Nanotechnology has revolutionized the field of medicine, particularly in targeted drug delivery and diagnostics. The integration of nanosensors into point-of-care devices has enabled the detection of multiple biomarkers simultaneously, allowing for more accurate diagnosis and monitoring of complex diseases. This technology has shown great promise in detecting various diseases, including diabetes, cardiovascular disease, and cancer.
Advances in microfluidics and lab-on-a-chip technology are driving the development of nanotechnology-based medical products. These technologies enable the miniaturization of complex laboratory procedures onto a single chip, allowing for rapid and accurate analysis of biological samples. Nanosensors used in point-of-care devices are typically fabricated using materials such as gold, silver, or carbon nanotubes, which provide high sensitivity and specificity.
Nanotechnology in Medicine
The regulatory frameworks for nanomedicines are complex and multifaceted, involving various international and national agencies. The European Medicines Agency and the Food and Drug Administration have established specific frameworks for the regulation of nanomedicines, including guidelines for development and authorization. Despite challenges and concerns regarding safety and efficacy, the integration of nanosensors into point-of-care devices has the potential to significantly improve patient care by enabling healthcare professionals to make informed decisions at the point of care.
Nanoparticles For Targeted Therapy
Nanoparticles for targeted therapy have shown significant promise in the field of medicine, particularly in cancer treatment. These tiny particles, typically measuring between 1-100 nanometers in size, can be engineered to selectively target and destroy cancer cells while sparing healthy tissue (Peer et al., 2007). This is achieved through various mechanisms, including passive targeting, where nanoparticles accumulate in tumor tissues due to the enhanced permeability and retention effect, and active targeting, where nanoparticles are conjugated with ligands that specifically bind to receptors overexpressed on cancer cells (Huang et al., 2018).
One of the key advantages of nanoparticle-based targeted therapy is its ability to overcome the limitations of traditional chemotherapy. Conventional chemotherapeutic agents often lack specificity, leading to significant side effects and damage to healthy tissues. In contrast, nanoparticles can be designed to release their therapeutic payload in a controlled manner, reducing systemic toxicity and improving treatment outcomes (Kumar et al., 2017). Furthermore, nanoparticles can be engineered to carry multiple therapeutic agents, allowing for combination therapy approaches that can enhance treatment efficacy.
The design of nanoparticles for targeted therapy involves careful consideration of several factors, including particle size, shape, surface chemistry, and targeting ligands. The choice of these parameters can significantly impact the biodistribution, pharmacokinetics, and therapeutic efficacy of the nanoparticles (Decuzzi et al., 2009). For example, smaller nanoparticles tend to have longer circulation times and greater tumor accumulation, while larger particles may be more readily cleared by the immune system.
In addition to their use in cancer therapy, nanoparticles are also being explored for targeted delivery of therapeutics to other diseases, including cardiovascular disease, neurological disorders, and infectious diseases. In these applications, nanoparticles can be designed to target specific cell types or tissues, allowing for localized delivery of therapeutic agents and minimizing systemic side effects (Moghimi et al., 2012).
The clinical translation of nanoparticle-based targeted therapy is an active area of research, with several formulations currently in various stages of human trials. While significant challenges remain, including scalability, regulatory approval, and cost-effectiveness, the potential benefits of this approach make it an exciting and promising area of investigation (Bawa et al., 2019).
Mechanisms Of Cellular Uptake
Cellular uptake is the process by which cells internalize molecules, such as nutrients, hormones, and drugs, from their surroundings. This process is crucial for maintaining cellular homeostasis and regulating various cellular functions. There are several mechanisms of cellular uptake, including receptor-mediated endocytosis, phagocytosis, pinocytosis, and passive diffusion.
Receptor-mediated endocytosis is a specific type of endocytosis that involves the binding of molecules to specific receptors on the cell surface, which triggers the internalization of the molecule-receptor complex. This process is highly regulated and requires the coordination of multiple proteins and lipids . For example, the epidermal growth factor receptor (EGFR) is a transmembrane receptor that undergoes endocytosis upon binding to its ligand, leading to the activation of downstream signaling pathways .
Phagocytosis is another type of cellular uptake that involves the engulfment of large particles, such as bacteria and dead cells, by specialized cells called phagocytes. This process requires the coordination of multiple proteins, including actin, myosin, and paxillin, which work together to extend pseudopodia around the particle and internalize it . Phagocytosis is an important mechanism for removing pathogens and debris from tissues.
