Enzyme-Immobilized Nanomaterials for Endocrine Disorders: Therapeutic Innovation and Translational Challenges

Abstract

Endocrine disorders result from chronic disturbances in the hormonal regulation and feedback control, which cause metabolic, reproductive, and systemic complications. Despite the advances in disease management with current therapies such as hormone replacement or multi-pathway pharmacological therapies, problems with variable patient response, side effects and the need for continuous monitoring and dose adjustment remain. To overcome some of these limitations, enzyme-immobilized nanomaterials have become attractive platforms for providing controlled biochemical interactions, catalytic activity and enzyme stability for therapeutic and diagnostic purposes. Enzymes can be immobilized using adsorption, covalent binding, cross-linking and entrapment to minimize denaturation, enhance reusability and improve operational performance. At the nanoscale level, other advantages of having high surface area and tunable interfacial chemistry are the better orientation of the enzyme, mass transfer and control over enzyme activity. There are other benefits for magnetic and field responsive nanomaterials in recovery, re-use, scalable processing and regenerable biosensing systems. In this review, the therapeutic potential of enzyme-immobilized nanomaterials for endocrine diseases has been discussed, focusing on the concepts of nanobiocatalyst design, biosensing for endocrine diseases, and the promise of biomanufacturing in the same field. It also points to some of the main hurdles facing the clinical translation of these technologies, such as problems with enzyme leaching, diffusion limitations, loss of interfacial activity, reproducibility issues, long-term stability, and safety evaluation requirements.

Introduction

Endocrine disorders are a long-standing clinical problem, as hormonal imbalance is not often limited to a specific gland, pathway, or symptom domain.¹ The fact that endocrine dysregulation represents a system-level phenotype but not the simplistic linear malfunction is supported by the mechanistic role of adipose-tissue signaling, systemic inflammation, and the intestinal microbiota in the pathogenesis of insulin resistance. But despite efficacy, there are still difficulties in tolerability, response variability, adherence to treatment and the engineering problem of how to administer and monitor therapy in a manner that maintains accuracy and safety in the long run.² The complexity of the field is highlighted by reproductive endocrine disorders. The endocrine dysregulation is usually manifested by polycystic ovary syndrome (PCOS), thyroid dysfunction, luteal phase defects, hyperprolactinemia, and adrenal involvement in female infertility which requires a multi-marker diagnostic strategy instead of depending on a single laboratory parameter.³ The extent of application of hormonal profiling and imaging to clinical care pathways is demonstrated by observational studies that indicate that a collective approach to diagnosis can enhance conception rates in a cohort of patients.⁴ At the same time, recent literature documents novel diagnostic and curative developments, in particular, personalized treatment plans based on endocrine pathophysiology and individual biological characteristics. The identified trends highlight an essential need in the field of endocrinology, ie, therapeutic developments should be supported by platforms that stabilize bioactive constituents, carefully control their presentation under functional forms, and enable measurement-based decision-making.^5,6

The global burden of endocrine disorders is increasing, particularly emphasizing their clinical relevance. In 2022, diabetes alone was the cause of almost 830 million people worldwide and is growing at a higher rate in countries with low and middle income. IDF also estimates that there are currently 589 million adults aged 20–79 years with diabetes, a figure that will grow to 853 million by 2050.⁷ Reproductive endocrine disorders also make a significant contribution to burden with Polycystic ovary syndrome estimated to affect 10–13% of women of reproductive age and as many as 70% of women with the disorder may not be diagnosed.⁸ In addition, thyroid conditions are very common, it is estimated that 20 million people in the United States alone suffer from thyroid conditions.⁹ The size of these numbers shows that endocrine disorders are a significant diagnostic, treatment and monitoring burden. Thus, there is real-world relevance of platforms which enhance the detection of biomarkers, enzyme stability, catalytic control and long-term monitoring in endocrine medicine.

The correlation between enzyme biology and endocrine disease is the motivation to study enzyme-immobilized nanomaterials. The enzymatic reactions that are needed for hormone biosynthesis, peripheral conversion, degradation and signal modulation are characterized by high degrees of regulation and are crucial for endocrine homeostasis. Several endocrinological diseases involve dysregulated reactions in steroid hormone production, activation of thyroid hormone, glucose metabolism, incretin signaling and reproductive hormone balance. So, enzymes are not only factors peripheral to endocrine disease but are important determinants of the amount of hormone available and the effects of hormones on the biological system. The therapeutic and diagnostic implications involve the development of enzyme based systems that are able to modulate biochemical pathways of interest in disease or can be used to detect soluble endocrine markers with high specificity. However, the use in clinical and technological applications is restricted because of the lack of stability, limited functional lifetime, sensitivity to environmental conditions and the inability to be used in repeated and controlled applications. One of the methods to overcome these barriers is by immobilizing enzymes onto the nanomaterials. When immobilized on engineered nanoscale supports, immobilization can enhance the structural stability of enzymes, maintain catalytic activity, allow for reusability and enable the use of the enzyme in biosensors, responsive therapeutic platforms, and scalable biomanufacturing systems. The added value of these benefits is especially important in endocrine diseases, as the requirement for continuous monitoring and dose adjustment and biochemical control can be extended for long periods.^10–12

The immobilization of enzymes on nanomaterials has become a solid engineering tool to promote the stability, working capacity, and reutilization of biocatalysts in harsh environments.¹³ Immobilization methods, including adsorption, covalent attachment, entrapment and cross-linking may provide resistance to pH and temperature variation and enable the removal of catalysts and reuse in a batch or continuous process. The benefits of nanomaterial supports are the high surface area, fine-tunable interfacial chemistry, and in reaction kinetics, which lead to high enzyme loading capacities and rate acceleration, respectively.¹⁴ Simultaneously, there is no passive interface between the enzyme and the nanomaterial. Adsorption of proteins can change conformation and activity, and the orientation of the enzyme with respect to the support may block or preserve the active site and rational design and characterization are consequently essential to achieve translational applicability.¹⁵

Magnetic nanoparticles have received special interest among nanomaterial platforms due to the ability to integrate catalytic stabilization with easy separation and reuse. The literature on immobilization suggests that magnetic nanomaterials exhibit superparamagnetic characteristics and thus allow rapid recovery in the presence of an external magnetic field and redispersion in the absence of that field. This permits the intensification of processes as well as reducing downstream handling demands.^16,17 Magnetite-based systems are also commonly mentioned due to their beneficial physicochemical characteristics and wide usage. To increase stability, reduce aggregation, and introduce reactive groups, surface coatings and functionalization strategies are used. However, there are still drawbacks, which include diffusion constraints in high loading, the tendency of enzyme leaching in the case of noncovalent attachment, and reproducibility issues during scale-up.¹⁷

Although the techniques of enzyme immobilization and nanomaterials-based biomedical platforms are becoming more popular, their applications in the field of endocrine diseases are still not well correlated in the literature. Much work has been done on nanobiocatalysis, biosensor devices, and drug delivery, yet these platforms have not been specifically linked to endocrine-related needs like hormone control, metabolic control by enzyme, biomarker monitoring, long time stability, and safety evaluation in disease-relevant settings. This review aims to fill in that void and highlights the applications of enzyme-immobilized nanomaterials in endocrine therapy, diagnosis, and translation. It marks a difference from the current literature, which focuses on general enzyme immobilization and nanomaterial engineering to the endocrine specific relevance of the platforms. The novelty of the present review is that it connects the endocrine pathophysiology with the design principles for enzyme immobilization, selection of nanomaterial platform, biosensing potential and practical challenges that need to be addressed to meaningfully advance enzyme immobilization-based systems in the clinical and biomanufacturing fields. It highlights the potential of immobilized enzymes to facilitate the detection of biomarkers related to the hormones, the metabolic pathways regulated by enzymes, localized catalytic activity, regenerable biosensing and translation development under endocrine-relevant biological conditions.

Conceptual Framework and Scope of This Study

Perspectives on Hormonal Imbalance in Endocrine Disorders

According to this narrative review, hormonal imbalance is a system-level phenotype that is a result of interactions between endocrine axes and tissue-derived signals, and not a defect in a single pathway.¹⁸ This approach is consistent with the current endocrine literature that describes obesity and type 2 diabetes as multifactorial disorders under the influence of adipose endocrine signalling, inflammation, and gut-related modulators, as well as with therapeutic considerations that emphasise the need to use multifactorial approaches to support biological heterogeneity.¹⁹ Conversely, the engineering rationale directly follows that if endocrine disease trajectories are network-driven and patient responses are variable, then enabling technologies that stabilize biochemical function, improve controllability, and support measurement-aligned decision-making become relevant complements to pharmacology.^20,21

Enzymes and Endocrine Disorders

Enzymes are central to endocrine disorders because they set hormone availability at three levels: (1) biosynthesis, (2) activation/inactivation, and (3) prohormone processing.²² Perturbation to the activity or expression of the enzymes in the cytochrome P450 can redirect the total hormonal production in steroidogenic tissues. As an example, CYP17A1 dysregulation causes disproportion of the precursor flux towards androgen production, whereas the estrogen-androgen balance is impaired by abnormal CYP19A1 (aromatase), thus adding to estrogen-sensitive pathophysiology.^19,23,24 Thyroid disorders are good examples of such enzyme bottlenecks. The reduced activity of thyroid peroxidase (TPO) and/or inadequate DUOX2-derived H2O2 reduces the iodination and coupling reactions required in thyroid biosynthesis of T3/T4, resulting in hypothyroidism, which is caused by a faulty hormone production phenomenon, not limited to defective glandular signaling.²⁵ Mainly, endocrine phenotypes can arise even when circulating hormone levels appear near-normal. As an example, the altered thyroid hormone activity can be caused by the changes in deiodinases (DIO1/DIO2/DIO3) which regulate the metabolic and developmental processes by changing local T3 production or breakdown.²⁶

