**1. Introduction**

Gene therapy is the treatment of diseases at molecular level by "switching genes on or off" through the use of Nucleic Acid Based Drugs (NABDs) which, including oligodeoxynucleotides, plasmid DNA, ribozymes, siRNA, miRNA and related chemically-synthesized molecules, are highly degradable therapeutics that need to be loaded into a vehicle in order to reach the patient target cell/tissue where the defective or mutated gene has to be corrected [1,2]. Indeed, naked genetic materials cannot be easily systemically administered due to their toxicity, low stability in serum, rapid renal clearance, reduced uptake by target cells, phagocyte uptake and their ability in activating the immune response, all features that preclude their clinical development [3]. Therefore, choosing the right vector represents one the most critical steps in attaining a successful gene therapy, and carriers based on lipid materials seem to be the most promising, as they have similar structures to cell membranes [4]. To date, lipid-based NABDs delivery systems represent a novel approach for the treatment of different diseases, mostly including advanced cancers, amyloidosis, fibrosis, hypercholesterolemia and various virus infections, as evidenced by the numerous products under clinical trials, ongoing or planned [3,5–7].

One of the most significant advances in lipid-based NABDs therapies dates back only to August 2018, and is undoubtedly represented by Patisiran (ALN-TTR02), the first ever approved RNAi therapeutic by the Food and Drug Administration (FDA) and by the European Commission (EC). Produced by Alnylam Pharmaceuticals Corporation and sold under the trade name Onpattro™, ALN-TTR02 is an siRNA formulation based upon Stable Nucleic Acid Lipid Particle (SNALP) transfecting technology, and is indicated for the treatment of the Hereditary Transthyretin-mediated Amyloidosis (hATTR), a progressively incapacitating and often fatal genetic disorder. Thus, after 20 years from the first publication about the RNAi-mechanism discovery [8], in August 2018 the gene-silencing technology has had its first drug approval as stated in an article published by Nature and just entitled "Gene-silencing technology gets first drug approval after 20-year wait" [9]. It appears evident how the clinical translation of lipid-based NABDs delivery systems shelf has progressed [10–17], leading to the formulation of several lipid-based carriers (see schematization of Figure 1), including micelles, solid lipid nanoparticles and liposomes [18].

Among these systems, liposomes are the most effective system. They are spherical particles composed of phospholipids, encapsulating a volume of aqueous medium showing several favorable features, such as an excellent biocompatibility, biodegradability, low toxicity, low immunogenicity, an ability to deliver a large piece of nucleic acids, structural flexibility and easiness of handling. Moreover, cationic liposomes, made by positively-charged lipids, are especially indicated for NABDs delivery, being this last characterized by negatively-charged phosphate groups. Their association gives compacted structures which can also electrostatically interact with the negatively-charged cell membrane, facilitating their cellular uptake [19]. In particular, in recent years, cationic liposomes have gained a lot of attention for siRNA delivery, improving their uptake into tumor tissues and their stability and bioavailability [20,21]. The siRNAs are 21–23 nucleotides long, double-stranded RNA, which, binding at specific sequences of messenger RNA (mRNA), "interfere" with the translation of proteins. Their therapeutic potential as a next generation medicine has been recently reviewed [22], together with the major delivery strategies currently adopted for their effective in vitro and in vivo delivery [23].

In this work an overview on the advances in NABDs-lipid systems delivery is presented. In particular, the attention is before focused upon liposomes as a lipid carrier system and on siRNA as NABDs. After, a discussion about liposomes structures, the formulations and production techniques suitable for NABDs delivery are reported. In the last part, the medical applications of siRNA-liposomes currently under investigation in clinical trials are then treated in detail.

#### **2. Liposomes: Structures and Basic Formulations**

Liposomes are vesicles characterized by a lipid bilayer that surrounds an aqueous core. They are mainly composed of phospholipids recognizable by a polar hydrophilic head and two apolar hydrophobic chains [24].

When dispersed in aqueous solutions, their polar heads interact with the aqueous environment, due to the hydrogen bonds and polar interactions, while their aliphatic chains interact with each other due to the van der Waals forces, leading to a lipid bilayers formation of which they constitute the lipophilic inner compartment [24,25]. During their formulation process, water-soluble drugs can be dissolved in the aqueous compartment while hydrophobic materials can be entrapped into the lipid bilayer [26–28]. Joined by being composed of biocompatible materials, liposomes show different structures, dimensions, lipid composition and surface charge. They can be composed by several concentric bilayers separated by aqueous compartments, with an external lipid bilayer containing other ever smaller bilayers separated by water cavities, like an onion structure. In this case liposomes are called Multilamellar Vesicles (MLVs) and show a size range of 500 nm to 5 μm, or by only one phospholipid bilayer surrounding an aqueous compartment. In this case liposomes can be differentiated in small, large and giant vesicles depending upon their dimension: They are called Small Unilamellar Vesicles (SUVs) if they have a 20 to 200 nm range size, Large Unilamellar Vesicles (LUVs) with a 200 to 1 μm range size and Giant Unilamellar Vesicles (GUVs) with a size larger than 1 μm. Finally, similar in dimension to MLVs there are multi-compartmental structures constituted by vesicles surrounded by other vesicles called Multi Vesicular Vesicles (MVVs) [19,29,30]. In Figure 2 a schematization of liposomes classification based on their structure, size and composition is presented.

