Activity In Human Blood Research Articles

A comparative study on biosynthesized silver nanoparticles from H. undatus fruit peel and their therapeutic applications

The green synthesis of nanoparticles (NPs) gained significant impacts in various fields due to the use of eco-friendly approaches. In this study, silver nanoparticles (AgNPs) were synthesized from the aqueous extract of Hylocereus undatus fruit peel. The presence of AgNPs was analysed using characterization methods such as UV‒Vis, FTIR, GCMS, XRD, EDAX, and FESEM. The synthesized AgNPs showed greater antibacterial activity against Escherichia coli than against Streptococcus pneumoniae. The antifungal activity against Candida albicans was greater than that against Candida tropicalis. The IC50 value for the antibiofilm activity of the AgNPs was 2.81 µg/mL, whereas that of the H. undatus peel extract was 1.34 µg/mL. The invitro antioxidant activity of the AgNPs was evaluated using two different methods. The DPPH radical scavenging activity of the AgNPs and fruit peel extract was observed with IC50 values of 3.8 and 2.03 µg/mL respectively. On the other hand, nitric oxide radical scavenging activities were recorded and the IC50 values were calculated to be 2.8 and 2.3 µg/mL. The AgNPs demonstrated thrombolytic activity in human blood with 10, 32.36, and 56.25% lysis. The cytotoxicity of the AgNPs was minimal, with an IC50 of 0.2 µg/mL and the peel extract had the greatest cytotoxicity with an IC50 of 0.3 µg/mL. The findings of this study demonstrated that the synthesized AgNPs from H. undatus peel extract could be potential candidates for treating prostate cancer.Graphical abstract

Phytochemical analyses and in vitro evaluation of antioxidant, antihemolytic and antibacterial activity of methanolic extract of aerial parts of marjoram (Origanum majorana L)

ABSTRACT This work reports an in vitro study of the biological activities of the methanolic extract of the aerial parts of marjoram (Origanum majorana), including antibacterial, anti-haemolytic and antioxidant activity. The extract showed strong antibacterial activity against three bacterial strains, E. Coli, Pseudomonas aeruginosa, and Staphylococcus aureus. It also demonstrated antihaemolytic activity in human blood, inhibiting haemolysis induced by sodium chloride and temperature. The phytochemical screening revealed the presence of cathechic tannins, flavonoids, saponosids, mucilagos, and glycosids. These results suggest that the methanolic extract of marjoram could be a potential source of antimicrobial agents, and natural antioxidants.

Human blood markers of cholinergic neurotoxicity and neuropathy: A useful guide for laboratory applications

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are useful markers to assess the effects of exposure to anticholinesterase insecticides (Anti-AChE). In addition, lymphocyte neuropathy target esterase (LNTE) has been used as biomarker of neuropathic organophosphate compounds (OPs). Thus, this study evaluates the main types of circulating biomarkers related to the cholinergic system and to the neuropathy induced by OPs in standardized human samples. To achieve this objective, total protein of human plasma, erythrocytes and lymphocytes were first standardized, and then AChE, BChE and LNTE activities in human blood were evaluated in the presence of inhibitors. The acceptance criteria of the regulatory agency were respected with coefficients of regression of curves of 0.9972 for cholinesterase and 0.9956 for LNTE analyses. The wavelength established to perform cholinesterase assay was 450 nm and the time of incubation of the enzymes with inhibitors was 30 min. Differences were observed among the IC50 values regarding the in vitro inhibition of AChE, BChE and LNTE in the presence of OPs. In conclusion, the procedures demonstrated by the present work were simple, fast, inexpensive, sensitive, easy to be replicated and suitable to make conclusions about the neurotoxicity induced by Anti-AChE and neuropathic OPs.