Similarly, cardiometabolic effects of glucocorticoid excess may be increased at the tissue level through an enzyme, 11β-HSD1, which changes cortisone into cortisol. On the other hand, mineralocorticoid receptors are protected by 11β-HSD2, which inhibits cortisol; interactions between the two isoforms can thus put individuals at risk of hypertension and metabolic risk due to their modified receptor access.²⁷ In peptide endocrinology, a defective post-translational processing may cause a so-called hidden hormone deficiency, where hormone production is not defective but they do not fully mature. The mutations that result in defects of PCs include PCSK1/3, PCSK2,²⁸ or carboxypeptidase E (CPE), disabling the transformations between prohormones and mature peptides, therefore reorganizing the pancreatic and enteroendocrine hormone environment relevant to the metabolic disease.²⁹ Finally, circulating enzymatic cascades form the basis of endocrine systems, such as the renin -angiotensin system. Angiotensin II is synthesized as a result of one cleavage by renin followed by a complementary transformation by angiotensin-converting enzyme (ACE); then the enzyme activity directly regulates the concentrations of vasoconstrictive hormones and, therefore, blood-pressure phenotypes.³⁰ Collectively, the examples above highlight the idea that many endocrine pathologies are enzyme synthesis-, enzyme activation-, or enzyme processing-related intrinsic enzyme nodes that are prevalent diagnostic biomarkers and treatment targets, as outlined in Table 1.

Table 1.

Examples of Enzyme and Endocrine Disorders

Endocrine Hormones	Enzyme Examples	Endocrine Role of the Enzyme	Mechanism of Dysfunction	References
Thyroid (T3/T4 synthesis)	TPO, DUOX2	TPO catalyzes iodide oxidation, iodination, and coupling; DUOX2 supplies H2O2 required for these reactions	Reduced enzyme function leads to impaired iodination and coupling, resulting in decreased thyroid hormone synthesis and hypothyroid phenotypes	[26]
Thyroid (local activation/inactivation)	DIO1/DIO2/DIO3	DIO1 and DIO2 activate T4 to T3, whereas DIO3 inactivates T4 and T3	Altered deiodinase balance can drive tissue-specific hypo- or hyperthyroid signaling even when serum hormone levels change only modestly	[25]
Glucocorticoids (tissue cortisol availability)	11β-HSD1, 11β-HSD2	11β-HSD1 regenerates cortisol locally; 11β-HSD2 inactivates cortisol to protect MR in the kidney and other tissues	Increased 11β-HSD1 promotes local cortisol excess and metabolic risk, whereas reduced 11β-HSD2 permits cortisol to inappropriately activate the mineralocorticoid receptor, contributing to hypertension-like effects	[27]
Steroid sex hormones (estrogen/androgen balance)	CYP19A1 (aromatase); also, steroidogenic pathway enzymes broadly (eg, CYP17A1)	Aromatase converts androgens to estrogens, and downstream pathway enzymes determine overall steroid output patterns	Dysregulated enzyme expression or activity leads to estrogen–androgen imbalance, contributing to estrogen-related disorders and reproductive endocrinopathies	[23]
Peptide hormones (maturation/processing)	PCSK1/3, PCSK2, CPE, PAM	Convert prohormones to mature active peptides; trimming (CPE) and amidation (PAM) finalize bioactivity for many peptides	Impaired processing results in reduced bioactive hormone availability despite preserved synthesis, leading to a “functional deficiency” and altered gut and pancreatic peptide profiles relevant to metabolic disease	[28]
RAAS (angiotensin II production)	Renin, ACE	Proteolytic cascade: renin makes Ang I; ACE makes Ang II	Increased cascade activity drives elevated angiotensin II signaling, leading to vasoconstriction and blood pressure dysregulation, and positioning key enzymatic steps as therapeutic targets	[28]
Catecholamines (dopamine/NE/E synthesis)	Tyrosine hydroxylase (TH)	Rate-limiting synthesis of catecholamines; rapid regulation via phosphorylation and feedback	Enzyme regulation changes catecholamine output under stress/endocrine-adrenal signaling; dysregulation can alter neuroendocrine tone	[31]

Enzyme Immobilized Nanomaterials as Enabling Platforms

Enzyme immobilization here is conceived of as a platform strategy that reconfigures soluble biocatalysts into a form of heterogeneous architecture, which increases stability of operation and allows reuse,³² while nanomaterial supports contribute high surface availability, and tunable interfacial chemistry that can influence loading, microenvironment, and functional durability.³³ The fact is supported in the literature that orientation effects and conformational transitions can be used to regulate enzyme activity using nanomaterial interfaces, thus highlighting the importance of performance sensitivity to the rational design of enzyme-support interactions, other than via carrier selection.³⁴ When choosing the immobilization method, it is essential to consider high retention of catalytic activity and strong enzyme retention. Loading, orientation, leaching, stability, reusability and access of the substrate are influenced by functional groups, reaction pH, temperature, spacer length, and concentration of the cross-linker and coupling chemistry. Too much covalent bonding or cross-linking can enhance retention by restricting the activity conformationally.^35,36

The nanoparticles can serve as delivery vehicles for enzymes: they can prevent enzymes from denaturation, from being broken down by enzymes, from being cleared by the immune system and from losing their activity too early. They can have a high surface area, tunable pores, and modifiable surfaces for enzyme loading, targeted delivery, cellular uptake, and controlled/stimuli-responsive release. There are four main factors that are used to optimize release behavior: composition of matrix, pore size, surface coating, and rate of degradation and pH sensitivity. For biomedical applications, however, the issues of biocompatibility, toxicity, biodistribution, and off-target effects must be carefully evaluated. Particular properties to be assessed for a potential biomedical, industrial or environmental application should include particle size, morphology, zeta potential, loading efficiency, release kinetics, retention of enzyme activity, stability and cytotoxicity.^10,37

The immobilized enzyme performance is affected by nanoscale properties which govern the enzyme orientation, accessibility of active sites, diffusion of substrates and conformational stability. Enzyme-support interactions can be influenced by surface curvature, charge, porosity, functional groups, and hydrophobic/hydrophilic balance, which in turn can result in changes in Km, Vmax, activity retention, and stability. Excessive binding or an unfavorable orientation may enhance retention, but decrease catalytic efficiency.^38,39 Immobilized enzymes are affected by nanoscale properties in terms of orientation, accessibility of the active site, diffusion of substrate and conformational stability. Any change in surface curvature, charge, porosity, functional groups, and hydrophilic/hydrophobic balance, can change the enzyme-support interaction, which can impact Km, Vmax, activity retention, and stability.^39,40

Endocrine as Clinical and Translational

Two major areas of clinical concern in endocrine literature are described by a diffuse dysregulation of hormones, which can be studied with increasingly dynamic and changing paradigms of therapy. The initial area is metabolic endocrinology, where modern trends in hormone-based pharmacotherapy and polypharmacy interventions have expanded the pharmacologic repertoire; however, the sustainability of clinical performance depends on how well the drug is tolerated, how well the patient adheres to the therapy, and how individuals differ in terms of pharmacodynamic reactions.⁴¹ The second domain is related to reproductive endocrine dysfunction, where conditions such as polycystic ovary syndrome (PCO) and thyroid-related disorders are often explored within the framework of new methods of diagnostic approaches and the approach of precision-medicine treatment, thus outlining the overall need to apply systematic phenotyping and constant monitoring of patient outcomes. Collectively, these illustrations support the fact that endocrine heterogeneity gives rise to a pressing demand to have robust platforms that guarantee the presence of trustworthy biochemical stability and quantitative fidelity.⁴²

Nanomaterials Classes

The major nanomaterials used for enzyme immobilization are carbon nanotubes, graphene/graphene oxide, metal and metal oxide nanoparticles, silica and mesoporous silica, polymeric nanoparticles, lipid-based carriers, metal-organic frameworks and magnetic nanoparticles. Their performances are highly reliant on their size, morphology, porosity, surface area, charge, functionalization, hydrophilicity/hydrophobicity and magnetic behavior. The high surface area and porosity leads to higher enzyme loading and functional groups and surface charge leads to better attachment and orientation. Some hydrophilicity might help retain enzyme conformation whereas too much hydrophobicity can lead to denaturation. Magnetic nanoparticles are particularly useful as they can easily be separated, recovered, reused and render the operation more stable in repeated cycles of catalysis or biosensing.^43,44

The nanomaterials scope is concerned with the carrier classes that are dominant in the immobilization literature and provide unique operational benefits that can be applied in the context of translational research. Carbon-based materials, metal and metal-oxide nanoparticles, polymeric supports, and magnetic nanoparticles can be regarded as major categories, since they all represent the main material design space disclosed in the existing literature. Magnetic systems have been highlighted as a high-utility platform due to the ability to recover fields mediated to allow useful reuse loops and easy separation, and due to the ability to recover and redispersing quickly, repeatedly discussed as operationally favorable in the immobilization literature.^45,46

