PLGA-Based Composites for Various Biomedical Applications.

Polymeric materials have been extensively explored in the field of nanomedicine; within them, poly lactic-co-glycolic acid (PLGA) holds a prominent position in micro- and nanotechnology due to its biocompatibility and controllable biodegradability. In this review we focus on the combination of PLGA with different inorganic nanomaterials in the form of nanocomposites to overcome the polymer's limitations and extend its field of applications. We discuss their physicochemical properties and a variety of well-established synthesis methods for the preparation of different PLGA-based materials. Recent progress in the design and biomedical applications of PLGA-based materials are thoroughly discussed to provide a framework for future research.

Organ-on-a-Chip: A Preclinical Microfluidic Platform for the Progress of Nanomedicine.

Despite the progress achieved in nanomedicine during the last decade, the translation of new nanotechnology-based therapeutic systems into clinical applications has been slow, especially due to the lack of robust preclinical tissue culture platforms able to mimic the in vivo conditions found in the human body and to predict the performance and biotoxicity of the developed nanomaterials. Organ-on-a-chip (OoC) platforms are novel microfluidic tools that mimic complex human organ functions at the microscale level. These integrated microfluidic networks, with 3D tissue engineered models, have been shown high potential to reduce the discrepancies between the results derived from preclinical and clinical trials. However, there are many challenges that still need to be addressed, such as the integration of biosensor modules for long-time monitoring of different physicochemical and biochemical parameters. In this review, recent advances on OoC platforms, particularly on the preclinical validation of nanomaterials designed for cancer, as well as the current challenges and possible future directions for an end-use perspective are discussed.

Magnetic Hybrid Wax Nanocomposites as Externally Controlled Theranostic Vehicles: High MRI Enhancement and Synergistic Magnetically Assisted Thermo/Chemo Therapy.

To fight against cancer, smarter drugs and drug delivery systems are required both to boost the efficiency of current treatments while reducing deleterious side effects, and combine diagnosis/monitoring with therapy (theranosis) in the search for the final goal of personalized medicine. This work presents the design, preparation, and proof-of-principle validation of a novel hybrid organic-inorganic nanocomposite joining together non-invasive imaging capabilities through magnetic resonance imaging and externally actuated therapeutic properties through a combination of chemo- and thermotherapy. The lipidic matrix of the nanocomposite was composed of carnauba wax, which was simultaneously dual loaded with magnetite nanoparticles and the anticancer drug Oncocalyxone A. Obtained formulations were fully characterized and showed outstanding performances as T2 -contrast agents in magnetic resonance imaging (r2 >800 mm-1 s-1 ), heat generating sources in magnetic hyperthermia (specific absorption rate, SAR>200 W g-1 Fe ), and magnetically responsive drug delivery vehicles. The potential of the designed formulations as theranostic agents was validated in vitro and results indicated a synergistic thermo/chemotherapeutic effect derived from heat generation and controlled drug delivery to cancer growth. Thereby, this external control over the drug delivery profile and the integrated imaging capability open the door to personalized cancer medicine and real-time monitoring of tumor progression.

Recent Progress on Manganese-Based Nanostructures as Responsive MRI Contrast Agents.

Manganese-based nanostructured contrast agents (CAs) entered the field of medical diagnosis through magnetic resonance imaging (MRI) some years ago. Although some of these Mn-based CAs behave as classic T1 contrast enhancers in the same way as clinical Gd-based molecules do, a new type of Mn nanomaterials have been developed to improve MRI sensitivity and potentially gather new functional information from tissues by using traditional T1 contrast enhanced MRI. These nanomaterials have been designed to respond to biological environments, mainly to pH and redox potential variations. In many cases, the differences in signal generation in these responsive Mn-based nanostructures come from intrinsic changes in the magnetic properties of Mn cations depending on their oxidation state. In other cases, no changes in the nature of Mn take place, but rather the nanomaterial as a whole responds to the change in the environment through different mechanisms, including changes in integrity and hydration state. This review focusses on the chemistry and MR performance of these responsive Mn-based nanomaterials.

Tunable Performance of Manganese Oxide Nanostructures as MRI Contrast Agents.

