Because the membrane-bound enzyme alkaline phosphatase (ALP) can etch phosphate substructures from drugs, adjuvants, and even nanocarrier surfaces, ALP makes it possible to engineer drug delivery systems with in vivo modifications that can be changed on demand. When at the site, the biologically nonfunctional anionic nanocarriers can switch surfaces to cross over because ALP causes anionic phosphate chains of their surface charge to become cations and enhance cellular binding, among other things. ALP-induced target-site nanocarrier accumulation or drug-specific release can also be added. ALP is also applied to bolster the efficacy of a large number of diagnostic systems. Most physiological and pathological processes are necessarily accompanied by ALP activity and thus it is a clinical and industrial biomarker of great significance.
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Alkaline Phosphatase (ALP)
ALP is a homodimeric protease, which mediates many aspects of biology including molecular transport, metabolism, and gene expression. It is an important biomarker for other biological systems and tests. The main activity of ALP in these reactions is to catalyze the dephosphorylation of several critical biological phosphate components to hydroxyl groups: proteins, nucleic acids, and small organic phosphate groups. In its most optimal alkaline state, ALP's photocatalytic activity comes from its very specific organometallic complex composition: two identical monomers, each with five cysteine residues, two zinc, and one magnesium atom. Because ALP enzymes catalyze so much of biology, they are found in the tissues and organs of both prokaryotic and eukaryotic cells.
As of now 6 ALP isozymes have been described: AKP1, AKP2, AKP3, AKP4, AKP5, and AKP6. The liver produces AKP1, AKP2, and AKP6, osteocytes make AKP3, cancer cells and the placenta make AKP4, and fibroblasts make AKP5.
Distribution and Physiology of Alkaline Phosphatase
Four genes in humans correspond to four ALP isozymes. Three of them are tissue-specific ALP (TSAP), namely placental ALP (PLAP), germ cell ALP (GCAP), and intestinal ALP (IAP). The fourth is tissue-nonspecific ALP (TNAP), the most widespread isozyme and its major expression areas are bones, liver, kidneys, and the central nervous system. ALPs are membrane-bound glycoproteins that are located on the outside of the plasma membrane and are highly expressed in the extracellular environment, very few in the cytoskeleton, mitochondria, peroxisomes, nuclei, endoplasmic reticulum or lysosomes, and tiny amounts in endosomes and Golgi apparatus. ALP can be found in human serum too and can be used as a biomarker for liver disease, bone disease, and cancer.
Biochemistry and Structure of Enzyme Active Site
ALP is a glycoprotein bound to the cell wall with a glycan-phosphatidylinositol anchor. The carbohydrate side chains of ALPs are ended with sialic acid, with the exception of IAP. Deletion of the carbohydrate unit didn't decrease the catalytic capacity of ALP. Because the enzyme active site is very dense with positive charges, substrate charge, electron-removal capacity of the leaving group, pH, and leaving group pKa all influence binding efficiency and overall catalytic/inhibitory activity. We know that the divalent ion form of the monophosphate is an ALP-suitable substrate and that the divalent ion form of free phosphate Pi binds as strongly or more strongly to the enzyme active site than the trivalent anion Pi.
Alkaline Phosphatase Activity
It is imperative to know in a clear way alkaline phosphatase (ALP) activity within the target organ or tissue, as ALP activity directly contributes to the activity of prodrugs and phosphate-functionalized DDS and diagnostics. So, too, ALP function – age, sex, hormones, disease, and diet. Children's ALP activity is correlated with height and weight, and up to puberty bone ALP contributes 77%-87% of all ALP activity. ALP is found in most cell lines used in in vitro experiments. ALP hydrolyzes phosphate monoesters but also S-phosphorothioates phosphoramidates and phosphorothioates. It hydrolyses sulfate monoesters and phosphonate monoesters with little interest in phosphate monoesters.
Blockers, Activators, and Regulators of Alkaline Phosphatase
The phosphatase inhibitors have also been used extensively in several in vitro studies (both on cultured cells and freshly removed tissues) to assess the influence of ALP on the activity of phosphate-functionalized DDS. Even inorganic phosphate produced by the dephosphorylation reaction competitively and irreversibly inhibits ALP. ALP is a protein, and its three-dimensional structure is what matters. Because disulfide bonds are what control the tertiary conformation of proteins, any chemical or physical modification of SH groups will alter the protein's conformation and cause irreversible inhibition.
