Biological Model Research Articles

Integrating Diversity, Equity, and Inclusion into Preclinical, Clinical, and Public Health Mathematical Models.

Mathematical modelling applied to preclinical, clinical, and public health research is critical for our understanding of a multitude of biological principles. Biology is fundamentally heterogeneous, and mathematical modelling must meet the challenge of variability head on to ensure the principles of diversity, equity, and inclusion (DEI) are integrated into quantitative analyses. Here we provide a follow-up perspective on the DEI plenary session held at the 2023 Society for Mathematical Biology Annual Meeting to discuss key issues for the increased integration of DEI in mathematical modelling in biology.

Functional predictability of universal gene circuits in diverse microbial hosts

AbstractAlthough the principles of synthetic biology were initially established in model bacteria, microbial producers, extremophiles and gut microbes have now emerged as valuable prokaryotic chassis for biological engineering. Extending the host range in which designed circuits can function reliably and predictably presents a major challenge for the concept of synthetic biology to materialize. In this work, we systematically characterized the cross‐species universality of two transcriptional regulatory modules—the T7 RNA polymerase activator module and the repressors module—in three non‐model microbes. We found striking linear relationships in circuit activities among different organisms for both modules. Parametrized model fitting revealed host non‐specific parameters defining the universality of both modules. Lastly, a genetic NOT gate and a band‐pass filter circuit were constructed from these modules and tested in non‐model organisms. Combined models employing host non‐specific parameters were successful in quantitatively predicting circuit behaviors, underscoring the potential of universal biological parts and predictive modeling in synthetic bioengineering.

Bioinspiration from bats and new paradigms for autonomy in natural environments

Achieving autonomous operation in complex natural environment remains an unsolved challenge. Conventional engineering approaches to this problem have focused on collecting large amounts of sensory data that are used to create detailed digital models of the environment. However, this only postpones solving the challenge of identifying the relevant sensory information and linking it to action control to the domain of the digital world model. Furthermore, it imposes high demands in terms of computing power and introduces large processing latencies that hamper autonomous real-time performance. Certain species of bats that are able to navigate and hunt their prey in dense vegetation could be a biological model system for an alternative approach to addressing the fundamental issues associated with autonomy in complex natural environments. Bats navigating in dense vegetation rely on clutter echoes, i.e. signals that consist of unresolved contributions from many scatters. Yet, the animals are able to extract the relevant information from these input signals with brains that are often less than 1 g in mass. Pilot results indicate that information relevant to location identification and passageway finding can be directly obtained from clutter echoes, opening up the possibility that the bats’ skill can be replicated in man-made autonomous systems.

Specific nanoprobe design for MRI: Targeting laminin in the blood-brain barrier to follow alteration due to neuroinflammation.

Chronic neuroinflammation is characterized by increased blood-brain barrier (BBB) permeability, leading to molecular changes in the central nervous system that can be explored with biomarkers of active neuroinflammatory processes. Magnetic resonance imaging (MRI) has contributed to detecting lesions and permeability of the BBB. Ultra-small superparamagnetic particles of iron oxide (USPIO) are used as contrast agents to improve MRI observations. Therefore, we validate the interaction of peptide-88 with laminin, vectorized on USPIO, to explore BBB molecular alterations occurring during neuroinflammation as a potential tool for use in MRI. The specific labeling of NPS-P88 was verified in endothelial cells (hCMEC/D3) and astrocytes (T98G) under inflammation induced by interleukin 1β (IL-1β) for 3 and 24 hours. IL-1β for 3 hours in hCMEC/D3 cells increased their co-localization with NPS-P88, compared with controls. At 24 hours, no significant differences were observed between groups. In T98G cells, NPS-P88 showed similar nonspecific labeling among treatments. These results indicate that NPS-P88 has a higher affinity towards brain endothelial cells than astrocytes under inflammation. This affinity decreases over time with reduced laminin expression. In vivo results suggest that following a 30-minute post-injection, there is an increased presence of NPS-P88 in the blood and brain, diminishing over time. Lastly, EAE animals displayed a significant accumulation of NPS-P88 in MRI, primarily in the cortex, attributed to inflammation and disruption of the BBB. Altogether, these results revealed NPS-P88 as a biomarker to evaluate changes in the BBB due to neuroinflammation by MRI in biological models targeting laminin.

