Repetitive drug delivery using Light-Activated liposomes for potential antimicrobial therapies

Abstract

Overuse or misuse of antibiotics and their residues in the environment results in the emergence and prevalence of drug-resistant bacteria and leads to serious health problems. Notable progress in liposome research has been made in drug delivery and several liposomal drugs have been approved for clinical use owing to its biocompatibility and improved efficacy. Recently, liposomes have been engineered further to release encapsulated drugs on the target of interest in a dose-controlled fashion in response to external stimuli such as light, pH, and heat. Among those, light-activated liposomal drug delivery gained a lot of attention because drug release at the targeted sites can be precisely controlled by varying laser/light duration, energy and beam area. We envision potential applications of the light-activated liposomal delivery systems for effective drug-resistant antimicrobial therapies. The use of light-activated liposomes will be widely spread in antimicrobial therapies if the amount of drug is precisely controlled for a prolonged time at a target location. In this review, we discussed the breadth and depth of various light-activated liposomal drug delivery technology. Emphasis was given to repetitive release mechanism and applications of light-activated liposomes because the repeatability provides stability and precise control of the drug delivery system to prevent overdose of antimicrobials and treat with minimal doses. We described limitations on translation from pre-clinical to clinical settings and strategies to overcome the limitations. Careful consideration of light-responsive materials, lipid composition, laser parameters and laser safety is important when selecting and designing the drug delivery system for successful applications.

Introduction

Biofilm, immobilized microbial colonies, causes infections and serious diseases, leading to one-third of global mortality.[1] Roughly>60% of hospital-associated infections are attributed to biofilms. [2] Although various antibiotics have been developed, overuse or misuse of antibiotics in the clinics has led to the emergence and spread of antibiotic-resistant bacteria. Infection by antibiotic-resistant bacteria is expected to become the number one cause of death by the year 2050.[3], [4] Because overuse of antibiotics can cause antibiotic-resistant bacteria, which is difficult to eradicate, it is critical to develop antimicrobial strategies, ideally with a minimal dose of antibiotics.

Over the last three decades, therapeutic drug delivery via the utilization of nanocarriers has received attention because of the unique advantages, including higher surface area, minimal side effects, desired pharmacological effects and enhanced therapeutic efficacy. Among other different nanocarriers, liposomes are one of the most widely used drug delivery materials because of inherent biocompatibility, low toxicity and ability to encapsulate both hydrophilic and hydrophobic drugs. Liposomal drug delivery systems have been used for treating diverse bacterial infections, including staphylococcus aureus and methicillin-resistant staphylococcus aureus (MRSA), with successful outcomes being recorded when compared to the conventional dosage forms. However, passive delivery of encapsulated drugs from liposomes often limits the efficacy because of slow accumulation of the drug at the targeted site or the negative effects on healthy cells. [5] In addition, traditional direct drug application or liposomal drug delivery may result in overdose, which often leads to drug-resistant bacteria issues. Thus, liposomes have been recently engineered to release encapsulated drugs on the target of interest in a controlled fashion in response to external stimuli such as pH, light, and high temperature. [6].

Light-triggered liposomal drug delivery is an attractive method to control the drug release rate at the targeted sites by varying duration, energy and beam area of laser. Several studies have reported drug release from light-activated liposomes by laser irradiation in a precise spatial-, temporal-, and dosage-controlled fashion. The technology was also successfully applied to antimicrobial therapies. For example, Ferro et al. demonstrated antibacterial photoinactivation via poly-cationic liposomes loaded with porphyrin effectively. The rate of survival of the MRSA cells reduced by 4.5 and 6 log upon exposure to light for five and ten minutes, respectively, compared to free porphyrin which showed a reduced rate of survival of less than 3 log. [7] Light can be various forms, including direct laser, direct UV light (100–400 nm), direct blue light (400 – 500 nm), and optical waveguide (fibers). [8].