The development of responsive magnetic resonance imaging contrast agents opens the door to a highly sensitive and specific diagnosis of altered physiological conditions. In this field, manganese dioxide (MnO2 ) is starting to be a leading contributor due to its susceptibility to conditions relevant to human diseased states, such as cancer. So far, the preclinical application of MnO2 has mainly been in the form of nanosheets, with enhancements of magnetic resonance imaging signals up to 50-fold upon activation. Herein, we thoroughly investigate, through a simple reaction, a series of Mnx Oy samples and correlate their phase composition and structure/morphology to the performance as classic/responsive MRI contrast agents in response to redox changes. Signal enhancements as high as 140-fold were obtained from MnO2 nano-urchins, and their capability as responsive magnetic resonance imaging contrast agents was demonstrated in vitro.

Orthogonal Clickable Iron Oxide Nanoparticle Platform for Targeting, Imaging, and On-Demand Release.

A versatile iron oxide nanoparticle platform is reported that can be orthogonally functionalized to obtain highly derivatized nanomaterials required for a wide variety of applications, such as drug delivery, targeted therapy, or imaging. Facile functionalization of the nanoparticles with two ligands containing isocyanate moieties allows for high coverage of the surface with maleimide and alkyne groups. As a proof-of-principle, the nanoparticles were subsequently functionalized with a fluorophore as a drug model and with biotin as a targeting ligand towards tumor cells through Diels-Alder and azide-alkyne cycloaddition reactions, respectively. The thermoreversibility of the Diels-Alder product was exploited to induce the on-demand release of the loaded molecules by magnetic hyperthermia. Additionally, the nanoparticles were shown to target cancer cells through in vitro experiments, as analyzed by magnetic resonance imaging.

Hybrid, metal oxide-peptide amphiphile micelles for molecular magnetic resonance imaging of atherosclerosis.

BACKGROUND: Atherosclerosis, a major source of cardiovascular disease, is asymptomatic for decades until the activation of thrombosis and the rupture of enlarged plaques, resulting in acute coronary syndromes and sudden cardiac arrest. Magnetic resonance imaging (MRI) is a noninvasive nuclear imaging technique to assess the degree of atherosclerotic plaque with high spatial resolution and excellent soft tissue contrast. However, MRI lacks sensitivity for preventive medicine, which limits the ability to observe the onset of vulnerable plaques. In this study, we engineered hybrid metal oxide-peptide amphiphile micelles (HMO-Ms) that combine an inorganic, magnetic iron oxide or manganese oxide inner core with organic, fibrin-targeting peptide amphiphiles, consisting of the sequence CREKA, for potential MRI imaging of thrombosis on atherosclerotic plaques. RESULTS: Hybrid metal oxide-peptide amphiphile micelles, consisting of an iron oxide (Fe-Ms) or manganese oxide (Mn-Ms) core with CREKA peptides, were self-assembled into 20-30 nm spherical nanoparticles, as confirmed by dynamic light scattering and transmission electron microscopy. These hybrid nanoparticles were found to be biocompatible with human aortic endothelial cells in vitro, and HMO-Ms bound to human clots three to five times more efficiently than its non-targeted counterparts. Relaxivity studies showed ultra-high r2 value of 457 mM-1 s-1 and r1 value of 0.48 mM-1 s-1 for Fe-Ms and Mn-Ms, respectively. In vitro, MR imaging studies demonstrated the targeting capability of CREKA-functionalized hybrid nanoparticles with twofold enhancement of MR signals. CONCLUSION: This novel hybrid class of MR agents has potential as a non-invasive imaging method that specifically detects thrombosis during the pathogenesis of atherosclerosis.

Magnetite Nanoparticles for Stem Cell Labeling with High Efficiency and Long-Term in Vivo Tracking.

Superparamagnetic iron oxide nanoparticles (SPIO-PAA), ultrasmall iron oxide nanoparticles (USPIO-PAA), and glucosamine-modified iron oxide nanoparticles (USPIO-PAA-GlcN) were studied as mesenchymal stem cell (MSCs) labels for cell tracking applications by magnetic resonance imaging (MRI). Pronounced differences were found in the labeling performance of the three samples in terms of cellular dose and labeling efficiency. In combination with polylysine, SPIO-PAA showed nonhomogeneous cell internalization, while for USPIO-PAA no uptake was found. On the contrary, USPIO-PAA-GlcN featured high cellular uptake and biocompatibility, and sensitive detection in both in vitro and in vivo experiments was found by MRI, showing that glucosamine functionalization can be an efficient strategy to increase cell uptake of ultrasmall iron oxide nanoparticles by MSCs.