Organization of Synthesis and Interpretation Discipline

The synthesis is performed in terms of merging endocrine requirements with platform capabilities in three translational domains. The first area deals with biocatalysis, which enables biomanufacturing steps where biocatalytic immobilization allows repeated cycles and improved process management.⁴⁷ The second area is the biosensing domain, focusing on monitoring, such that nanomaterials can enhance the stability and sensitivity of enzyme-based interfaces, provided they are controlled by orientation and conformational stability. The third area concerns the localized catalytic modulation, which is considered to be an experimental frontier and not a well-explored therapeutic modality.⁴⁸ In these directions, an interpretive discipline is maintained by allocating equivalent appraisal significance to reported constraints and benefits, especially in the critical case of diffusion constraints at high loading circumstances and the danger of leaching enzyme, which can significantly diminish quantitative reliability over time.¹⁴

Core Concept of Enzyme Immobilization and Nanomaterials

Enzyme immobilization has become an inseparable part of the biochemical engineering field as a way to overcome the main shortcomings of the free enzymes, such as structural instability, rapid inactivation, and limited workability.⁴⁹ Enzyme immobilization refers to the process of immobilizing enzymes in physical form by confining them on a solid surface or immobilizing them by means of chemical bonds, in this respect stabilizing enzymes and allowing their reuse, and in any case realizing that the immobilization process may alter kinetic behavior and introduce transport limitations.⁵⁰ Over the last several years, nanomaterials have received significant attention as a novel nanoscale-based enzyme immobilization support. Such materials provide benefits compared to traditional carriers, which is due to their large surface area, surface chemistry capability, mechanical strength, or biocompatibility.¹⁴ Enzyme-nanostructure interaction has been associated with a number of advantages, such as enhancement of enzyme loading, minimization of conformational distortion, and prolonged catalytic stability.⁵¹ These advantages are attributed to the close contact between the enzyme and the nanomaterial surface that may stabilize the tertiary structure of the protein, as well as suppress denaturation pathways. Examples of nanomaterials that have been considered in this application include carbon-based nanostructures, magnetic nanoparticles, metal-organic frameworks, and polymeric nanoparticles.⁵² The most promising type is magnetic nanomaterials, which, due to the magnetic responsiveness of magnetism, are easily recoverable and recycleable in magnetic fields. This feature is often mentioned as an advantage, as it contains ease of separation operation and makes the immobilization of enzyme systems more operationally practicable.⁵³

The immobilization of enzymes on nanoparticle supports can be categorized as reversible and irreversible (covalent). Reversible methods, such as physical adsorption, ionic binding and affinity binding, involve weak interactions such as hydrophobic, electrostatic, hydrogen-bonding or ligand-specific interactions. They are simple, mild, and sometimes maintain the catalytic activity, but may result in enzyme leaching and decreased operational stability. Stronger retention, higher thermal stability or storage stability, and greater reusability are achieved with irreversible methods, such as covalent coupling, cross-linking or entrapment/encapsulation, although a decrease in activity may occur as a result of conformational restriction or mass transfer limitations. These factors of enzyme structure, active-site orientation, support chemistry, substrate size, diffusion behavior, stability requirements, toxicity, cost, scalability, and application context should all be taken into account in the selection of a strategy.^36,54

Nanoparticles can disrupt the endocrine balance if they induce the production of reactive oxygen species, inflammation, and mitochondrial damage and apoptosis in hormone-sensitive organs. Oxidative stress can cause a decrease in the antioxidant capabilities, and an activation of the NF-κB/cytokine pathways, while the damage to mitochondria decreases the ATP levels and affects the steroidogenic machinery. Nanoparticle exposure in reproductive endocrine cells has been linked to changes in caspase activity, BAX/BCL2 ratio, and a decrease in the expression levels of steroidogenic enzymes, which can result in altered synthesis of oestrogen, progesterone or testosterone. These effects can disrupt the biosynthesis, secretion, feedback regulation and tissue response to hormones. So dose, surface chemistry, corona and long-term safety should be carefully considered in the application of endocrine.^55–57

Besides increasing reusability and stability, enzyme immobilization increases catalytic properties by altering the immediate microenvironment of the active site. The reduction of product-inhibition, aggregation, and, in specific systems, increased binding affinity of substrates can be effected by alteration of surface charge, by alteration of hydration state, and accessibility of the substrate.⁵⁸ Specialized nanomaterial further optimizes the orientation and intermolecular spacing of enzymes and enhances catalytic activity and the reduction of steric hindrance.⁵⁹ In turn, immobilization is gradually gaining recognition as not only a stabilization method but also a tool of functional optimization in therapeutic and diagnostic practices.^60,61

Significantly, immobilization has enabled the development of advanced therapeutic enzyme systems, which have increased circulatory duration, reduced immunogenicity, and enhanced clinical results.¹⁰ As an example, asparaginase, which is also widely used in the treatment of acute lymphoblastic leukemia, is immobilized on the polymeric nanoparticles and porous silica matrices to stabilize the serum and reduce hypersensitivity reactions.^62,63 PEGylated nanocarriers or magnetic particles conjugated with immobilized uricase used in the treatment of hyperuricemia and gout show a longer half-life and continue to degrade uric acid.⁶⁴

Endocrine Disorders and the Therapeutic Innovation Landscape

Instability, inaccuracy and inadequate monitoring are the limitations of current endocrine therapies. Many hormone and protein therapies are repeatedly administered and must be adjusted for individual patients; pharmacokinetics, absorption, metabolism and tissue responsiveness differ from patient to patient. Insulin therapy in diabetes enhances glycemic control; however, it is associated with risks of hypoglycemia, glycemic variability and failure of adherence. The conventional monitoring is also flawed: HbA1c is an average of glycemia over time and not a reflection of the short-term variability and hypoglycemia; SMG is only giving “snapshot” information. Factors affecting thyroid hormone absorption, comorbidities, age, body weight, pregnancy, and drug interactions also affect thyroid hormone requirements, requiring ongoing monitoring with thyroid hormone replacement.^65,66

Metabolic and Endocrine Disorders as Multi-Axis Network Dysregulation

There is a growing consideration of obesity and type 2 diabetes as disorders of integrated endocrine signalling as opposed to isolated defects in glucose handling. Obesity-related endocrine abnormalities can include impairments in hypothalamic-pituitary axes and disturbed adipose-derived hormone signalling, and adipose tissue is an endocrine gland that secretes substances like leptin and adiponectin.⁶⁷ Endocrine functional remodeling is of great clinical significance as it coordinates the energy storage process with the mechanisms of appetite, insulin sensitivity, inflammatory conditions, and cardiometabolic risk. In such a conceptual paradigm, insulin resistance is not limited to one lesion, but is a multifactorial phenotype with inflammatory, gut microbiome, and adipose tissue biology components that interact in a manner that eventually disrupts glucose homeostasis and gradually stresses β-cells. As a result, therapeutic innovation is more likely to address numerous nodes on this network than single endpoints.⁶⁸

Hormone-Based Pharmacotherapy and Combination Strategy Evolution

Hormonal-based therapeutic strategies have expanded at an increased rate, and incretin biology has become the central translational pathway that connects the signaling pathways in the intestine to the control of metabolic processes in the system. Salient pharmacological innovations, such as glucagon-like peptide-1 (GLP-1) receptor agonists and sodium-glucose cotransporter-2 (SGLT-2) inhibitors, play a role in the improvement of glycaemic control and weight management support, along with bariatric surgery, which remains a highly effective treatment for properly selected patients.⁶⁹ At the same time, endocrine pharmacology is becoming paradigm-based, with combinatorial approaches that aim to act on synergistic pathways, such as co-agonist regimens and multi-hormone interventions that seek to not only work harder to achieve better therapeutic effects but also to solve the tolerability problems. The reason behind this change is the fact that metabolic disease is often characterized by heterogeneous etiological drivers, such as a difference in the regulation of appetite, the ability to secrete insulin, the distribution of adipose tissue, and the presence of inflammatory burden, in such a way that monolithic mechanistic therapies are likely to have heterogeneous effects on different patient groups.^70,71

Precision Endocrinology and Biomarker-Guided Therapeutics

The concept of precision endocrinology has been developed in response to the realization that therapeutic responses are dependent on the personal biological variable, comorbidity, and pathway heterogeneity. Empirical research in obesity and diabetes has led to an emphasis on the need to integrate personalized medicine paradigms that explicitly target individual biological pathways and genetic alleles to improve the predictability of outcomes, as well as reduce the use of trial-and-error prescribing approaches.⁷² The practical importance of systematizing observation and reporting of physiological parameters is that it is most often more important not only because it can give information on glycemic indices, but also because it can provide information on the upstream factors, including appetite regulation, flexibility in metabolism, and adverse effect susceptibility. In this context, stabilization of bioactive constituents, controlled delivery, and solid sensing are multiplicative determinants of therapeutic efficacy, since they improve reliability and decision level without extending the cognitive spectrum of therapeutic genus.⁷³

Reproductive Endocrine Dysfunction and Infertility Hormone Axes

Endocrine-related infertility illustrates how hormonal imbalance can emerge from interacting disturbances in ovarian function, thyroid signaling, pituitary regulation, and adrenal contributions. The cross-sectional studies of infertile patients indicate that the common factors are polycystic ovary syndrome (PCOS) and thyroid dysfunction, but luteal phase defects are as well common; the diagnostic processes nearly always include ultrasonography in addition to hormonal profiling. The therapeutic approaches used in this area of study often include multi-layer interventions that include pharmacologic drugs, assisted reproductive technologies (ART) and lifestyle modifications. It has also been reported that combination protocols increase conception rates in a subset of patients. These results support a broad rule of endocrine therapeutics: the most successful results are conditional on the presence of specific interventions based on the individual endocrine phenotype and the monitoring of the therapeutic response with the help of appropriate biomarkers.^74,75