New Approach Methods to Assess the Enteropathogenic Potential of Strains of the Bacillus cereus Group, including Bacillus thuringiensis

Bacillus cereus (Bc) is a wide group of Gram-positive and spore-forming bacteria, known to be the etiological agents of various human infections, primarily food poisoning. The Bc group includes enteropathogenic strains able to germinate in the digestive tract and to produce enterotoxins such as Nhe, Hbl, and CytK. One species of the group, Bacillus thuringiensis (Bt), has the unique feature of producing insecticidal crystals during sporulation, making it an important alternative to chemical pesticides to protect crops from insect pest larvae. Nevertheless, several studies have suggested a link between the ingestion of pesticide strains and human cases of food poisoning, calling their safety into question. Consequently, reliable tools for virulence assessment are worth developing to aid decision making in pesticide regulation. Here, we propose complementary approaches based on two biological models, the human intestinal Caco-2 cell line and the insect Drosophila melanogaster, to assess and rank the enteric virulence potency of Bt strains in comparison with other Bc group members. Using a dataset of 48 Bacillus spp. strains, we showed that some Bc group strains, including Bt, were able to induce cytotoxicity in Caco-2 cells with concomitant release of IL-8 cytokine, a landmark of pro-inflammatory response. In the D. melanogaster model, we were able to sort a panel of 39 strains into four different classes of virulence, ranging from no virulence to strong virulence. Importantly, for the most virulent strains, mortality was associated with a loss of intestinal barrier integrity. Interestingly, although strains can share a common toxinotype, they display different degrees of virulence, suggesting the existence of specific mechanisms of virulence expression in vivo in the intestine.

Efficient, rapid, and high-yield synthesis of aryl Schiff base derivatives and their in vitro and in silico inhibition studies of hCA I, hCA II, AChE, and BuChE.

This study reports a rapid and efficient synthesis of four novel aryl Schiff base derivatives. Biological activity and molecular modeling studies were conducted to evaluate the inhibitory effects of these compounds on human carbonic anhydrases (hCA) and cholinesterases. The results indicate that the triazole-ring-containing compounds have strong inhibitory effects on hCA I, hCA II, acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) targets. Besides comparing the Schiff bases synthesized in our study to reference molecules, we conducted in silico investigations to examine how these compounds interact with their targets. Our studies revealed that these compounds can occupy binding sites and establish interactions with crucial residues, thus inhibiting the functions of the targets. These findings have significant implications as they can be utilized to develop more potent compounds for treating the diseases that these target proteins play crucial roles in or to obtain drug precursors with enhanced efficacy.

Bayesian approach for evaluation of lactation curve in cross bred cattle based on monthly and bimonthly test day milk yield.

In field progeny testing program milk recording at monthly or bimonthly intervals and prediction of first lactation 305-day milk yield (FL305DMY) from these test day yields have been adapted as an alternative to daily milk recording. Wood's incomplete gamma function is the one of the commonly used nonlinear lactation curve model. In recent years Bayesian approach of fitting nonlinear biological models is gaining attention among researchers. In this study Wood's incomplete gamma function was fitted using Bayesian approach using monthly (MTDY) and bimonthly test day (BTDY) yields. The lactation curve parameters thus obtained were used for prediction of FL305DMY. Efficiency of prediction based on monthly and bimonthly test day milk yield were compared using error of prediction. It was found to be 5.78% and 7.59% as root mean square error (RMSE) based on MTDY and BTDY respectively.The Breeding values of 97 Karan Fries sires were estimated using BLUP-AM based on actual and predicted FL305DMY thus obtained. The RMSE was calculated as the difference between estimated breeding values based on actual and predicted yield. It was found that RMSE calculated based on MTDY showed only a marginal superiority of 0.79% over BTDY and showed high degree of correlation with actual yield. Therefore, recording at bimonthly intervals could be an economical alternative without compromising the efficiency.

On stability of a reaction diffusion system described by difference equations

This work investigates the dynamics of discrete reaction-diffusion Gierer-Meinhardt system as mathematical models of biological pattern formation. We study the system's local asymptotic behaviour with and without the diffusion once developing the discrete integer variant of the well-known Gierer-Meinhardt model and proving that the model has a unique equilibrium. The requirements for the steady-state solution's local and global stability are found with the help of relevant approaches and the Lyapunov technique. Two large biological models and simulations are used throughout the work to validate the utility of the suggested technique.

