Project

Background

Problem

Challenge

Solution

Goals

Feasibility

Reference

Background

Drug delivery systems (DDS) are technologies designed to control the pharmacokinetics of therapeutic agents in the body with the goal of enhancing therapeutic efficacy [1]. The strategies of DDS can generally be divided into three categories: (i) improving absorption through optimized administration routes, (ii) regulating drug release from formulations, and (iii) enabling selective transport to target sites [1]. Consequently, targeted carriers such as antibodies and liposomes have come to play increasingly important role.

Liposomes, first reported in 1965 as vesicles composed of phospholipid bilayers [2], immediately attracted attention as drug carriers. Since then, extensive research and development has been carried out, including remote drug loading [3–6], size control technologies [7–8], PEGylation to prolong circulation time [9–14], stimulus-responsive release systems [15–16], hybridization with nucleic acid polymers [17–18], ligand modification for targeting [19–20], and even multifunctional liposomes capable of carrying multiple drugs simultaneously [21–22]. These liposomal technologies have been applied in clinical trials not only for anticancer, antifungal, and antibiotic agents, but also for antibiotics, gene therapeutics, anesthetics, and anti-inflammatory drugs [23].

Fig.1 Schematic representation of various liposomal structures.

In recent years, liposomes have also been combined with DNA origami for DDS applications. DNA origami is a technique that is folded into nanometer-scale structures with high precision by hybridization with a set of designed short “staple” strands, taking advantage of complementary base pairing [24]. By introducing hydrophobic modifications such as cholesterol, DNA origami can be anchored onto liposomal membranes and function as nanopores or stimulus-responsive meshes. Indeed, insertion of DNA origami pores into membranes has enabled size-selective molecular permeability [25] (Fig. 2A), while external stimuli or specific DNA sequences have been used to alter membrane permeability and thereby regulate drug release [26] (Fig. 2B).

Fig.2 Representative designs combining liposomes and DNA origami. (A) Liposomes with DNA origami pores conferring size selectivity. (B) Triggered liposomes releasing encapsulated drugs via self-contraction of DNA meshes on the membrane surface.

The major advantage of integrating liposomes with DNA origami is in imparting programmable functions to otherwise conventional lipid carriers. Combining the high biocompatibility and drug encapsulation capacity of liposomes with the structural precision and stimulus-responsiveness of DNA origami will enable the development of DDS that offer both controllability and flexibility.

Problem

However, even with the integration of liposomes and DNA origami, several challenges toward their full realization in DDS remain to be addressed. Among these, we focused on the control of drug release patterns in combination therapy. To maximize therapeutic efficacy while minimizing systemic toxicity, it is not sufficient to merely accumulate drugs at local sites such as tumors; precise control over the order and timing of drug release is indispensable.

Indeed, in combination therapies for ovarian and breast cancer using platinum-based drugs and PARP inhibitors, both preclinical and clinical studies have demonstrated that treatment outcomes are strongly influenced by the administration sequence and interval. For example, pretreatment with olaparib before platinum administration reduces cytotoxic activity [27], whereas a 48-hour interval between initiation of chemotherapy and PARP inhibitor administration significantly improved therapeutic outcomes [28]. These findings clearly demonstrate that, when multiple drugs are used, their order and timing of release are directly linked to therapeutic success.

Nevertheless, current DDS faces significant limitations in enabling such spatiotemporal precision. Although it is possible to encapsulate multiple drugs within a single liposome, in practice they are often released simultaneously in a disordered manner, mixing indiscriminately within the system. As a result, controlled sequential release cannot be achieved. Moreover, existing systems lack mechanisms to independently modulate the release timing of individual drug, making precise interval control extremely difficult.

Fig.3 In conventional liposomes loaded with multiple drugs, all cargos leak out simultaneously in a disordered fashion.

You may wonder whether encapsulating drugs in separate liposomes and introducing them at staggered times would be a straightforward solution. Yet, in practice, each liposome exhibits distinct pharmacokinetics once inside the body, which results in uncontrolled distribution across multiple sites—including the intended target—and mismatched arrival times. Consequently, achieving the desired synergistic effect becomes difficult [29].

Fig.4 Loading drugs into separate liposomes results in divergent biodistribution and prevents synergistic effects.

