Liposomal formulations for treating lysosomal storage disorders q

Lysosomal storage disorders (LSD) are a group of rare life-threatening diseases caused by a lysosomal dysfunction, usually due to the lack of a single enzyme required for the metabolism of macromolecules, which leads to a lysosomal accumulation of speciﬁc substrates, resulting in severe disease manifestations and early death. There is currently no deﬁnitive cure for LSD


Introduction
Drug therapy is continuously advancing towards a more precise delivery of biomolecules and drugs of interest; more specifically, towards an ''a la carte" delivery of active pharmaceutical ingredients (APIs) to the intended site of action [1].Biomedically applied nanotechnology has become a successful tool for achieving this goal, seeking to improve conventional drug therapies, especially by introducing the concept of drug delivery systems (DDS) in which biomolecules or drugs of interest are conjugated, complexed, or encapsulated (in)to a carrier molecule/system (usually a nanocarrier, i.e., with sizes in the nanometric scale) and transported to the place of action via active or passive mechanisms.This process can open the door to a large number of alternative strategies for drug transport and delivery, overcoming the limiting factors of conventional drug administration.
Despite the considerable progress in recent years in treating a large number of diseases (e.g., cancer, hematological disorders, immune disease, metabolic disease) [2], there are a considerable number of constraints in conventional treatments, such as a low sensitivity, limited specificity, poor biodistribution, high drug toxicity, and severe adverse effects.For example, numerous therapeutic drugs present a narrow therapeutic window in which the therapeutic dose (i.e., the quantity of drug required to produce the optimal effect) is not much lower than the toxic level.A change in the drug's temporal and spatial biodistribution, e.g., using an appropriate DDS (taking into account DDS parameters such as structure, size, composition, targeting, type of drug loading, and intended administration route), can lead to reduced toxicity and improved efficacy [3].Moreover, DDS can provide additional functionality, such as protection from rapid degradation or clearance, improvement of the drug's solubility, and passive or active targeting, which enhances the drug concentration in the desired tissue, requiring lower doses of the drug [4].
Among lipid-based nanovesicles, liposomes are one of the most promising carriers for nanomedical applications.Liposomes were discovered by Alec Bangham in the 1960s after observing that phospholipids in aqueous systems can form self-assembled, closed, bilayered structures [8].Liposomes have been extensively investigated and have shown excellent potential as pharmaceutical carriers due to their biocompatibility and biodegradability, and their versatility in terms of size, composition, surface properties, and the ability to entrap hydrophilic and hydrophobic compounds [9,10].Liposomes can entrap water-soluble pharmaceutical agents inside their internal water cavities, as well as water-insoluble pharmaceuticals into the lipophilic membrane and can covalently bond a targeting group at their external membranes.These abilities can meet the frequent demands in conventional drug therapy, such as increasing drug solubilization, offering protection against degradation, decreasing nonspecific side-effects, and improving the selectivity to targeted cells and organs [9].
Liposomes are spherical nanovesicles composed of amphiphilic molecules (predominantly phospholipids) that self-assemble in one or several concentric closed lipid bilayers.Like phospholipids, amphiphilic molecules have a hydrophilic region (e.g., polar head group) and a hydrophobic section (e.g., non-polar tail) that can spontaneously organize into vesicles, exposing their polar groups to the water phase and protecting their hydrophobic tails in the bilayer [11].However, not all lipid nanoparticles have a bilayer lipid structure that would qualify them as lipid vesicles or liposomes.This spontaneous organization of phospholipids adopting bilayer structures when dispersed in an aqueous medium is the main difference between liposomes and other lipid nanoparticles.For example, non-bilayer structures are formed between synthetic cationic lipids and anionic nucleic acids, which yield stable complexes and are widely used as a nonviral delivery system for nucleic acid drugs [12].Other lipid nanoparticles include solid lipid nanoparticles (SLN) and nanostructured lipid carriers, which consist of solid lipids or a mixture of a solid and liquid lipids, respectively.These solid lipid nanoparticles and nanostructured lipid carriers therefore possess a solid lipid core matrix that can solubilize lipophilic molecules.The lipid core is mainly stabilized by surfactants [13].Lipid vesicles (and liposomes among them) can be classified according to their size and lamellarity (i.e., number of bilayers).Vesicles can be small (less than200 nm), large (200-1000 nm), or giant (greater than 1000 nm) and can be unilamellar (single bilayer) or multilamellar (several concentric bilayers).When several small non-concentric vesicles are entrapped inside a larger one, they are known as multivesicular vesicles [14][15][16].Of these types of vesicles, small unilamellar vesicles (SUVs) are of particular interest for medical use.Small liposomes (usually in the 100-200 nm range) are compatible with intravenous administration, are large enough to avoid rapid clearance through the kidneys, and are small enough to show a minimal uptake by reticuloendothelial system (RES) cells, leading to an extended circulation time [14,17].
An important feature of liposomes is their compatibility with intravenous administration, which is the most efficient route for DDS administration, because many bioactive molecules such as biological agents have low permeation if they are administered extravascularly (e.g., orally) [2,3].Once in circulation, liposomes can take advantage of passive and active targeting.Passive mechanisms include the accumulation of liposomes in vascular tissue in specific medical conditions, such as tumors, mainly due to enhanced permeability and retention (EPR) effect, a phenomena based on increased permeability in inflamed tissues or tumor environments, leading to the passive accumulation of small nanoparticles (especially in the range of 100-200 nm), which are then able to cross these vessels and accumulate in the affected tissue [7,[18][19][20].Active targeting can be achieved by the attachment of ligands or molecules (e.g., peptides, antibodies, nucleic acids) on the nanoparticle surface, with specific affinity for certain cellular receptors.Recognition of these ligands by target cells and tissues can result in enhanced uptake at target expression sites [13,21,22].Unlike small molecules and biological drugs, the predominant elimination route for DDS is their clearance by the RES of certain tissues, such as the liver, spleen, bone marrow, and lungs [3,9,23].The phagocytic cells (e.g., macrophages) of these tissues can remove nanocarriers from circulation because the cells recognize DDS as foreign or undesirable entities.Moreover, opsonization of the nanoparticles by serum proteins (e.g., antibodies and complement proteins) can enhance this process [23].These elimination pathways are serious drawbacks for the use of liposomes as effective DDS to target cells other than phagocytic cells, and, consequently, significant efforts have been made to overcome these problems in recent years.

Evolution of liposomal structures for drug delivery
Since their discovery, liposomes for drug delivery have evolved (Fig. 1).Early classical plain liposomes composed exclusively of phospholipids with certain water-or lipid-soluble drugs led to the ''first-generation" of liposomal DDS [9].These liposomes showed evident limitations, especially regarding stability and rapid clearance from the bloodstream.To improve liposomes' physical stability, cholesterol was added as a membrane component because cholesterol induces a denser packing of phospholipids, thereby increasing membrane rigidity, which enhances liposomal colloidal stability, decreasing membrane permeability and preventing drug leakage [3,14].
Additionally, other molecules, such as polyethylene glycol (PEG), can be anchored to membrane liposome components (e.g., cholesterol).Modifying the nature and ratio of phospholipids, adding cholesterol, and further liposomal surface decoration (e.g., with PEG) strongly affects the biochemical and physicochemical properties of liposomes and their in vitro and in vivo behavior, resulting in ''second-generation" liposomal DDS [11,25].With the addition of PEG, ''stealth" liposomes or ''long-circulating" liposomes are obtained.PEG is a hydrophilic, inert, low immunogenicity, biocompatible polymer widely used for protecting nanoparticles and prolonging the in vivo circulation time [26].PEGylation of liposomes (i.e., coating the external liposomal surface with PEG polymer) grants protection against recognition by opsonins, i.e., the adsorption of blood proteins to the particle surface, thereby preventing subsequent clearance of liposomes from the bloodstream by the RES [9,27].Currently, PEGylated liposomes are widely used in the majority of liposomes that have reached the clinical phase.However, the PEG coating of liposomal surface not only protects the nanoparticle but can also limit the cellular uptake and endosomal escape, reducing the final therapeutic effect [28].Another disadvantage of PEG-coated systems is the possibility of inducing complement activation-related pseudoallergic reactions, as has been described for liposomes and lipid nanoparticles [29][30][31][32].Therefore, the amount of accessible PEG chains must be controlled to counter these drawbacks.Another strategy for overcoming the limitations of conventional liposomes is the use of stimuli-responsive liposomes, aiming to obtain a triggered drug release profile.A triggered chemical component sensitive to an external (e.g., heat, light, ultrasound, magnetic field) or internal (e.g., pH, enzymes) stimulus is incorporated into the liposome composition to control the spaceand time-delivery of the drug.Stimuli-responsive liposomes protect drugs from degradation and unspecific delivery and only release the compound of interest in response to the stimuli, which generally destabilizes the liposomal membrane provoking the drug release [25].
The ''third-generation" or ''ligand-targeted liposomes" combine several additional functionalities to produce higher and more selective therapeutic activity, by incorporating specific ligands onto the liposome surface [26].Targeting moieties include pep- tides, receptor ligands, growth factors, glycoproteins, carbohydrates, monoclonal antibodies and fragments and can be added directly to the nanoparticle surface or through a PEG molecule acting as a linker [9].When ligand-targeted liposomes arrive at the target location, they interact by their complementary affinity with the ligand receptor, contributing to cell-specific retention and uptake [26].
Theranostic liposomes can be considered the ''fourthgeneration" of liposomes, which refers to the approach of simultaneously facilitating the therapy and diagnosis.These new and complex liposomes attempt to combine several additional functionalities, such as delivering a therapeutic agent by encapsulating/entrapping a drug, site-specific targeting by incorporating a targeting ligand, and complementary bioimaging capability by adding an imaging agent [25].