Translational Implications for Platform Stabilization and Monitoring

There are two recurrent translational imperatives in the fields of metabolic and reproductive endocrinology. The former is the concept of durability, which is the ability to have long-term biological action without the rapid attenuation, adverse effects, or untimely discontinuation. The second one relates to controllability, which is the ability to control the dosing intensity and timing, test the biological response, and adjust therapy in accordance with quantifiable indicators.⁷⁶ These requirements create an innate interface with enzyme immobilized nanomaterials that were designed to increase stability, regulate activity through supporting chemistry and allow reuse or lifelong activity in engineered environments. These principles are relevant in industrial catalysis as well as in other fields. The same processes that stabilize enzymes and regulate their activity, including surface functionalization, control of orientation, and denaturation resistance, are also necessary to support strong biosensing and robust biochemical conversion systems that have the potential to support future endocrine biosensing or therapeutic adjuncts.⁷⁷

Nano-Biocatalyst Platforms for Endocrine Innovation

Nano-biocatalyst platforms can offer an important pathway for endocrine innovation that merges the specificity of an enzyme with the stability, high surface area and tunable surface chemistry of the nanoscale platform, thus supporting the concept of investigation of their potential use as immobilization matrices.⁷⁷ Enzymes immobilized on nanomaterials can exhibit enhanced operational stability, reusability, catalytic activity and tolerance to severe biological and/or processing conditions. In the endocrine area, they are particularly significant for biosensing of glucose, detection of hormones/metabolic biomarkers, explorative localized biochemical modulation and regenerable diagnostic systems. They have potential ability to connect the activity of the enzymes with controlled interfaces and signal amplification, which is however limited by the need to be safe, reproducible and free of protein corona. Considering the increasing attention in endocrine medicine to stability, personalisation, and measurement-directed care, a targeted evaluation of enzyme-immobilised nanomaterials is justified as of now.⁷⁸

Principles of Enzyme Immobilization on Nanomaterials

Immobilization of enzymes is designed in a manner that soluble catalysts are converted into heterogeneous systems, which allows an increase in operational control. The idea behind it is that immobilized enzymes may exhibit greater functional stability in conditions that normally affect native structure, that is, thermal stress, pH changes, and solvent exposure, and also allow catalyst recovery and reuse in a series of iterations. To be translationally relevant, these improvements must be determined by quantifiable parameters, including retention of catalytic activity after repeated operations, activity over longer environmental distances and reproducible activity during storage and handling.⁴⁹ Immobilization of the enzyme to nanomaterial-based systems can achieve much more than simple enzyme stabilization since nanoscale carriers, due to their large surface area and tunable interfacial chemistry, can modify the enzyme microenvironment, control its orientation, and impact on substrate accessibility.⁷⁹

Immobilization-Induced Stability and Reusability

Immobilization methods can be classified into physical, Physicochemical and chemical modes of attachment. Physical adsorption is a technique often preferred due to its simplicity of operation and the low amounts of ancillary reagents needed. This strategy relies on weak interfacial interaction, such as H-bonding, Van der Waals force, hydrophobic interactions, and electrostatic interactions. Its major weakness lies in the fact that these interactions are reversible in nature and therefore, although they do not alter the native conformation of the enzyme, allow desorption and resulting performance drift to changes in ionic strength, temperature or pH.^80,81 Covalent coupling, used to chemically immobilize the enzyme, is mainly used when long-term retention and resistance to leaching are necessary; however, covalent immobilization can decrease apparent enzyme activity by altering the geometry of the active site or inhibiting the conformational activity necessary to cause catalysis.¹³ Entrapment and crosslinking techniques can also be used to enhance enzyme retention, in which case the crosslinking process helps to form enzyme networks that do not rely on carrier surfaces; however, in dense matrices, the mass-transfer resistances can become overwhelming. In the range of possible strategies, the decision is not wholly chemical; instead, it is an engineering choice that trades stability, activity, diffusivity, and manufacturability to the application context of interest.⁸¹ Enzyme immobilization technologies can broadly be categorized into reversible and irreversible methods, with each possessing a unique stability profile, activity profile, and reusability profile as shown in Figure 1.

Figure 1.

Classification of enzyme immobilization strategies using nanoparticle supports, illustrating reversible and irreversible attachment modalities, including adsorption, ionic and affinity binding, covalent coupling, cross-linking, and entrapment-based encapsulation.⁷⁷

Immobilization Strategy Taxonomy and Structure Function Tradeoffs

Nanomaterials present promising carriers of biocatalysts due to their large surface-area-volume ratios, which thus provide greater enzyme loading and consequently high catalytic productivity per gram of carrier and at the same time reduce the volume of reactors needed.⁸² Nonetheless, this advantage is not universal; an increase in enzyme concentration can give rise to diffusion constraints that hinder the delivery of substrate and product adjacent or inside the immobilization support, which work against the apparent activity despite a high loading.¹⁴ In this regard, nanomaterials should be selected based on parameters, which include porosity, surface chemistry and dispersion. In practice, performance arises from the interaction between nanoscale geometry and enzyme microenvironment rather than from surface area alone, which is why comparative studies often emphasize the need for matrix design optimization rather than simple carrier substitution.⁸³ Nanoparticle carriers used for enzyme immobilization and delivery exhibit distinct physicochemical properties that determine loading capacity, stability, biocompatibility, and transport behavior.⁵³ A comparative overview of major nanoparticle classes is presented in Table 2. Though the classes of nanoparticles listed here do not deal exclusively with endocrine disorders, they are relevant in the context of immobilized enzymes and their performance in therapeutic, diagnostic and biomanufacturing applications, as their physicochemical properties directly impact the performance of the immobilized enzymes.

Table 2.

Overview of the Benefits and Limitations of Different Nanoparticle Classes for Enzyme Delivery and Immobilization

Nanoparticle Type	Characteristics	Benefits	Limitations	References
Liposomes	Phospholipid bilayer vesicles incorporating cholesterol, capable of encapsulating hydrophilic and hydrophobic molecules, and adaptable to multiple administration routes	Biodegradable, low toxicity, versatile delivery, suitable for protein and enzyme encapsulation	Larger particle size limits tissue penetration, reduces deformability, stability and scale-up challenges	[84]
Transfersomes	Elastic bilayer vesicles containing edge activators, designed primarily for transdermal transport	High flexibility and deformability; enhanced skin penetration; efficient enzyme permeation under non-occlusive conditions	Limited capacity for hydrophobic compounds; reduced efficiency under occlusive conditions	[85]
Ethosomes	Vesicles composed of phospholipids, cholesterol, ethanol, and water; optimized for transdermal and systemic delivery	Enhanced skin penetration; suitable for hydrophilic and lipophilic compounds; improved systemic absorption	Ethanol may reduce enzyme stability; long-term safety requires further validation	[84]
Niosomes	Vesicles formed from non-ionic surfactants and cholesterol; single or multilamellar structures	Cost-effective alternative to liposomes; improved stability; suitable for enzyme and protein delivery; enhanced transdermal permeation	Requires optimization for large-scale production; aggregation during storage	[85]
Solid lipid nanoparticles	Nanocarriers composed of solid lipids at physiological temperature; mainly used in topical formulations	Biocompatible; controlled release; effective entrapment of lipophilic molecules	Low loading capacity due to crystallinity; risk of drug expulsion during storage	[86]
Nanostructured lipid carriers	Hybrid systems combining solid and liquid lipids have been developed to improve drug retention	Higher loading efficiency than solid lipid nanoparticles; improved physical stability; prolonged shelf life	Long-term stability and scale-up challenges require further investigation	[86]
Synthetic nanoparticles	Polymeric or inorganic matrices prepared by adsorption, encapsulation, or dispersion; tunable physicochemical properties	High structural integrity; thermal stability; adaptable composition; suitable for diverse payloads	Biodegradability and clearance require evaluation; potential polymer-dependent toxicity	[87]
Metallic nanoparticles	Rigid nanostructures composed of metals such as gold, silver, and iron oxide; customizable surfaces	High stability; recyclability; optical and catalytic properties; suitability for theranostic platforms	Potential toxicity; limited long-term in vivo safety data	[85]

Nanomaterial Classes for Enzyme Immobilization

A variety of nanomaterials have been suggested as supports of enzymes; each has its advantages and disadvantages.⁸⁸ Carbon-based materials, eg, carbon nanotubes and graphene, can freely provide a large specific surface area, as well as conductive properties, and thus must be considered applicable in biosensing applications. Conversely, metallic and metal-oxide nanoparticles are tunable with respect to surface chemistries and selectivity; empirical evidence suggests that scaffolds composed of nanomaterials can stabilize biocatalysts as well as raise their reuse possibilities. The use of magnetic nanoparticles also provides an additional separation mechanism supported through external magnetic fields and thus simplifies the recovery and re-utilization of the catalyst entities during operations.⁸⁹ Several studies indicate that these nanomaterial platforms can not only enhance the stability and reusability of biocatalysts, but can also result in diffusional mass-transfer resistance and enzyme leaching depending on the immobilisation chemistry. Additionally, they require increased preparation complexity and are not scalable because of the problems of reproducibility and environmental sensitivity. These constraints are neither marginal nor ancillary; they are the key determinants of whether a nano-biocatalyst system is simply a laboratory experiment or whether it is translationally plausible.⁷⁷