**Figure 2.** Liposomes classification by structure/size and lipid composition.

Liposomes are mainly made of phospholipids, which contain two major categories including glycerophospholipids and sphingomyelins. Composed by a hydrophilic head group, a hydrophobic side chain and a glycerol backbone, glycerophospholipids are those that characterized the eukaryotic cells, and are used in liposomes production by varying their head group i.e., phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG) [31].

#### *2.1. Conventional Liposomes*

Conventional liposomes are neutrally-charged vesicles made primarily from PC and cholesterol; the incorporation of this last into the lipid bilayer, with its hydroxyl group oriented towards the aqueous surface, and the aliphatic chain aligned parallel to the acyl chains [2], enhances the stability of the vesicles, reducing their permeability, and increasing their in vivo and in vitro performance [24,32–34].

Although reducing the in vivo drugs toxicity, being recognized as foreign substances by serum opsonines and destroyed by the reticuloendothelial system (RES), conventional liposomes are characterized by a short circulation time when intravenously administered [17,35].

To overcome this limit, the strategy of a vesicle surface modification by hydrophilic inert polymers has been adopted for the development of stealth liposomes, and the approach is discussed in the "Long circulating liposomes" paragraph. Moreover, the use of conventional liposomes for NABDs delivery usually results in a scarce encapsulation efficiency due to the lack of electrostatic interactions between the positive nucleic acids and the uncharged vesicles bilayer. To avoid this problem, cationic liposomes can be used, as discussed below.

#### *2.2. Cationic Liposomes*

Vesicles can be prepared by using positively-charged lipids, i.e., the 1,2-DiOleoyl-3- TrimethylAmmonium Propane (DOTAP), achieving cationic liposomes which can electrostatically attract the negative phosphate groups of NABDs, increasing their encapsulation efficiency. Moreover, the cationic liposomes also facilitate the intracellular uptake of NABDs and their endosomal escape, more efficiently if "helper" lipids are included in the formulation [19].

Indeed, neutral and zwitterionic lipids, mainly 1,2-dioleoylsn-glycero-3-phosphatidylcholine (DOPC), Di-Oleoyl-Phosphatidyl-Ethanoalamine (DOPE) and 1,2-DiStearoyl-sn-glycero-3- PhosphoCholine (DSPC), being more fusogenic than the cationic one, can affect the polymorphic features of the liposome–siRNA complexes, promoting the transition from a lamellar to a hexagonal phase, thus inducing fusion and a disruption of the membrane [36]. In particular the fusogenicity increases with the decrease of the degree of the saturation of the lipids hydrophobic tails, and with the decrease of their polar heads size (i.e., lipids with large polar heads and highly saturated tails prefer the lamellar phase, and thus are not fusogenic; lipids with small polar heads and highly unsaturated tails prefer the reverse hexagonal phase, and thus are fusogenic) [15]. DOPC lipid has been successfully used by the M.D. Anderson Cancer Center to produce siRNA-EphA2-DOPC, which is currently in a Phase I clinical trial for the treatment of advanced cancers, as discussed later in the "Current applications of lipid-based siRNA delivery" Section. Some of the most common cationic and helper lipids used for NABDs delivery are reported in Figure 3.

Cationic liposomes formulation is in constant improvement. In order to optimize siRNA encapsulation efficiency, Santel and collaborators have synthesized the cationic lipid β-l-Arginyl-2,3-l-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride (AtuFECT), which when complexed with siRNA is termed AtuPLEX [37]. Atu027 is an AtuPLEX produced to treat advanced solid tumor [38] which has completed the Phase I and the Phase I/II of clinical trials (see Section 4).

Cationic liposomes can be also formulated by adding to the basic vesicles composition the lipidoids, lipid-like materials produced through the conjugate addition of an amine to an acrylate or acrylamide [4,39–43]. Akinc and collaborators have produced a complex called "LNP01" which is made by 98N12-5:cholesterol:PEG-lipid in a molar ratio of 42:48:10 (mol:mol:mol) respectively, with a total lipid:siRNA ratio of about 7.5:1 (wt:wt), a C14 alkyl chain length on the PEG lipid and a mean particle size of roughly 50–60 nm. The developed formulation is liver targeted (with a >90% injected dose distributed to the liver) and can induce, after repeated administrations, a long-duration gene silencing without any loss of activity [42,43].