Clinical application of liposomes
Breakthroughs over the past decades have resulted in a number of approved nanomedicines.The first liposomal pharmaceutical product, known as Doxil Ò , was approved in 1995 by the US Food and Drug Administration (FDA).Doxil Ò consists of a liposomal product containing the doxorubicin chemotherapy drug for treating ovarian cancer and AIDS-related Kaposi's sarcoma [33].This nanomedicine was followed by other liposomal products that reached the marked in the following years.Cancer was the most widely explored area in terms of clinically approved liposome products, by means of entrapping chemotherapy drugs into liposomes for several cancer therapies (e.g., daunorubicin, DaunoXome Ò for AIDS-related Kaposi's sarcoma, approved in 1996; cytarabine, Depocyt Ò for neoplastic meningitis, 1999; doxorubicin, Myocet Ò for breast cancer, 2000; mifamurtide, Mepact Ò for osteosarcoma, 2004; vincristine, Marqibo Ò for leukemia, 2012; and irinotecan, Onivyde Ò for pancreatic adenocarcinoma, 2015) [8,34,35].The entrapment of drugs inside liposomal systems leads to improved bioavailability and selectivity over non-entrapped drugs and decrease the drugs' side-effects.Liposomal products have also been explored for diseases other than cancer, although in smaller numbers, such as for fungal infections, viral infections, vaccine developments, and anesthetics [34].
There are currently approximately 15 liposomal-based drugs that have been approved for clinical use, and more than one hundred are in various stages of clinical trials, representing more than 50 % of clinical trials involving nanoparticles [8,34,36].These numbers illustrate liposomes' ability to overcome the limitations of conventional therapy and justify the intense research on liposomal applications for other types of diseases.However, nanomedicine applied in pediatrics remains a significant challenge.Although nanomedicines may solve many needs of overall population diseases and medicines, the unavailability of data on pharmacokinetics, safety and efficacy of both drugs and nanoparticles in pediatric patients limits the development and clinical translation of new pediatric nanomedicines and, thus, among them those based on liposomes [37].Clinical trials of liposomes in children's anticancer therapy represents only about 4.77 % of clinical trials on liposomes in cancer therapy, which is the most widely explored area [38].
This review addresses the use of liposomes for their specific application in lysosomal storage disorder (LSD) therapy and the current development status of these liposomal formulations.Additionally, the study reviews the various physicochemical characterization methods and production methodologies of each liposomal formulation to highlight the importance of these issues for future clinical translation.The quality of the liposomal products must be ensured so that they can be translated into pharmaceutical liposomal products.

Lysosomal storage disorders
LSDs are a group of more than 50 diseases caused by a lysosomal dysfunction, usually due to the lack or malfunction of a single enzyme required for metabolizing macromolecules such as lipids, glycoproteins, and mucopolysaccharides, resulting in accumulation of undegraded substrates [39,40].The deficiency of an active specific enzyme typically occurs due to genetic defects, both monogenic autosomal and X-linked diseases [41].The majority of LSDs are characterized by a progressive course, usually resulting in severe disease manifestations and early death [39].Individually, LSDs are considered rare diseases, because their individual incidence is very low (less than 1 in 100,000 live births).However, as a disease group, the cumulative incidence of LSDs has been estimated at 1:7000-8000, representing a serious global health problem [39,42].
Lysosomes are generally spherical and membrane-limited subcellular organelles in eukaryotic cells that are involved in numerous cellular processes particularly related to the intracellular digestion and degradation of biomacromolecules, such as proteins, nucleic acids, glycosphingolipids, mucopolysaccharides, and glycogen, as well as other foreign substances [39,41].Lysosomes contain more than 200 lysosomal-resident proteins, approximately 60 of which are part of a mixture of acidic hydrolytic enzymes [43].These glycosylated hydrolytic enzymes are produced in the endoplasmic reticulum, modified with mannose-6-phosphate (M6P) recognition markers in the Golgi apparatus, packed into secretory vesicles, and delivered to the late endosomes/lysosomes [44][45][46].After their passage through Golgi apparatus, a variable fraction of the newly synthesized glycosylated enzyme can also be secreted from the cell and be endocytosed and transported to the lysosomes of neighboring cells, through plasma membrane-located M6P receptors [45].Glycosylation is essential for enzyme watersolubility, activity, stability, and correct transport to the lysosome [47].
In LSD, lysosomal hydrolases are one of the most affected enzymes, resulting in an accumulation of specific substrates due to the inability to degrade them.Clinically, lysosomal diseases are generally classified according to the major undegraded and stored compound.Thus, LSDs mostly include mucopolysaccharidoses (MPS) (e.g., Hunter disease and Sanfilippo disease), sphingolipidoses (e.g., Fabry disease and Gaucher disease), oligosaccharidosis and glycoproteinosis, and glycogenosis (e.g., Pompe disease) [48].

Current strategies for lysosomal disease treatment
The low prevalence of each individual lysosomal disease imposes evident limitations on the development of therapies (e.g., low patient recruitment for trials, insufficient financial incentives, and poor understanding of the metabolic and biological basis of the disease) resulting in few approved drug therapies [49].Besides, patients with the infantile form are the most severely affected and, generally, recruitment of children is a persistent challenge for researchers attempting to include this population in clinical trials [37,38].Overall, drug development for rare diseases is not financially viable without the support of regulatory agencies.As a consequence, several nations have promoted incentives to stimulate the development of orphan drug therapies, offering tax incentives, research subsides, market exclusivity, and extended patent protection, with the first of these incentives introduced in the United States in 1983 and later in the European Union (EU) in 2000 [50][51][52].The EU has extensively supported this field through its research and innovation framework programs, with more than €1.8 billion made available over the past 14 years under the Seventh Framework Programme (FP7) and Horizon 2020 to more than 320 projects on interdisciplinary research related to rare diseases [53].With the new research and innovation funding program Horizon Europe (2021-2027), EU will continue to support these efforts.In addition to EU-funded collaborative projects, specific initiatives offer a solid framework for enhanced cooperation in the area (e.g., the European Joint Programme on Rare Diseases, the European Rare Disease Research Coordination and Support Action Consortium, and the International Rare Diseases Research Consortium).The FDA funds critical rare disease research through a variety of programs, such as the congressionally mandated Orphan Products Grants Program, which has supported rare disease clinical trial research since 1983 and has facilitated over 70 product approvals [54].
Although there is no definitive cure for LSD, several specific treatments have been developed to correct the metabolic defect and reduce the pathophysiological effects of substrate accumulation into lysosomes (Fig. 2A) [42,45].
One treatment approach includes small-molecule therapies, based on substrate inhibitors, chemical chaperones, or cytosolic molecule modulators [49].This substrate reduction therapy (SRT) is based on reducing the accumulated substrate by inhibiting the enzyme responsible for its synthesis.The first substrate-reducing compound was miglustat, an inhibitor of the glucosylceramide synthase, which affects the glycosphingolipid biosynthetic pathway.Miglustat was approved in 2002 for the treatment of Gaucher disease (OMIM ID #230800) [70].Other SRTs include eliglustat, approved for Gaucher disease by the FDA in 2014.A number of substrate-reducing molecules are still in clinical development, such as venglustat (in phase II trials for Gaucher disease [Clini-calTrials.govID NCT02843035] and for Fabry disease [OMIM ID #301500], NCT02228460 and NCT02489344) and lucerastat (in phase II/III trials for Fabry disease) [43,71].
Another strategy is pharmacological chaperone therapy (PCT), based on the use of small-molecule drugs that improve the stability and activity of the defective enzyme, although it is only useful for mutant enzyme proteins with residual activity [43].Currently, the only approved PCT for LSD is migalastat (Galafold Ò ).Migalastat can enhance the activity and stability of the a-galactosidase (GLA) enzyme and was approved in 2016 for the treatment of patients with Fabry disease with amenable GLA gene mutations [71][72][73].Other PCTs in clinical development include ambroxol (in phase II trials for Gaucher disease, NCT03950050), arimoclomol (in phase II/III trials for Niemann-Pick disease type C [OMIM ID #257220], NCT02612129, and in phase II trials for Gaucher disease, NCT03746587) [73].
Another therapy that can provide constant delivery of a therapeutic protein to the entire body is gene therapy (GT), which includes hematopoietic stem cell transplantation (HSCT) and gene transfer [43].Historically, the first approach in this direction was hematopoietic cell transplantation (HCT) (i.e., bone marrow transplantation) from healthy compatible donors.Over the past few decades, early studies were performed on certain patients with LSD treated with allogeneic HCT [74].HCT can be indicated for a small number of LSDs in presymptomatic or very early symptomatic phases, although the indication for transplantation in the other LSDs is not as strong as in MPS I (OMIM ID #607014) [74,75].HCT therapy could be improved by GT-based approaches (e.g., HSCT and gene transfer).In HSCT, also called ex vivo GT, hematopoietic stem cells are extracted, genetically modified in vitro, and transplanted into the patient.In vivo gene transfer is based on injecting the transgene carried in a vector directly into the desired tissue or into the blood circulation [76].Therefore, the major advantages are obtained in hematopoietic stem cell GT (HSC-GT).One advantage of HSC-GT for LSD compared with conventional HSCT is the capacity to overexpress the therapeutic gene in HSC and their progeny, as well as the ability to perform autologous transplantation from the patient's own HSC [73].The modification of a relatively small number of cells can lead into the correction of a wider range of cell types thanks to the crosscorrection phenomena [76], characterized by the secretion of a small number of lysosomal enzymes from the cell, which can be trafficked and uptaken by other cells by M6P recognition.Although all GT-based treatments for LSD are currently in clinical trials [73,76], the results for other diseases and the approval of the first GT-based products by the European Medicines Agency (EMA) and FDA are encouraging the continued exploration of this strategy [77,78].
Enzyme replacement therapy (ERT) is currently considered the gold-standard treatment for several LSDs and consists of the exogenous administration of a deficient lysosomal enzyme.All ERT drugs are produced using recombinant DNA methods, and the resulting enzymes are administered by intravenous infusion [41].Currently, there are approximately 14 FDA and/or EMAapproved ERT products for LSDs, especially intended to treat Gaucher disease (4 ERT products), Fabry disease (2 ERT products), Pompe disease (OMIM ID #232300) (3 ERT products), several types of mucopolysaccharidoses (5 ERT products in total), and lysosomal acid lipase deficiency (OMIM ID #278000) (1 ERT product) [49].Additionally, there are several ERT drugs currently in clinical trials, a number of which are in an advanced stage, such as bglucuronidase (UX-003, NCT02230566) for MPS VII (OMIM ID #253220), and acid sphingomyelinase (Olipudase alfa Ò , NCT02004691) for Niemann-Pick type B (OMIM ID #607616), which are all in phase III [49].
Although there has been remarkable success with ERT drug products in effectively reducing substrate accumulation in certain tissues [79,80] no ERT seems to completely reverse the disease, especially in patients with an advanced stage of the disease.Moreover, ERT presents several potential limitations including 1) limited efficacy, generally due to poor biodistribution (liver sequestration) and the development of antidrug antibodies with neutralizing effect; 2) no crossing of the blood-brain barrier (BBB), complicating the treatment of brain impairment; 3) low stability of the enzyme, resulting in its rapid degradation and short circulation time; 4) high immunogenicity issues, leading to infusion-associated reactions; and 5) inconvenience of life-long therapy, usually requiring weekly or biweekly intravenous administration and resulting in expensive treatment costs [43,72,[81][82][83][84].
Several strategies to overcome these limitations are currently under study, in particular those related to developing and improving new forms of ERT.Second-generation recombinant enzymes, created by chemically modifying the exogenous recombinant enzyme (e.g., by covalently linking a PEG coating, additional glycosylation, targeting moieties, or antibodies to the recombinant enzyme) were designed to improve their targeting properties and pharmacodynamic behavior [43].For example, pegunigalsidase alfa (PRX-102, Protalix Biotherapeutics) is a chemically modified version of the GLA enzyme with attached PEG chains and is currently being evaluated for efficacy in clinical trials (phase III, NCT03018730) on Fabry disease [85].Another example is avalglucosidase alfa, a glycoengineered recombined a-glucosidase (GAA) enzyme with increased bis-M6P levels, which was recently been approved by the FDA after positive phase III data in Pompe disease [43,86].Another chemical modification is the conjugation of the recombinant enzyme with various targeting peptides or antibodies, e.g., the iduronate-2-sulfatase enzyme fused with antihuman transferrin receptor antibody for MPS II (OMIM ID #309900) treatment (IZCARGO Ò , currently approved in Japan) [87] and the a-Liduronidase (IDUA) enzyme fused with the antihuman insulin receptor for MPS I treatment (valanafusp alpha, currently in phase I/II, NCT03053089) [88].
Another strategy for overcoming some of the abovementioned limitations of current ERT is the encapsulation of the recombinant enzyme into a nanocarrier.Robust enzyme delivery nanoformulations are an attractive strategy for improving ERT formulations, for which liposomes are an interesting approach.