Nanoparticle Carrier for Enzyme Delivery

The enzyme nanomaterial interface can alter enzyme structure and function through adsorption-induced conformational transitions, formation of protein coronas, or steric blocking of catalytic sites. An immobilization orientation is a decisive factor, and an unfavorable orientation can block the active site or limit the access of substrates even when the enzyme is in its structural integrity. Interfacial phenomena depend on the physicochemical conditions of the surrounding medium, geometrical configuration, dimensional characteristics, surface functional groups, and the physicochemical attributes of nanomaterials.^34,90 Therefore, the effectiveness of immobilization methods requires dedicated attention to the control of the orientational processes and architecture of the microenvironment, especially in the case of translating these systems into sensor platforms or allowing catalytic processes to occur over time; any insignificant structural anomalies can trigger large changes in signal transduction or product synthesis.⁷⁷

Limitations for Endocrine Applications

Two major constraints are worth mentioning in the enzyme immobilization of nanomaterials used in endocrine applications.⁹¹ The key to these challenges lies in how to maintain the temporal stability of the enzymatic activity, which can be accomplished through reducing enzyme leaching, preventing enzyme aggregation, and maintaining the catalytic or sensing activity of the enzyme through repeated operation cycles and over extended periods.⁹² One of the secondary, salient constraints is in control of mass transport processes. It is of utmost importance to put all of these under dense control of diffusion constraints and microenvironmental determinants such that the signals that are recorded would be a result of endogenous biochemical activity, instead of being skewed by mass-transfer artefacts.⁹³ As endocrine applications often require accurate quantitative results to be delivered, be it in securing manufacturing consistency or in accurately detecting biomarkers, these limitations can cause concern at the design stage.⁹⁴ Depending on size, surface charge, coating, dose, duration of exposure and biological environment, nanoparticles can cause oxidative stress, inflammation, mitochondrial damage, apoptosis and/or steroidogenic/metabolic signal transduction. Biological variability, lack of long-term safety data, lack of disease-relevant models, scale-up problems, and regulatory uncertainty are also limitations for translation.⁵⁵

Magnetic Nanomaterials as High Utility Platforms for Enzyme Immobilization

Functional Rationale for Magnetic Recovery and Reusability

Magnetic nanomaterials are among the most widely studied supports in enzyme immobilization because of their high surface area as well as the availability of an external-field-responsive separation mechanism.⁹⁵ These nanoparticles provide a route for recoverable enzyme immobilized systems where their responsiveness allows rapid separation from reaction media, repeated reuse and easier integration into the regenerable biosensing and catalytic workflows. It is beneficial when the design goal is to attain repeated catalyst recovery, recycling, and process intensification without turning to advanced filtration or centrifugation processes.⁹⁶ The magnetic nanoparticle platforms allow repeated or continuous catalytic operation, as immobilization of enzymes ensures rapid and easy recovery of the catalysts by an external magnetic field, which results in a decreased loss of catalysts and an improved reusability. Their performance depends on magnetic strength, stability, recovery efficiency, resistance to aggregation and surface functionalization. These factors have an impact on the enzyme retention, catalytic accessibility, separation efficiency and long term operational stability.⁴⁴ Many studies have found magnetic nanomaterials to be convenient immobilization matrices with high enzyme stability and catalytic activity as well as the ability to recover enzymes rapidly by an external magnetic field. These properties are consistent to the demands of industrial biocatalysis, and are progressively discussed as permissive principles of more translational use, where the functionality of reproducibility and manageability is of the essence.⁵³

Superparamagnetism and Field-Responsive Separation Dynamics

Superparamagnetism is the phenomenon which describes the properties of the magnetic nanoparticles when they are small enough to become strongly magnetized in the presence of an external magnetic field, with minimal remanence once the field is removed. Rapid field responsive separation along with minimized permanent aggregation in re-use is essential for enzyme-loaded magnetic nanoparticles. The factors affecting recovery efficiency include magnetic properties of the magnetic field, magnetic field gradient, particle size, magnetic susceptibility, coating stability, aggregation properties of the particles, and medium viscosity. The higher the magnetic response, the better the separation, while too much aggregation may lead to decreased magnetic accessibility and redispersion. Thus, the use of optimized superparamagnetic carriers can enhance the recovery, operational stability, reusability, and repeated catalytic/biosensing performance of enzymes.^44,97

This property supports two mainly competing operation goals: (1) the successful isolation of immobilised enzymes during recoveries, and (2) their successful redistribution to support further catalytic steps.⁵³ Superparamagnetic properties of the material allow the easy separation of the immobilised enzymes with the help of an external magnetic field and also the redispersion of the catalysts when the field is removed which has made it possible to reuse the catalyst and operate continuously. With respect to process engineering, this strategy helps to eliminate losses at the downstream, decreases mechanical stress on the biocatalyst, and simplifies the designs of scale-up compared to non-magnetic supports.⁴⁴

Magnetite Platforms and Surface Chemistry

Among the most used magnetic supports are the magnetite-based systems.⁹⁸ Understanding of existing literature indicates that magnetite is a widely used material because it possesses the desirable properties, including good magnetic susceptibility, good practical recovery, and the ability to employ it as an immobilization matrix in a vast range of applications in catalysis.⁹⁹ Magnetic nanoparticles are characterized as preferable immobilization aids, and it is suggested that recovery in the aid of a magnetic field amplifies the recyclability and also reduces the operation costs involved in carrying out a multi-cycle catalytic reaction. In this sense, magnetite-based supports are utilized as a reference system in design schemes and can be optimized to the target enzyme category and other target environmental conditions by modifying the surface chemistry.⁵³

Coating and Functionalization Strategies for Stability and Binding Control

Magnetic nanoparticles are reported to be very surface-reactive, easy to aggregate, and are reported to degrade under particular chemical conditions that may interfere with immobilization, reproducibility, and enzyme stability. To address these shortcomings, different strategies of coating and functionalization have been widely used to introduce reactive groups, to improve dispersion, and to improve biomolecule compatibility.¹⁰⁰ The literature suggests the use of polymeric coatings like polyethylene glycol, polyvinylpyrrolidone, poly(lactic-co-glycolic acid), and polyvinyl alcohol, naturally occurring and plentiful organic coatings, including chitosan and other polysaccharide-based layers, to enhance physicochemical properties and allow a variety of immobilization strategies. These types of surface treatments may provide a controlled microenvironment, suppress non-specific adsorption, and enable covalent binding chemistries, which minimize leaching.¹⁰¹

Immobilization Through Magnetite

The immobilization on magnetic supports involves the same general principles as the rest of the matrices, but the selection of an appropriate procedure would be done based on the expected useful life, the permissible loss of enzyme, and the stability concerns.⁴⁴ One of the most common is referred to as physical adsorption because it is relatively easier to be experimented and typically, it exploits non-covalent forces such as the electrostatic attraction and hydrogen bond. But its stability is sensitively dependent on the pH, ionic strength, and temperature variations, which may encourage desorption in the working condition.¹⁰² Covalent attachment is commonly required in cases where long-term retention and low leaching are needed, albeit at the expense of an established risk of low activity caused by enzyme structural distortions or inhibition of key motions.¹⁰³ The trade-off is supported by comparative studies of immobilization techniques, which show that covalent attachment and crosslinking significantly inhibit enzyme leakage, whereas adsorption selectively maintains enzyme activity at the same time, having an increased susceptibility to desorption and release. In the framework of magnetic systems, design preferences are generally focused onto functionalised surfaces enabling robust binding and at the same time access to the catalytic site by carefully balancing spatial arrangement and orientation.^104,105 The magnetic response of these nanoparticles can be used for rapid and efficient separation, recovery and re-use in enzyme immobilization as a reusable solid support. Surface coatings and functional groups enhance enzyme attachment, orientation, dispersibility, stability and retention of enzymatic activity, but aggregation, leaching, diffusion limits, coating stability, and sensitivity to reaction conditions need to be optimized.^44,97 The dispersibility, attachment strength, enzyme orientation, enzyme activity retention, enzyme stability, and magnetic separation efficiency are enhanced by surface coatings or by the presence of groups like –OH, –COOH, –NH₂, or –CHO.¹⁰⁶

Stability and Magnetic Performance

In spite of these significant benefits, the magnetic nanomaterial platforms are limited by the repeatability of recurrent factors that govern reproducibility and scalability of reported performance. The first limitation is the mass transfer limitation, ie, large enzyme loading or thick layers of surface coverings hinder the diffusion of substrates and, by extension, reduce the apparent catalytic activity, which is well-reported in the literature of nanomaterial immobilization. The other major limitation is stability during consecutive cycles, which occurs due to leaching of the enzyme, aggregation, or changes in magnetic characteristics after coating and immobilization; this drift may affect the separation efficiency and could make the process uncontrollable.^107,108 These issues surpass technicalities. They have a direct impact on the viability of endocrine-based applications, such as reproducible biocatalytic production and robust biosensing applications, where quantitative precision is essential.

