L-asparaginase (L-ASP) is one of the key enzymes used in therapeutic applications, mainly to treat Acute Lymphocytic Leukemia (ALL) and Lymphosarcoma cancer. In food industries, especially for the reduction of acrylamide (carcinogen) from starch-rich foods which are cooked at temperatures above 90°C [1,2]. It is produced by cells as protein to catalyze the deamination process of L-asparagine amino acid into aspartic acid and ammonia [3,4].

It has been almost 70 years since the L-ASP is known for anti-tumor activity, particularly ALL. In 1953, L-ASP was first reported that the guinea pig serum had activity against rat lymphoma [4]. Later, in 1961, Broome identified that the anti-tumor activity of guinea pig serum was due to the presence of L-ASP [5].

The differences in metabolism between healthy cells and tumor cells explain the mode of action of L-ASP. Healthy cells can independently synthesize many amino acids, including asparagine, while leukemic cells cannot synthesize asparagine, but they need a large amount for protein synthesis and cell division processes to ensure their vital functions. L-Asparaginase, an amidohydrolase, catalyzes the breakdown of L-asparagine into aspartic acid and ammonia, thereby depriving leukemic cells of asparagine. This leads to decreased protein synthesis and cell division in tumor cells [6,7].

So far, various organisms, including bacteria, fungi, actinomycetes, plants, microalgae, and animals, have been used to extract L-ASP [8]. As the L-ASP gene is present in all types of life, researchers have been continuously screening for L-ASP with superior features [9,10].

Recently Sobat et al. [11] used next-generation sequence and in-silico screening for L-ASP with anti-leukemia properties from the Caspian Sea using 27,000 publicly available prokaryote genomes. The most commonly used bacterial sources include Erwinia caratovora, Pseudomonas stutzeri, Bacillus sp, Corynebacterium glutamicum and E. coli etc. However, only Erwinia and E. coli were used on a manufacturing scale. For example, Elspar®, isolated from E.Coli was the first L-ASP available on the market since 1978 and was approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with acute lymphoblastic leukemia (ALL) [3].

Enzymes as therapeutic agents for cancer treatment have a low risk of lysine resistance due to selective interactions with cell wall components like enzymes acting on bacterial cell wall peptidoglycan and therefore unlikely to affect mammalian cells negatively [12].

However, using L-ASP has side effects, such as hypersensitivity or allergic reaction, antigenicity, temporary blood clearance, and toxicity [13]. The main limitation of L-ASP from E. coli is hypersensitivity, which is reported in 15 to 73% of cases [14]. Additional side effects, such as hepatotoxicity, thrombosis, hyperglycemia, pancreatitis, and abnormalities of lipid metabolism, are commonly linked with using L-ASP [15]. Furthermore, the activity of most of the L-ASPs is limited by high temperatures, narrow pH range, and by its short half-life [16].

Therefore, there is a trend in minimizing or circumventing its side effects and improving the performance parameters of the native L-ASP, such as selectivity, stability against degradation, shelf-life and affinity [7,17]. The enrichment of L-ASP and their therapeutic properties can be achieved by various immobilization techniques within several supports with consideration of quality aspects of the product [18]. Enzyme immobilization can improve the L-ASP activity, stability, and catalytic half-life, allow their reusing at a broad range of temperatures and pH, and improve enzyme pharmacokinetics with minimum side effects [19,20].

To date, five types of L-ASP immobilization techniques are used to stop the immune system from attacking nonhuman enzymes and improve the performance of the enzyme [18,[21], [22], [23]]. Generally, they can be divided into two strategies [24]. The first strategy is based on the chemical or physical modification of the enzyme molecule. It includes the technique of covalent binding (conjugation) with polymers, such as polyethylene glycol (PEG) [[25], [26], [27]]; physical adsorption to the support material via van der Waals forces, ionic interactions and hydrogen bonds [28]; and cross-linking of enzyme molecules [29,30], which often improves its stability [31].