Especially, light-activated liposomes that enable repetitive drug releases multiple times can be of great interest in antimicrobial therapies. The repeatability is important because it provides stability and tight dose-controllability that allow us to avoid overuse of antimicrobials. One can monitor and determine the exact dose released by tuning laser parameters, such as duration and power. The repeatability has potential not only to prevent antibiotic-resistant bacteria problems but also treat the infections. Furthermore, this feature can save the drug for the long-term use. One can apply the light-activated liposomes once and control the dose later again by laser irradiation without adding more doses. To summarize, repetitive light-activated liposomes have broadly three advantages over conventional liposomes: 1. prolonged delivery, 2. dose-controlled combination therapy, and 3. synergistic chemo-photodynamic-photothermal effect. Because drug is stored in the liposomes until it is needed, prolonged delivery is possible via the light-activated liposomes. Specifically, when the liposomes are incorporated into matrix or capsules, retention of liposomes at the site of application can be enhanced, attributed to improved physical stability of the liposomes and repetitive drug release properties. [9], [10] Therefore, it has potential to be applied for long-term delivery of antimicrobials repetitively, especially for treatment of chronic and recurrent infections. In vivo investigation of liposomal mupirocin-in-chitosan hydrogel on healing of burden wounds demonstrated the efficacy of formulation comparable to the marketed product of mupirocin. This prolonged release technology helps to dispense drug with even tissue distribution and proportional interaction with target bacteria.

Combinational therapy made of antibiotic-antibiotic or antibiotic-adjuvant can improve antimicrobial efficacy. The antibiotic-adjuvant therapy increases the life expectancy of antibiotics, either by a synergistic effect, or by the inhibition of antibacterial resistance. [11] However, the pharmacokinetics of different drugs leads to the complexity in the dosage and schedule of drug combinations, which consequently affects the synergy of antibiotics in vivo. To address the challenge, light-activated liposomes can be a well-established platform for the co-encapsulation of multiple drugs. [12] By designing and tuning lipid membranes, the release kinetics can be controlled depending on the molecules having different physicochemical properties, such as charge, size, and hydrophobicity.[13], [14] For example, mechanosensitive channel of large conductance (MscL) from L. lactis and E. coli has been known as a novel means of antimicrobial streptomycin delivery. [15], [16] At the same time, MscL can be reconstituted in lipid bilayers composed of light-sensitive lipids as shown in Iscla et al. [15] Light-induced isomerization of an azobenzene moiety of 4-Azo-5P on one of the lipids from trans to cis was used to add stresses in the membrane and activate MscL. [17] Thus, combining the two methods, delivery of antimicrobials can be tightly controlled via light-activated liposomes, to manage dosage and release time and kinetics.

Last but not least, light-triggered liposomal delivery has advantages because of its synergistic antimicrobial effects of chemo and hyperthermia. For instance, Zhang et al. showed when liposomes containing vancomycin and photo-responsive materials were irradiated with NIR, the antimicrobial effect significantly increased compared to other testing groups. As shown in Fig. 1b and c, when MRSA was exposed to PBS or and NIR light, the number of MRSA stayed stationary till 15 h, indicating that NIR irradiation itself has no effect on bacterial activity. The vancomycin-loaded liposome (denoted as vanco@liposome) also caused no obvious delay to the growth of MRSA either with or without NIR irradiation. However, when the BPQDs@liposome group is irradiated with the NIR laser, the bacteria growth is seriously delayed, compared to no laser. The results showed that the NIR-triggered photothermal effect has a pronounced effect on inactivation of bacteria. In addition, photosensitizers used for drug release trigger also have ability to exert a antimicrobial effect by producing reactive oxygen species (ROS) when irradiated by light of an appropriate wavelength. Several studies have shown the efficacy of light-activated antimicrobial agents including indocyanine green (ICG) with a near-infrared laser (808 nm). Omar et al. showed the cytotoxicity of ICG with an 808 nm laser (1.37 W cm⁻²) on Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa up to 99.99%. [18].