Liposomal formulations for treating lysosomal storage disorders
Liposomes' potential as drug carriers in the context of LSD was recognized early.The first studies to attempt using liposomes to carry LSD enzymes were conducted in the 1970s-1990 s and involved loading several LSD enzymes, such as b-galactosidase, bglucosidase, b-glucuronidase, b-fructofuranosidase, and amannosidase [89][90][91][92][93][94][95][96].As expected, enzyme behavior and biodistribution differ when administered free or loaded into liposomal carriers.For example, varying tissue biodistribution in rats was observed for liposomes containing b-galactosidase [95], bfructofuranosidase [93], and a-mannosidase [92].These early studies showed that differing liposomal characteristics (e.g., surface charge, phospholipid composition, surface coating) lead to differing in vivo outcomes when the liposomes were administered to mice, e.g., the loading of b-glucuronidase in positively charged liposomes leads to more prolonged enzyme activity than when loaded into negatively charged liposomes [96], and the PEG coating of liposomes loaded with dextranase results in differing enzyme delivery and activity [94].Various liposomal surface coatings using antibodies (e.g., immunoglobulins such as IgM and IgG) or apoproteins (e.g., apolipoprotein E) were also explored in lysosomal disease cell models [89,90].
Each individual LSD affects a very low percentage of the population, which affects the current research efforts and is directly reflected in the low number of drugs currently in development.Indeed, an intelligence database search (Cortellis Drug Discovery Intelligence TM ) lists, at the time of this writing, only 1317 search results for the LSD group, compared to more than 200,000 for cancer and 60,516 for cardiovascular disease [97][98][99].For state-of-theart liposomal formulations in the drug development stage, there are approximately 975 search results for liposomes regarding drugs and biologics and more than 3000 results for liposomes regarding clinical studies worldwide, although fewer than half are for diseases other than cancer [100,101].
To the best of our knowledge, there are no liposomal drug products currently being evaluated in the clinical stages for any disease in the LSD group [102,103].Therefore, liposomes have not yet reached the clinical phase for testing in humans with LSD.Nevertheless, Table 1 lists a number of promising liposomal formulations in preclinical development for treating LSD.These liposomal formulations were selected after a search in the Web of Science and Cortellis Drug Discovery Intelligence TM databases for original scientific articles from the last 20 years; only 11 different liposomal formulations addressing LSD therapy were found.This review analyzed the 11 formulations from various perspectives such as treatment strategy, characterization techniques, and production methods.

Treatment strategies
The liposomal formulations under development operate under two distinct treatment strategies: 1) the ERT strategy, in which liposomes are used as nanocarriers to entrap and deliver the deficient enzyme and 2) the gene strategy, in which the delivered gene encodes for the deficient enzyme (GT) (Fig. 2B).Due to their strikingly distinct basis, these formulations deserve detailed discussion.