Smart magnetic poly(N-isopropylacrylamide) to control the release of bio-active molecules.

Thermo switchable magnetic hydrogels undoubtedly have a great potential for medical applications since they can behave as smart carriers able to transport bioactive molecules to a chosen part of the body and release them on demand via magneto-thermal activation. We report on the ability to modify the lower critical solution temperature (LCST) of poly(N-isopropylacrylamide) (PNIPAM) on demand from 32 °C to LCST ≥ 37 °C. This was achieved by the absorption of controlled amounts of magnetite nanoparticles on the polymer chains. We show, through the effect on cell viability, that the resulting magnetic PNIPAM is able to trap and to release bio-active molecules, such as cell growth factors. The activities of the released bio molecule are tested on human umbilical vein endothelial cells culture. We demonstrate that the LCST of the magnetic PNIPAM can be reached remotely via inductive heating with an alternating magnetic field. This approach on magnetic PNIPAM clearly supports appealing applications in safe biomedicine.

Machine Learning-Assisted Optimization of Drug Combinations in Zeolite-Based Delivery Systems for Melanoma Therapy.

Two independent artificial neural network (ANN) models were used to determine the optimal drug combination of zeolite-based delivery systems (ZDS) for cancer therapy. The systems were based on the NaY zeolite using silver (Ag+) and 5-fluorouracil (5-FU) as antimicrobial and antineoplastic agents. Different ZDS samples were prepared, and their characterization indicates the successful incorporation of both pharmacologically active species without any relevant changes to the zeolite structure. Silver acts as a counterion of the negative framework, and 5-FU retains its molecular integrity. The data from the A375 cell viability assays, involving ZDS samples (solid phase), 5-FU, and Ag+ aqueous solutions (liquid phase), were used to train two independent machine learning (ML) models. Both models exhibited a high level of accuracy in predicting the experimental cell viability results, allowing the development of a novel protocol for virtual cell viability assays. The findings suggest that the incorporation of both Ag and 5-FU into the zeolite structure significantly potentiates their anticancer activity when compared to that of the liquid phase. Additionally, two optimal AgY/5-FU@Y ratios were proposed to achieve the best cell viability outcomes. The ZDS also exhibited significant efficacy against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus); the predicted combination ratio is also effective against S. aureus, underscoring the potential of this approach as a therapeutic option for cancer-associated bacterial infections.

Magnetic nanoparticle-based hyperthermia for cancer treatment.

Nanotechnology involves the study of nature at a very small scale, searching new properties and applications. The development of this area of knowledge affects greatly both biotechnology and medicine disciplines. The use of materials at the nanoscale, in particular magnetic nanoparticles, is currently a prominent topic in healthcare and life science. Due to their size-tunable physical and chemical properties, magnetic nanoparticles have demonstrated a wide range of applications ranging from medical diagnosis to treatment. Combining a high saturation magnetization with a properly functionalized surface, magnetic nanoparticles are provided with enhanced functionality that allows them to selectively attach to target cells or tissues and play their therapeutic role in them. In particular, iron oxide nanoparticles are being actively investigated to achieve highly efficient carcinogenic cell destruction through magnetic hyperthermia treatments. Hyperthermia in different approaches has been used combined with radiotherapy during the last decades, however, serious harmful secondary effects have been found in healthy tissues to be associated with these treatments. In this framework, nanotechnology provides a novel and original solution with magnetic hyperthermia, which is based on the use of magnetic nanoparticles to remotely induce local heat when a radiofrequency magnetic field is applied, provoking a temperature increase in those tissues and organs where the tumoral cells are present. Therefore, one important factor that determines the efficiency of this technique is the ability of magnetic nanoparticles to be driven and accumulated in the desired area inside the body. With this aim, magnetic nanoparticles must be strategically surface functionalized to selectively target the injured cells and tissues.

Enhanced alpha-amylase inhibition activity of amine-terminated PAMAM dendrimer stabilized pure copper-doped magnesium oxide nanoparticles.

