Therapeutic Enzymes Research Articles

Remodeling the tumor microenvironment by vascular normalization and GSH-depletion for augmenting tumor immunotherapy

Remodeling tumor microenvironment (TME) is a very promising and effective strategy to enhance the effects of chemotherapy, photodynamic therapy, and immunotherapy. Normalization of tumor vasculature as well as depletion of glutathione (GSH) can improve the TME. Here, we developed a novel therapeutic nanoparticle functional enzyme ultra QDAU5 nanoparticles (FEUQ Nps) based on a fluorescence-on and releasable strategy by combining a vascular normalization inducer, a GSH depleting agent, and an activated fluorophore. In which the cleavage of disulfide bonds releases active molecules that induce vascular normalization and improve the hypoxic microenvironment. In addition, it may deplete GSH in cancer cells, thus inducing the production of reactive oxygen species (ROS) and lipid peroxide (LPO) and promoting iron toxicity. It may also lead to endoplasmic stress and release of calmodulin, which activates the immune system. Meanwhile, quenched fluorophores are turned on in the presence of galactosidase (GLU) for tumor-specific labeling. In summary, we developed novel therapeutic agent nanoparticles with the function of vascular normalization inducers to achieve specific labeling of hepatocellular carcinoma while exerting efficient antitumor effects in vivo.

Remote Activation of Enzyme Nanohybrids for Cancer Prodrug Therapy Controlled by Magnetic Heating.

Herein, we have developed nanohybrids (nHs) to remotely activate a therapeutic enzyme for its use in Directed Enzyme Prodrug Therapy (DEPT). The coencapsulation of magnetic nanoparticles (MNPs) with horseradish peroxidase (HRP) using biomimetic silica as an entrapment matrix was optimized to obtain nanosized hybrids (∼150 nm) for remote activation of the therapeutic enzyme. HRP converts indole-3-acetic acid (3IAA) into peroxylated radicals, whereas MNPs respond to alternating magnetic fields (AMFs) becoming local hotspots. The AMF application triggered an increase in the bioconversion rate of HRP matching the activity displayed at the optimal temperature of the nHs (Topt = 50 °C) without altering the temperature of the reaction media. This showed that enzyme nanoactuation is possible with MNPs even if they are not covalently bound. After an extensive physicochemical/magnetic characterization, the spatial location of each component of the nH was deciphered, and an insulating role of the silica matrix was suggested as critical for introducing remote control over HRP. In vitro assays, using a human pancreatic cancer cell line (MIA PaCa-2), showed that only upon exposure to AMF and in the presence of the prodrug, the enzyme-loaded nHs triggered cell death. Moreover, in vivo experiments showed higher reductions in the tumor volume growth in those animals treated with nHs in the presence of 3IAA when exposed to AMF. Thus, this work demonstrates the feasibility of developing a spatiotemporally controlled DEPT strategy to overcome unwanted off-target effects.

Gene therapies for mucopolysaccharidoses.

Current specific treatments for mucopolysaccharidoses (MPSs) include enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT). Both treatments are hampered by several limitations, including lack of efficacy on brain and skeletal manifestations, need for lifelong injections, and high costs. Therefore, more effective treatments are needed. Gene therapy in MPSs is aimed at obtaining high levels of the therapeutic enzyme in multiple tissues either by engrafted gene-modified hematopoietic stem progenitor cells (ex vivo) or by direct infusion of a viral vector expressing the therapeutic gene (in vivo). This review focuses on the most recent clinical progress in gene therapies for MPSs. The various gene therapy approaches with their strengths and limitations are discussed.

Two-Dimensional Supramolecular Polymerization of DNA Amphiphiles is Driven by Sequence-Dependent DNA-Chromophore Interactions.