Translational Opportunities for Enzyme Immobilized Nanomaterials in Endocrine Therapeutics

Endocrine Pharmacology to Nano-Biocatalyst Design

Therapeutic innovation in endocrinology has two complementary imperatives characterizing it. The first necessity is biological breadth because it is shown that metabolic and reproductive endocrine diseases are a result of the failure of distributed networks and not a result of single pathway malfunctions. The second imperative concerns the reliability of operations because the long-term benefit is tied to consistent delivery, predictable kinetics, quantifiable response, and controllable adverse-effect profiles.¹⁰⁹ The immobilization of enzymes on nanomaterials offers a platform that meets these needs through a means of controlled catalytic activity, denaturation driver stabilization, and reproducible functionality through recoverable heterogeneous structures. Accordingly, the translational utility of these platforms cannot be limited to a single concept of clinical device; instead, it can be considered in a variety of applications that link endocrine treatment requirements with immobilization-based control of biochemical action.^77,103

Endocrine Therapeutics

Endocrine medicine is moving towards more complex forms of therapeutic modalities, including the use of peptide-based agonists and multi-pathway pharmacology, thus putting additional strain on manufacturing pipelines to provide a steady, high-quality output at cost-effective and scalable production. Enzyme systems that are immobilized together are an established and flexible methodology to optimize the economics of processes, through the facilitation of catalyst recovery and reuse, reduction of the quantity of enzymes used, and facilitation of constant operation.¹¹⁰ Nanomaterials also enhance this possibility by providing a better surface area and adjustable surface chemistry, which subsequently increases the enzyme loading and, in certain conditions, increases the catalytic activity and stability of operation compared to traditional carriers.

In the context of translation, the key advantage of the immobilization of nanomaterials does not relate to the synthesis of novel hormonal entities; instead, it is the increase in controllability, strength, and stability of biocatalytic processes that support the synthesis and modification of pharmaceutical intermediates and active compounds, and in parallel, enable environmentally favorable transformation pathways, which can be scaled up.^14,111 Simultaneously, industrial bioprocessing is increasingly using an increasingly diverse range of waste-derived feedstocks to initiate an ever-expanding range of enzyme-mediated transformations.

In Table 3, lignocellulosic biomass, lipids, glycerol, carbon dioxide, and methane are some of the key renewable feedstocks in the production of value-added chemicals, biofuels, polymers, and pharmaceutical precursors through the use of specific enzymatic systems. Such feedstock platforms are further enhanced by the addition of immobilized enzyme technologies, which further enhance process stability, catalyst life, and cost-effectiveness, thus justifying their significance in endocrine therapeutic production and related biotechnological uses.

Table 3.

Major Renewable and Waste-Derived Substrates Associated with Enzymatic Pathways, and Their Industrial Applications in Bioprocessing

Waste Category	Primary Sources	Decomposition Products	Industrial Utilization	Key Enzymes Involved	References
Lignocellulosic biomass	Agricultural residues such as straw and bagasse, agro-industrial by-products such as paper waste, fruit industry by-products such as wine and coffee processing residues	Sugars and phenolic compounds	Food additives, fragrances, nutraceuticals, pharmaceuticals, materials	Cellulases, including endoglucanases, exoglucanases, β-glucosidases, hemicellulases, including xylanases, lignin-degrading enzymes, including laccases	[112]
Triglycerides	Animal fats, vegetable oils, used cooking oils, food processing waste, sewage scum	Biodiesel range products, including alkanes and alkenes	Biofuels, polymers	Lipases, including pancreatic lipase and hormone-sensitive lipase	[113]
Fatty acids	Food and agro-industrial waste, including animal fat and cooking oil, palm oil mill effluent	Hydrocarbons, including alkanes and alkenes, fatty alcohols, fatty acid esters, methyl ketones	Biofuels, chemicals, polymer monomers	Lipoxygenases, alcohol dehydrogenases, esterases	[113, 114]
Glycerol	Food waste, byproducts of biodiesel and bioethanol production	Short-chain alcohols, short-chain acids, and hydrogen gas	Polymers, chemicals, biofuels, and pharmaceuticals	Glycerol dehydrogenase, glycerol kinase, glycerol 3-phosphate dehydrogenase	[115]
Carbon dioxide (CO2)	Emissions from anaerobic digestion, such as landfills, industrial heating, and petroleum refining	Short-chain acids, alcohols, aldehydes, ketones, and isoprene derivatives	Chemicals, polymer monomers, biofuels, preservatives, pharmaceuticals	RuBisCO, carbonic anhydrase	[116]
Methane (CH4)	Anaerobic digestion, decomposition of food waste, animal digestion, and agricultural activities	Methanol, polyhydroxybutyrate, lactic acid	Biofuels, polymers	Methane monooxygenase, methanol dehydrogenase	[117, 118]

Enzyme-Based Biosensing for Precision Endocrinology

Therapeutic uses include nanomaterials containing enzymes either for loading or immobilization to effect change in biochemical activity associated with disease, to enhance enzyme stability, or to provide for controlled and/or localized delivery. However, enzymes are currently more advanced to be used in biosensing and diagnostic applications, such as detection of biomarkers, rapid testing and monitoring, where the enzymes are immobilized as recognition, or signal-generating components.¹¹⁹ Precision-oriented endocrine care requires measurement frameworks that can track disease drivers and treatment response over time.¹¹¹ The endocrine disorders literature characterizes insulin resistance as a systems-level disturbance influenced by inflammatory tone and gut microbial ecology, supporting a shift from one-off measurements toward repeated, multi-dimensional biomarker strategies when feasible.¹²⁰ In the parallel literature on enzyme nanomaterials, it is shown that nanoparticles and other nanostructures have the potential to improve both stability and sensitivity of enzyme-based biosensing platforms by providing favorable physicochemical properties and by providing strong immobilization interfaces. Translationally, the evidence to this effect has been applied to a platform paradigm in which immobilized enzymes are the recognition or transduction components of sensors, with the complementary nanomaterials delivering interface stability, signal quality, and mechanical strength.⁹¹ The most important parameter is preservation of enzymatic orientation and accessibility to active-sites since improper adsorption will trigger conformational rearrangements or will block catalytic pockets, which will trigger drift or loss of specificity. At the cellular level, nanoparticle-based systems can adjust endocrine-related signaling through mechanisms such as oxidative stress, mitochondrial perturbation and activation of inflammatory pathways, which can subsequently regulate processes of steroidogenesis, apoptosis and metabolic regulation.¹²¹ These multilevel interactions are summarized in Figure 2.

Figure 2.

Schematic representation of nanoparticle-mediated modulation of cellular signaling pathways involved in oxidative stress, inflammation, mitochondrial dysfunction, apoptosis, and steroidogenic regulation, highlighting potential interfaces between nanomaterial platforms and endocrine homeostasis.⁵⁵ The red lines show blockage/downregulation and the blue lines show activation/upregulation.

Magnetic Nano-Biocatalyst for Catalysis and Sensing

Magnetic nanomaterials represent a highly useful category, as a result of their ability to provide a field-responsive handling property to immobilized enzymatic systems.¹²² Superparamagnetic behavior supports rapid separation by external magnetic fields and re-dispersion after field removal, enabling repeated use and practical recovery workflows.¹²³ In catalytic reactions, such a strategy can reduce downstream demand, promote the recycling of materials; in defined sensing regimes, it can simplify the process of regenerating and reconditioning the bioactive interface.¹²⁴ Surface coating and support materials are widely used, often based on magnetite (which can be derivatized), and surface coating is more often used to facilitate dispersion and to allow reactive moieties to be introduced to facilitate stable immobilization. The magnetic platforms are especially attractive in their translational perspective, as they enable immobilized enzymes that can easily be incorporated into process equipment, significantly improving the reliability of their operations.¹²⁵

Catalytic and Mechanistic Feasibility Boundaries

The study of enzyme immobilization has focused on localized catalytic strategies, in which immobilised enzymes are applied to localise microenvironmental chemistry within spatially confined systems. This is an up-and-coming idea that has yet to be combined with even those established clinical therapeutics, but is grounded in the principles of enzyme immobilization. The immobilization enables denaturation protection, enables the enzyme activity to be sustained under a wider temperature span, and moreover enables creation of the microenvironment, which controls the local reaction kinetics.¹²⁶ Nanomaterials provide organized surfaces and controllable surface chemistry that enable the control of the spatial organization and accessibility of the enzyme.¹²⁷ Nevertheless, translation requires the consideration of the previously identified limitations, like diffusion limitation due to high enzyme loading, as in confined structures, transport barriers can dominate apparent activity. Therefore, localized catalytic paradigms should be presented as the research opportunities that predetermine clear-cut engineering requirements, instead of the full-fledged therapeutic strategies.¹⁴

Endocrine Therapy Platforms

Combination therapies in metabolic endocrinology are an expression of the recognition of heterogeneity amongst patients, possibly not being adequately met by unimodal interventions. The urgency of the innovations that will enable multi-pathway pharmacodynamics and the urgency of addressing the multifaceted determinants of obesity and diabetes have been highlighted by a considerable body of literature.¹²⁸ Conceptually, the immobilised nanomaterial platforms fit into this trend because immobilisation allows one to present the bioactive catalytic functions with high specificity, and nanomaterials can be tailor-made through surface functionalisation to be able to fit in a given operational milieu.⁷⁷ However, the concept of modularity alone does not guarantee the best performance. The common problems include instability, enzyme loss, and reproducibility, being particularly acute in noncovalently immobilized systems, where enzymatic leakage can pollute culture media and affect the effectiveness in the long-term. The desired translational approach must then be towards platform standardization, regulation of interfaces and reproducible fabrication processes, but not necessarily on the more complex.¹²⁹