Potential anti-inflammatory biomarkers from Myrtaceae essential oils revealed by untargeted metabolomics

Many species from Myrtaceae have traditionally been used in traditional medicine as anti-inflammatory, antimicrobial, antidiarrheal, antioxidant and antirheumatic, besides in blood cholesterol reduction. In the present work, the anti-inflammatory activity of essential oils from eighteen Myrtaceae spp. were evaluated according to their ex-vivo anti-inflammatory activity in human blood, and the corresponding biomarkers were determined using untargeted metabolomics data and multivariate data analysis. From these studied species, six displayed anti-inflammatory activity with percentage rates of inhibition of PGE2 release above 70%. Caryophyllene oxide (1), humulene epoxide II (2), β-selinene (3), α-amorphene (4), α-selinene (5), germacrene A (6), β-bisabolene (7), α-muurolene (8), α-humulene (9), β-gurjunene (10), myrcene (11), β-elemene (12), α-cadinol (13), α-copaene (14), E-nerolidol (15) and ledol (16) were annotated as potential anti-inflammatory biomarkers. The results obtained in this study point to essential oils from species of the Myrtaceae family as a rich source of anti-inflammatory agents.

A novel RP-HPLC method for quantification of cholinesterase activity in human blood: An application for assessing organophosphate and carbamate insecticide exposure.

Several methods have been reported to estimate Acetylcholinesterase (AChE) enzyme activity in blood samples. The Ellman assay is the most important among all but with several shortcomings, and there is a need to develop a method which is accurate, sensitive and quick for analyzing AChE. Therefore, we have developed an assay utilizing RP-HPLC with UV detection for the determination of AChE activity. This method measured the conversion of 1-naphthol acetate to 1-naphthol to estimate AChE activity in blood samples. Performance was judged on the basis of reproducibility, sensitivity, accuracy, and the ability to screen enzyme activity within 20minutes. A series of experiments were performed, varying the concentration of blood and substrate, with optimal sensitivity using 50 μM substrate and 10μL blood. The validation parameters such as linearity (R2 of ≥ 0.9842 for 1-naphthol and ≥ 0.9897 for 1-naphthol acetate), precision (94.21-96.41%), accuracy (85.2%-99.6% and 82.6%-99.3% for 1-naphthol and 1-naphthol acetate respectively), and robustness were validated according to International Conference on Harmonization (ICH) guidelines. Blood samples were collected from healthy people, farmers exposed to spraying of pesticides, and suicidal patients who ingested pesticides and were hospitalized and were analyzed by the developed method. The AChE level was approximately 21 units/mL compared to 24units/mL in controls, whereas suicidal patients showed the least AChE levels of 1 unit/mL. The employment of this method is recommended for estimating AChE level on various matrices.

A dual-signal sensing platform based on nanosheet materials for ratiometric fluorescence and colorimetric detection of enzyme activities in human blood

A dual-signal sensing platform comprising g-C3N4/MnO2 nanosheet composite and a routine chromogenic reagent o-phenylenediamine (OPD) was constructed for activity evaluation of several enzymes including Alkaline phosphatase (ALP), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) in human blood. MnO2 nanosheets was employed as a catalyst for oxidizing OPD to fluorescent 2,3-diaminophenazine (oxOPD), while g-C3N4 nanosheets served as a fluorescent indicator. MnO2 nanosheets also exhibited inner filter effect (IFE) on the fluorescence of g-C3N4 nanosheets due to the overlap of their respective absorption band and fluorescence emission band. Similarly, oxOPD would quench the fluorescence of g-C3N4 nanosheets via photoinduced electron transfer (PET) process. By using L-ascorbic acid-2-phosphate (AAP) and acetylthiocholine iodide (ATCh) as substrates, products of target enzyme-catalyzed hydrolysis reactions would reduce MnO2 nanosheets to Mn2+ cations, resulting in the inhibition of OPD oxidation and the fluorescence recovery of g-C3N4 nanosheets. Based on these reactions and effects, activities of target enzymes were indicated by the intensity ratio of fluorescence emitted from g-C3N4 nanosheets and oxOPD (F440/F560), as well as by the absorbance decrease of oxOPD at 420 nm (ΔA420). Limits of detection (LODs) obtained by the sensing platform for ALP, AChE, and BChE were respectively 0.28 U L-1, 0.19 U L-1, and 0.15 U L-1 for ratiometric fluorescence mode, and 1.3 U L-1, 1.0 U L-1 and 0.9 U L-1 for colorimetric mode. This dual-signal sensing platform can be extended for testing other enzymes after applying appropriate substrates for target enzyme-catalyzed hydrolysis reactions and demonstrates good potential for clinical diagnosis.