Two-dimensional (2D) assemblies of water-soluble block copolymers have been limited by a dearth of systematic studies that relate polymer structure to pathway mechanism and supramolecular morphology. Here, we employ sequence-defined triblock DNA amphiphiles for the supramolecular polymerization of free-standing DNA nanosheets in water. Our systematic modulation of amphiphile sequence shows the alkyl chain core forming a cell membrane-like structure and the distal π-stacking chromophore block folding back to interact with the hydrophilic DNA block on the nanosheet surface. This interaction is crucial to sheet formation, marked by a chiral "signature", and sensitive to DNA sequence, where nanosheets form with a mixed sequence, but not with a homogeneous poly(thymine) sequence. This work opens the possibility of forming well-ordered, bilayer-like assemblies using a single DNA amphiphile for applications in cell sensing, nucleic acid therapeutic delivery and enzyme arrays.

Two‐Dimensional Supramolecular Polymerization of DNA Amphiphiles is Driven by Sequence‐Dependent DNA‐Chromophore Interactions

AbstractTwo‐dimensional (2D) assemblies of water‐soluble block copolymers have been limited by a dearth of systematic studies that relate polymer structure to pathway mechanism and supramolecular morphology. Here, we employ sequence‐defined triblock DNA amphiphiles for the supramolecular polymerization of free‐standing DNA nanosheets in water. Our systematic modulation of amphiphile sequence shows the alkyl chain core forming a cell membrane‐like structure and the distal π‐stacking chromophore block folding back to interact with the hydrophilic DNA block on the nanosheet surface. This interaction is crucial to sheet formation, marked by a chiral “signature”, and sensitive to DNA sequence, where nanosheets form with a mixed sequence, but not with a homogeneous poly(thymine) sequence. This work opens the possibility of forming well‐ordered, bilayer‐like assemblies using a single DNA amphiphile for applications in cell sensing, nucleic acid therapeutic delivery and enzyme arrays.

Therapeutic Potential of Microbial Metabolites: New Insights and Perspectives

Microbes release important metabolites that regulate various physiological activities inside and outside of organisms. The human gastrointestinal tract is a reservoir of microbes that play important regulatory roles in modulating the immune system and numerous other physiological functions. Thus, there is substantial interest in these microbial products and their clinical significance. These microbial metabolites have shown promise as therapies for cancer, inflammation, neurological disorders, and many other diseases. Here, we discuss microbial metabolites with substantial therapeutic potential, including proteasome inhibitors, therapeutic enzymes, bacteriocins, polyamines, and flavonoids.

Profiling the disintegration of BRPs released by massive wasp stings using serratiopeptidase: An in-silico insight

Serratiopeptidase is a multifaceted therapeutic enzyme renowned for its anti-inflammatory, analgesic, anti-biofilm, fibrinolytic, and anti-edemic properties. It is vital to uncover more about the assets of such efficacious enzyme in order to facilitate their contribution in all health-related issues, notably inflammatory ailments. The current study sought to determine whether serratiopeptidase would disintegrate bradykinin related peptides (BRPs) from wasp venom in the same manner as it does with human bradykinin. To accomplish this objective, we docked selected BRPs onto the binding pocket of wild and previously identified mutant (N412D) of serratiopeptidase. Based on their docked scores, the top two BRPs were selected, and their conformational behavior was analyzed employing molecular dynamics studies. Additionally, thermodynamics end-state energy analysis reported that both the complexes exhibited higher stability and identical ΔG values when compared to the reference complex. Further, we condemned the external pulling forces on both peptides to observe the force needed in the disassociation process to endorse the binding affinity findings in terms of unbinding mechanism. This analysis suggested that BRP-7 (Wasp kinin PMM1) peptide was tightly anchored and laid out the highest pulling force to get detach from the active pocket of serratiopeptidase in contrast to the BRP-6 peptide. The current study endorses up the present findings and paves the way for serratiopeptidase to be used as an anti-angioedemic peptidase as well as a fixed-dose combination (FDC) in hypotensive drugs.