Global research on centenarians: A historical and comprehensive bibliometric analysis from 1887 to 2023.

Centenarians are considered the most successful human biological aging model. However, the characteristics and patterns of research among centenarians have not been described or analyzed. Thus, this study aimed to disclose the historical landscape of global research on centenarians. This bibliometric study investigated historical evidence on centenarian research published in the Scopus database. The bibliometrix package in R was used to perform visual and quantitative analyses of research metrics, trends, and patterns. Of the 2061 documents included between 1887 and 2023, 84.2% (n = 1736) were published as articles with primary data. We identified international collaboration and annual growth rates of 21.4% and 3.15%, respectively. The United States published the highest number of papers on centenarians (n = 786), whereas the publications from Italy had the highest impact (h-index = 90). Based on the frequency of keywords, mortality, genetics, dementia, Alzheimer's disease, and immunosenescence are a few of the most studied topics among centenarians, with emerging research related to mitochondrial DNA and comparison of results between nonagenarians and centenarians. Italy, the United States, and China lead the global research collaboration network, collaborating most frequently with Japan and European countries. Global research on centenarians has grown over the last 20 years, primarily led by Italy, the United States, and China. Latin American and African countries have conducted little or no research on centenarians. The most widely studied topics include mortality, cognition, immunosenescence, and genetics.

Single-Step Low-Temperature Synthesis of Carbon Dots for Advanced Multiparametric Bioimaging Probe Applications.

Carbon dots (CDs) have recently emerged in biomedical and agricultural domains, mainly for their probe applications in developing efficient sensors. However, the existing high-temperature approaches limit the industrial level scaling up to further translate them into different products by mass-scale fabrication of CDs. To address this, we have attempted to lower the synthesis temperature to 140 °C and synthesized different CDs using different organic acids and their combinations in a one-step approach (quantum yield 3.6% to 16.5%; average size 3 to 5 nm). Further, sensing applications of CDs have been explored in three different biological models, mainly Danio rerio (zebrafish) embryos, bacterial strains, and the Lactuca sativa (lettuce) plant. The 72 h exposure of D. rerio embryos to 0.5 and 1 mg/mL concentrations of CDs exhibited significant uptake without mortality, a 100% hatching rate, and nonsignificant alterations in heart rate. Bacterial bioimaging experiments revealed CD compatibility with Gram-positive (Bacillus subtilis) and Gram-negative (Serratia marcescens) strains without bactericidal effects. Furthermore, CDs demonstrated effective conduction and fluorescence within the vascular system of lettuce plants, indicating their potential as in vivo probes for plant tissues. The single-step low-temperature CD synthesis approach with efficient structural and optical properties enables the process as industrially viable to up-scale the technology readiness level. The bioimaging of CDs in different biological models indicates the possibility of developing a CD probe for diverse biosensing roles in diseases, metabolism, microbial contamination sensing, and more.

Compact meta-differentiator for achieving isotropically high-contrast ultrasonic imaging

Ultrasonic imaging is crucial in the fields of biomedical engineering for its deep penetration capabilities and non-ionizing nature. However, traditional techniques heavily rely on impedance differences within objects, resulting in poor contrast when imaging acoustically transparent targets. Here, we propose a compact spatial differentiator for underwater isotropic edge-enhanced imaging, which enhances the imaging contrast without the need for contrast agents or external physical fields. This design incorporates an amplitude meta-grating for linear transmission along the radial direction, combined with a phase meta-grating that utilizes focus and spiral phases with a first-order topological charge. Through theoretical analysis, numerical simulations, and experimental validation, we substantiate the effectiveness of our technique in distinguishing amplitude objects with isotropic edge enhancements. Importantly, this method also enables the accurate detection of both phase objects and artificial biological models. This breakthrough creates new opportunities for applications in medical diagnosis and nondestructive testing.