Pinocytosis is a type of endocytosis that involves the non-specific uptake of small molecules and fluids by cells. This process occurs through the formation of small vesicles called pinosomes, which are generated by the invagination of the plasma membrane . Pinocytosis is an important mechanism for regulating cellular volume and maintaining cellular homeostasis.
Passive diffusion is a type of cellular uptake that involves the movement of molecules across the cell membrane without the need for energy or specific transport proteins. This process occurs through the random motion of molecules, which allows them to cross the cell membrane by simple diffusion . Passive diffusion is an important mechanism for regulating the concentration of ions and small molecules within cells.
Designing Nanocarriers For Drugs
Nanocarriers are designed to target specific cells or tissues, increasing the efficacy of drug delivery while minimizing side effects. The design of nanocarriers involves the selection of materials with suitable properties, such as biocompatibility, biodegradability, and non-toxicity (Kumar et al., 2017). For instance, liposomes are a type of nanocarrier that can be engineered to target specific cells by modifying their surface chemistry (Torchilin, 2005).
The size and shape of nanocarriers also play a crucial role in determining their ability to target specific sites within the body. Research has shown that nanoparticles with diameters between 10-100 nm are optimal for targeting tumors due to their ability to accumulate in tumor tissues through the enhanced permeability and retention (EPR) effect (Maeda et al., 2000). Furthermore, studies have demonstrated that nanocarriers with a rod-like shape exhibit improved circulation times and targeting efficiency compared to spherical nanoparticles (Geng et al., 2007).
The surface chemistry of nanocarriers is another critical factor in determining their targeting ability. Functionalization of the surface with specific ligands or antibodies can enhance the binding affinity of nanocarriers for target cells, thereby improving their therapeutic efficacy (Huang et al., 2010). For example, research has shown that nanocarriers conjugated with folic acid exhibit enhanced targeting efficiency towards cancer cells overexpressing folate receptors (Low et al., 2008).
In addition to passive targeting strategies, active targeting approaches have also been explored. These involve the use of external stimuli, such as magnetic fields or light, to guide nanocarriers to specific sites within the body (Pankhurst et al., 2003). For instance, studies have demonstrated that magnetically responsive nanocarriers can be guided to target tissues using an external magnetic field, resulting in improved therapeutic outcomes (Chertok et al., 2010).
The design of nanocarriers also involves consideration of their pharmacokinetics and biodistribution. Research has shown that the size, shape, and surface chemistry of nanocarriers can significantly influence their circulation times, tissue distribution, and clearance rates (Longmire et al., 2008). Understanding these factors is crucial for optimizing the design of nanocarriers to achieve efficient targeting and therapeutic efficacy.
The development of nanocarriers has also been influenced by advances in imaging technologies. For example, research has shown that nanocarriers can be designed to serve as contrast agents for imaging modalities such as MRI or CT scans (Liu et al., 2011). This enables the visualization of nanocarrier distribution and targeting efficiency in real-time, facilitating the optimization of their design.
Active And Passive Targeting Strategies
Active targeting strategies in nanotechnology-based drug delivery systems involve the use of specific molecules or ligands that bind to target cells or tissues, allowing for precise delivery of therapeutic agents . This approach is based on the principle of molecular recognition, where the targeting molecule recognizes and binds to a specific receptor or antigen expressed by the target cells . For example, antibodies or antibody fragments can be used as targeting molecules to deliver drugs specifically to cancer cells expressing specific tumor-associated antigens.
Passive targeting strategies, on the other hand, rely on the inherent properties of nanoparticles, such as their size and surface chemistry, to accumulate preferentially in certain tissues or organs . This approach is based on the enhanced permeability and retention (EPR) effect, where nanoparticles tend to leak out of blood vessels and accumulate in tumor tissues due to the increased vascular permeability and impaired lymphatic drainage .
One example of active targeting strategy is the use of folate-conjugated nanoparticles to deliver chemotherapeutic agents specifically to cancer cells expressing folate receptors . Folate receptors are overexpressed on the surface of many types of cancer cells, making them an attractive target for nanoparticle-mediated drug delivery. In contrast, passive targeting strategies can be used to deliver drugs to tumors without the need for specific targeting molecules.
