Mechanisms of light‐induced liposome permeabilization

Abstract Liposomes have been widely studied for drug delivery applications. The inclusion of photoactive molecules into liposomes opens the possibility of light‐controlled cargo release to enhance drug biodistribution or bioavailability at target sites. Membrane permeabilization induced by light can be an effective strategy for enhancing cargo delivery with spatial and temporal control, which holds potential for chemophototherapy approaches. Several diverse mechanisms have been reported including light‐induced oxidation, photocrosslinking, photoisomerization, photocleavage, and photothermal release. Here, we review selected recent reports of light‐triggered cargo release from liposomes.


| I N T R O D U C T I O N
Liposomes have been used extensively for numerous applications, including as drug delivery systems. They have been used as carriers for anti-cancer chemotherapeutic drugs, as well as for several other indications, such as for treatment of fungal and bacterial infections. 1 Liposomes can vary in size, ranging from the nano-to the micron-scale, and are generally formed by self-assembly of amphiphilic lipids in an aqueous solution. Liposomes can load and retain cargo in their hydrophilic core or within the hydrophobic region of the bilayer. This can lead to extended circulation times and improved biodistribution. 2,3 Adverse side effects from traditional chemotherapies can potentially be reduced in this way. 4,5 The ability to release a drug, on-demand, in a controlled and selective manner should increase the efficacy of liposomal therapies by increasing the local concentration or local bioavailability at a specific site of interest. Such approaches make use of internal or external triggers to induce the nanocarrier to release its content whenever and wherever necessary. Several triggers have been demonstrated to release liposomal cargo. A common example of an external trigger is the use of heat to induce membrane permeability, [6][7][8] and other demonstrated triggers include ultrasound 9 and magnetic fields. 10 Alternatively, endogenous triggers at the target sites aim to exploit differential properties of tumors such as lower pH [11][12][13] and enzymes found in cancer tissues. 14,15 Light is an intriguing stimulus to remotely trigger cargo release. [16][17][18][19][20] Chemophototherapy, which has been explored preclinically and clinically, combines chemotherapy and phototherapy and stands to benefit from nanomaterials that release their cargo in response to light. 21 The advantages of using light as a trigger is that various parameters, such as exposure time, wavelength, beam diameter, and laser intensity can be readily externally modified to adapt to different purposes. Different light wavelengths can be used for lasertriggered release of cargo. Ultraviolet (UV) and visible light are not ideally suited for treatments that require deep tissue penetration due to high scattering and absorption by tissue components, 22,23 thus are often restricted to topical applications. [24][25][26] Conversely, wavelengths in the near-infrared (NIR; generally 650-900 nm) can penetrate better into tissues (e.g., up to 1-2 cm) and are more suitable for light-triggered cargo release in deeper tissues. 27,28 Although NIR light penetration is relatively limited in tissues, most areas in the body can be reached with careful treatment planning and with the use of interstitial fibers.

| Light-sensitive liposomes
Liposomes have been studied extensively as biocompatible nanocarriers for a plethora of purposes, including drug and gene delivery, vaccines adjuvants, diagnostic agents, and ingredients for cosmetics and vitamins, among many others. 29,30 They are generally composed of phospholipids and cholesterol and can be sterically stabilized with polyethylene glycol (PEG) to give "stealth" abilities to delay clearance by the reticuloendothelial system. As liposomes are formed mostly from naturally occurring lipids, they present good biocompatibility and low toxicity. Furthermore, due to their small size and high flexibility, liposomes can passively accumulate in tumor sites via the enhanced permeability and retention effect. Although these features make liposomes an attractive and clinically used drug delivery system, some limitations should still be overcome depending on their formulation; for example, short circulating half-life, poor entrapment stability, passive leakage and fusion, uptake by undesired organs, and manufacturingrelated problems such as high production costs and time. 30 Incorporation of photoactive molecules into liposomes has potential to enable light-triggered cargo release of entrapped molecules. As will be discussed here, several different photoactive molecules are capable of inducing membrane destabilization and permeabilization.
Their localization in the liposome is dependent on their intrinsic polarity. Photoactive molecules can also be conjugated to phospholipids, either at the hydrophobic tail or at the head.
Several mechanisms have been reported as a mean for lightinduced membrane destabilization to promote cargo release. These include light-induced oxidation, photocrosslinking, photoisomerization, photocleavage, and photothermal release. We note that several excellent reviews exploring common mechanisms of cargo release from liposomes, micelles, polymer-based nanoparticles and other inorganic nanoparticles exist in the literature. 30