Challenges and Limitations

Translation for Nano-Biocatalyst

Enzyme-immobilized nanomaterials are commonly used because of performance advantages, such as enhanced stability and reuseability, catalytic performance; nonetheless, practice must be implemented despite the fact of these advantages in real operating conditions, reproducible manufacturability, and plausible safety profiles.¹³⁰ In endocrine-oriented applications, the tolerance to variability is low, since production of therapeutics is batch-like, and biosensing needs quantitative reliability. It follows that constraint analysis should not be regarded as a relatively marginal discourse; a decisive variable that would inform whether an immobilised nanobiocatalyst remains an experimental artefact or can be an enabling technology that can be deployed in a controlled fashion.¹³¹

Enzyme Leaching, Functional Drift, and Contamination Risk

A major limiting factor that has consistently been encountered in the use of nanomaterials in enzyme immobilization is the loss of activity at the carrier surface during the working of enzymes. This is most severe in noncovalent immobilization methods, where adsorption-based binding is precarious to changes in ionic strength, temperature, or pH, and causes a gradual decrease in catalytic efficiency together with contamination of the surrounding medium.¹³² In adsorption- or weak ionic-binding systems, enzyme leaching may be experienced during storage or operation, where the immobilized enzyme is released from the supports as nanoparticles. Leaching decreases the lifetime of the catalyst, signal drift for biosensors, decreases reusability, and potentially increases safety concerns for biomedical applications. Enzymes can be lost, but this loss can be minimized through stronger covalent coupling, cross-linking, surface activation and optimized coating.¹²⁹ Another main issue regarding the endocrine-based approaches is that the smallest drift can undermine the dose-invariance or sensor-calibration; hence, the results of retention and stability tests should be considered the key ones instead of the auxiliary ones.¹³³

Diffusion Limitations and Mass Transfer Control

Higher surface area nanomaterials promote a higher loading of enzymes, though excess loading can result in transport constraints, reducing the perceived catalytic activity by limiting diffusion of substrates and diffusion of products near the immobilized enzyme layer.¹⁴ In the framework of nanomaterial-based immobilization, the diffusion limitations are often observed, particularly when using immobilization matrices with a large enzyme loading density.¹⁴ This is not only restricted to kinetic aspects; reduced apparent activity can be caused by mass-transfer artefacts and not necessarily by true catalytic inhibition, which can complicate interstudy comparisons.¹³⁴ Diffusion limitations in endocrine biosensing are indicated by a slow response time and signal hysteresis, whereas in biomanufacturing, they are indicated by reduced space-time yields. In this regard, the underlying design methods, to increase porosity, interfacial distance, or to choose support materials that diminish the diffusion barrier, play a central role in supporting believable performance indicators.¹³⁵

Preparation Complexity, Reproducibility, and Scale-Up Barriers

The immobilization of enzymes with nanomaterials usually requires several steps involving synthesis and surface functionalisation thus increasing the complexity of the process compared to standard immobilization supports. Thus, the complexity of the preparation processes highlights the possible barrier of the specialised method and equipment, which can prevent large-scale applications. Additionally, scalability is also a problem due to the lack of reproducibility and uniformity in large-scale production, and the inherent sensitivity of nanomaterials to environmental conditions that may promote aggregation or degradation. Reproducibility is a big challenge for translation as the loading of the enzymes, the size of the particles, surface chemistry, immobilization efficiency, activity retention, and leaching behavior may differ among batches. Even minor changes in pH, temperature or support preparation can affect the catalytic results, therefore standardized characterization, activity assays and stability testing are necessary. These constraints have a direct negative effect on translational feasibility because regulatory and industrial environments require uniform physicochemical characteristics across batches of production, such as particle size distribution, surface chemistry and measures of enzyme loading.¹³⁶

Magnetic Nanoparticles Stability Issues

Magnetic nanomaterials have operational advantages due to recovery and reuse of the material with the help of external magnetic fields, but this can be affected by surface reactivity and instability in certain environmental conditions. High reactivity and degradation in some situations may cause a decrease in efficiency, which leads to the large-scale development of surface modification approaches aimed at maximizing carrier stability and dispersion. Coatings and functional layers can also increase compatibility, but also, at the same time, reduce magnetic saturation and change separation efficiency, particularly in situations in which the diamagnetic polymer coatings are large in thickness or aggregation mechanism changes the effective behaviour of the particles. Translation, therefore, requires a combined optimization strategy that retains sufficient magnetic response for recovery while also providing stable enzyme immobilization and reducing aggregation with each operational cycle.^137,138

Protein Corona Effects, and Functional Uncertainty

On entering biological fluids, enzyme-immobilized nanomaterials immediately interact with proteins, lipids, metabolites and other biomolecules present at the surface. Of these interactions, the adsorption of proteins is of particular interest, as it results in the formation of a protein corona on the surface of the nanomaterial. The protein corona can alter the biological identity of the nanomaterial. In the case of enzyme-immobilized nanomaterials, protein corona poses a functional uncertainty as it can change the accessibility, orientation and catalytic activity of immobilized enzymes. Adsorbed proteins can partially block the enzyme active sites, can slow down the diffusion of substrates, can alter the local pH or charge environment, or can cause conformational changes at the enzyme-nanomaterial surface. These effects can diminish catalytic activity, lead to signal drift in biosensing devices, and cause loss of in vitro to in vivo reproducibility.^139–141

The enzyme-nanomaterial interaction may initiate uncertain changes in the enzymatic structure and activity. Depending on the type of nanomaterial, the surface chemistry, orientation, and environmental conditions, enzymes may adsorb onto the surfaces of nanoparticles and experience conformational alterations that, consequently, prevent or stimulate catalytic activity. These interfacial effects limit the extrapolation of results on different systems. Any surface change that gives one enzymatic form stability can render the other form unstable, and the effects of orientation can block active sites when the binding affinity is high. In endocrine use, where specificity and accuracy are the most important, these considerations require systematic characterization of orientation, maintained activity, and functional stability of each enzyme-support combination, and not the assumption that any one model system will be predictable to behave in a different situation.¹²⁷

Reporting Standards and Characterization Requirements

In order to improve comparability and translational credibility, those studies that use immobilized nanomaterials must report at least a set of characterization data. The required data include the particle size distribution and stability of dispersion, the identification of the surface functional groups or coating chemistries, the enzyme loading, the proportions of the activity relative to free enzyme, stability of the operation over a specified set of pH-temperature conditions, leaching measurements over time, and reusability numbers over a given series of cycles.^127,142 When applied to magnetic systems, it is a requirement that reporting must contain measurements of magnetic saturation or separation efficiency before and after coating the system and the immobilization of the coating and immobilization layers because they can suppress magnetic responsiveness and inhibit recovery mechanisms. Standardization of reporting procedures is essential to integrating the field of science out of contingent demonstration research into platforms that are reproducible and applicable in endocrine-related production and monitoring measures.¹⁴²

Translational readiness is characterized by the effects of enzymatic leaching, the limitations of diffusion, the uncertainty in the interface-based functions and the ability to reproducibly scale up. These barriers are also focused on immobilization to reduce leaching and maintain catalytic function; these barriers seek to design porous matrices, standardize characterization procedures, and assess the stability of such systems in different applications.

Safety and Biocompatibility Considerations

The safety of nanomaterials in endocrine applications should not be assessed only with regard to general cytotoxicity, as nanoparticles can interact with the hormone sensitive tissues and disrupt the endocrine signaling. The nanoparticles may cause oxidative stress, inflammation, mitochondrial dysfunction, apoptosis, organ accumulation and possible endocrine disrupting effects, these adverse effects are however dependent on size, charge, dose, coating and exposure period. Biocompatibility is another challenge. The properties that determine biocompatibility are composition, surface chemistry, degradability, colloidal stability, protein corona formation, immune recognition and clearance behavior of nanoparticles. Materials used for endocrine applications should maintain the enzymatic activity meanwhile avoiding toxicity, off target accumulation, immune activation or interference with the hormonal pathways. So, prior to clinical translation, long-term testing in disease-relevant biological models is essential.^143–145

Future Direction

Enzyme-Immobilized Nanomaterials for Endocrine Disorders

An effective future roadmap requires the placement of enzyme-immobilized nanomaterials as facilitative technologies and not as standalone experimental systems.^91,142 The main reason to assume this arrangement is the fact that immobilization provides reproducible approaches to biocatalytic activity stabilization, enhancing operational lifetime and increasing efficiency of handling in heterogeneous systems. Nanomaterials have remarkably high surface area and surface chemistries that are adaptable, which dictate the easy loading of enzymes and the creation of an enabling microenvironment. When these methodologies are applied to a system to perform endocrine measurements, it is necessary to set the parameters of the system design to conform to standardized protocols needed to allow a consistent evaluation of system stability, reactive leaching phenomena, mass-transfer dynamics, and indicators of reproducibility. This orientation redefines progress as going beyond an increase in the novelty of nanomaterials to augmenting reliability, comparability, and real-world operability of nanomaterials.¹⁴⁶

Immobilization for High Retention and Activity

One major challenge to the development of the field is the heterogeneity of the characterization and reporting of immobilized nanomaterial systems.¹⁴⁶ A significant amount of literature highlights the long-standing limitations created by diffusion, leaching and scalability, which cannot be meaningfully measured without a standardized reporting framework. A pragmatic agenda, therefore, involves establishing a minimal dataset to publish, which records the properties of particles, retention of activity in the face of clearly defined stresses and the ability to reuse across a set number of cycles.¹⁴⁷ In the case of magnetic systems, magnetic response measures before and after coating and immobilization also should be included as the minimum dataset, as magnetic response modifications can weaken magnetic properties and cause changes in recovery efficiency.¹⁴⁸