The choice between active and passive targeting strategies depends on various factors, including the type of disease being targeted, the properties of the nanoparticles, and the characteristics of the target cells or tissues . For example, active targeting may be more suitable for diseases where specific molecular markers are expressed by the target cells, while passive targeting may be more effective for diseases where the EPR effect is pronounced.
In addition to these two main strategies, there are also hybrid approaches that combine elements of both active and passive targeting. For instance, nanoparticles can be designed to have a passive targeting component, such as a lipophilic coating, which allows them to accumulate in tumor tissues through the EPR effect, while also having an active targeting component, such as a ligand or antibody, which binds specifically to cancer cells .
Imaging Techniques For Tracking Nanoparticles
Imaging techniques play a crucial role in tracking nanoparticles, which is essential for understanding their behavior, distribution, and interactions within biological systems. One of the most commonly used imaging techniques for nanoparticle tracking is fluorescence microscopy. This technique involves labeling nanoparticles with fluorescent dyes or proteins, allowing researchers to visualize and track their movement using specialized microscopes . Fluorescence microscopy offers high spatial resolution and sensitivity, making it an ideal tool for studying nanoparticle dynamics at the cellular level.
Another powerful imaging technique used for nanoparticle tracking is magnetic resonance imaging (MRI). MRI utilizes strong magnetic fields and radio waves to generate detailed images of internal structures. By incorporating contrast agents or labeling nanoparticles with magnetic materials, researchers can visualize and track their distribution within tissues and organs . MRI offers excellent spatial resolution and the ability to image deep tissue structures, making it a valuable tool for studying nanoparticle biodistribution.
In addition to fluorescence microscopy and MRI, other imaging techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are also used for nanoparticle tracking. TEM provides high-resolution images of nanoparticles at the nanoscale, allowing researchers to study their morphology and interactions with cellular structures . SEM offers similar resolution and is often used in conjunction with energy-dispersive spectroscopy (EDS) to analyze the elemental composition of nanoparticles.
Optical coherence tomography (OCT) is another imaging technique that has been used for nanoparticle tracking. OCT uses low-coherence interferometry to generate high-resolution images of internal structures, allowing researchers to study nanoparticle distribution and dynamics within tissues . This technique offers excellent spatial resolution and the ability to image deep tissue structures without the need for contrast agents.
In recent years, there has been a growing interest in using photoacoustic imaging (PAI) for nanoparticle tracking. PAI uses laser pulses to generate acoustic waves, which are then detected by ultrasound sensors to produce high-resolution images of internal structures . This technique offers excellent spatial resolution and the ability to image deep tissue structures without the need for contrast agents.
Biocompatibility And Toxicity Concerns
Biocompatibility concerns are a crucial aspect of nanotechnology in medicine, particularly when it comes to targeted drug delivery and diagnostics. The interaction between nanoparticles and biological systems can lead to unintended consequences, such as toxicity, inflammation, and immune responses . For instance, studies have shown that certain types of nanoparticles, like silver and zinc oxide, can cause oxidative stress and DNA damage in human cells .
The size and shape of nanoparticles also play a significant role in determining their biocompatibility. Research has demonstrated that smaller particles tend to be more toxic than larger ones due to their increased surface area-to-volume ratio, which can lead to greater interactions with biological molecules . Furthermore, the shape of nanoparticles can influence their internalization by cells and subsequent toxicity. For example, rod-shaped nanoparticles have been found to be more toxic than spherical ones due to their ability to penetrate deeper into tissues .
Another critical factor in determining biocompatibility is the material composition of nanoparticles. Certain materials, such as gold and silicon dioxide, are generally considered biocompatible, while others, like cadmium selenide, can be highly toxic . The surface chemistry of nanoparticles also plays a crucial role in their interactions with biological systems. Functionalization of nanoparticles with biocompatible molecules, such as polyethylene glycol, can improve their stability and reduce toxicity .
In addition to material composition, the route of administration is another essential factor in determining biocompatibility. Nanoparticles administered via injection or inhalation may have different toxicological profiles compared to those administered orally or topically . For instance, studies have shown that nanoparticles administered via inhalation can cause inflammation and oxidative stress in lung tissues .
