both ends. So when it binds to the aptamers at the both ends, the middle part remains single-stranded and becomes a loop.
As an aptamer is cholesterol-conjugated and has high affinity for a liposome, it floats on a liposome and enables the aptamer-loop strand complex attach to the liposome. In other words, a loop strand hybridizes with a liposome via two aptamers.
Fig.9 Make loops on the surface of a liposome
2. Next, we add a trigger strand corresponding (complementary) to the loop strand on the liposome. The trigger strand hybridizes with the loop part, making it change to be straight.
3. The double-stranded part keeps straight (though it was originally a loop part), because the trigger strand is designed to be shorter than its persistence length.
Fig.10 How to straighten loop4.In this process, some stress is placed on the liposome, and it is collapsed.
Fig.11 Liposomal burst
It is considered if some triggers are kept inside a liposome, and the liposomal membrane is collapsed by the above i) and ii) methods from the outside, it would be much easy to trigger "Chain-reactive burst", because the released triggers serve as new triggers for neighbor liposomes.
We designed the DNA sequences for this approach by DNA design, software for designing DNA sequences.
We arranged three kinds of loop strands.
Each loop strand has a 40nt, 20nt, or 10nt loop part (shown below in black and blue), which becomes a loop after the hybridization of the whole loop strand with aptamers.
The blue part of a loop strand is complementary to a corresponding trigger strand (also shown in blue). So a loop strand and a trigger strand are expected to hybridize with each other, place some stress on a liposome, and collapse it.
The red part of a loop strand is complementary to an aptamer (shown in red). Cooperating with aptamers, it enables the whole loop strand to attach to the surface of a liposome.
Aptamers are the same strands as those used in i)Bending approach.
40nt loop DNA
Trigger DNA for 40 nt loop DNA
20nt loop DNA
Trigger DNA for 20 nt loop DNA
10nt loop DNA
Trigger DNA for 10 nt loop DNA
温度上昇を感知して割れて始まりのトリガーを放出するイニシエーターとして私たちはPNIPAM付リポソームを使用した。 Hydrophobicity of NIPAM varies at temperatures. NIPAM is hydrophilic at less than 32 ºC, but it become hydrophobic and shrinks at > 32 ºC. Therefore, liposomes containing a modified NIPAM (poly(NIPAM-co-AA-co-ODA) in their membranes become unstable at high temperature (temperature-sensitive liposomes). Consequently, increasing temperature disrupt the liposomes.