ERRATUM: Evaluation of the Anti-inflammatory, Antioxidant, and Protease Inhibitory Activity of the Crude Methanol Extract of Portulaca oleracea

Protease inhibitors have considerable applications in biomedicine, biotechnology, and therapy. The aim of the study was to evaluate the methanol crude extracts of aerial portions of Portulaca oleracea (P. oleracea) for anti-inflammatory, antioxidant, and protease inhibitor activities, and to evaluate their inhibitory action against various therapeutically significant proteases. A modified colorimetric assay was used to analyze the protease inhibitors on trypsin and other therapeutic enzymes. P. oleracea crude leaf extract exhibited 79.92% antioxidant and 75% anti-inflammatory activity. Analysis of the effects of crude P. oleracea extract on the values of enzyme kinetics (Km, Vmax) was determined by Lineweaver-Burk reciprocal plot. It was found that the extract exhibited a potent inhibitory effect on the Vmax (µmole/min) of the chymotrypsin 26.0, trypsin 18.0, papain 8.33, and elastase 10.0, when compared to control 41.0, 73.0, 11.11, and 20.3, respectively. While no effect on Km except for elastase which was decreased from 0.026 mM for the control to 0.01 mM. It was found that this effect showed a powerful inhibition in a different way. There is a mixed non-competitive inhibition on elastase as well as pure non-competitive inhibition on chymotrypsin, trypsin, and papain. The crude extract contained 53.3%, 55.7%, 14.9%, and 26.9%, respectively, of protease inhibitors for the therapeutic protease enzymes trypsin, papain, chymotrypsin, and elastase. Therefore, trypsin, papain, chymotrypsin, and elastase have been inhibited by the crude extract of P. oleracea. Characterization of the crude extract indicated potent anti-inflammatory and antioxidant effects.

RBC Membrane-coated Nanoparticles: A Comprehensive Review on the Preparation Methods, Characterisations and Applications

Abstract:Natural cells have become an area of interest due to their biocompatibility, nonimmunogenicity, biodegradability, and targeting specificity. The human vascular system retains distinctive physiological features that can be developed for enhanced and effective targeted drug delivery. Red blood cells (RBCs) have unique features and properties that make them potential natural carriers for numerous substances. Recently, the RBC membrane has become a unique biological carrier and it has been extensively studied due to its long-circulating half-life, low toxicity, high stability and the ability to transport various biologically active substances with higher drug release efficiency. Among the benefits of the RBC membrane as a drug delivery carrier in medical and biological fields is the use of this system in anticancer therapy. Antitumor drugs are loaded in gold NP, magnetic NPs, or mesoporous silica NPs. Then, the loaded NP is used as a core and coated with an RBC membrane to protect the NP from immune attack and enhance drug targeting. Moreover, RBCs have been used for encapsulating different enzymes to overcome the undesirable outcomes associated with enzyme replacement therapy. This review highlighted the most recent RBC membrane preparation methods, such as Membrane coating technology and Osmotic Loading Procedures. The recent advances in the design of RBC membrane carriers and discuss the applications of RBCs in different fields such as therapeutic enzymes, immunotherapy and anti-tumour therapy. Given the potential risks and challenges in the development of any treatment protocol, this review elucidated the problematic aspects and prospects, describing new modalities to overcome these problems. RBCs as a drug carriers are among the most interesting topics as a novel drug delivery system as they are convenient, effective, safer, biocompatible and have good properties to deliver and administrate the drug specifically to the target site of action with fewer side effects and interference with therapeutic aspects.

Diamine Oxidase as a Therapeutic Enzyme: Study of Germination from Vegetal Sources and Investigation of the Presence of β-N-Oxalyl-L-α,β-diaminopropionic Acid (β-ODAP) Using LC-MS/MS.