Temperature Effects on Plasma Li-heparin and Transaminase Activity in Children Blood with Tetralogy of Fallot

Tetralogy of Fallot (TOF) is a congenital heart disease that is caused by four heart defects, namely VSD, overriding aorta, obstruction of the right ventricular outflow tract and right ventricle hypertrophy. These abnormalities, which affect the anatomic structure of the heart, cause poor oxygenated blood to be pumped out of the heart. Children have smaller veins than adults, which make obtaining blood sample much harder. An experimental design was utilized, which allows the researchers to manipulate one or more variables and measure the resultant effects on the dependent variables. The population in this study was all vein blood sample of paediatric patients under the age of ten in National Cardiovascular Centre Harapan Kita Jakarta with TOF. Eleven sample were choose randomly as the sample in this study. We took 3 millilitres of blood from each sample and gave Li-heparin. The samples were then stored for 30 minutes at 5° C, 22-25° C and 37 ° C. We then used these sample to conduct ALT and AST tests. Based on the results of these tests we conducted descriptive statistical analysis, the normality test, homogeneity test and ANOVA test. We found that there is no significant difference in the results of AST and ALT tests using the three different samples. This study aimed to examine the effects of different temperatures on the volume of Li-heparin plasma and transaminase activity in human blood with Tetralogy of Fallot. The highest plasma volume was obtained from the sample that was put 30 minutes at 37°C.

Microwave assisted synthesis of Fe3O4 stabilized ZrO2 nanoparticles – Free radical scavenging, radiolabeling and biodistribution in rabbits

AimsIn vivo biodistribution of radio labeled ZrO2 nanoparticles is addressed for better imaging, therapy and diagnosis. Nanoparticles are synthesized by microwave assisted sol-gel technique using Fe3O4 as a stabilizer. Antioxidant assay, hemolytic activity in human blood and biodistribution in rabbits was explored to study the therapeutical as well as in vivo targeted diagnostic applications of as synthesized nanoparticles. Main methodsFe3O4 stabilized zirconia nanoparticles are synthesized using microwave assisted sol-gel method. Microwave (MW) powers are varied in the range of 100 to 1000 W. As synthesized nanoparticles are evaluated using different characterizations such as X-ray diffractometer, scanning electron microscope, Raman spectroscopy, impedance analyzer, Vickers micro hardness indenter, FTIR, and UV–Vis spectroscopy. In vitro activity of synthesized nanoparticles is checked in freshly extracted human blood serum. To study biodistribution of Fe3O4 stabilized zirconia nanoparticles in rabbit, technetium-99 m was used for labeling purpose. The labeling efficacy and stability of labeled nanoparticles are also measured with instant thin layer chromatography (ITLC) method. Intravenous injection of 99mTc-Fe3O4 stabilized zirconia nanoparticles (0.2 ml), containing 110 MBq of radioactivity, is performed to study the biodistribution; nanoparticles are injected into the ear vein of animal (rabbit). Key findingsZirconia (ZrO2) nanoparticles (NPs) are stabilized using Fe3O4 that were prepared by means of microwave assisted sol-gel method. Crystallite size (~20 nm) agrees well with the values required to stabilize tetragonal zirconia (t-ZrO2). Volume shrinkage results in high value of hardness (~1369). Dielectric constant values, compatible for biomedical application, are observed for tetragonally stabilized samples. Low value of hemolytic response is observed for Fe3O4 stabilized ZrO2 NPs. 99mTc radio labeled ZrO2 NPs proved to be potential candidate to study biodistribution. Biodistribution studies show stability of radiolabeled NPs in the original suspension as well as in blood serum. CT scan of rabbit is performed for several times to check the biodistribution of NPs with time and survival of rabbit. Results suggest that these NPs can also be used as targeted nanoparticles as well as variants of drug payload carrier. SignificanceResults signify that Fe3O4 stabilized ZrO2 nanoparticles synthesized by microwave assisted sol-gel method may be considered as “all-rounder” nanoplatform and are safe enough to be used in diagnostic as well as therapeutic purposes.

