Efficient gram-scale continuous production of near-infrared-sensitive liposomes for light-triggered delivery of polyinosinic-polycytidylic acid

Highlights

• Production of infrared sensitive liposomes using a novel supercritical assisted process.

• A supercritical assisted process for the controlled production of liposomes at micro and nano level.

• Successful encapsulation of light sensitive agents for liposomes triggered delivery.

• High biocompatibility of liposomes produced with SuperLip technique.

Abstract

Liposomes can be used as drug carriers, biocompatible with human cells. However, efficient on-demand release of the liposome payload still remains a challenge. Also, sophisticated liposome formulations, such as those encapsulating one or more drugs together with a suitable compound to facilitate triggered release, are usually prepared in complex multistep batch procedures with low (e.g. milligram) production rates. The aim of this work is to develop a continuous supercritical CO₂ based scalable process for the production of near-infrared (NIR) light-sensitive liposomes. Hollow Gold Nanospheres (HGN), about 5 nm large, have been synthesized as light sensitive agents for triggered release and, then, they were embedded in controlled size liposomes (from 0.75 ± 0.19 μm to 1.76 ± 0.71 μm) together with biological active compounds Polyinosinic-polycytidylic Acid (PIC) and Bovine Serum Albumin (BSA), using a supercritical assisted process. High encapsulation efficiency of all components, up to 95%, with a gram-scale production of 1 g/h of HGN loaded liposomes have been obtained. Drug release tests verified that NIR light could be efficiently used as a stimulus on these vectors to achieve drug release on-demand. Produced liposomes presented high biocompatibility.

Introduction

Liposomes are spherical vesicles formed by a double layer of phospholipids that entrap an inner aqueous core. They are often used as drug carriers for pharmaceutical and nutraceutical applications [[1], [2], [3]]. In particular, the efficacy of these structures is linked to their ability to interact with human cells [[4], [5], [6]] due to their high similarity with lipidic membranes [7,8]. An example is represented by the Enhanced Permeability and Retention (EPR) effect, used for the direct deposition of liposomes inside tumor cells, thanks to their increased vascularization with respect to the surrounding tissues [9,10]. Recently, the developments in the liposomes field resulted in the production of carriers able to improve intracellular delivery of water-soluble drugs and high molecular weight compounds, such as peptides, oligonucleotides and plasmids [11,12]. However, the efficacy of a liposomal drug delivery is often limited, due to an inefficient cellular uptake of liposomes. Lysosomal degradation is the most common fate of internalized liposomes, unless specific release mechanisms have been incorporated to facilitate drug escape from these cellular compartments [13,14]. These evidences stimulated investigations into the development of new approaches [15] using physical and chemical triggering mechanisms [16]. Different kinds of triggers have been investigated, some remote such as ultrasound, heat or light, and some local that are related to the target cells or tissues, such as enzymes and pH changes [17,18].

Light activation [19] is one of the most promising and well known approaches to artificially trigger drug release in pharmaceutical applications. The release of vesicle contents can be controlled by adjusting several parameters such as pulse duration and cycle, irradiance and wavelength of irradiation [20,21]. To obtain this result, photosensitizing agents are usually entrapped into Temperature-Sensitive Liposomes (TSL) in order to induce drug release. Nanoparticles absorbing light can be encapsulated into liposomes and convert external light stimuli into thermal energy, for drug release activation [22]. Micro-cavitation phenomena generated and thermal expansion generated by local temperature gradients contributes to disrupt lipidic double layers of liposomes, allowing the fast release of drugs [23,24]. Gold nanospheres or nanoparticles are very attractive photosensitive devices thanks to their biocompatibility [25], optical properties and high absorbance in the visible-near infrared spectra. According to the technologies proposed in the literature [26,27], their production is also scalable to industrial level. Moreover, they can be functionalized with the addition of ligands and fluorophores to provide them with additional features [28,29].