For example, the Michaelis-Menten constant (Km) for immobilized L-ASP was 1.6-fold lower than the free form, indicating a higher affinity for the asparagine substrate. In addition, immobilized L-ASP exhibited superior stability against elevated temperature, freeze-thaw cycles, and proteolysis, yielding a novel stabilized preparation with promising biomedical applications [25]. For example, PEG-L-ASP was approved for the treatment of acute lymphoblastic leukemia (ALL) patients allergic to L-ASP by the US Food and Drug Administration (FDA) in 1994, and it has been used as the first-line treatment for adult and pediatric ALL since 2006 [32]. Additionally, in 2019 Young et al. [26] based on wide pieces of evidence from literature reported that intramuscular (IM) or intravenous (IV) administration of PEG L-ASP is an effective first-line treatment for pediatric and adult patients with ALL those who are hypersensitive to E. coli L-ASP.

In a recent systematic review, Сamile et al. 2020 [27] compared the efficacy and safety of PEG-L-ASP with native E. coli L-ASP for the treatment of ALL in both children and adults using the wide range of literature data (PubMed, Web of Science, Science Direct, Cochrane Library, Scopus, LILACS (Latin American and Caribbean Health Sciences Literature) and EMBASE databases). They used the Cochrane recommendation tool and the GRADE system and their results show that evidence is inadequate to evaluate the effects of PEG-L-asp due to the limited number and power of studies and essential flaws in their design or conduct in classifying PEG-L-ASP as a superior drug or not, in the therapy of ALL.

Nevertheless, some side effects of PEG have been identified, such as the presence of PEG antibodies in the blood due to immune response [33]. In addition, PEGylated L-ASP, which has been approved for treating Acute Lymphoblastic Leukemia (ALL), can give allergic reactions, and reports hypersensitivity reactions to Oncaspar® have also been attributed to anti-PEG antibodies that develop CARPA [34].

Also, the chemical modification of residues, often nonspecific, changes the enzyme's hydrophobicity and surface charge, lowering the enzyme's activity [35]. In this strategy, the enzyme's surface is exposed to the environment. This eliminates its performance and does not allow targeted delivery of the enzyme.

The second strategy is based on the entrapment of the enzyme inside the protecting structure or encapsulation [22]. This strategy offers the same benefits as the first but also enables a targeted delivery and controlled release of the enzyme [36]. It also resulted in a longer circulation half-life, reducing toxicity and antigenicity, enhancing stability, activity, etc. Furthermore, compared to the first strategy, encapsulation does not modify the chemical structure of the enzyme, since L-ASP is only effective against leukemia in its native tetrameric form and very significantly doesn't disrupt the L-ASP conformation [37].

Among other techniques, an encapsulation technique is one of the easiest to implement and shows high enzyme activity at a low cost of immobilization [21]. Encapsulation can be commonly implemented via three basic procedures: solvent extraction/evaporation, phase separation (coacervation) and spray-drying [[38], [39], [40]]. Numerous encapsulating systems include polymers, polyelectrolytes, solid inorganic nanoparticles, liposomes, phytosomes etc. (Fig. 2) [41]. However, it is worth noting that not many reports are available on L-ASP immobilization. (See Fig. 1.) (See Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11.)

During immobilization, the support materials can change the enzyme properties; therefore the activity of immobilized L-ASP should be thoroughly studied and evaluated before therapeutic use to know that the steric availability of the enzyme binding site did not change after immobilization [43]. Hence, the following section focuses on the encapsulation systems used for L-ASP delivery to improve L-ASP performance and reduce its side effects.

This review will mainly discuss the second approach for delivering L-ASP, including encapsulation and entrapment techniques. Both these techniques will be generally called encapsulation. The study of encapsulation of asparaginase in different protective structures, its effect on release kinetics and the enzyme activity is of great interest to researchers since this approach brings crucial benefits for asparaginase performance.