In-Vitro Evaluation of Biofilm and Hemolysis activity of Candida albicans Isolated from Oral Cavity

Candida albicans is a member of the healthy human microflora, colonizing several niches in the body and can cause opportunistic infection under host debilitated and immunocompromised condition. The present study aimed to investigate the in-vitro hemolytic activity of C. albicans isolated from oral cavity and screen biofilm through three different methods. During the study, 200 oral rinse samples from general human population were analyzed in microbiology laboratory of Central Campus of Technology, Tribhuvan University, Hattisar, Dharan. Nepal. Candida albicans were isolated and identified by conventional microbiological procedures. The hemolytic activity was evaluated through two different Sabouraud dextrose broth media (SDB) containing 7% defibrinated human blood, one supplemented with 3% glucose (SDBwG) and the other without glucose (SDBwoG). The biofilm formation was screened through congo red agar, tube method and tissue culture plate method. In this present study, 42 (21%) isolates of Candida albicans were isolated from 200 oral rinse samples. Isolated Candida albicans exhibited mean hemolysis activity of 28.66% on human blood SDB without glucose and 43.55% on human blood SDB with 3% glucose. Tissue culture plate method was considered sensitive, specific and accurate method for quantitative screening of biofilm in comparison to tube method and congo red agar method. This research concluded that Candida albicans exhibited greater hemolytic activity in human blood with glucose (SDBwG) than in human blood without glucose (SDBwoG). This finding explains that an increased blood glucose concentration may contribute to increased hemolysis activity of Candida albicans that could play pathogenic role for inducing infection like oral candidiasis in debilitated host like diabetic patients. Tissue culture plate method can accurately screen biofilms than tube and congo red agar method.
 Int. J. Appl. Sci. Biotechnol. Vol 8(4): 394-399

Integrin αIIbβ3 Regulates Platelet-Procoagulant Activity in the Lung

Pulmonary thrombosis is a major complication associated with high morbidity. Despite advances in diagnosis and treatment, the pathophysiology of pulmonary thrombosis remains incompletely understood. New clinical evidence suggests that in situ platelet activation resulting in enhanced procoagulant activity may promote pulmonary thrombosis. Improved understanding of the etiological mechanism would enable the development of new therapies for pulmonary thrombosis. Collagen and thromboplastin (TF) were administered intravascularly (IV) to C57BL/6 (WT) mice and the pulmonary microcirculation was visualized using quantitative fluorescence intravital fluorescence lung microscopy (qFILM). Fluorochrome-conjugated anti-mouse CD49b Ab and dextran was administered IV for in vivo staining of circulating platelets and visualization of blood vessels, respectively. Pulmonary thrombosis was defined as occlusion of blood vessels with platelet aggregates leading to pulmonary ischemia. Additionally, quantitative microfluidic fluorescence microscopy (qMFM) was used to study the effect of platelet αIIbβ3 inhibition on platelet procoagulant activity in human blood under vascular mimetic flow conditions. Collagen and TF triggered dose-dependent pulmonary thrombosis in mice in vivo, which involved development of platelet-rich thrombi in the pulmonary arteriolar bottlenecks (junction of pulmonary arteriole and capillaries), resulting in a transient ischemia in the arteriole and the down-stream capillary tree. The pulmonary arteriole thrombosis triggered by IV collagen or TF was protracted, lethal and completely abrogated following IV administration of αIIbβ3 receptor inhibitor (eptifibatide). Inhibition of platelet αIIbβ3 also significantly reduced platelet procoagulant activity, fibrin formation and thrombus formation in human blood flowing through microfluidic channels ex vivo. Our current findings suggest that αIIbβ3-dependent platelet procoagulant activity promotes pulmonary thrombosis. Both our models have potential application in investigating the molecular determinants of pulmonary thrombosis in diverse pulmonary disorders as well as evaluating efficacy of new antithrombotic drugs. Disclosures No relevant conflicts of interest to declare.