The development of standardized testing protocols is essential for evaluating the biocompatibility and toxicity of nanoparticles. Regulatory agencies, such as the US FDA, have established guidelines for the evaluation of nanomaterials in medical products . However, more research is needed to develop comprehensive testing frameworks that can accurately predict the behavior of nanoparticles in complex biological systems.
The use of alternative methods, such as in vitro and in silico models, can also aid in the assessment of nanoparticle biocompatibility. These approaches can provide valuable insights into the interactions between nanoparticles and biological systems, reducing the need for animal testing .
Personalized Medicine Through Nanotechnology
Personalized medicine through nanotechnology involves the use of nanoparticles to tailor medical treatment to individual patients’ needs. This approach has shown promise in targeted drug delivery, where nanoparticles can be engineered to target specific cells or tissues, reducing side effects and improving efficacy (Freitas Jr, 2005; Ferrari, 2005). For instance, researchers have developed nanoparticles that can selectively target cancer cells, delivering chemotherapy directly to the tumor site while sparing healthy tissue (Peer et al., 2007; Davis et al., 2010).
Nanoparticles can also be designed to respond to specific stimuli, such as changes in pH or temperature, allowing for controlled release of therapeutic agents. This has led to the development of “smart” nanoparticles that can adapt to changing physiological conditions (Bae et al., 2007; Gao et al., 2010). Furthermore, nanotechnology has enabled the creation of personalized diagnostic tools, such as biosensors and lab-on-a-chip devices, which can detect biomarkers for specific diseases at the molecular level (Wang et al., 2011; Zhang et al., 2012).
The use of nanoparticles in personalized medicine also raises important questions about toxicity and biocompatibility. Researchers have shown that certain types of nanoparticles can cause oxidative stress and inflammation in cells, highlighting the need for careful design and testing of nanomaterials (Nel et al., 2006; Oberdörster et al., 2005). However, advances in materials science have led to the development of biodegradable and non-toxic nanoparticles that can be safely used in medical applications (Kumar et al., 2013; Liu et al., 2014).
Personalized medicine through nanotechnology also relies on advanced imaging techniques, such as MRI and PET scans, to monitor nanoparticle distribution and therapeutic efficacy. Researchers have developed nanoparticles that can be visualized using these imaging modalities, allowing for real-time tracking of treatment response (Louie et al., 2000; McCarthy et al., 2007). This has enabled clinicians to adjust treatment protocols based on individual patient responses, leading to more effective and targeted therapy.
The integration of nanotechnology with other emerging fields, such as genomics and proteomics, is also expected to play a major role in the development of personalized medicine. For instance, researchers have used nanoparticles to deliver gene editing tools, such as CRISPR-Cas9, to specific cells or tissues (Kim et al., 2014; Zetsche et al., 2015). This has opened up new avenues for treating genetic diseases and has the potential to revolutionize the field of personalized medicine.
Overcoming Blood-brain Barrier Challenges
The blood-brain barrier (BBB) is a highly selective semipermeable border that separates the circulating blood from the brain’s extracellular fluid in the central nervous system (CNS). It is composed of endothelial cells that line the brain’s capillaries, which are tightly joined together by tight junctions, and pericytes that surround the capillaries. The BBB restricts the passage of various substances, including drugs, from the bloodstream into the brain, making it a significant challenge for targeted drug delivery to the CNS.
The main function of the BBB is to maintain the brain’s internal environment, regulating the exchange of essential nutrients and waste products between the blood and the brain. However, this barrier also hinders the delivery of therapeutic agents to the brain, limiting their efficacy in treating neurological disorders. To overcome this challenge, researchers have been exploring various strategies to enhance drug delivery across the BBB. One approach is to use nanoparticles that can interact with the BBB, allowing for targeted and controlled release of therapeutic agents.
Nanoparticles can be engineered to target specific cells or tissues in the brain, increasing the efficacy of drug delivery while minimizing side effects. For example, liposomes, a type of nanoparticle, have been shown to cross the BBB and deliver drugs to the brain in animal models. Additionally, nanoparticles can be designed to release therapeutic agents in response to specific stimuli, such as changes in pH or temperature, allowing for controlled release.
Another approach to overcome the BBB is to use molecular Trojan horses, which are molecules that can cross the BBB and carry therapeutic agents into the brain. For example, certain peptides have been shown to cross the BBB and deliver drugs to the brain in animal models. Furthermore, researchers have also explored the use of focused ultrasound to temporarily disrupt the BBB, allowing for the delivery of therapeutic agents to the brain.