The present work aims to prepare copper-doped MgO nanoparticles via a sol-gel approach and study their antidiabetic alpha-amylase inhibition activity with undoped MgO nanoparticles. The ability of G5 amine-terminated polyamidoamine (PAMAM) dendrimer for the controlled release of copper-doped MgO nanoparticles to exhibit alpha-amylase inhibition activity was also evaluated. The synthesis of MgO nanoparticles via sol-gel approach and optimization of calcination temperature and time has led to the formation of nanoparticles with different shapes (spherical, hexagonal, and rod-shaped) and a polydispersity in size ranging from 10 to 100 nm with periclase crystalline phase. The presence of copper ions in the MgO nanoparticles has altered their crystallite size, eventually modifying their size, morphology, and surface charge. The efficiency of dendrimer to stabilize spherical copper-doped MgO nanoparticles (ca. 30 %) is higher than in other samples, which was confirmed by UV-Visible, DLS, FTIR, and TEM analysis. The amylase inhibition assay emphasized that the dendrimer nanoparticles stabilization has led to the prolonged enzyme inhibition ability of MgO and copper-doped MgO nanoparticles for up to 24 h.

Optimization of iron-ZIF-8 catalysts for degradation of tartrazine in water by Fenton-like reaction.

Optimization of iron zeolitic imidazole framework-8 (FeZIF-8) nanoparticles, as heterogeneous catalysts, were synthesized and evaluated by the Fenton-like reaction for to degrade tartrazine (Tar) in aqueous environment. To achieve this, ZIF-8 nanoparticles were modified with different iron species (Fe2+ or Fe3O4), and subsequently assessed through the Fenton-like oxidation. The effect of different parameters such as the concentration of hydrogen peroxide, the mass of catalyst and the contact time of reaction on the degradation of Tar by Fenton-like oxidation was studied by using the Box-Behnken design (BBD). The BBD model indicated that the optimum catalytic conditions for Fenton-like reaction with an initial pollutant concentration of 30 ppm at pH 3.0 were T = 40 °C and 12 mM of H2O2, 2 g/L of catalyst and 4 h of reaction. The maximum Tar conversion value achieved with the best catalyst, Fe1ZIF-8, was 66.5% with high mineralization (in terms of decrease of total organic carbon - TOC), 44.2%. To assess phytotoxicity, the germination success of corn kernels was used as an indicator in the laboratory. The results show that the catalytic oxidation by Fenton-like reaction using heterogeneous iron ZIF-8 catalysts is a viable alternative for treating contaminated effluents with organic pollutants and highlighted the importance of the validation of the optimized experimental conditions by mathematical models.

Targeted treatment of triple-negative-breast cancer through pH-triggered tumour associated macrophages using smart theranostic nanoformulations.

Triple-negative breast cancer (TNBC) represents 15-25 % of the new breast cancer cases diagnosed worldwide every year. TNBC is among the most aggressive and worst prognosis breast cancer, mainly because targeted therapies are not available. Herein, we developed a magnetic theranostic hybrid nanovehicle for targeted treatment of TNBC through pH-triggered tumour associated macrophages (TAMs) targeting. The lipid core of the nanovehicle was composed of a Carnaúba wax matrix that simultaneously incorporated iron oxide nanoparticles and doxorubicin (DOX) - a chemotherapeutic drug. These drug-loaded wax nanovehicles were modified with a combination of two functional and complementary molecules: (i) a mannose ligand (macrophage targeting) and (ii) an acid-sensitive sheddable polyethylene glycol (PEG) moiety (specificity). The TAMs targeting strategy relied on the mannose - mannose receptor recognition exclusively after acid-sensitive "shedding" of the PEG in the relatively low tumour microenvironment pH. The pH-induced targeting capability towards TAMs was confirmed in vitro in a J774A.1 macrophage cell line at different pH (7.4 and 6.5). Biocompatibility and efficacy of the final targeted formulations were demonstrated in vitro in the TNBC MDA-MB-231 cell line and in vivo in an M-Wnt tumour-bearing (TNBC) mouse model. A preferential accumulation of the DOX-loaded lipid nanovehicles in the tumours of M-Wnt-tumour bearing mice was observed, which resulted both on an efficient tumour growth inhibition and a significantly reduced off-target toxicity compared to free DOX. Additionally, the developed magnetic hybrid nanovehicles showed outstanding performances as T2-contrast agents in magnetic resonance imaging (r2 ≈ 400-600 mM-1·s-1) and as heat generating sources in magnetic hyperthermia (specific absorption rate, SAR ≈ 178 W·g-1Fe). These targeted magnetic hybrid nanovehicles emerge as a suitable theranostic option that responds to the urgent demand for more precise and personalized treatments, not only because they are able to offer localized imaging and therapeutic potential, but also because they allow to efficiently control the balance between safety and efficacy.