Liposomal formulations for enzyme replacement therapy
There are currently seven formulations that follow the strategy of entrapping lysosomal enzymes of interest inside a liposomal vehicle for intravenous infusion (Fig. 3).Thekkedath et al. [55] developed the first formulation by entrapping glucocerebrosidase (velaglucerase alfa, e.g., VPRIV TM ) in phosphatidylcholine-based liposomes as a method for improving the treatment of Gaucher disease, which is characterized by deficiency of the bglucocerebrosidase lysosomal enzyme, resulting in the accumulation of glucocerebroside substrate especially in macrophages from the liver, spleen, and bone marrow [104].The main treatment currently employed for Gaucher disease is ERT, which was approved in the early 1990s by the FDA and consists of intravenous administration of free glucocerebrosidase enzyme.ERT of type 1 Gaucher disease results in hematological, visceral, and skeletal improvements, e.g., decreased severity and frequency of bone pain and crises [105,106].In 2002, the EMA also approved the SRT miglustat, an enzyme inhibitor of glucosylceramide synthase, one of the initial enzymes involved in the reaction pathways that lead to glucosylceramide synthesis [70,104].However, these therapies for addressing Gaucher disease still share certain limitations.The recombinant glucocerebrosidase enzyme in ERT has a short half-life (9.8 min) and therefore requires a periodic injection of high doses (up to 60 units/kg), resulting in high treatment costs [107].Thekkedath et al. [55] studied the use of a liposomal system for the formulation of glucocerebrosidase enzyme to overcome the current ERT limitations (e.g., the need for high doses and associated side effects) by means of improved lysosomal targeting and glucocerebrosidase enzyme accumulation in the affected cells, because one of the critical aspects of ERT success is ensuring that the enzyme can reach the affected tissue.The authors' strategy was based on increasing the specific intracellular targeting to lysosomes as the therapeutic target by modifying liposomes with lysosomotropic octadecylrhodamine B (RhB), given that the authors' previous studies achieved an increase in the delivery of a liposome's model cargo to lysosomes in HeLa cells when using liposomes modified by lysosomotropic octadecyl-RhB [108].Glucocerebrosidase-loaded RhBmodified liposomes showed a neutral/slightly negative surface charge, and the presence of Rh-B induced higher enzyme loading.Thekkedath et al. evaluated the specific intracellular delivery into lysosomes in vitro using Gaucher fibroblasts and monocytederived macrophages, a representative cell model of Gaucher macrophages [55].The results showed an improvement in glucocerebrosidase-loaded liposomes in intralysosomal compartment localization of both cell lines, evaluated by flow cytometer and confocal microscopy (Fig. 3A).The authors concluded that although the results of these studies are still far from being of clinical use, the development of liposomes for delivering therapeutic enzymes into lysosomes has the potential to improve the treatment of LSDs and illustrates the importance of incorporating targeting modifications (e.g., lysosomotropic octadecyl-rhodamine B for lysosomal targeting).
Sun et al. [56] entrapped functional acid b-glucosidase (GCase) in nanovesicles based on saposin C (SapC) and dioleoylphosphatidylserine (DOPS) to improve the treatment of neuronopathic Gaucher disease, which differs from Gaucher disease type I in that the former presents several primary brain conditions and central nervous system involvement [104].None of the approved ERTs for Gaucher disease result in a reduction in the signs and symptoms in the central nervous system and brain, mainly due to the failure of conventional ERT to cross the BBB.In fact, numerous drugs (e.g., therapeutic enzymes) currently used for treating central nervous system disorders fail due to their poor penetration of the BBB [109,110], which acts as a very selective permeable barrier, regulating the entrance of substances (e.g., nutrients) but restricting penetration by most drugs (e.g., large molecular weight proteins such as therapeutic enzymes), limiting the treatment of neurological LSD symptoms [84,111].Sun et al. [56] proposed the formulation of rh-GCase enzyme in SapC and DOPS-based liposomes to cross the BBB and provide a vehicle for delivering the enzyme into the central nervous system.SapC is a small lysosomal glycoprotein that physiologically acts as an optimizer of GCase activity [112]; therefore, its addition to the liposomal composition can provide enhanced GCase enzyme activity and brain targeting.The cellular uptake and in vitro efficacy of this formulation were studied in mouse fibroblasts derived from GCase knockout (KO) mice.Moreover, the in vivo tissue biodistribution of GCaseloaded nanovesicles was evaluated in a neuronopathic Gaucher mouse model.GCase protein was detected by immunolabeling techniques and enzyme activity in brain tissue (Fig. 3B).The results from these studies indicated that GCase-loaded nanovesicles colocalize with inflammatory cells from the brain, suggesting a new mechanism for targeting the central nervous system by a phosphatidylserine-mediated and lymphatic circulation system.Overall, the authors demonstrated the successful transport of rh-GCase into neuropathic Gaucher-like mice brains when rh-GCase was formulated in a liposomal vehicle, resulting in therapeutic effects on the brain and lymph nodes and illustrating the potential ability of liposomes to overcome some of the limitations of current ERT.
A few years later, Aldosari et al. [57] entrapped the recombinant human acid sphingomyelinase (rh-ASM) in liposomes to improve the treatment of Niemann-Pick disease type B. This disease is characterized by an absence (in type A disease) or a marked decrease (in type B) of ASM enzyme activity, resulting in progressive accumulation of non-degraded lysosomal sphingomyelin in almost every cell type and with special involvement of the spleen and lungs [113].Unfortunately, there is currently no effective treatment for this condition.Unlike a number of the previously presented LSDs, the ERT for this LSD is based on administering free rh-ASM and is still in phase II/III clinical trials (NCT02004691) [114].Preclinical studies in animal models have also found that the direct intravenous administration of rh-ASM can present potential toxicity due to the narrow therapeutic window associated with elevated ceramide release [114,115].Aldosari et al. [57] proposed the formulation of rh-ASM in bis(monoacylgly cerol)phosphate (BMP)-based liposomes to prevent the systemic off-target degradation of sphingomyelin in the circulation, prevent toxicity signs and expand the treatment's therapeutic window.The ASM-loaded liposomes showed a negative surface charge, due to the incorporation of BMP lipids as membrane components.The cellular uptake was studied in vitro using wild-type and Niemann-Pick type B (NPD-B) fibroblasts and macrophage-like cell lines.
The efficacy in sphingomyelin degradation was also evaluated in NPD-B fibroblasts.The results of these in vitro studies indicated that rh-ASM was more effective in reducing accumulated sphingomyelin when formulated in liposomes than as free enzyme.
Although these results are clearly far from being used in real clinical applications, they suggest that ASM-loaded liposomes might provide a safer profile than the free enzyme, given that the liposomal carrier might prevent systemic sphingomyelin degradation (Fig. 3C).
Hamill et al. [58,59] encapsulated recombinant human IDUA enzyme in guanidinylated neomycin (GNeo)-targeted liposomes to develop an ERT for mucopolysaccharidosis type I (MPS I) disease, which is characterized by IDUA enzyme deficiency, resulting in the lysosomal accumulation of non-degraded glycosaminoglycans [116].Currently, there are two approved treatments available for MPS I: 1) an ERT, approved in 2003 by the EMA and FDA and con-  -E) or the free enzyme (VPRIV TM ) in Gaucher's fibroblasts, expressed as a % increase in mean fluorescent intensity (MFI) normalized to control cells.Adapted from [55], with permission.(B) Successful transport of rh-GCase when associated to saposin C-modified liposomes in brain cells of Gaucher-like mice.GCase protein (green) detected in astrocytes, neurons and microglia (red).Adapted from [56], with permission.(C) Better prevention of sphingomyelin degradation (expressed as % reduction in sphingomyelin conversion relative to free rh-ASM) when rh-ASM is loaded in BMP-based liposomes versus free.Adapted from [57], with permission.(D) Uptake and lysosomal delivery increase of rh-IDUA in IDUA-deficient MPS I fibroblasts when loaded in GNeo-modified liposomes (GNeo-Lip) compared to unmodified liposomes (Lip), measured by IDUA enzymatic activity of lysate cells.Adapted from [58], with permission.(E) rh-GAA immunogenicity reduction when the enzyme is loaded in PI-based liposomes versus free.Anti-rh-GAA antibodies in plasma were measured in GAA-KO mice (20 mg/kg), after 4 weeks of intravenous injections.Adapted from [60], with permission.(F) Major efficacy in the Gb3 reduction when the enzyme is loaded in RGD-liposomes than when it is free, evaluated in vitro in primary endothelial cells derived from Fabry KO mice.Adapted from [62], with permission.(G) Better localization of liposomes functionalized with the H16 targeting peptide (H16-Lipo) in lysosomes after cellular uptake.Adapted from [64], with permission.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)sisting of the intravenous administration of rh-IDUA enzyme and 2) an HSCT.However, these therapies also had several limitations, such as limited effect in neurological symptoms with ERT and poor efficacy in treating bone deformities and cognitive defects with HSCT therapy [117,118].Hamill et al. [58,59] studied the use of liposomes functionalized with GNeo derivatives (termed GNeosomes), which are molecular transporters that have previously been shown to target the lysosomes through heparan sulfate pathways [119].The IDUA-loaded GNeosomes showed an overall positive surface charge due to the nature of GNeo.Cell uptake, lysosomal targeting, and intracellular delivery of the IDUA enzyme into lysosomes were assessed in Chinese hamster ovary cells and MPS I fibroblasts, resulting in a significant increase in the uptake and lysosomal delivery of the IDUA enzyme when loaded into GNeosomes compared with unmodified liposomes (Fig. 3D).These findings highlight the importance of adding specific targeting moieties to the liposome, not only to enhance cell uptake but also to increase their delivery to well-defined intracellular compartments such as lysosomes, an important feature for more efficient and specific drug delivery.
More recently, Schneider et al. [60] entrapped recombinant human acid GAA (rh-GAA) in phosphatidylinositol-based liposomes to improve the treatment of Pompe disease, which is characterized by decreased GAA enzyme activity, leading to the accumulation of non-degraded lysosomal glycogen [120].The current main treatment for Pompe disease is ERT consisting of intravenous administration of free rh-GAA enzyme.This treatment received marketing approval in 2006 by the FDA, and resulted in several benefits for patients with Pompe disease, such as a longer survival rates and remarkable improvements in cardiac function [121].However, ERT with free rh-GAA enzyme presented drawbacks regarding the formation of anti-rh-GAA antibodies, pharmacokinetic issues, and poor targeting of skeletal muscles, leading to a general decrease in the efficacy over time [122].Schneider et al. [60] studied the use of a liposomal system for the GAA enzyme formulation to overcome some of the abovementioned limitations.The entrapment of GAA into liposomes could protect the enzyme from rapid degradation, improving their PK profile and reducing the formation of anti-GAA antibodies, compared with standard ERT.GAA-loaded liposomes showed a negative surface charge due to the use of phosphatidylinositol as a membrane component.The pharmacokinetic behavior of GAA-loaded liposomes was evaluated in GAA KO mice.Additionally, the glycogen reduction efficacy and the production of anti-GAA antibodies were also studied, the results of which showed a slight improvement in reducing rh-GAA immunogenicity when the enzyme is formulated with the liposomal vehicle (Fig. 3E), although the improvement in PK parameters and efficacy were minimal.The authors indicated that these results could be improved by increasing the association efficiency between GAA and the liposome, which in this study was low.
The next system refers to the entrapment of recombinant human GLA (rh-GLA) enzyme in peptide-targeted liposomes to improve the treatment of Fabry disease [61][62][63], which is characterized by deficient GLA enzyme activity, resulting in the lysosomal accumulation of neutral glycosphingolipids (mainly Gb3) with multisystemic organ involvement (e.g., kidneys, heart, and central nervous system) [123].The main treatment is currently ERT, in which free rh-GLA enzyme is administered intravenously.There are two enzymes currently approved for ERT in Fabry disease: Fabrazyme Ò and Replagal Ò , which have been available since 2001 [79,124].Although both compounds reduce Gb3 accumulation in tissues, neither treatment seems to completely reverse the disease, especially in advanced stages.Both treatments present several drawbacks including poor biodistribution, low stability, limited efficacy, high immunogenicity, and low capacity to cross biological barriers, such as cell membranes and the BBB [79,80].In this context, we developed a liposomal system containing an rh-GLA, within the framework of a multidisciplinary project (Smart-4-Fabry, European Commission H2020, ID 720942) [61][62][63]125].The GLA-loaded liposomes (''nanoGLA") contain a targeting peptide (i.e., c-RGDfk) linked to the cholesterol moiety through a PEG chain to favor the recognition of a v b 3 -integrins, expressed in injured endothelial cells [126].These GLA-loaded liposomes are produced by DELOS-susp, a compressed CO 2 -based technology patented and owned by Nanomol Technologies SL [127,128].DELOS is a robust and green nanoformulation production platform that will be explained in greater detail later in this review.The physicochemical characteristics, critical quality attributes (CQA), enzyme entrapment efficiency, enzyme activity, and biocompatibility of GLAloaded liposomes were assessed in vitro, as was their in vivo efficacy in reducing Gb3 deposits in GLA KO mice [61][62][63].The results were promising in terms of the excellent physicochemical properties, bioactivity (Fig. 3F), and high entrapment efficiency.The in vivo results also showed a positive behavior for nanoGLA when compared with the free enzyme, given that GLA-loaded liposomes induced higher Gb3 reduction in mice tissues.These results helped obtain the orphan medicinal product designation (EU/3/20/2396) [61] by the EMA for treating Fabry disease, demonstrating the strong potential of targeted liposomal systems for drug delivery applications.
Hayashi et al. [64] developed polyhistidine peptide-modified liposomes to entrap rh-GLA, as a model of lysosomal disorders such as Fabry disease, which is characterized by the lack of GLA.The authors entrapped rh-GLA in neutral DOPC/cholesterol liposomes functionalized with a polyhistidine peptide, consisting of a lineal chain of 16 histidines (i.e., H16 peptide: HHHHHHHHHHHHHHHH-NH 2 ).H16 is known to act as a cellpenetrating peptide against mammalian cells [129].The cellular uptake of H16-modified liposomes and their co-localization in cell organelles were examined in human fibrosarcoma (i.e., HT1080) cells, the results of which showed that the H16-modified liposomes were internalized into cells and were localized in endogenous lysosomes (Fig. 3G), even when loaded with GLA.The ERT efficiency of H16-modified GLA-loaded liposomes was assessed in an LSD-like cell line (an in-house GLA-KO cell line) characterized by the inhibition of endogenous GLA protein expression though small interfering RNA to silence the gla gene.The ERT efficiency of the H16-modified liposomes was evaluated by measuring cell proliferation recovery, given that GLA-KO cells have been reported to slow cell proliferation relative to normal cells [130].The authors observed increased cell proliferation when using the H16-modified GLA-loaded liposomes, indicating the liposomes' successful delivery of GLA to the lysosomes to restore enzymatic activity.The authors demonstrated the successful transport of rh-GLA in a GLA-KO cell culture model, resulting in increased cell proliferation relative to normal cells.However, the authors indicated that an increase in enzyme encapsulation in the H16-modified liposomes is required for their practical use in the future, given that the GLA concentration in the H16-modified liposomes used in the study (4.21 lunits/nmol of lipid) was too low and did not fully restore the cell proliferation levels of GLA-KO cells in vitro.Additionally, more representative biomarkers of Fabry disease should be considered in addition to cell proliferation, such as quantification of Gb3 levels, GLA enzymatic activity, and gene expression.