Alkaline Phosphatase Related Drug Delivery Systems
In recent decades, enzyme-responsive DDS have been widely researched as means of improving drug solubility, delivering drugs to pathology, improving absorption and controlled, targeted drug delivery. Of all the endogenous enzymes that have been able to provide drug delivery, including matrix metalloproteinases, hyaluronidase, lipase and trypsins, phosphatases are special. Such enzymes are important for most physiologic functions because they cut through phosphate esters involved in many of biology's most essential processes: information storage and transfer, energy transfer, and membrane fluidity. This discovery and function of ALP in many cancers and diseases became very popular, which spawned new ALP detection techniques, disease therapies and diagnostic tools.
Fig. 1 Alkaline phosphatase for drug delivery (Le‐Vinh, B., et al. 2022).
Charge-Switching Delivery Systems
The surface charge is also relevant for the coupling of DDS with the biological world. A promising strategy for charge switching at the destination site is based on ALP. An enzyme that's membrane bound, it shifts charge from negative to positive on the cell membrane of the target by breaking down and dissociating anionic phosphates from various drug carriers.
Self-Assembling Drug Delivery Systems
ALPs regulate up to 30% of both intracellular and extracellular biology and have been utilised as an induction mechanism for the production of self-assembled biomaterials. In phosphorous-containing precursors, ALPs are converted into self-assembling molecules that have been employed to induce the self-assemblement of aromatic peptide derivatives. Dephosphorylation by ALP leads to more hydrophobic end groups, which promote self-assemblement into micelles and lower the LCST as close as possible to physiological temperature.
Polymeric Drug Delivery Systems
Polymer dephosphorylation causes hydrophilic change which in turn leads to aggregation or depolymerisation. The ALPs break up these phosphate groups, and the resulting particles accumulate to create nanoassemblies by non-covalent bonding. These redox complexes get taken up internally mostly through caveolae/raft-dependent endocytic channels, selectively killing mitochondria and cancer cells.
Disaggregative Drug Delivery Systems
Besides causing drug delivery system self-assemble and collect, ALPs can also depolymerise nanocarriers. It can also be an attractive way to tailor drug delivery systems. After ALP dephosphorylation, TPP cross-links in the particle network are broken and nanoparticles break up and release the active component controlled. Since -galactosidase is sensitive to the intestinal metabolic milieu, -galactosidase can be incorporated into chitosan/TPP nanoparticles to retain the enzyme integrity up until the time it reaches the targeted mucosal tissue.
Alkaline Phosphatase Targeting Strategies
ALP can be an early warning sign for many illnesses, and its levels are linked to the liver, prostate, bone, and endocrine systems. We find ALP in the gut, liver, kidney, bone, and placental tissues, too, and it is a popular biomarker for a host of diseases in medicine. So, measuring ALP levels is very critical for the diagnosis and treatment of the above diseases. ALP is also widely deployed as a clinical biomarker, but in immunoassay systems, it can also be employed as a reporter enzyme for other potential biomarkers.
ALP is an enzyme, which means that other technologies will need to be designed to detect its catalytic activity to satisfy the general desire in academia and industry for non-invasive inexpensive, simple, and rapid detection. Yet targeted ALP detection needs the sensing technology to satisfy the usual criteria for reliable quantitative assessment of chemical sensors: sensitivity and selectivity.
Conclusion
Alkaline phosphatase (ALP) has always been a partner for medication and diagnosis. When phosphate groups are cut from phosphate prodrugs by ALP, their membrane partitioning is altered. These modifications not only enhance drug absorption across the gastrointestinal barrier but facilitate drug localization on tumor cells and minimize toxicity. As phosphate substructures are evicted from their surface, ALP-activated systems alter their surface charge, shape, aggregate state, or opacity for improved and controllable mucosal drug delivery, targeted drug delivery, tissue and organ imaging, cellular uptake, and gene transfection.
References
- Vimalraj, S., et al. Alkaline phosphatase: Structure, expression and its function in bone mineralization. Gene. 2020, 754: 144855.
- Le‐Vinh, B., et al. Alkaline phosphatase: a reliable endogenous partner for drug delivery and diagnostics. Advanced Therapeutics. 2022, 5(2): 2100219.
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