Redox double-switch cancer theranostics through Pt(IV) functionalised manganese dioxide nanostructures.

Manganese dioxide (MnO2)-based nanostructures have emerged as promising tumour microenvironment (TME) responsive platforms. Herein, we used a one-pot reaction to prepare MnO2 nanostructures with Pt(IV) prodrugs as redox- (and thus TME-) responsive theranostics for cancer therapy, in which the Pt(IV) complexes act as prodrugs of cisplatin (Pt(II)), a clinical chemotherapeutic drug. The cytotoxicity of these MnO2-Pt(IV) probes was evaluated in two and three dimensional (2D and 3D) A549 cell models and found to be as effective as active drug cisplatin in 3D models. Moreover, MnO2-Pt(IV) nanoparticles exhibited strong off/ON magnetic resonance (MR) contrast in response to reducing agents, with the longitudinal relaxivity (r1) increasing 136-fold upon treatment with ascorbic acid. This off/ON MR switch was also observed in (2D and 3D) cells in vitro. In vivo MRI experiments revealed that the nanostructures induce a strong and long-lasting T1 signal enhancement upon intratumoral injection in A549 tumour-bearing mice. These results show the potential of MnO2-Pt(IV) NPs as redox responsive MR theranostics for cancer therapy.

Antibiofilm Efficacy of the Pseudomonas aeruginosa Pbunavirus vB_PaeM-SMS29 Loaded onto Dissolving Polyvinyl Alcohol Microneedles

Resistant bacteria prevail in most chronic skin wounds and other biofilm-related topical skin infections. Bacteriophages (phages) have proven their antimicrobial effectiveness for treating different antibiotic-resistant and multidrug-resistant bacterial infections, but not all phages are effective against biofilms. Phages possessing depolymerases can reach different biofilm layers; however, those that do not have depolymerase activity struggle to penetrate and navigate in the intricate 3D biofilm structure and mainly infect bacteria lodged in the outer biofilm layers. To address this, Pseudomonas aeruginosa phage vB_PaeM-SMS29, a phage with poor antibiofilm properties, was incorporated into polyvinyl alcohol (PVA, Mowiol 4:88) supplemented with 0.1% (v/v) of glycerol, and cast onto two different microneedle arrays varying in geometry. The dissolving microneedles were thoroughly characterized by microscopy, force-displacement, swelling, phage release and stability. Furthermore, 48 h-old biofilms were formed using the colony biofilm procedure (absence of broth), and the antibiofilm efficacy of the phage-loaded microneedles was evaluated by viable cell counts and microscopy and compared to free phages. The phages in microneedles were fairly stable for six months when stored at 4 °C, with minor decreases in phage titers observed. The geometry of the microneedles influenced the penetration and force-displacement characteristics but not the antimicrobial efficacy against biofilms. The two PVA microneedles loaded with phages reduced P. aeruginosa PAO1 biofilms by 2.44 to 2.76 log10 CFU·cm-2 at 24 h. These values are significantly higher than the result obtained after the treatment with the free phage (1.09 log10 CFU·cm-2). Overall, this study shows that the distribution of phages caused by the mechanical disruption of biofilms using dissolving microneedles can be an effective delivery method against topical biofilm-related skin infections.

A Review of the Advances and Challenges in Measuring the Thermal Conductivity of Nanofluids.

Fluids containing colloidal suspensions of nanometer-sized particles (nanofluids) have been extensively investigated in recent decades with promising results. Driven by the increase in the thermal conductivity of these new thermofluids, this topic has been growing in order to improve the thermal capacity of a series of applications in the thermal area. However, when it comes to measure nanofluids (NFs) thermal conductivity, experimental results need to be carefully analyzed. Hence, in this review work, the main traditional and new techniques used to measure thermal conductivity of the NFs are presented and analyzed. Moreover, the fundamental parameters that affect the measurements of the NFs' thermal conductivity, such as, temperature, concentration, preparation of NFs, characteristics and thermophysical properties of nanoparticles, are also discussed. In this review, the experimental methods are compared with the theoretical methods and, also, a comparison between experimental methods are made. Finally, it is expected that this review will provide a guidance to researchers interested in implementing and developing the most appropriate experimental protocol, with the aim of increasing the level of reliability of the equipment used to measure the NFs thermal conductivity.