Nano Matrices Design

Enzyme leaching is another constant drawback in nanomaterial-based platforms, especially in cases where immobilization is practiced by adsorption-based methods.¹⁴⁹ A wide range of literature on immobilization recommends covalent attaching or crosslinking to increase retention but these methods can suppress enzymatic activity when the geometry of the active site, or essential conformational dynamics, is disturbed.³⁴ Therefore, the research agenda should focus on attachment chemistries that can allow high retention and still maintain access to the active site.¹⁵⁰ This includes controlled procedures and orientation-specific immobilization strategies that are purposefully designed to consider the structural characteristics of the enzyme and the spatial positioning of the binding locus and recognize orientational effects as possibly determining whether immobilization enhances or hinders catalytic competence.¹⁵¹ In the context of endocrine-responsive perception and biomanufacturing, the retention parameters are to be considered as the main performance standards; quantitative leaching tests need to be reported along with the initial activity examinations.^152,153

Magnetic Platform for Continuous Operation and Recovery Efficiency

In cases where high enzyme loadings are used, or the development of dense immobilization layers that hinder the transfer of mass, diffusion limitations can dominate to determine apparent activity.¹⁴ Diffusion restrictions represent an existing problem and requirement to implement porous supports or to optimize the matrix to improve transport properties.¹⁵⁴ Based on this, a design approach that puts an emphasis on hierarchical porosity, control of enzyme spacing, and matrix architectures, rationalizes high loading and low transport resistance.¹⁵⁵ Biosensing has a direct effect on transport optimisation that affects the time response of a system. It also influences the space-time yield and reaction efficiency of biocatalysis. It is therefore proposed that diffusion-conscious characterizations, including substrate concentration gradients, response kinetics, and the characterization of mass-transfer artefacts not available in terms of mass-transfer proxies, must be incorporated into future studies.¹⁵⁶

Biosensors for Endocrine Monitoring

Biosensors can facilitate real-time or near real-time monitoring of endocrine biomarker enzymes, such as glucose, hormones, cortisol, TSH, insulin and metabolic indicators through electrochemical, optical or colorimetric conversion of the enzymatic, antibody, aptamer or receptor based recognition of the analyte. Enzyme-immobilized nanomaterials can enhance enzyme loading, maintain enzyme activity, facilitate electron transfer and amplify signal response to improve the sensitivity and selectivity. Nanobiosensors that are based on glucose oxidase can be used for continuous glucose monitoring in diabetes and hormone biosensors with nanoparticles can enhance the detection of TSH and cortisol and facilitate more rapid and convenient point-of-care testing, personalized monitoring and more effective endocrine management of thyroid and adrenal disorders.^157–159

Magnetic nanomaterials still occupy one of the most operationally desirable supports through the ability of the external field recovery in enabling rapid separation and re-dispersion and thus allowing numerous reuse cycles of the same material and simplifying the downstream handling abilities.¹⁶ The highest research need is the ability to maintain magnetic separability and at the same time improve surface stability and enzyme retention.⁴⁴ Functionalizations and surface coatings are already commonly utilized, such as polymeric and biopolymeric coating which enhance dispersion and provide reactive groups to immobilize. However, these types of coatings can reduce magnetic saturation, and aggregation can alter the effective magnetic response.⁸¹ Future studies, therefore, must include the measurement of magnetic performance during the immobilization cycles and in real-world conditions, and not only to report on magnetism during the synthesizing stage. These are the considerations within the framework of endocrinology that outline the operational limits of reuse and the development of approaches that maintain activity and recovery performance.

Biosensing for Precision Endocrinology

Decision-making based on measurement,¹⁶⁰ is becoming more accepted in endocrine care, and appropriate possibilities of enzyme-nanomaterial interfaces represent a plausible route to richer biosensing elements in the settings where interfacial influences are tightly mediated.¹⁶¹ It has been shown that conformation and catalytic activity of enzymes can be modified during adsorption and orientation under certain condition thus losing specificity and stability. Based on this, the design of systems that maintain enzymatic activity over the long duration of operation, reduce the adverse effects, and remain stable in the presence of physiologically relevant conditions should be the priority of future studies.⁵⁸ The validation of mechanisms that can distinguish between enzyme deactivation, leaching, and surface chemistry rearrangements of nanomaterials should also feature on the research agenda. Conclusively, enzyme-immobilized nanomaterials need this high level of consideration in order to support a monitoring framework that will enhance endocrine pharmacotherapy.^77,162

Translational Roadmap for Clinical and Manufacturing Integration

The translational pathway requires the rational integration of safety and environmental concerns, such as the persistence of nanomaterials, the possibility of toxicity, and exposure pathways.¹⁶³ Health and environmental hazards have been stressed over and over again in the literature on nanomaterial biotechnology as correlated with the functional benefits of these nanomaterials.¹⁶⁴ Finally, in endocrine targeted milieus, especially those in which equipment is in direct contact with biological fluids, the research agenda must embrace a standardized biocompatibility assessment protocol and reveal transparent records of degradation kinetics and by-products.¹⁶⁵ In addition, in endocrine targeted milieus, especially those in which equipment is in direct contact with biological fluids, the research agenda must embrace a standardized biocompatibility assessment protocol and reveal transparent records of degradation kinetics and by-products. By incorporating safety considerations into the platform architecture at an early stage, the likelihood of late failures will be reduced and research results can be aligned with the expectations of high-impact academic journals and translational investment organizations.^166,167

Five actionable priorities can be used to formulate a coherent research agenda. To begin with, standardization of characterization and reporting models should be developed to facilitate useful cross-study comparisons. Second, immobilization chemistries are designed in such a way that they reduce the phenomenon of leaching yet maintain catalytic performance. Third, it is the mandate of the researchers to determine how to carry optimized matrices in a way that will reduce the diffusion constraints. Fourth, in the case of magnetic platforms, a systematic study of magnetic recovery performance in consecutive cycles and environmental conditions needs to be carried out, accompanied by optimization of the surface coatings. Fifth, safety and environmental assessment as a pathway of platform development cannot be ignored. All of these are aimed at advancing the field to reproducible, scalable enzyme-immobilized nanomaterials, which can plausibly support future innovation in endocrine therapeutics by means of critical processes, such as production, biosensing, and the creation of future controlled catalytic ideas.^168,169

Technology Driven Innovation

AI-assisted enzyme design could speed up the time-consuming process of developing biocatalysts that are specific, stable, and compatible with immobilization and are relevant to endocrine applications. Prior to experimental validation, machine learning and generative models can be used to predict enzyme structure–function relationships, screen for beneficial mutations, and optimize catalytic pockets. This may help to minimize the trial-and-error approach to designing enzymes for biosensing, biomanufacturing, or therapeutic support.^170,171 Moreover, smart nanomaterials with responsive enzyme immobilization could be used to achieve controlled activity in response to pH, redox state, certain enzymes, glucose, temperature, magnetic field, or other disease-relevant triggers. This can be to control enzyme exposure or access to substrates, or to control release behaviour, which can increase precision and reduce off target activity, only under specific biological conditions.¹⁷² Additionally, fusion of enzyme-immobilized biosensors, continuous monitoring and patient-specific data analytics can prove useful in personalized endocrine therapy. In the endocrine arena, continuous glucose monitoring already shows how real-time biomarker data can help to inform personalized decision making in treatment. This could be expanded to include monitoring of hormones and metabolic markers in thyroid, adrenal, reproductive and metabolic conditions.¹⁷³

Conclusion

Endocrine disorders represent complex dysregulations in hormonal regulation, enzymatic activity, feedback control, and responsiveness of tissues. These diseases are enzyme-dependent, whether in the production of hormones, hormone activation/inactivation, metabolic conversion, or biomarker production. For the treatment of endocrine disorders, enzyme-immobilized nanomaterials present an effective strategy. These are not only passive carrier systems but also engineered interfaces where catalytic function, materials chemistry, biological recognition, and translational design are all converging and integrated. The significance of enzyme immobilization is more general in that it can overcome the practical limitations of using free enzymes in therapeutic, diagnostic and biomanufacturing applications. Immobilization can provide increased enzyme stability, decreased enzyme denaturation, the possibility of enzyme re-use, the opportunity to control the local catalytic environment, and the ability to attach the immobilized enzyme to magnetic, electrochemical, optical, or responsive nanoplatforms. Such benefits are important for endocrine applications which require long term monitoring, very sensitive biochemical detection, controlled activity and reproducibility. But the field has to go beyond just new nanomaterial compositions. Standardized procedures and techniques are required, however, for comparison of immobilization efficiency, enzyme loading, activity retention, enzyme leaching, reusability, storage stability and performance in complex biological fluids. Before considering clinical translation as a reality, the protein corona formation, drift of interfacial activity, immune recognition, toxicity and long term biodistribution should be evaluated further. Future development will rely on whether these systems are able to show that they retain the enzyme, that the substrate can be transported in a predictable way, that the enzyme retains its catalytic properties, biocompatibility and stability under biological conditions relevant to the endocrinological applications.

Funding Statement

There is no funding to report.

Data Sharing Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Ethical Approval and Consent to Participate

Not Applicable. This is a review paper and does not involve direct research on humans or animals.

Consent for Publication

“Not applicable” as this manuscript does not contain data from any person.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. All the authors listed meet the criteria for authorship as per the ICMJE guidelines, read the final manuscript and agree to publish this work.

Disclosure

The Authors declare that they have no competing interests financial or non-financial or any other interests that might be perceived to influence the results and/or discussion reported in this paper.