An identification algorithm of switched Box-Jenkins systems in the presence of bounded disturbances: An approach for approximating complex biological wastewater treatment models

This paper focuses on the development of linear Switched Box–Jenkins (SBJ) models for approximating complex dynamical models of biological wastewater treatment processes. We discuss the adaptation of these processes to the SBJ framework, showing the model's generality and flexibility as a class of switched systems that can offer accurate predictions for complex and nonlinear dynamics. This approach of modeling enables real-time data reconciliation from experiments and allows the design of model-based control strategies. Through the extension of the Outer Bounding Ellipsoids (OBEs) algorithm, the paper introduces an online two-stage parameter identification algorithm that effectively handles bounded disturbances for SBJ models. Using the OBE method relaxes the stochastic assumptions on disturbances, which may not be satisfied in practice, particularly for biological and environmental fluctuations. The proposed decomposed OBE algorithm separately identifies the switching patterns and parameters of linear submodels, conducting parameter identification in two distinct phases for input/output and disturbance/output submodels. The efficacy of this approach is shown via simulation results validated against both ADM1 and PBM models, demonstrating the proposed algorithm's capability to accurately predict outputs from different biological wastewater treatment models.

Suitable Cottonseed Protein Concentrate Supplementation in Common Carp (Cyprinus carpio) Serves as an Effective Strategy for Fish Meal Sparing Based on Improvement in Intestinal Antioxidant Capacity, Barrier and Microbiota Composition

The application of cottonseed protein concentrate (CPC) is an effective strategy to moderate the shortage of fish meal (FM) for the aquafeed industry. However, little attention has been paid to the effects of replacing fishmeal with CPC on cyprinid fish. This study used common carp (Cyprinus carpio) as the biological model and assessed the potential of applying CPC as a substitute for fishmeal in the diet of common carp. The proportion of fish meal substituted with CPC in the six diets was 0% (CPC0), 25% (CPC25), 50% (CPC50), 75% (CPC75), and 100% (CPC100). Each diet was fed to three replicate groups of common carp (4.17 ± 0.02 g) for 56 days. Results revealed that the CPC50 group significantly increased the growth indexes via up-regulating the genes of the GH/IGF axis and the TOR pathway. The intestinal digestive ability was also elevated in the CPC50 group via markedly increasing intestinal villus height, protease and lipase activities in the whole intestine, and the amylase activity of the foregut and midgut. The CPC50 group captured significantly higher activities and gene expressions of antioxidant enzymes and lower malonaldehyde contents via evoking the Nrf2/Keap1 signal pathway. The CPC50 group enhance the intestinal mechanical barrier via up-regulating the gene expressions of tight junction proteins and heighten the intestinal biological barrier by increasing the probiotics (Lactococcus) and decreasing the harmful bacteria (Enterococcus). But excessive substitution levels (75% and 100%) would compromise growth performance, intestinal antioxidant capacity, and immune function. The optimum substitution level was estimated to be 46.47%, 47.72%, and 46.43% using broken-line regression analyses based on mass gain rate, protein efficiency ratio, and feed conversion rate. Overall, the fishmeal in common carp feed could be substituted up to 50% by CPC without negative influence on growth, feed utilization, and or intestinal health.

BIOLOGICAL MODEL OF LUNG CANCER COMBINATION AND TUBERCULOSIS: DEVELOPMENT FOR PRECLINICAL STUDY OF RATIONAL COMBINATIONS OF TARGETED ANTITUMOR AND ANTITUBERCULOSIS THERAPY

Introduction. Lung cancer occupies a leading position in the structure of mortality from cancer. Chronic inflammation characteristic of tuberculosis increases the risk of lung cancer. Currently, more and more information is emerging confirming the cause-and-effect relationship between tuberculosis and cancer. The need to develop recommendations for public health regarding screening and treatment of tuberculosis in the tumor process determines the relevance of experimental studies on biological models of the combination of cancer and tuberculosis.. Aim. Creation а biological model of the combination of lung cancer and tuberculosis for preclinical study of rational combinations of antitumor and antituberculosis therapy. Material and methods: The biological model was implemented on C57BL/6 mice at the age of two months. Lewis epidermoid lung carcinoma was used to reproduce the tumor process. Modeling of tuberculosis was carried out using the reference strain Mycobacterium Tuberculosis H37RV. During the study, the following groups were formed: “intact mice” (healthy, uninfected with the Mycobacterium Tuberculosis (MBT) H37Rv strain without tumor cell transplantation); “control of MBT infection” (animals infected with Mycobacterium Tuberculosis strain H37Rv), “tumor control” (animals that were transplanted with epidermoid Lewis lung carcinoma) and “main group” (animals that were transplanted with epidermoid Lewis lung carcinoma simultaneously with MBT infection). Results. During the experiment, several models for creating the combined pathology of lung cancer and tuberculosis were identified. In the first (simultaneous infection and tumor inoculation), carcinoma developed more slowly in infected animals than in the tumor control group, and lung damage occurred with a predominance of the tuberculous process over the tumor process. The second (staged infection) also showed minimal metastatic manifestations with pronounced secondary changes in the primary tumor node. Analysis of the choice of model showed that the model with simultaneous infection and tumor inoculation most adequately ensures the development of the tumor process and tuberculosis infection, which allows maintaining the viability of the animal, fully developing the tumor process with metastasis to the lungs and obtaining the development of morphologically verified pulmonary tuberculosis.