Vegetal diamine oxidase (vDAO), an enzyme proposed to relieve symptoms of histaminosis, shows better reactivity with histamine and aliphatic diamines, as well as higher enzymatic activity than DAO of animal origin. The objective of this study was to evaluate the enzyme activity of vDAO from germinating grains from Lathyrus sativus (grass pea) and Pisum sativum (pea), and to verify the presence of a neurotoxin, β-N-Oxalyl-L-α,β-diaminopropionic acid (β-ODAP), in the crude extract obtained from their seedlings. A targeted liquid chromatography-multiple-reaction monitoring mass spectrometry method was developed and used to quantify β-ODAP in the analysed extracts. An optimized sample preparation procedure, involving protein precipitation with acetonitrile followed by mixed-anion exchange solid-phase extraction, allowed for high sensitivity and good peak shape for β-ODAP detection. The Lathyrus sativus extract exhibited the highest vDAO enzyme activity of the extracts, followed by the extract from pea cultivar Amarillo from the Crop Development Centre (CDC). The results have also shown that even though β-ODAP was present in the crude extract from L. sativus, its content was far below the toxicity threshold (300 mg of β-ODAP/kg body/day). CDC Amarillo showed 5000-fold less β-ODAP than the undialysed L. sativus extract. It was concluded that both species can be considered as convenient sources of vDAO for potential therapeutic use.

A universal GlycoDesign for lysosomal replacement enzymes to improve circulation time and biodistribution.

Currently available enzyme replacement therapies for lysosomal storage diseases are limited in their effectiveness due in part to short circulation times and suboptimal biodistribution of the therapeutic enzymes. We previously engineered Chinese hamster ovary (CHO) cells to produce α-galactosidase A (GLA) with various N-glycan structures and demonstrated that elimination of mannose-6-phosphate (M6P) and conversion to homogeneous sialylated N-glycans prolonged circulation time and improved biodistribution of the enzyme following a single-dose infusion into Fabry mice. Here, we confirmed these findings using repeated infusions of the glycoengineered GLA into Fabry mice and further tested whether this glycoengineering approach, Long-Acting-GlycoDesign (LAGD), could be implemented on other lysosomal enzymes. LAGD-engineered CHO cells stably expressing a panel of lysosomal enzymes [aspartylglucosamine (AGA), beta-glucuronidase (GUSB), cathepsin D (CTSD), tripeptidyl peptidase (TPP1), alpha-glucosidase (GAA) or iduronate 2-sulfatase (IDS)] successfully converted all M6P-containing N-glycans to complex sialylated N-glycans. The resulting homogenous glycodesigns enabled glycoprotein profiling by native mass spectrometry. Notably, LAGD extended the plasma half-life of all three enzymes tested (GLA, GUSB, AGA) in wildtype mice. LAGD may be widely applicable to lysosomal replacement enzymes to improve their circulatory stability and therapeutic efficacy.

Enhanced uricase production using novel Escherichia marmotae strain (DJDSS001): Characterization and optimization

Uricase is a diagnostic and therapeutic enzyme used to detect the levels of purine end product namely uric acid in the biological samples and it also promotes the conversion of uric acid to a soluble allantoin. In this study, a potential extracellular uricase-producing Escherichia sp. was isolated from canal water near dairy and molecular characterization using16 S rRNA sequence was performed and the data showed 98.95% similarity with Escherichia marmotae. Morphological and biochemical characteristics of the isolated bacterium were determined. The effect of medium components on uricase production was studied and optimized medium enhanced the uricase production to 27.47 U/ml ± 0.44 which is 1.37 fold greater than initial uricase production. The enzyme was purified using ammonium sulphate precipitation followed by ion exchange chromatography and the resulted enzyme is stable over wide pH range from 6.0 to 9.0 and temperatures ranging from 30 °C to 37 °C retaining more than 94% activity. The enzyme activity and specific activity of the purified enzyme were 40.02 U/mL and 87.76 U/mg respectively with an overall purification fold of 4.04 and the yield obtained is 14.57%.