Love and coworkers have also made complexes by combining lipidoid, cholesterol, and a polyethylene glycol modified lipid in a yield weight fractions of 52:20:28 (wt:wt:wt), obtaining an siRNA-directed liver gene silencing in mice at doses below 0.01 mg/kg [41]. A lipidoid nanoparticle which mediates potent gene knockdown in hepatocytes and immune cell populations after IV administration to mice, with siRNA EC50 values as low as 0.01 mg/kg, was successfully produced also by Whitehead and collaborator [39]. The lipidoids structures used in the cited works for siRNA-lipid complexes production are given in Figure 4.

**Figure 3.** Structures of cationic and helper lipids used as components of various siRNA liposomal delivery systems.

**Figure 4.** Lipidoids synthetized by different research groups and used in cationic liposomal formulations to increase siRNA encapsulation efficiency.

Finally, several commercial products based on cationic liposomes for NABDs transfection during in vitro experiments are available on the market, the most used ones are Lipofectine and LipofectAMINE, produced and sold by the Invitrogen Company.

Despite the advantages in strongly attracting NABDs, the use of cationic liposomes presents several drawbacks due to their instability, rapid systemic clearance, toxicity and their induction of immunostimulatory responses [44,45]. Alternatively, in order to ameliorate cationic liposomes performances in gene therapy, different techniques were developed for the production of relatively new cationic lipid structures, including Stable Nucleic Acid Lipid Particles (SNALP), as discussed in Section 3.

#### *2.3. Long Circulating Liposomes*

The rapid decrease of liposomal drug complexes in blood with the consequent accumulation in liver, spleen and other organs has led to the development of the long circulating liposomes, lipid vesicles characterized by a surface covered with inert polymeric molecules, such as oligosaccharides, glycoproteins, polysaccharides and synthetic polymers [28]. Among these, covering liposomes with the polyethylene glycol (PEG) represents an effective strategy to increase the repulsive forces between liposomes and serum-components, thus avoiding their elimination by the RES, while improving their stability and enhancing their circulation times in the blood [46–48]. Such surface modified liposomes have been called "PEGylated" or "stealth" liposomes, since they can evade recognition by T cells and macrophages, and avoid rapid clearance by the immune system [49,50]. In particular, the polymeric chain adsorbed on the liposomes surface consisting of tails, loops and trains, can form several structures on the bases of the polymeric layer size formed. Polymeric "pancake", "mushroom" or "brush" structures are formed with a PEG surface thickness of about 1.5 nm, 3.5 nm and 5 nm, respectively [18,51]. Doxil® represents the first FDA-approved drug carrier based on PEGylated liposome technology for the treatment of advanced ovarian cancer, multiple myeloma and HIV-associated Kaposi's sarcoma [52]. Moreover, several PEGylated liposomal products for NABDs delivery are in preclinical development, i.e., the lipidoid-based siRNA formulation 98N12-5 [53] or the DACC cationic lipoplex [54].

However, the long-circulating liposomes exhibit some drawbacks: (1) The PEGylation has to be optimized by modulating PEG length and density in order to control the interactions between the liposomal, the plasmatic and the endosomal membranes, which can prevent the vesicles' cellular uptake/escape from the endosome. This problem, known as "PEG dilemma", can be avoided by using cleavable PEG-lipids such as the "pH-responsive" or "enzyme-responsive" ones [47]; (2) covering with PEG does not guarantee the selectivity of the drug delivery to the target tissue. This problem can be overcome by using the targeting ligands conjugation approach [55] discussed below.

#### *2.4. Ligand-Targeted Liposomes*

Tumor tissues/blood vessels are characterized by specific and overexpressed receptors which can bind to antibodies, polysaccharides, proteins, polypeptides, aptamers and other molecules. The attachment of these specific targets to the NABDs-liposomes surface guarantees an active targeting of the carriers to the diseased tissue [28]. Recently, a Dual-Targeting Ligands approach was developed by Riaz and collaborators [49], and a combination of peptide and antibody ligands was used to fuctionalize a single liposomal formulation, obtaining an increased nanoparticles cell uptake [56].

In 1995 folic acid was conjugated for the first time onto the surface of nucleic acids liposomal carriers (folate-PEG-liposomes) by Wang and collaborators [57]. To date, the folate receptor is one of the most used targets for siRNA liposomal delivery, being overexpressed in several malignancies, such as ovarian and uterine cancer, osteosarcoma, meningioma and other cancer cells, but not in normal tissues [28].