Economic Analysis of Forest Management for Timber Production and Agricultural Production in Hillside Watershed Areas of Madagascar: A Case Study of the Itasy Region

In hillside watersheds, forests play a crucial role in protecting against soil erosion. Not only does forest vegetation cover serve as a biological measure to prevent soil erosion, it also generates income through sustainable management practices. However, there is a scarcity of research that evaluates and compares the profitability of perennial woody crops with that of other agricultural crops by integrating biological aspects within an economic analytical framework in Central Madagascar. In this study, we address this gap by combining a biological growth model that captures complex forest dynamics with economic management data to utilize the discounted cash flow method. We applied this approach to evaluate and compare the profitability of forest management and agricultural crop production in the hillside watershed areas of Central Madagascar, which have experienced severe human-induced soil erosion. Although our results showed that timber production is a profitable option, other agricultural crops generate much higher profits, indicating that financial subsidies alone may not be enough to shift land use toward perennial woody crops. In addition, growing fruit trees such as oranges and mangoes appears to be a much more financially attractive option for local farmers than annual agricultural crop production.

Aquarium fish and temperature neuropharmacology: Update

At the end of the 20th century in Russia, research on temperature dependence of specific pharmacological activity of antihypoxants and some neurotropic drugs under normo- and hypothermia of various biological objects in experimental and clinical conditions was started. At the beginning of the 21st century, an original biological model of acute hypoxia in aquarium fish was developed. This model made it possible to start the study of fish resistance to hypoxia, screen biologically active substances, and evaluate the activity of antihypoxants under normo- and hypothermia. The initial results of using this model allowed us to conclude that the dynamics of motor activity of fish under acute hypoxic conditions are similar to that of motor activity of fetuses inside the uterus under diagnostic intrauterine hypoxia created by voluntary apnea in a pregnant woman. The period of immobility of fish and fetuses under hypoxia was directly proportional to the value of their resistance to hypoxia. The reduction in conditions of acute hypoxic water temperature with floating fish in it at 10°C lengthens the duration of the immobile state of fish and preservation of their viability more than two times, and the preliminary introduction of hydrogen peroxide in the water in therapeutic dose along with hypothermia lengthens the corresponding periods by four times. Neurotropic drugs such as ethyl alcohol and local and general anesthetics will completely eliminate the pain syndrome that develops during local hypothermia in healthy patients. In addition, immersing the hands of people under alcohol intoxication and/or surgical anesthesia in water with melting ice for 2 min alleviates pain in the hands and accelerates the development of subsequent hyperemia in the skin of the hands by two times compared with the norm.

Establishment and molecular characterization of HCB-541, a novel and aggressive human cutaneous squamous cell carcinoma cell line.

Cutaneous squamous cell carcinoma (cSCC) is a common type of skin cancer that can result in significant morbidity, although it is usually well-managed and rarely metastasizes. However, the lack of commercially available cSCC cell lines hinders our understanding of this disease. This study aims to establish and characterize a new metastatic cSCC cell line derived from a Brazilian patient. A tumor biopsy was taken from a metastatic cSCC patient, immortalized, and named HCB-541 after several passages. The cytokeratin expression profile, karyotypic alterations, mutational analysis, mRNA and protein differential expression, tumorigenic capacity in xenograft models, and drug sensitivity were analyzed. The HCB-541 cell line showed a doubling time between 20 and 30h and high tumorigenic capacity in the xenograft mouse model. The HCB-541 cell line showed hypodiploid and hypotetraploidy populations. We found pathogenic mutations in TP53 p.(Arg248Leu), HRAS (Gln61His) and TERT promoter (C228T) and high-level microsatellite instability (MSI-H) in both tumor and cell line. We observed 37 cancer-related genes differentially expressed when compared with HACAT control cells. The HCB-541 cells exhibited high phosphorylated levels of EGFR, AXL, Tie, FGFR, and ROR2, and high sensitivity to cisplatin, carboplatin, and EGFR inhibitors. Our study successfully established HCB-541, a new cSCC cell line that could be useful as a valuable biological model for understanding the biology and therapy of metastatic skin cancer.