| Light-induced oxidation
One of the most common mechanism used for disruption of nanocarriers is light-induced oxidation by reactive oxygen species (ROS). ROS have unpaired electrons or unstable bonds. In tissues, high concentrations of ROS leads to several types of damage due to oxidative stress, which includes DNA damage, oxidation of lipids, amino acids and proteins, inflammation, and ultimately, cell apoptosis. 37,38 Common ROS resulting from chemical reactions include: singlet oxygen ( 1 O 2 ), hydroxyl radicals (HO·), superoxide (O2· 2 ), and hydrogen peroxide (H 2 O 2 ). 39 Photosensitizers (PS) are known to produce singlet oxygen when excited by light at specific wavelength ranges. This mechanism has been extensively explored in photodynamic therapy, in which singlet oxygen derived from excitation of a PS induces oxidative stress, disruption of cellular membranes and cell death. [40][41][42] Light-induced generation of singlet oxygen has been used to release cargo from nanoparticles by oxidation of different lipids. The mechanism behind the release is thought to occur via membrane permeabilization caused by oxidization of unsaturated lipids and concomitant formation of pores in the bilayer, which causes the cargo to leak from the nanoparticle, as previously shown by Pashkovskaya et al. 43 In this study, liposomes formed by unsaturated lipids were shown to release 5,6-carboxyfluorescein (CF), sulforhodamine B (SRB) and cal-cein in such manner that the permeability of the dye increased as molecular weight decreased (calcein < SRB < CF).
The permeability of the membrane can be influenced by the molar percentage of unsaturated lipids, as shown by Luo et al., where 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was used as a "helper" lipid when combined with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). 44 Increasing DOPC concentration, up to a maximum of 10 mol. %, was found to increase doxorubicin (Dox) release rates when liposomes were permeabilized with NIR at 665 nm ( Figure 1A). This study showed that both DOPC and cholesterol are oxidized by singlet oxygen resulting from excitation of porphyrin-phospholipid (PoP). PoP is a lipid-like molecule that has a PS (e.g., the chlorophyll-derived pyropheophorbide-a (Pyro)) covalently linked to a phospholipid side chain, and can readily be used to form biocompatible and optically active liposomes. PoP is currently being investigated for numerous multimodal imaging and phototherapy purposes. [45][46][47][48][49][50][51][52] Porphyrins themselves possess intrinsic qualities that make them attractive as theranostic agents. 53 Figure 1C). 44 PoP liposomes were able to release variable cargos such as Dox, calcein, SRB, and gentamicin in response to NIR light. 19 Rwei et al. demonstrated a novel liposome strategy that could be used as an injectable drug delivery system to allow patients to modulate the timing, duration, and intensity of local anesthesia. 55

| Photocrosslinking
The mechanism by which photocrosslinking induces content release typically occurs via polymerization of unsaturated bonds located in the hydrophobic domain of the bilayer. When photosensitive polymerizable moieties are irradiated with a specific wavelength of light, the crosslink reaction between them causes the bilayer to shrink in the surrounding domain where the sensitizers are present. This causes the bilayer natural packing to undergo conformational changes, which in turn correlates with local pore formation, increasing membrane permeability and content leakage.

| Photoisomerization
Photoisomerization relies on the propensity for certain molecules to undergo a conformational change upon light stimulation. In those molecules, often based on azobenzenes, isomerization occurs when their spatial orientation switches from the trans to cis state. When incorporated into liposomal membranes, for example, this transition is associated with bilayer disruption followed by cargo release. 59 molecule changing from having a more apolar (trans) conformation to a more polar (cis) one ( Figure 3). 61,62 Azobenzenes are, in most cases, sensitive to light in the UV spectra, although their activation has also been reported with wavelengths in the range of 530-560 nm. 63 The transition from trans to cis can be triggered by light irradiation at wavelengths ranging from 320-350 nm and the reverse reaction can be triggered by irradiation at 400-450 nm.
The reversible reaction can also be achieved by heat increase, although this process is slower than the photochemical counterpart. 62 This reversible transition is important, because it enables nanocarriers to be more dynamic by allowing, on-demand, external control of cargo release through changes in membrane permeability. The reverse switch from cis to trans state decreases membrane permeability, "locking" the nanocarrier once again.
Azobenzene moieties were also reported to act as "host-guest" In general, azobenzene photoisomerization is a well-known mechanism and the attachment of azobenzene moieties to phospholipids has been explored. 60 The versatility for its use and the reversible character of the reaction can be explored in different manners, which makes it a promising candidate to be used as a light-triggered mechanism.

| Photothermal release
Photothermal approaches are based on the conversion of light into heat to induce liposome permeabilization. Several materials can be used as photothermal transducers to trigger on demand drug delivery.
For instance, gold nanoparticles (AuNPs) can be used to enhance lighttriggered release due to their suitability for photothermal conversion based on surface plasmon resonance and hot electron mechanisms.
AuNPs are able to rapidly and efficiently absorb visible, UV 72 and NIR light 73 and release energy as heat on the scale of picoseconds. 74 This effect is enhanced especially if the light wavelength matches AuNP absorption bands. This phenomenon generates a heated electron gas that rapidly exchanges energy with the particle structure, which then, dissipates this energy in the surrounding medium. 36,75 When proximal to liposomes, the high temperatures achieved by AuNPs can induce membrane stress and rupture, followed by cargo release. In some cases, instead of membrane rupture, the photothermal effect induces a phase transition in the bilayer, which makes it leakier and leads to an increased cargo release. 72 The location of AuNPs or any other light absorber is variable and dependent on its hydrophobicity and charge.
They can be encapsulated within the liposomal core, 76,77 tethered to the membrane, 73,77,78 inserted within the bilayer, 72 free in liposome solution 77,79 or assembled as aggregates with liposomes ( Figure 6). 80 Thermosensitive liposomes (TSL) can be activated with photothermal transducers and NIR light is an appealing wavelength for clinical use due to its deeper tissue penetrance. Kwon 4 A strategy for the use of azobenzene moieties to control cargo release from liposomes. In this example, azobenzene moieties are covalently attached to b-cyclodextrin to act as "host-guest" molecules. After light irradiation, the transition from the trans to cis state leads azobenzene to leave the transmembrane cavity due to its larger size when compared with the trans isomer. Figure  system tethering gold clusters on liposome bilayers with NIR sensitivity. 73 Light absorbed by the gold cluster is converted into heat and transferred to the TSL, causing membrane destabilization. The system was effective in releasing Dox and decreasing cell viability and promoting antitumor effect. TSLs have also been shown to release Dox following photothermal activation from non-tethered nanorods located in the tumor vicinity. 81

CONFLICT OF INTERESTS
The authors declare that they have no conflicts of interest with the contents of this article.