The development of effective strategies to overcome the BBB is crucial for the treatment of neurological disorders, such as Alzheimer’s disease, Parkinson’s disease, and brain cancer. Researchers continue to explore new approaches to enhance drug delivery across the BBB, including the use of nanoparticles, molecular Trojan horses, and focused ultrasound.
Stimuli-responsive Nanosystems For Delivery
Stimuli-responsive nanosystems have emerged as a promising approach for targeted drug delivery, enabling the controlled release of therapeutic agents in response to specific physiological or environmental stimuli. These systems are designed to respond to various triggers, such as temperature, pH, light, or enzymes, allowing for precise control over the release of drugs at the site of action . For instance, thermoresponsive nanosystems can be engineered to release their payload in response to elevated temperatures, which is particularly useful for treating cancerous tissues that exhibit higher temperatures than normal tissues .
The design and development of stimuli-responsive nanosystems involve the use of various materials and technologies, including polymers, liposomes, and nanoparticles. These systems are often fabricated using techniques such as self-assembly, emulsion polymerization, or sol-gel processing, which enable the creation of complex nanostructures with tailored properties . The incorporation of stimuli-responsive elements, such as thermoresponsive polymers or pH-sensitive linkages, allows for the controlled release of drugs in response to specific triggers .
One of the key advantages of stimuli-responsive nanosystems is their ability to reduce side effects and improve therapeutic efficacy. By releasing drugs only at the site of action, these systems can minimize exposure to healthy tissues and reduce the risk of adverse reactions . Additionally, stimuli-responsive nanosystems can be designed to release multiple drugs in a sequential or simultaneous manner, enabling the treatment of complex diseases that require combination therapy .
The use of stimuli-responsive nanosystems for targeted drug delivery has shown promising results in various preclinical studies. For example, thermoresponsive liposomes have been used to deliver doxorubicin to cancerous tissues, resulting in improved antitumor efficacy and reduced cardiotoxicity compared to free doxorubicin . Similarly, pH-sensitive nanoparticles have been employed to deliver siRNA to cancer cells, leading to enhanced gene silencing and inhibition of tumor growth .
Despite the promising results, there are still several challenges that need to be addressed before stimuli-responsive nanosystems can be translated into clinical practice. These include the need for improved stability and shelf-life, as well as the development of more efficient and cost-effective fabrication methods . Additionally, further research is required to fully understand the in vivo behavior of these systems and to optimize their design for specific applications .
Diagnostic Applications Of Nanotechnology
Nanotechnology has revolutionized the field of diagnostics, enabling the detection of diseases at an early stage with high accuracy. One of the key diagnostic applications of nanotechnology is in the development of biosensors. Biosensors are devices that use biological molecules to detect specific biomarkers associated with diseases. Nanoparticles, such as gold nanoparticles and quantum dots, have been used to enhance the sensitivity and specificity of biosensors (Wang et al., 2011; Katz & Willner, 2004). These nanoparticles can be functionalized with antibodies or other biomolecules that specifically bind to target biomarkers, allowing for the detection of diseases such as cancer and infectious diseases.
Another diagnostic application of nanotechnology is in the development of imaging agents. Nanoparticles can be designed to accumulate in specific tissues or cells, allowing for the visualization of diseases using imaging modalities such as MRI and CT scans (Liu et al., 2013; Cormode et al., 2010). For example, iron oxide nanoparticles have been used as contrast agents for MRI imaging of cancer tumors. These nanoparticles can be functionalized with targeting ligands that specifically bind to cancer cells, allowing for the visualization of tumor growth and response to treatment.
Nanotechnology has also enabled the development of point-of-care diagnostic devices. These devices are designed to detect diseases in a rapid and cost-effective manner, making them ideal for use in resource-poor settings (Yager et al., 2008; Chin et al., 2012). For example, lateral flow assays have been developed using nanoparticles to detect biomarkers associated with infectious diseases such as malaria and tuberculosis. These devices are simple to use and do not require specialized equipment or training.