What Feathers Flock Together? Examining College Students’ Perceptions of Social Model Similarity

Educators often try to motivate students for learning science, technology, engineering, and math (STEM) topics by exposing them to social models with similar characteristics. However, it is not clear what dimensions of similarities are most optimal for motivating students in STEM. The present study investigated different dimensions in which college students perceived similarity to their social models in chemistry (N = 265) and biology (N = 143) and how different dimensions of perceived similarity were related to students’ competence-related beliefs and task values for learning in those fields. Academic similarities—particularly similar abilities, efforts, and interests—were the most frequent dimensions of similarity college students reported perceiving with respect to their STEM social models. Additionally, perceiving similar academic abilities to a social model was associated with greater competence-related beliefs in both studies. The relative salience of different academic similarities and their relations to STEM motivation also differed by study, suggesting that the importance of academic similarity perceptions may differ across learning contexts. Educators should consider emphasizing academic similarities when using social models to recruit or retain college students in STEM, while also acknowledging contextual nuances that may attenuate how different perceptions of similarities motivate students.

An inverse problem for nonlocal reaction-diffusion equations with time-delay

The reaction-diffusion equations have been studied in various aspects of nature, such as heat transfer and biological dynamic modelling. In this paper, we study a nonlocal reaction-diffusion model with time delay associated with the density and diffusion behaviour of biological populations. Based on our previous conclusions and numerical strategy of the direct problem, we continue to study the inverse problem about the diffusion coefficient as well as the parameters in the birth function, and also perform numerical simulations with error analysis. In addition, we further generalize the constant diffusion coefficient to the position-dependent diffusion rate, which is intended to improve the generalizability to multiple organisms diffusion in nature.

A Microfluidics-Assisted Double-Barreled Nanobioconjugate Synthesis Introducing Aprotinin as a New Moonlight Nanocarrier Protein: Tested toward Physiologically Relevant 3D-Spheroid Models.

Proteins are promising substances for introducing new drug carriers with efficient blood circulation due to low possibilities of clearance by macrophages. However, such natural biopolymers have highly sophisticated molecular structures, preventing them from being assembled into nanoplatforms with manipulable payload release profiles. Here, we report a novel anticancer nanodrug carrier moonlighting protein, Aprotinin, to be used as a newly identified carrier for cytotoxic drugs. The Aprotinin-Doxorubicin (Apr-Dox) nanobioconjugate was prepared via a single-step microfluidics coflow mixing technique, a feasible and simple way to synthesize a carrier-based drug design with a double-barreled approach that can release and actuate two therapeutic agents simultaneously, i.e., Apr-Dox in 1:11 ratio (the antimetastatic carrier drug aprotinin and the chemotherapeutic drug DOX). With a significant stimuli-sensitive (i.e., pH) drug release ability, this nanobioconjugate achieves superior bioperformances, including high cellular uptake, efficient tumor penetration, and accumulation into the acidic tumor microenvironment, besides inhibiting further tumor growth by halting the urokinase plasminogen activator (uPA) involved in metastasis and tumor progression. Distinctly, in healthy human umbilical vein endothelial (HUVEC) cells, drastically lower cellular uptake of nanobioconjugates has been observed and validated compared to the anticancer agent Dox. Our findings demonstrate an enhanced cellular internalization of nanobioconjugates toward breast cancer, prostate cancer, and lung cancer both in vitro and in physiologically relevant biological 3D-spheroid models. Consequently, the designed nanobioconjugate shows a high potential for targeted drug delivery via a natural and biocompatible moonlighting protein, thus opening a new avenue for proving aprotinin in cancer therapy as both an antimetastatic and a drug-carrying agent.