Magnetic Solid Nanoparticles and Their Counterparts: Recent Advances towards Cancer Theranostics.

Cancer is currently a leading cause of death worldwide. The World Health Organization estimates an increase of 60% in the global cancer incidence in the next two decades. The inefficiency of the currently available therapies has prompted an urgent effort to develop new strategies that enable early diagnosis and improve response to treatment. Nanomedicine formulations can improve the pharmacokinetics and pharmacodynamics of conventional therapies and result in optimized cancer treatments. In particular, theranostic formulations aim at addressing the high heterogeneity of tumors and metastases by integrating imaging properties that enable a non-invasive and quantitative assessment of tumor targeting efficiency, drug delivery, and eventually the monitoring of the response to treatment. However, in order to exploit their full potential, the promising results observed in preclinical stages need to achieve clinical translation. Despite the significant number of available functionalization strategies, targeting efficiency is currently one of the major limitations of advanced nanomedicines in the oncology area, highlighting the need for more efficient nanoformulation designs that provide them with selectivity for precise cancer types and tumoral tissue. Under this current need, this review provides an overview of the strategies currently applied in the cancer theranostics field using magnetic nanoparticles (MNPs) and solid lipid nanoparticles (SLNs), where both nanocarriers have recently entered the clinical trials stage. The integration of these formulations into magnetic solid lipid nanoparticles-with different composition and phenotypic activity-constitutes a new generation of theranostic nanomedicines with great potential for the selective, controlled, and safe delivery of chemotherapy.

Magnetic lipid nanovehicles synergize the controlled thermal release of chemotherapeutics with magnetic ablation while enabling non-invasive monitoring by MRI for melanoma theranostics.

Nowadays, a number of promising strategies are being developed that aim at combining diagnostic and therapeutic capabilities into clinically effective formulations. Thus, the combination of a modified release provided by an organic encapsulation and the intrinsic physico-chemical properties from an inorganic counterpart opens new perspectives in biomedical applications. Herein, a biocompatible magnetic lipid nanocomposite vehicle was developed through an efficient, green and simple method to simultaneously incorporate magnetic nanoparticles and an anticancer drug (doxorubicin) into a natural nano-matrix. The theranostic performance of the final magnetic formulation was validated in vitro and in vivo, in melanoma tumors. The systemic administration of the proposed magnetic hybrid nanocomposite carrier enhanced anti-tumoral activity through a synergistic combination of magnetic hyperthermia effects and antimitotic therapy, together with MRI reporting capability. The application of an alternating magnetic field was found to play a dual role, (i) acting as an extra layer of control (remote, on-demand) over the chemotherapy release and (ii) inducing a local thermal ablation of tumor cells. This combination of chemotherapy with thermotherapy establishes a synergistic platform for the treatment of solid malignant tumors under lower drug dosing schemes, which may realize the dual goal of reduced systemic toxicity and enhanced anti-tumoral efficacy.

Insights into the Effect of Magnetic Confinement on the Performance of Magnetic Nanocomposites in Magnetic Hyperthermia and Magnetic Resonance Imaging.

The combination of superparamagnetic iron oxide nanoparticles (SPIONs) and lipid matrices enables the integration of imaging, drug delivery, and therapy functionalities into smart theranostic nanocomposites. SPION confinement creates new interactions primarily among the embedded SPIONs and then between the nanocomposites and the surroundings. Understanding the parameters that rule these interactions in real interacting (nano)systems still represents a challenge, making it difficult to predict or even explain the final (magnetic) behavior of such systems. Herein, a systematic study focused on the performance of a magnetic nanocomposite as a magnetic resonance imaging (MRI) contrast agent and magnetic hyperthermia (MH) effector is presented. The effect of stabilizing agents and magnetic loading on the final physicochemical and, more importantly, functional properties (i.e., blocking temperature, specific absorption rate, relaxivity) was studied in detail.