Lactate oxidase/vSIRPα conjugates efficiently consume tumor-produced lactates and locally produce tumor-necrotic H2O2 to suppress tumor growth

Aggressive tumor formation often leads to excessive anaerobic glycolysis and massive production and accumulation of lactate in the tumor microenvironment (TME). To significantly curb lactate accumulation in TME, in this study, lactate oxidase (LOX) was used as a potential therapeutic enzyme and signal regulatory protein α variant (vSIRPα) as a tumor cell targeting ligand. SpyCatcher protein and SpyTag peptide were genetically fused to LOX and vSIRPα, respectively, to form SC-LOX and ST-vSIRPα and tumor-targeting LOX/vSIRPα conjugates were constructed via a SpyCatcher/SpyTag protein ligation system. LOX/vSIRPα conjugates selectively bound to the CD47-overexpressing mouse melanoma B16-F10 cells and effectively consumed lactate produced by the B16-F10 cells, generating adequate amounts of hydrogen peroxide (H2O2), which induces drastic necrotic tumor cell death. Local treatments of B16-F10 tumor-bearing mice with LOX/vSIRPα conjugates significantly suppressed B16-F10 tumor growth in vivo without any severe side effects. Tumor-targeting vSIRPα may allow longer retention of LOX in tumor sites, effectively consuming surrounding lactate in TME and locally generating adequate amounts of cytotoxic H2O2 to suppress tumor growth. The approach restraining the local lactate concentration and H2O2 in TME using LOX and vSIRPα could offer new opportunities for developing enzyme/targeting ligand conjugate-based therapeutic tools for tumor treatment.

Novel dual promoter AAV gene therapy platform ensures production of therapeutic soluble lysosomal enzymes with high M6P content to enable broad cellular uptake and cross correction

Novel dual promoter AAV gene therapy platform ensures production of therapeutic soluble lysosomal enzymes with high M6P content to enable broad cellular uptake and cross correction

Delivering therapeutic lysosomal enzyme to the CNS: A rapid, large-scale screen identifies optimal characteristics of BBB-crossing TFRC antibodies

Delivering therapeutic lysosomal enzyme to the CNS: A rapid, large-scale screen identifies optimal characteristics of BBB-crossing TFRC antibodies

Portable Alkaline Phosphatase-Hydrogel Platform: From Enzyme Characterization to Phosphate Sensing.

Here, we present a study on the incorporation and characterization of the enzyme alkaline phosphatase (ALP) into a three-dimensional polymeric network through a green protocol to obtain transparent hydrogels (ALP@AETA) that can be stored at room temperature and potentially used as a disposable biosensor platform for the rapid detection of ALP inhibitors. For this purpose, different strategies for the immobilization of ALP in the hydrogel were examined and the properties of the new material, compared to the hydrogel in the absence of enzyme, were studied. The conformation and stability of the immobilized enzyme were characterized by monitoring the changes in its intrinsic fluorescence as a function of temperature, in order to study the unfolding/folding process inside the hydrogel, inherently related to the enzyme activity. The results show that the immobilized enzyme retains its activity, slightly increases its thermal stability and can be stored as a xerogel at room temperature without losing its properties. A small portion of a few millimeters of ALP@AETA xerogel was sufficient to perform enzymatic activity inhibition assays, so as a proof of concept, the device was tested as a portable optical biosensor for the detection of phosphate in water with satisfactory results. Given the good stability of the ALP@AETA xerogel and the interesting applications of ALP, not only in the environmental field but also as a therapeutic enzyme, we believe that this study could be of great use for the development of new devices for sensing and protein delivery.

A quaternary amine cryogel column for chromatographic capture of l-asparaginase

L-asparaginase is a therapeutic enzyme used in therapies of acute lymphoblastic leukemia. Its purification conventionally uses several unit operations, and usual protocols lead to low yields and extensive processing times, which reduce productivity and increases process costs. In this study, we produced a supermacroporous cryogel activated with polyethyleneimine, followed by quaternization with epichlorohydrin resulting in a quaternary ammonium anion-exchanger. The anionic cryogel had a ligand density of 121.6 mmol/L and the HETP ranged from 0.18 to 0.27 cm. The activated cryogel was applied to capture L-asparaginase from filtered broth. Yield and purification fold (PF) were optimized using the response-surface methodology, resulting in optimum values of 56 % and 7.6, respectively. The effect of flow velocity over PF was also analyzed, and the results showed that PF had an average value of 8.5 and did not vary with the fluid velocity, confirming the convective mass transfer property of the activated cryogel. The results showed that the activated cryogel shows advantages in using unclarified broth without pretreatment and works at a high flow rate with the perspective of reducing unit production cost.