Liposomes loaded with gold nanoparticles can be useful in achieving controlled release of drugs in specific areas such as eyes and skin, that are easily accessible to light irradiation [30,31]. Several studies have explored the triggered release of liposomes or nanocomposites delivery devices based on a photothermal effect mediated by near infrared (NIR) light [[32], [33], [34], [35], [36]]. Singh et al. [37] entrapped curcumin into liposomes together with gold nanoparticles to help in situ release of the drug for heat treatment of carcinogenic tissues. Chauhan et al. [38] studied the behavior of light triggered gold nanorods for liposomal drug therapy as well. Agarwal et al. [39] and Ji-Ho Park et al. [40] employed doxorubicin (DOX) loaded liposomes with gold nanorods, used to localize the tumor region thanks to biochemical receptors linked on liposomes surface and programmed to reach target carcinogenic tissue. In this manner, a triggered release of DOX from liposomes has been obtained under NIR light irradiation. Leung et al. studied a method for NIR light-induced content release from gold-coated liposomes [41,42], demonstrating that drug release from these vesicles can be controlled modifying the location of the plasmon resonance band. You et al. [43] developed a near-infrared (NIR) light-sensitive liposomal formulation in which lipidic vesicles contained hollow gold nanospheres (HGNs) and DOX. Fast and repeatable DOX release from liposomes was reported upon NIR laser irradiation. In most of the reported works, HGNs were incorporated in the lipid bilayer of liposomes using the thin layer hydration method [44]. To increase the encapsulation efficiency of HGNs into liposomes, the surface of HGNs can be functionalized and the drug can be loaded by the passive loading method gradient. However, these batch wise preparation methods are very complex since they require several preparation and post-processing steps to obtain purified liposomal suspensions. Indeed, high amounts of non-encapsulated drugs and also organic solvents used during liposomes formulation are present in the final suspensions and have to be removed from the final product. Furthermore, from process engineering point of view, the overall production rate is low (milligram-scale) and, consequently, the process layout is difficult to scale-up to industrial level.

Regarding the methods of production of liposomes, the most well-known is the conventional thin layer hydration, proposed by Bangham et al [45]. However, this process suffered of low replicability, high solvent residue and low encapsulation efficiencies [46]. More conventional and supercritical assisted methods, such as microfluidic, reverse phase evaporation, supercritical anti-solvent, DESAM and DELOS suspension have been proposed in the literature [47]. However, there still some drawbacks in vesicles stability and particle size distribution control due to their semi-continuous configuration [48]. Recently, Reverchon and coworkers proposed a new process based on the use of supercritical CO₂ for the production of liposomes [49]. This process has been named SuperLip (Supercritical Assisted Liposome Formation; it allows continuous the 1-shot production of liposomes loaded with hydrophilic or hydrophobic drugs. The major hypothesis on the mechanism at the basis of this method is that, first, water droplets are produced and, then, they are rapidly covered by phospholipids. The high mass transfer rate of carbon dioxide makes lipids coverage faster than in conventional techniques for the production of liposomes. In this way, it is possible to produce vesicles with a good control of particle size distribution and high encapsulation efficiency (EE). Indeed, several hydrophilic compounds have been encapsulated inside the aqueous core, with high encapsulation efficiencies, using this innovative process [[50], [51], [52], [53], [54]]. This process has another advantage over conventional liposome production methods, it allows the production of several grams of liposomes depending on the duration of the lab-scale experiment, thanks to the continuous layout.

In this work, SuperLip has been tested for the production of engineered near-infrared (NIR) light-sensitive liposomes in order to provide a manufacturing route able of large and reproducible scale production.

In previous works, SuperLip has been used for the production of liposomes loaded with one payload. In this work, SuperLip is employed for the first time to create a more complex liposomal system in which both drug and NIR-absorbing nanoparticles have been loaded. Indeed, liposomes loaded with hollow gold nanospheres (HGNs) in the vesicle inner core, used as triggering agents [55], together with polyinosinic-polycytidylic acid (PIC) used in tumor immunotherapy [56] will be produced. Anionic molecules such as PIC do not easily cross the cell membrane; therefore, PIC delivery using liposomes, has been proposed to facilitate the delivery of this drug into living cells [57]. Bovine serum albumin (BSA) will also be entrapped in the liposomal inner core to protect PIC from degradation. The hybrid vehicles containing the drug (PIC), protective agent (BSA) and triggering agent (HGNs) will be, then, used for the controlled delivery of PIC activated using NIR.