Therefore, the light-activated liposomal drug delivery can be successfully applied to tackle current challenges in antimicrobial therapies because of its unique capabilities of precise controls in time, location, and dosage as well as repetitive use. Recently, several studies demonstrated repetitive antimicrobial therapies using light-activated liposomes (Table 1). Other studies that have not focused on repetitive release but utilizing light-activated liposomal delivery against S. aureus and MRSA are summarized in Ref. [12]. Table 1 includes studies that clearly demonstrated repeatability and reliability of applying the liposomal delivery systems to antimicrobial therapies. For example, tungsten sulfide quantum dots-encapsulated liposomes released antibiotic vancomycin via near-infrared (NIR) laser (808 nm, 1 W/cm², 10 min). The quantum dots had photothermal capability and triggered release vancomycin when irradiated by laser. The temperature increases up to 47 °C upon laser irradiation was observed (Fig. 2i). In addition, the quantum dots were used as peroxidase activity agents for synergistic antibacterial therapy. The study also showed that multiple repetitive release of FITC by the NIR laser (Fig. 2j). The platform exerted antibacterial effects against Mu50 (gram-positive) and E-coli (gram-negative) and disrupted biofilms. Note that several other studies that applied the light-activated liposomal drug delivery to antibacterial therapies demonstrated feasibility of repetitive treatment by showing multiple cycles of temperature increase and decrease as in Fig. 2i. [19], [20].

Because of the advantages listed above, including tunability, versatility, and stability, the applications of the repetitive on-demand technology is highly expected in the near future. However, there are few light-activated liposomal drugs available in clinical settings to date. The first FDA-approved light-activated liposome is Visudyne® (liposome for injection, Novartis AG).[24] It was approved by FDA in 2000 as a photodynamic therapy drug for the treatment of pathologic myopia, ocular histoplasmosis and age-related macular degeneration (AMD). This liposome was formulated with lipids DMPC (1,2-dimyristoyl-sn-glycero-3-phosphorylcholine) and egg phosphatidyl glycerol loading photosensitizer verteporfin (VP) (molar ratio, VP: total lipids = 1:8), with the size between 150 and 300 nm.[25] Under light illumination at 690 nm, VP is activated to generate the highly toxic reactive oxygen species, leading to the local damage to neovascular endothelium. [26] Site-specific pharmaco-laser therapy using light-activated liposomal formulation was tested to treat port wine stains in clinical trials in 2017. [27] Since then, no clinical trials has been tried to the best of our knowledge although there are many preclinical liposome formulations. This fact suggests that there are still many barriers to the clinical implementation and several strategies to design repetitive light-activated liposomal drug should be taken into account.

One of the major limitations that restrict the applications is the penetration depth of the light source to the targeted tissue. UV–Vis light that activates most photosensitizers has a short tissue penetration depth (less than1 cm) accompanied with potential tissue damage. On the other hand, NIR laser can penetrate soft tissue up to 2 cm and have low biological absorption; thus, it is more suitable for in vivo use. [28], [29] We will discuss later about a strategy of using upconverting nanoparticles that convert wavelengths of lights to activate molecules responsive to UV–Vis region (Section 2.3.3). Application of optical fibers also has been adapted to deliver the light source to deep-situated tumor or deep-tissue infections in some studies.[30], [31], [32], [8] Second, the stability of liposomes in various physiological media is a challenge for reliable dose-controllability. It is necessary to rationally design the liposomal formulations to avoid aggregation, self-fusion and the clearance by the systemic circulation. [24] In addition, to achieve repeated light-activations, a good photostability of the light-responsive materials is critical to minimize the inconsistency of dose released. Potential toxicity of synthetic lipids also needs to be considered. For instance, cationic lipids may cause cytotoxicity to the cells and also induce immune toxicity. Lastly, laser parameters such as power density and irradiation durations need to be carefully considered for repeatability and safety. Different devices from different manufacturers may induce variations in light output and power densities. [33] Also collateral damage to surrounding tissue from laser itself needs to be taken into account. Overall, these limitations of light-activatable liposomes need to be overcome before translation to clinical applications.

In order to overcome the current limitations and to use the repetitive light-activated liposomal drug delivery widely in antimicrobial applications, the fundamentals of the technology need to be understood. Thus, this review will focus on fundamentals of repetitive light-activated liposomal drug delivery with discussion of potential antimicrobial therapies. Various light-activated liposomes in biomedical applications are summarized to include strategies for improved liposome stability and repetitive dose-controllable liposomes based on lipid compositions and light-responsive materials. In contrast to other reviews which discussed conventional liposomes, photoresponsive lipids or photothermal liposomes alone, this review includes both photochemical and photophysical mechanisms for repetitive liposomal drug delivery. Overall, the review suggests broad impact of the technology for effective antimicrobial therapies.