The transferrin receptor-dependent mechanism is also used for liposomal NABDs internalization, i.e., Mendoça and coworkers have successfully developed a transferrin receptor-targeted liposome loaded with an anti-BCR-ABL siRNA or asODN for the treatment of chronic myeloid leukemia [58].

Recently, Zang and collaborators have developed a pH-sensitive cholesterol-Schiff base-polyethylene glycol (Chol-SIB-PEG)-modified cationic liposome–siRNA complex, conjugated with the recombinant humanized anti-EphA10 antibody (Eph). The Eph–PEG–SIB–Chol-modified liposome–siRNA complex (EPSLR) has shown a good endo-lysosomal escape, releasing siRNA into the cytoplasm after 4 h from in vitro transfection. Moreover, an in vivo study conducted in tumor-bearing mice shows that EPSLR can reach the diseased tissue more effectively [59].

Moreover, polysaccharides, i.e., galactose, mannose, dextran and hyaluronic acid can be also used as ligands to prepare glycosyl-liposomes targeting tissues overexpressing these receptors. The use of functionalization with polysaccharides and other targeting moieties for an effective NABDs delivery has been widely described in [18].

#### *2.5. Bubble Liposomes*

Lipid vesicles filled with gas are called "Bubble Liposomes". They are particles ranging from 1 to 10 micro meters, characterized by a gas core and a shell composed of several materials i.e., proteins, lipids, polymers, surfactants and galactose, used to deliver NABDs and other drugs into the cells and tissues [60]. In particular, the term "Bubble Liposomes" was coined by Suzuki, Maruyama and collaborators, who developed novel liposomes containing lipid nanobubbles loaded with perfluoropropane, used as ultrasound imaging agent. Briefly, "Bubble Liposomes" are prepared by producing at first polyethyleneglycol-modified liposomes (PEG-liposomes) through the reverse phase evaporation method, and placing them in vials with perfluoropropane gas, then sonicating in a bath sonicator. Upon exposure to ultrasound, these particles can induce cavitation, which supplies the energy required to deliver extracellular molecules into the cytosol, and this can thus be utilized as a gene delivery tool [61]. This phenomenon involves a greater cell/tissue permeability, improving NABDs penetration into the cells and ensuring, at the same time, their escape from the endosome with their consequent expression [62].

In that regard, it was demonstrated that bubble liposomes can deliver genes into cells even when the cells were exposed to ultrasound for only 1 s. They have also shown that this kind of liposome is more effective in delivering NABDs, i.e., the luciferase gene, into a tumor than are the conventional cationic liposomes used as transfection agents [63].

Recently, several research groups have opted for the use of bubble liposomes for NABDs delivery such as Negishi and collaborators, who have used bubble liposomes for a selective gene delivery to syndecan-2 overexpressing cancer cells [64], or also Endo-Takahashi and coworkers, in whose work bubble liposomes were successfully used to deliver miRNA (miR-126) for the cure of hindlimb ischemia by the systemic administration of miR-126-loaded bubble liposomes into mice coupled with US exposure [65].

#### **3. Liposomes Preparation Techniques**

There are many different methods for the preparation of liposomes [19,66–68], here the attention is focused on those available for NABDs encapsulation.

#### *3.1. Thin Film Hydration*

The most commonly used technique for liposomes preparation is the Thin Film Hydration (TFH) or the Bangham method, in which lipids are dissolved in an organic solvent, then evaporated through the use of a rotary evaporator leading to a thin lipid layer formation [69]. After the layer hydration by an aqueous buffer solution containing the hydrophilic drug to be loaded, Multilamellar Vesicles (MLVs) are formed, which can be reduced in size to produce Small or Large Unilamellar vesicles (LUV and SUV) by extrusion through membranes or by the sonication of the starting MLV [18,19,70]. Even if the method is not scalable, and toxic solvent traces could remain in the final formulation, its easiness to perform and low cost make this one of the most adopted techniques for liposomes preparation. The method is used also for the production of liposomes containing NABDs, whose encapsulation efficiency, however, is generally quite low, ranging from 3 to 45%, but can be increased by modulating the lipid mixtures [71,72]. In our previous works DOTAP cationic lipid was added to a cholesterol and phosphatidylcholine formulation in order to electrostatically attract siRNA molecules. Briefly, through the TFH method, followed by a duty cycle sonication, siCyD1-nanoliposomes [73] and siE2F1-nanoliposomes [74] with 100% siRNA encapsulation efficiency, able to reduce respectively colon CyD1 and E2F1 proteins expression after transfection in ex vivo human tissue cultures, were produced.

By modulating the lipid formulation adding DOTAP, Coated Cationic Liposomes (CCL) entrapping an antisense oligodeoxynucleotides (asODN), with a diameter of 188 nm and an encapsulation efficiency of 85–95% were also prepared through the TFH technique by Stuart and coworkers [75].