In addition to these applications, nanotechnology has also enabled the development of diagnostic platforms for detecting genetic disorders. Nanoparticles can be designed to bind specifically to DNA sequences associated with genetic disorders, allowing for the detection of diseases such as sickle cell anemia and cystic fibrosis (Kaittanis et al., 2010; Zhang et al., 2013). These nanoparticles can be used in conjunction with other diagnostic techniques, such as PCR and sequencing, to confirm the diagnosis.
The use of nanotechnology in diagnostics has also enabled the development of personalized medicine. Nanoparticles can be designed to detect specific biomarkers associated with an individual’s genetic profile, allowing for tailored treatment strategies (Huang et al., 2012; Lee et al., 2015). For example, nanoparticles have been used to detect biomarkers associated with cancer, allowing for the development of targeted therapies that specifically kill cancer cells.
Point-of-care Devices Using Nanosensors
Point-of-Care (POC) devices integrated with nanosensors have revolutionized the field of medicine by enabling rapid and accurate diagnosis at the patient’s bedside. These devices utilize nanotechnology to detect biomarkers, pathogens, or other molecules in bodily fluids, allowing for timely interventions and improved patient outcomes. According to a study published in the journal Biosensors and Bioelectronics, POC devices equipped with nanosensors have demonstrated high sensitivity and specificity in detecting various diseases, including cancer and infectious diseases .
The integration of nanosensors into POC devices has also enabled the detection of multiple biomarkers simultaneously, allowing for more accurate diagnosis and monitoring of complex diseases. For instance, a study published in the journal Lab on a Chip demonstrated the use of a nanosensor-based POC device to detect multiple biomarkers for cardiovascular disease . This technology has the potential to significantly improve patient care by enabling healthcare professionals to make informed decisions at the point of care.
Nanosensors used in POC devices are typically fabricated using materials such as gold, silver, or carbon nanotubes, which provide high sensitivity and specificity. These sensors can be functionalized with specific antibodies or aptamers to detect target biomarkers. According to a review article published in the journal Nanomedicine, the use of nanosensors in POC devices has shown great promise in detecting various diseases, including diabetes, cardiovascular disease, and cancer .
The development of POC devices integrated with nanosensors has also been driven by advances in microfluidics and lab-on-a-chip technology. These technologies enable the miniaturization of complex laboratory procedures onto a single chip, allowing for rapid and accurate analysis of biological samples. According to a study published in the journal Analytical Chemistry, the use of microfluidic-based POC devices has shown great promise in detecting biomarkers for various diseases .
The integration of nanosensors into POC devices has also raised concerns regarding their safety and efficacy. However, studies have shown that these devices can be designed to minimize potential risks and ensure accurate results. According to a review article published in the journal Journal of Biomedical Nanotechnology, the use of nanosensors in POC devices has been shown to be safe and effective in various clinical settings .
Regulatory Frameworks For Nanomedicines
The regulatory frameworks for nanomedicines are complex and multifaceted, involving various international and national agencies. The European Medicines Agency (EMA) has established a specific framework for the regulation of nanomedicines, which includes guidelines for the development and authorization of these products. According to the EMA, nanomedicines are considered as “new active substances” and must undergo rigorous testing and evaluation before they can be approved for use in humans.
In the United States, the Food and Drug Administration (FDA) has also established a regulatory framework for nanomedicines. The FDA considers nanomedicines to be a subset of combination products, which are subject to regulation under the Federal Food, Drug, and Cosmetic Act (FD&C Act). The FDA has issued guidance documents on the development and approval of nanomedicines, including guidelines for the submission of investigational new drug applications (INDs) and new drug applications (NDAs).
The International Organization for Standardization (ISO) has also developed standards for the characterization and testing of nanomaterials used in medical products. These standards provide a framework for the evaluation of the physical and chemical properties of nanomaterials, as well as their potential toxicity and environmental impact.
In addition to these regulatory frameworks, there are also various international guidelines and recommendations for the development and use of nanomedicines. For example, the World Health Organization (WHO) has issued guidelines on the evaluation of the safety and efficacy of nanomedicines, while the International Council on Harmonisation (ICH) has developed guidelines on the quality and non-clinical testing of these products.
The regulatory frameworks for nanomedicines are continually evolving as new scientific evidence emerges. For example, recent studies have highlighted the potential risks associated with the use of certain types of nanoparticles in medical products, leading to increased scrutiny by regulatory agencies.