Liposomal formulations for gene therapy
In addition to the use of liposomes to entrap the deficient LSD enzyme, liposomes could also be used to entrap genetic material with the genetic information to be further translated to the desired protein by the cell machinery.Currently, four liposomal formula-tions have been developed to load nucleic acid derivatives for LSD GT (Fig. 4).
The first example was from Yamaguchi et al. [65] who encapsulated plasmid DNA in liposomes to develop a GT for Sandhoff disease, a gangliosidosis family disorder characterized by mutations in the HEXB gene encoding the subunits of lysosomal hexosaminidases B, resulting in the accumulation of glycosphingolipids, such as GM2 ganglioside, inside cell lysosomes, causing severe neurodegenerative impairment [131,132].There is current no approved treatment for Sandhoff disease, especially given the challenge of getting therapeutic drugs to cross the BBB [133].Yamaguchi et al. conducted a study based on a GT approach by conjugating a b-hexosaminidase gene plasmid in commercial cationic liposomes.The expression levels of the b-hexosaminidase gene were evaluated after a single administration to Sandhoff disease mice (i.e., KO for HEXB gene), the results of which showed higher enzymatic activity in several organs (e.g., in liver Fig. 4A), although this increase was not observed in the brain, probably due to the lack of a targeting unit in the liposomal composition.This early study (2003) served as a proof of concept for the use of GT based on plasmid-conjugated liposomes for application in LSDs, taking into account the use of a commercial liposomal vehicle and not the full development of the formulation.
Zhang et al. [66] encapsulated plasmid DNA in liposomes to develop a GT for mucopolysaccharidosis type VII (MPS VII) disease (also called Sly syndrome), which is considered one of the rarest types of MPS and is characterized by b-glucuronidase enzyme deficiency, leading to non-degraded glycosaminoglycan accumulation [134].Currently, the only treatment available for MPS VII is ERT consisting of the intravenous administration of rh-bglucuronidase enzyme, which was approved by the FDA in 2017 [135].As an alternative approach, Zhang et al. [66] proposed a GT based on the entrapment of GUSB plasmid DNA into PEGylated liposomes targeted with monoclonal antibodies against the transferrin receptor (TfR).In a previous study, the authors developed these liposomal vehicles containing PEG 2k and a monoclonal antibody against TfR [136].This targeting aimed to transport the lipo-somes across the BBB.Liposomes were loaded with the GUSBplasmid DNA encoding for b-glucuronidase enzyme.The GUSB enzymatic activity was evaluated in vivo in various organs, including the brain, after a single-dose administration in GUSB KO mice.The results demonstrated that therapeutic levels of GUSB activity were achieved in several organs such as the brain (Fig. 4B), demonstrating again the potential of liposomes to overcome biological barriers (as in this case, the BBB) and improve some of the issues of conventional treatments.
Schuh et al. [67,68] encapsulated CRISPR/Cas9 plasmid in liposomes to develop gene editing-based therapy for mucopolysaccharidosis type I (MPS I) disease, which is characterized by IDUA enzyme deficiency, as previously explained (see Section 3.1.1).GT is one of the most promising therapies for overcoming the limitations of currently approved treatments (ERT and HSCT) because GT would permanently provide sufficient levels of the missing enzyme.A novel tool in GT is the CRISPR/Cas platform (i.e., clustered regularly interspaced short palindromic repeats (CRISPR) Cas-associated nucleases) for genome editing [137].Briefly, this technique consists of using an RNA guide to insert an additional sequence into the desired gene.The Cas9 nuclease then detects and cleaves the target DNA at a specific point.A homologous sequence, which is provided along with the system (i.e., donor sequence), could then be inserted into the cell's genome [78].Schuh et al. [67] proposed MPS I gene editing via the CRISP/Cas9 system to restore cellular IDUA production and enable glycosaminoglycan degradation by using a liposomal carrier complexed with the CRISPR/Cas9 system.CRISPR-loaded liposomes showed an overall positive surface charge due to the use of a cationic phospholipid (DOTAP) for the complexation of the negative nucleic acid.The liposomes also contained PEG2k to provide stability and prevent aggregation.The activity and efficacy of CRISPRloaded liposomes were evaluated in vitro using fibroblast cells from patients with MPS I, while the direct injection of CRISPRloaded liposomes was studied in an MPS I murine model (i.e., IDUA KO mice).The biodistribution, IDUA activity, and glycosaminoglycan accumulation levels were analyzed, showing promising results, given that the use of CRISPR-loaded liposomes led to increased in vitro IDUA enzyme activity and a reduction in glycosaminoglycan levels in the patients' fibroblasts.The in vivo results also demonstrated effective transfection of cells and long-lasting production of active IDUA enzyme [67], as well as improvements in bone, heart, and lung disease (Fig. 4C) [68].This case is another example of the benefits of using liposomes as non-viral vectors for use in LSDs.
Jiang et al. [69] encapsulated a plasmid DNA in liposomes to develop a GT for Niemann-Pick type C1 disease (NPC1), which is characterized by mutations in the gene encoding the NPC1 intracellular membrane transporter of non-esterified cholesterol, leading to unesterified cholesterol and glycosphingolipids accumulation in the form of inclusion bodies inside cells, with particular involvement of the liver, spleen, and brain [138].There is currently no effective treatment for this disease.In 2009, the EMA approved miglustat (previously approved in 2002 for Gaucher disease) as supporting and symptomatic therapy for NPC1 [139].Unfortunately, the classical ERT with the recombinant NPC1 protein was not feasible due to its insolubility given that it is a membrane protein [138].Therefore, there have been major efforts to develop an effective and secure GT for NPC1, with a number of viral-vector approaches currently in preclinical phases.Jiang et al. [69] proposed entrapping NPC1 plasmid DNA into PEGylated liposomes targeted with monoclonal antibodies against TfR, a liposomal vehicle previously described by Zhang et al. [66,136].In this case, however, the liposomes were aimed at treating a different LSD.The liposomal vehicle contained a monoclonal antibody against TfR to target BBB penetration, and the liposomes were loaded with the NPC1-plasmid DNA.The delivery of the plasmid DNA and NPC1 mRNA in certain target organs (brain, spleen, and liver) was evaluated in vivo after repeated doses in NPC1 KO mice.The results were promising, showing a reduction in inclusion body accumulation in certain organs (Fig. 4D) and demonstrating the proper delivery of plasmid DNA into target organs, such as brain.The mice's lifespan was not prolonged, but the authors attribute this finding to the disease stage at the start of therapy.This example illustrated the approach of using liposomes as vehicles for GT.Liposomes could present several benefits over viral vectors, such as no limitation in the nucleic acid size for packing and less immunogenicity [137].

Summary of treatment strategies based on liposomal formulations for LSD
The loading of lysosomal enzymes or genes in liposomes can overcome some limitations of current treatments, providing specific cell/organ targeting (e.g., allowing BBB crossing) and offering protection of the active ingredient from degradation, reduction of immunogenicity, and cell uptake improvement.Liposomes are also attractive nanocarriers due to their biocompatibility and biodegradability, and because of the possibility of large-scale liposomes production.However, insufficient drug loading to achieve therapeutic doses for treating LSD, as well as instability in storage, especially for those liposomes presenting neutral charge on the surface, are some issues to overcome for the developers that ultimately limit the clinical translation.
Potential concerns of the use of liposomes and other nanocarriers for such chronic condition deserve further investigation.For example, it has been reported that intracellular delivery of lipids, polymers or other material from carrier formulations could render side effects due to the accumulation of these foreign materials in the dysfunctional lysosomes, which are unable to degrade them [149].Lysosomal processing defects can produce cell and tissue damage, as in LSDs (e.g., endocytic and trafficking alterations due to this storage which aggravate disease and hinder treatment).Therefore, understanding the role of the lysosome in regulating endocytosis will improve therapeutic efficacy and decrease the toxicity of future drug delivery systems, including not only those aimed at treating LSDs, but also other diseases requiring cellular internalization mediated by endocytosis.