This raised several key questions for us:

Can we design new architectures by leveraging the controllability of liposome–DNA origami integration? Such architectures could encapsulate multiple drugs in a single liposome and release them sequentially over time.
If realized, could such DDS maximize the therapeutic efficacy of multidrug regimens in cancer therapy?
More broadly, could this approach enable precise temporal control not only in drug delivery for precision medicine but also in molecular robotics and artificial cells?

Challenge

Several challenges remain in addressing these questions.

First, multiple drugs must be stably compartmentalized within a liposome without mixing. Conventional liposomal DDS typically encapsulate a single drug in the aqueous lumen or incorporate hydrophobic drugs into the lipid bilayer. As a result, when multiple drugs are co-loaded, they tend to intermingle, substantially limiting flexibility in dosing design.

Fig.5 Drugs co-encapsulated in conventional liposomes mix together, limiting controlled release.

Second, a system must be established that enables selective release of compartmentalized drugs in response to temporally programmed signals. Although numerous DDS designs have been developed to release drugs in response to a single stimulus, they generally lack the ability to discriminate between different drugs and thus provide only uniform release control. To achieve sequential control, it is essential to establish a system that responds to multiple distinct signals introduced at different times, releasing different drugs in order.

Fig.6 A signal-specific system is required to enable selective and temporally ordered drug release.

Solution

To overcome these challenges, we propose a novel class of functional liposomes termed GIFTs (Gate-controlled Internal Facing Transport system), which regulate drug release in response to external signals.

Fig.7 Overview of the GIFTs structure.

The structural design of GIFTs is inspired by everyday cartridge systems. Cartridges allow the interchangeable loading of contents into a main body, enabling contamination-free and sequential use of multiple components.

GIFTs adapt this concept at the molecular scale. Multiple drugs are compartmentalized in separate inner liposomes encapsulated within a giant unilamellar vesicle (GUV, >1 µm in diameter). By mediating DNA origami nanopores arranged on both the outer and inner membranes, drug release can be selectively triggered in response to specific molecular signals. In effect, drugs can be released sequentially, much like exchanging cartridges, thereby adressing the challenges of compartmentalization, temporal control, and selective release.

Fig.8 Schematic of the cartridge replacement as inspiration for GIFTs.

Drug release in GIFTs is mediated by docking–undocking interactions between DNA origami nanopores on the outer and inner liposomal membranes. In the initial state, each inner liposome pore is capped with a lid, preventing exchange with the external environment. Upon introduction of a signal DNA strand, the lid dissociates, and the inner pore docks with the outer membrane pore, creating a transient channel for cargo release. A subsequent signal strand triggers undocking and lid re-capping, restoring the closed state. This strand-displacement reaction based process, allow reversible and programmable control of liposome opening and closing. The detailed mechanism is described in the Design section.

Fig.9 sequential drug release through distinct nanopore docking events triggered by different DNA signals.

Our proposed GIFTs are constructed solely from liposomes and DNA origami, without requiring for auxiliary enzymes or cofactors. This results in a simple yet programmable platform. Furthermore, GIFTs offer significant potential for integration with emerging technologies. For instance, combining them with photoresponsive DNA or chemical modifications could further optimize their performance.

Goals

While this research represents a highly original approach, many challenges remain before its effectiveness and practical applicability can be fully established—challenges that cannot be fully addressed within the limited duration of the BIOMOD competition. Liposomal technologies capable of time-sequenced molecular control are still rare, and establishing their validity as foundational platforms will require detailed experiments and continued verification.

With these constraints in mind, we prioritized demonstrating the fundamental feasibility of our concept rather than constructing a fully completed system. Specifically, our project focused on (1) fabricating GUV-in-GUV structures as the basic framework, and (2) designing a mechanism for selective molecular release using DNA origami.

Feasibility

Translating these research designs into real-world applications will require additional validation steps. In particular, assessing stability and long-term functionality in vivo are beyond the BIOMOD timeframe. Nonetheless, proof-of-concept demonstrations are feasible within current resources and constraints. Accordingly, this project focused on constructing the basic architecture and verifying its operation.

By referencing established design principles and prior reports, we minimized the need for entirely novel trial-and-error approaches. Additionally, being a part of a research group well-versed in molecular nanotechnology provided crucial support, enabling us to focus on design and validation rather than developing basic methodologies from scratch.

For these reasons, while full societal implementation remains as our long- term goal, our study demonstrates proof-of-concept feasibility and highlights its potential as a foundation for future applications.