Fibrin-Rhamnogalacturonan I Composite Gel for Therapeutic Enzyme Delivery to Intestinal Tumors.

Therapy of colorectal cancer with protein drugs, including targeted therapy using monoclonal antibodies, requires the preservation of the drug's structure and activity in the gastrointestinal tract or bloodstream. Here, we confirmed experimentally the fundamental possibility of creating composite protein-polysaccharide hydrogels based on non-degrading rhamnogalacturonan I (RG) and fibrin as a delivery vehicle for antitumor RNase binase. The method is based on enzymatic polymerization of fibrin in the presence of RG with the inclusion of liposomes, containing an encapsulated enzyme drug, into the gel network. The proposed method for fabricating a gel matrix does not require the use of cytotoxic chemical cross-linking agents and divalent cations, and contains completely biocompatible and biodegradable components. The process proceeds under physiological conditions, excluding the effect of high temperatures, organic solvents and ultrasound on protein components. Immobilization of therapeutic enzyme binase in the carrier matrix by encapsulating it in liposomes made from uncharged lipid made it possible to achieve its prolonged release with preservation of activity for a long time. The release time of binase from the composite carrier can be regulated by variation of the fibrin and RG concentration.

Pharmaceutical and Biopharmaceutical Industries: Revolutionizing Healthcare

The drug and biopharmaceutical enterprises play a pivotal part in transforming healthcare through the incident and delivery of creative cures and remedies. This item explores the key facets of these areas, stressing their impact on healthcare. Pharmaceuticals, outlined as wealthy secondhand in the diagnosis, situation, or stop of disease, aim to restore, correct, or refine everyday functions. On the other hand, biopharmaceuticals (or biologicals) circumscribe sugars, proteins, nucleic acids, living containers, or tissues and are curative devices that arise natural beginnings to a degree persons, animals, or microorganisms. In contrast to common pills combined with synthetic processes, biopharmaceuticals are primarily acquired through unaffected processes, containing extraction from living constructions or production utilizing alteration of genetic material Table 1. • Some usual biopharmaceuticals, originally gleaned from animals or persons, are immediately created through biotechnological advancements. • For instance, healing insulin, previously gleaned from porcine pancreatic islets, is immediately made utilizing alteration of genetic material in yeast (Saccharomyces cerevisiae) or E. coli. • Biopharmaceuticals caused by alteration of genetic material usually fall into three classifications: • Substances nearly equal to the body’s own key signaling proteins. • Monoclonal antibodies look like those caused by apiece human immune plan against bacteria. • Receptor builds (fusion proteins) established uniformly happening receptors connected to the immunoglobulin frame. Examples include From living systems: Whole blood and ancestry parts, organs and fabric transplants, stem containers, antibodies for inactive immunization, polluted microbiota, human bosom milk, and human reproductive containers. Produced by recombinant DNA: Blood determinants, fabric plasminogen activators, hormones, hematopoietic growth determinants, interferon, interleukin-located produce, vaccines, monoclonal antibodies, tumor loss determinants, therapeutic enzymes. • Key dispute Pharmaceutical manufacturing • Biopharmaceuticals • Healthcare strike • Innovative medicines • Therapeutic fragments • Recombinant DNA technologies • Personalized cure • Gene medicines • Regulatory processes

Quality by design approach implementation in media development for the production of therapeutic enzyme

Quality by design approach implementation in media development for the production of therapeutic enzyme