Techniques for preparing liposomal formulations
To successfully advance to the clinical stage and manufacture the drug product consistently at an industrial scale, the production process needs to allow for batch-to-batch reproducibility, involving a minimum number of steps and equipment and fulfilling the requirements of the pharmaceutical industry's good manufacturing practices (GMPs) [150,151].The use of manufacturing methods compatible with a fast and robust scale-up, from the beginning of a nanoformulation preclinical development, facilitates the further translation from the bench-scale to the preclinical, clinical, and commercial scale.Otherwise, adapting laboratory experimental methods to a larger-scale production will be one of the main limitations for developing new nanopharmaceuticals [152,153].
The main preparation methods used for liposomal formulations intended to treat LSDs are compiled in Table 1 and combine conventional and advanced techniques.Usually, multistep processes are needed to manufacture liposomal formulations, especially when using conventional methods.The first step consists of the liposome formation, usually followed by homogenization, sizereduction, and/or purification.The process of loading the active substance (i.e., the enzyme or gene) into the liposomal carrier, which can be performed during the liposome formation step or in a post-formation step, is a critical issue that novel techniques are trying to improve to obtain high entrapment efficiencies without compromising the stability of the liposomal formulation [154].Recently, authors such as Lombardo et al. [155], Andra et al. [156], and Leitgeb et al. [154] have reviewed conventional and novel techniques to produce liposomes.
In multistep manufacturing techniques, it is important to define when and at what stage the drug is incorporated into the system.There are two major loading strategies: 1) ''active loading", where the drug molecule is incorporated after liposome preparation, through incubation techniques driven by a transmembrane gradient involving buffer or ammonium sulfate salts and usually including a lyophilization or freeze-thaw process and 2) ''passive loading", such as mechanical dispersion, solvent dispersion, and detergent removal methods, where lipid films are first deposited on a substrate and subsequently hydrated to yield liposomes [157].The use of active loading approaches generally shows higher entrapment efficiencies than with passive methods.
Among the methods for integrating drug substances into the liposomal formulations for LSD shown in Table 1, passive loading is employed for 1) Gaucher disease, 2) neuronopathic Gaucher disease, 3) Niemann-Pick B disease, 4) MPS I, 6) Fabry and 7) Fabry disease treatment; incubation during liposome post-production is applied in 5) Pompe disease, 8) GM2 -Sandhoff disease, and 10) MPS I; and the freeze-thaw technique is used for 9) MPS VII and 11) Niemann-Pick C1 disease.

Preparation of liposomes by conventional methods
Conventional liposome production methods are based on dissolving lipidic membrane components in an organic solvent that is then removed, and the dried lipid is then hydrated with an aqueous phase.These methods usually involve working under harsh conditions (e.g., process temperatures above 35 °C, high shears, and the use of other direct energy supply like ultrasonication) and complex procedures, leading to poor entrapment efficiency [155,156].Most of the methods tend to show difficulties in precisely controlling the self-assembling of the molecules constituting the liposomal system, leading to nanomaterials with high structural heterogeneity [3,158].These conventional methods tend to require large amounts of organic solvents, with associated toxic residue in many cases, which can be difficult to remove not only at the laboratory level but also in large-scale production [159].Some of the most widely used conventional techniques for producing liposomes are listed below.
3.2.1.1.Thin-film hydration method.The thin-film hydration technique (TFH) or Bangham method consists of dissolving lipid components in organic solvents, obtaining a thin film by evaporating the organic solvent, followed lastly by rehydration of the thin film in an aqueous media.With this method, lipophilic molecules can be loaded by dissolving them prior to the thin-film formation; the hydrophilic molecules can be loaded into the liposome in the rehydration phase.A size and/or lamellarity reduction step is commonly applied in this process [160,161].The TFH technique followed by a post-formation process (e.g., manual extrusion, sonication) is the most widely used methodology for preparing liposomal formulations designed to treat 1) Gaucher disease, 3) Niemann-Pick B disease, 4) MPS-I, 5) Pompe disease, 7) Fabry disease, 9) MPS VII, 10) MPS I, and 11) Niemann-Pick C1 disease (Table 1).

Detergent removal (by dilution or dialysis).
The detergent removal (depletion) method consists of dissolving lipid micelles in an aqueous phase with the help of a detergent agent (e.g., sodium cholate).The methodology and fundamentals of lipid self-assembly have been previously described [162,163].With this technique, high homogeneity in particle size has been reported, depending on the detergent removal rate and lipid/detergent ratio.However, the technique's use in treating LSDs is likely restricted by the post-process residual detergent and the inconvenience of removing the organic solvent.
3.2.1.3.Solvent injection method.Solvent injection methods are based on dissolving membrane components in ethanol (or ether) and injecting them into an aqueous phase.Process parameters such as solvent concentration, flow rates for both phases, stirring, and lipid concentration are critical for this technique [164,165], which has been employed for encapsulating lipophilic and hydrophilic active compounds.The ethanol injection method is one of the preferred techniques for producing small unilamellar liposomes simply and rapidly due to its scalability potential.The pharmaceutical industry has led the development of several modifications to this technique to improve the productivity, physical properties, and uniformity of liposomes and to create a convenient and practical method that can be upscaled to an industrial level.Despite the advantages of this technique, it has not been used to produce liposomal formulations to treat LSDs.The need for large quantities of hydrophilic active compounds for acceptable entrapment efficiency and the possible degradation of certain bioactive compounds in ethanol could explain the lack of its application for treating LSD.
3.2.1.4.Reverse-phase evaporation.In the reverse-phase evaporation method, lipids are dissolved in organic solvent mixtures favoring the formation of inverted micelles.A certain quantity of aqueous media is then added, causing rearrangement of lipids between the water-oil interface, creating a water-in-oil microemulsion, which can be emulsified to obtain a homogeneous dispersion.The organic solvent is then removed by rotary evaporation, promoting the formation of large unilamellar vesicles.The large amount of aqueous phase integrated by the microemulsions increases the encapsulation of macromolecules within the liposomes.This technique is not being developed for treating LSDs, probably due to the difficulty in removing the residual solvent, the complexity of its scale-up, and the incompatibility of biomolecules such as proteins and peptides with the organic solvents employed [165,166].

Post-processing of liposomes
Particle size, homogeneity, and lamellarity are critical quality attributes of liposomal formulations dedicated to biomedical applications; controlling these attributes during manufacturing is therefore critically important [154][155][156].The low particle-to-particle homogeneity obtained by all conventional liposome formation techniques, especially in terms of size and lamellarity, is usually improved by additional post-formation processes (e.g., extrusion, sonication, and high-pressure homogenization) to achieve better vesicle-to-vesicle structural homogeneity [3].

Extrusion.
Liposome extrusion in several passes through membranes with pore sizes ranging from 25 nm to 1 mm is a common method for obtaining highly reproducible results, achieving liposome sizes according to the pore size being employed [167].The main disadvantage of this method is sensitive product loss, which is a limitation for large-scale productions.Liposome extrusion has been the preferred post-processing method used in most of the liposomal formulations listed in Table 1, including 1) Gaucher disease, 2) neuronopathic Gaucher disease, 3) Niemann-Pick B disease, 4) MPS I, 7) Fabry disease, 9) MPS VII, and 11) Niemann-Pick C1 disease.For the liposomal formulation 5) intended for Pompe disease, a modified method based on highpressure extrusion was used.
3.2.2.2.Sonication.Sonication is usually applied as a postprocessing method to downsize and standardize an existing liposome dispersion but can also be used as a starting point for preparing liposomes, mainly at the laboratory scale.This method is based on generating cavitations in the MLV liposome solution produced by applying an ultrasonic input.Depending on how this high energy is applied, it can be differentiated between bath sonication and probe sonication techniques [168].In the probe approach, a sonicator tip is introduced into the liposomal solution placed in a cold bath to prevent degradation that can be provoked by local overheating.Using this method, metal (titanium) particles can be released from the tip.In the bath technique, often used for large volumes of lipid solution, the liposome dispersion vessel is placed in an inert and temperature-controlled atmosphere [169].Despite this technique's disadvantages, such as low encapsulation efficiency, lipid degradation and high polydispersity in the size of the obtained liposomes, this approach is one of the most widely used for SUV formation.It is employed to obtain primary liposomes for 2) neuronopathic Gaucher disease (Table 1) and as a post-formation process in 7) Fabry disease and 10) MPS I.

3.2.2.3.
High-pressure homogenization.The homogenization method consists of the continuous injection of a multilamellar liposome dispersion through an orifice at constant high pressure.The stream collides with a stainless-steel wall, downsizing the liposomes impacting on it.The main homogenization mechanisms that occur are cavitation and shearing during laminar and turbulent flow.Pressure and the number of passages are key factors for this homogenization method to obtain high homogenous dispersions [170].However, the use of high operating pressures and metal contamination are the main drawbacks [170].

Microfluidization.
Microfluidizer technology uses a fixedgeometry, high-shear zone and constant pressure pumps.This combination enables the microfluidizer processor to achieve a constant process pressure, ensuring that every microliter of product receives the same treatment, resulting in a more homogeneous and reduced size using fewer passages than with the highpressure homogenization method.Liposomes can be previously manufactured by adding powdered lipids to aqueous buffers without organic solvents and then homogenized with the microfluidizer processor.As with other techniques, further purification and/or loading steps are still necessary.Lipid concentrations of up to 80 mg/mL can be processed using this manufacturing method [171].This technique has been used in preparing liposomes for 10) MPS I treatment and in combination with sonication after TFH (Table 1).
3.2.2.5.Freeze-drying and dehydration-rehydration methods.The freeze-drying method consists of freezing the aqueous solution containing the liposome formulation and then removing the ice by sublimation [172], a method used to overcome certain drawbacks such as leakage of active compounds during storage or degradation caused by oxidation or other chemical reactions.The method has also been employed to entrap active substances (e.g., small molecule drugs and enzymes).The freeze-drying technique is related to the dehydration-rehydration methodology to obtain dried reconstituted vesicles (DRVs), which present high entrapment capacity due to the fact that preformed small unilamellar vesicles are disrupted during the freeze-drying cycle in the presence of the active substance destined for entrapment.Subsequently and during controlled rehydration and in the presence of the active substance to be encapsulated, vesicles fuse into large oligolamellar (or multilamellar) vesicles, entrapping large amounts of the active substance.The fact that the DRV technique involves vesicle formation under mild conditions (e.g., conditions that do not cause decomposition or loss of activity of active substances) makes this technique the method of choice for preparing liposomal formulations of sensitive active substances such as peptides, proteins, and enzymes [173,174].For LSD, DRV is applied to produce primary liposomes in 3) Niemann-Pick B disease and to entrap the rh-IDUA enzyme in 4) MPS I formulations (Table 1).
3.2.2.6.Purification of liposomal formulations.Regardless of the preparation technique employed, the non-encapsulated compounds and residual organic solvents need to be removed from the final nanoformulation through a purification process.The most common methods are centrifugation (for purification or concentration, taking the pellet and discarding the supernatant with nonloaded substances) [175], ultracentrifugation (applying the same principle but for liposomes smaller than 100 nm [176], chromatography (size exclusion, gel permeation, or ion exchange), dialysis (using the same type of membrane as the extrusion method for removing most liposomes or substances smaller than a predetermined size) [177,178], and tangential flow filtration (in which the membranes' pore size and the shear applied to the loadedliposomal system can be controlled for easy scaling-up of the manufacturing process of purification and/or concentration) [62,63].Among the post-processing techniques in liposomes for LSD (Table 1), ultracentrifugation is used as a post-formation process in 2) neuronopathic Gaucher disease, 3) Niemann-Pick B disease and 7) Fabry disease; the dialysis method is used in 11) Niemann-Pick C1 disease; size exclusion chromatography in 4) MPS I; and tangential flow filtration is used in 6) Fabry case.

Novel methods for liposomes preparation
The development of new methodologies for processing nanoliposomes with greater control of molecular self-assembling, while being environmentally respectful, has gained significant interest in the field [179][180][181].The main drawbacks of conventional liposome formation approaches include the difficulty in achieving an easily scalable process for industrial production and in achieving high drug encapsulation efficiency.Conventional methods might not be suitable for the processing of many biomolecules, given that they use detergents, organic solvents, and high-shear homogenization processes, which might drastically affect the clinical applications.The novel methodologies being developed to produce liposomal formulations should allow for the preparation of these supramolecular nanostructures in a well-controlled manner and the obtainment of systems with well-established characteristics including multifunctionality.
3.2.3.1.Microfluidics.Microfluidic methods are based on injecting a lipid solution into an organic solvent (e.g., ethanol, isopropanol) between two aqueous streams in a microfluidic channel (5-500 lm section), creating a laminar flow and a diffusive mixing regime of the two phases, favoring lipid self-assembly into vesicles.By controlling the flow rates and mixing, the technique allows for the production of small and monodisperse nanoformulations with controlled sizes and distribution [182].Comparing these microfluidic methods to conventional procedures, the final liposomal formulation does not require post-formation processing and can be manufactured in continuous mode with compact and small equipment, although large-scale production capability is limited due to the particle size (mixing geometry and flow dependency).The current scale-up difficulty has been solved by a scale-out approach, i.e., increasing production capacity by increasing the equipment units, instead of scaling-up the equipment itself.New microfluidic methods have been developed for the formation of liposome nanoformulations for biomedical applications.The microfluidic hydrodynamic focusing method [183] can produce homogeneous (40-140 nm) SUVs and large unilamellar vesicles with excellent control of the flow and mixing conditions.In the microfluidic droplet method, two immiscible phases (such as water and oil) are forced to flow (under specific conditions) into a microchannel and generate small droplets of one phase, allowing the formation of giant (4-20 lm) liposomes.The microfluidic jetting method consists of drying the solution containing phospholipid in microtubes and hydrating the formed lipid film in a perfusion process that forms giant (200-500 lm) vesicles of uniform size with high encapsulation efficiency.These methods have been reviewed by Carugo et al. [184] and Funakoshi et al. [185].Recent innovations in the microfluidic technique include continuous flow liposome formation [186], but none of these techniques have been used to prepare liposomal formulations specifically for LSD.

Compressed fluid-based technologies.
As previously stated, conventional methods for preparing liposomes have certain shortcomings, such as poor monodispersion, poor stability, high residual organic solvent, and side effects [187].In addition, the organic solvents used for conventional liposome preparation can cause environmental pollution, can become toxic and can degrade the active ingredient, presenting a possible risk for human health.In the case of protein drug substances, the detergents and organic solvents employed can cause denaturation and affect the membrane properties.
Compressed fluid-based preparation processes have been considered an interesting alternative to conventional methods for producing vesicles, such as liposomes, in a sustainable, environmentally friendly manner.The use of compressed fluids (also known as dense gases) can overcome some of the concerns of conventional production methods, given that they are less toxic, more ambivalent, and economically and environmentally more friendly [188].Among compressed fluids, CO 2 is the most widely used, especially in the pharmaceutical industry because CO 2 is considered a safe solvent by the FDA, and their critical parameters (critical temp, 31.1 °C; critical pressure, 72.8 atm) make it easily accessible and user friendly when working with labile bioactive compounds, such as enzymes, proteins, and other biomolecules [159,189].
Several CO 2 -based methodologies are currently implemented to produce DDSs with better homogeneity in terms of nanoparticle size distribution, decreased number of process steps, and potential reduction in the amount of organic solvent required by conventional methods.Moreover, compressed fluid-based methodologies can provide sterile and soft operating conditions and the potential for transferring to larger-scale productions under GMP conditions [159,188,190].Liposomes produced with compressed CO 2 -based methods have enhanced intactness, sphericity and uniformity compared with liposomes produced by the TFH method [191].Compressed CO 2 enables the processing of phospholipid aggregates into nano/microparticles.Their characteristics can be controlled via tuning critical process parameters such as decompression rate and the nozzle's opening diameter [154].
Among the liposomal formulations for treating LSD, only the DELOS-susp method has been used in 6) Fabry disease.In this approach, the method incorporates the therapeutic enzyme during the formation of the liposomal self-assembled structure, thereby obtaining drug-loaded liposomes in a single step [61][62][63].This process has shown high batch-to-batch consistency and easy scalability, which are essential for clinical translation [128,153], although it has not yet been applied to a marketed product.

Summary of techniques for manufacturing liposomal formulations for LSD treatment
The preparation of loaded liposomes in multiple-step processes, such as TFH, usually leads to low API entrapment efficiency.In addition, the need for several freeze-thaw cycles or long incubations (several hours) of the liposomal formulation with the API to improve the loading capacity entails a significant increase in production time.However, compressed CO 2 -based techniques have reported the preparation of loaded liposomes in a single step, achieving enzyme entrapment efficiencies of over 90 % in the case of the DELOS-susp method.From the environmental point of view, conventional and compressed fluid-based approaches present a major difference: while conventional techniques such as TFH and DRV use halogenated organic solvents (e.g., chloroform and dichloromethane) to produce liposomes, novel methods such as compressed CO 2 -based techniques uses mainly ethanol and/or other class 3 organic solvents at low concentrations recommended by regulatory agencies [198].
In terms of the equipment needed to produce loaded liposomes, conventional methods depend on equipment such extruders, rotary evaporators, and bath sonicators, which are usually difficult to scale-up.On the other hand, compressed CO 2 -based methodologies (e.g., DELOS-susp platform) are based on equipments that are easy to scale-up, such high-pressure autoclaves.Among purification methods, tangential flow filtration is one of the current methods which is easily available at the industrial scale for pharmaceutical and biotechnological manufacturing.Although conventional methods for producing liposomes for LSD can be suitable at small scales (e.g., mL scale), they present certain limitations that complicate their industrialization.
An optimized production, together with an improved biodistribution and increased efficacy of the treatment might imply the use of less amount of API to obtain the same or better therapeutic effect.This would lead to cost-effective treatments, having a direct impact on the life-quality of patients and on the sustainability of health-care systems.

Techniques for physicochemically characterizing liposomal formulations
The importance of having well-established production methods and analytical techniques that accurately characterize critical formulation attributes that affect the safety and efficacy profile of the formulation are of utmost importance for enabling the translation from basic research to clinical applications.Unfortunately, less than 10 % of basic research reaches the clinical phase, comprising a real and significant challenge [199].There is a growing need for preclinical researchers to be much more involved in the proof-ofconcept process for new therapeutics and diagnostics and not only to explore all technical and scientific possibilities of a novel drug delivery formulation [153].
Therefore, this section summarizes how the formulation parameters should be accurately characterized to advance preclinical development.

Importance of identifying the critical quality attributes of a liposomal product
During the development of liposomal drug products, the identification and pertinent characterization of the CQAs of liposomal drug products is one of the main challenges from the quality point of view, together with the definition of proper control strategies.A CQA can be understood as a property, either physical, chemical, biological, or microbiological, that can affect the quality or performance of the finished product [150,200].Liposomal products are highly complex formulations, and small changes in their physicochemical attributes can have notable effects in their in vivo performance and, thus, in their safety and efficiency profile.A suitable identification of the CQAs and control strategies to ensure that these CQAs are within an appropriate limit, range, or distribution to ensure the desired product quality could therefore enable more efficient drug product development [200].The definition of a CQA for a liposomal product should consider the FDA and EMA guidelines [201] and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH Guidelines), developed by regulatory and pharmaceutical industry authorities, which aims to provide uniform standards on the technical requirements for pharmaceuticals for human use [151].
Based on these recommendations, the CQAs (Table 2) for a liposomal product intended for intravenous administration (the most widely used administration route for LSD treatments) are usually identified.These attributes are critical for ensuring the liposomal product's quality and should be monitored with adequate instrumental techniques because they can have a significant impact on the biodistribution, half-life and cell uptake [202][203][204][205].

Analysis of characterization techniques in liposomal formulations for LSD
After a general overview of the most common techniques used for characterizing and controlling the liposomes' CQAs, the current analysis aims to provide baseline understanding of how the previously presented liposomal formulations have been characterized.Table 3 summarizes the published information regarding the char-acterization of the liposomal formulations in the preclinical development stage for treating the various LSDs previously mentioned in Table 1.
According to the analysis, particle size is the most frequently reported physicochemical characteristic of the liposomal formulations, probably because this attribute is a frequently requested characteristic when discussing nanomaterials [207].It has been repeatedly proven that nanomaterial size is a major CQA because it plays an important role in determining absorption, biodistribution, drug loading, drug release, and excretion.In addition, particle size is one of the crucial parameters for further in vivo application of liposomes because it can be a potential health hazard in preparations intended for intravenous use.Although according to best practices recommendations various sizing methodologies should be used [16,207,208], dynamic light scattering has been widely employed in the physicochemical characterization of liposomal formulations, probably due to the fact that this is a relatively affordable, fully automated, non-invasive and fast analytical technique, with user-friendly operational procedures.In the nanometric range (1-1000 nm), the selection of appropriate methods becomes difficult because many instruments are reaching their upper or lower resolution limits.However, dynamic light scattering can detect nanoliposomes smaller than 100 nm and can be performed on concentrated samples.
Other attributes such as surface characteristics (e.g., surface charge [expressed as f-potential] and targeting ligand characterization), drug content, entrapment efficiency and bioactivity are mostly reported for these formulations using a diversity of direct or indirect methodologies for their control.In contrast, crucial parameters such as drug release from the liposomal core (given that it partly defines the pharmacological performance of the dosage form) are not available for these liposomal systems.This lack of information is probably due to the lack of standard methods specified for liposomes either in the United States Pharmacopeia or the European Pharmacopeia and the fact that regulatory guidelines released by the FDA and EMA contain non-binding recommendations [206].
Another important challenge faced by researchers is that method sensitivity remains a critical aspect in nanomedicine analysis, especially for those methods employed in determining free drug and release kinetics [208].Finding effective techniques for separating the free drug from carriers without impairing the drug release rate from liposomes is an arduous task.Standardized protocols and specific characterization strategies are of utmost importance to the clinical translation of nanomedicines.In this context, two state-of-the-art facilities for the preclinical characterization of nanomedicines, the European Union Nanomedicine Characterization Laboratory and the National Cancer Institute-  Nanotechnology Characterisation Laboratory (NCI-NCL), have supported the pharmaceutical community by developing standard operating procedures and test methods that help fill current methodological gaps (e.g., these two facilities have provided standard operating procedures for successfully applying multidetector-AF4 for the physicochemical characterization of the aforementioned CQAs [e.g., particle size distribution, shape, drug loading and stability of nanopharmaceuticals]) in this highly regulated field.This type of collaboration is contributing to the harmonization of the regulatory framework for nanomedicine characterization between Europe and USA [209,210].Clinical translation is not, however, the only goal.Standardization can facilitate the conduct and communication of research and can, consequently, increase the robustness, reproducibility, and usefulness of published research for the rest of the community.The development of the Minimum Information Reporting in Bio-Nano Experimental Literature (MIRIBEL) is the first proposal for standardization that suggests a ''minimum information standard" for scientific literature investigating bio-nano interactions [211,212].Other more specific standards have been developed (e.g., the recently suggested Minimum Information about Nanomaterial Biocorona Experiments) for biomolecular corona studies.The key is that MIRIBEL and other standards are viewed as guidelines for the community to improve research quality and preventing the omission of information, which could be vital for researchers in the same field [213].

Conclusions and final remarks
The design and development of liposomes containing biomolecules (''biologicals") open new opportunities for LSD treatment, with potentially more effective and safer formulations, with greater capability to cross biological barriers than the few currently approved drug therapies.Among the many current strategies for LSD treatment that aim to correct the metabolic defect and reduce the pathophysiological effect of substrate accumulation into the lysosomes (e.g., ERT, SRT, PCT, HSCT and GT), two are used with liposomal formulations: 1) ERT, in which liposomes are used as nanocarriers to entrap and deliver the deficient enzyme and 2) gene strategy, in which the delivered gene encodes for the deficient enzyme (GT).
However, despite many incentives promoted by the EU and USA to push the development of orphan drug therapies, the number of liposomal formulations under development for LSD remains low compared with the liposomal products for other disease groups (e.g., cancer, cardiovascular diseases) which integrate small molecules.A total of eleven promising liposomal formulations addressing LSD therapy are currently in preclinical development.The innovative and complex structures of these formulations represent a challenge for the transfer from the preclinical to clinical phase, but important milestones are being achieved.The identification, characterization, and control of the CQAs of these complex formulations are of utmost importance to ensure the desired quality, efficacy, and safety requirements for their medical use.Accordingly, the demanding regulatory requirements, which are far higher than required for a publication, must be considered in the drug's development.However, an important issue faced by researchers is the lack of standardized protocols and specific characterization strategies for nanomedicines, which results in significant heterogeneity of adopted models and generated data.Moreover, the use of manufacturing methods compatible with a fast and robust scale-up since the beginning of the preclinical development is another factor to consider to facilitate the further translation of these liposomal products to the clinical and commercial scale.An optimized production, together with an increased efficacy, will lead to cost-effective treatments, having a direct impact on the life-quality of patients and on the sustainability of health-care systems.
Although there are several challenges still to be addressed, it is clear that liposomes have been extensively studied in fields such as GT and drug delivery.In recent years, the development of nanomedicines based on the use of liposomes has gradually grown, and this extensive research has led to the successful translation of numerous liposomal formulations to clinical use, addressing broad health areas.Although all liposomal formulations developed for LSD therapy are currently in the preclinical stage, liposomes could represent a powerful delivery system for LSD treatment in the future, especially if translation issues are addressed from the beginning of the liposomal formulation development.

Declaration of Competing Interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors declare the following financial interests/personal relationships that might be considered potential competing interests: S.S., J.V., and N.V. are the inventors of patent WO/2014/001509 licensed to Biopraxis Research AIE and patent WO/2006/079889 owned by Nanomol Technologies SL and are stockholders in Nanomol Technologies SL. J.T-M., J.M-M., S.S., A.C., J.V., E.G-M., and N.V. are the inventors of patent application EP21382062.4.

Fig. 3 .
Fig. 3.Most relevant results obtained with liposomal formulations in preclinical development for treating LSD, based on ERT: (A) Increased lysosomal delivery of glucocerebrosidase (VPRIV TM ) when associated to octadecyl-rhodamine B modified liposomes (RhB-Lip-E) versus the unmodified liposomes (Lip-E) or the free enzyme (VPRIV TM ) in Gaucher's fibroblasts, expressed as a % increase in mean fluorescent intensity (MFI) normalized to control cells.Adapted from[55], with permission.(B) Successful transport of rh-GCase when associated to saposin C-modified liposomes in brain cells of Gaucher-like mice.GCase protein (green) detected in astrocytes, neurons and microglia (red).Adapted from[56], with permission.(C) Better prevention of sphingomyelin degradation (expressed as % reduction in sphingomyelin conversion relative to free rh-ASM) when rh-ASM is loaded in BMP-based liposomes versus free.Adapted from[57], with permission.(D) Uptake and lysosomal delivery increase of rh-IDUA in IDUA-deficient MPS I fibroblasts when loaded in GNeo-modified liposomes (GNeo-Lip) compared to unmodified liposomes (Lip), measured by IDUA enzymatic activity of lysate cells.Adapted from[58], with permission.(E) rh-GAA immunogenicity reduction when the enzyme is loaded in PI-based liposomes versus free.Anti-rh-GAA antibodies in plasma were measured in GAA-KO mice (20 mg/kg), after 4 weeks of intravenous injections.Adapted from[60], with permission.(F) Major efficacy in the Gb3 reduction when the enzyme is loaded in RGD-liposomes than when it is free, evaluated in vitro in primary endothelial cells derived from Fabry KO mice.Adapted from[62], with permission.(G) Better localization of liposomes functionalized with the H16 targeting peptide (H16-Lipo) in lysosomes after cellular uptake.Adapted from[64], with permission.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4 .
Fig. 4. Relevant results obtained with liposomal formulations in preclinical development for treating LSD, based on GT: (A) Reduction of the GM2 ganglioside accumulation in the liver of Sandhoff disease (SD) mice, after single administration of plasmid b-hexosaminidase gene-loaded liposomes GM2 levels evaluated by immunohistochemistry (green).Adapted from [65], with permission.(B) Achievement of therapeutic levels of GUSB enzyme activity in several organs, such as brain, after single intravenous administration of liposomes modified with anti-transferrin receptor antibodies and encapsulated with a non-viral GUSB expression plasmid DNA (Lipo treated) into GUSB null mice.Adapted from [66], with permission.(C) Reduction of glycosaminoglycan accumulation levels (i.e., dermatan sulfate in the figure) in MPS I mice treated with liposomes loaded with IDUA expression plasmid (glycosaminoglycan's quantification by tandem mass spectrometry).Adapted from [68], with permission.(D) Visible reduction of vacuolated cells number in brain of NPC1 mice after repeated intravenous administration of liposomes modified with anti-transferrin receptor antibodies, and encapsulated with NPC1 gene (Lipo treated).Adapted from [69], with permission.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1
Liposomal formulations in the preclinical development stage for treating a specific disease belonging to the lysosomal storage disorder (LSD) group and the methods used to produce those liposomal formulations.

Table 2
Several major critical quality attributes of liposomal products and common techniques for their monitoring.

Table 3
Characterization checklist regarding the quality attributes of liposomal formulations listed in Table1.
N/A, not applicable.* Liposomes intended for intravenous administration must comply with the requirements for injectable pharmaceuticals.