Engineering of Long-Circulating Peptidoglycan Hydrolases Enables Efficient Treatment of Systemic Staphylococcus aureus Infection

Staphylococcus aureus is a human pathogen causing life-threatening diseases. The increasing prevalence of multidrug-resistant S. aureus infections is a global health concern, requiring development of novel therapeutic options. Peptidoglycan-degrading enzymes (peptidoglycan hydrolases, PGHs) have emerged as a highly effective class of antimicrobial proteins against S. aureus and other pathogens. When applied to Gram-positive bacteria, PGHs hydrolyze bonds within the peptidoglycan layer, leading to rapid bacterial death by lysis. This activity is highly specific and independent of the metabolic activity of the cell or its antibiotic resistance patterns. However, systemic application of PGHs is limited by their often low activity in vivo and by an insufficient serum circulation half-life. To address this problem, we aimed to extend the half-life of PGHs selected for high activity against S. aureus in human serum. Half-life extension and increased serum circulation were achieved through fusion of PGHs to an albumin-binding domain (ABD), resulting in high-affinity recruitment of human serum albumin and formation of large protein complexes. Importantly, the ABD-fused PGHs maintained high killing activity against multiple drug-resistant S. aureus strains, as determined by ex vivo testing in human blood. The top candidate, termed ABD_M23, was tested in vivo to treat S. aureus-induced murine bacteremia. Our findings demonstrate a significantly higher efficacy of ABD_M23 than of the parental M23 enzyme. We conclude that fusion with ABD represents a powerful approach for half-life extension of PGHs, expanding the therapeutic potential of these enzybiotics for treatment of multidrug-resistant bacterial infections.IMPORTANCE Life-threatening infections with Staphylococcus aureus are often difficult to treat due to the increasing prevalence of antibiotic-resistant bacteria and their ability to persist in protected niches in the body. Bacteriolytic enzymes are promising new antimicrobials because they rapidly kill bacteria, including drug-resistant and persisting cells, by destroying their cell wall. However, when injected into the bloodstream, these enzymes are not retained long enough to clear an infection. Here, we describe a modification to increase blood circulation time of the enzymes and enhance treatment efficacy against S. aureus-induced bloodstream infections. This was achieved by preselecting enzyme candidates for high activity in human blood and coupling them to serum albumin, thereby preventing their elimination by kidney filtration and blood vessel cells.

Non-typical nucleoside analogs as fluorescent and fluorogenic indicators of purine-nucleoside phosphorylase activity in biological samples

Several novel non-typical nucleoside analogs were examined as potential fluorescent indicators of purine-nucleoside phosphorylase (PNP) activity in human blood. The substrates included N7-riboside of 8-aza-2,6-diaminopurine, N6-riboside of 1,N6-etheno-adenine and N2-riboside of N2,3-etheno-2-aminopurine. Reaction rates and apparent Michaelis’ constants were determined in 1000-fold blood lysates and compared with those for reference compounds, guanosine and 7-methylguanosine. It was concluded that the most promising for assaying human PNP in biological material was N6-riboside of 1,N6-etheno-adenine and N2-riboside of N2,3-etheno-2-aminopurine was optimal for the E. coli PNP, both offering at least 10-fold improvement in sensitivity relative to conventional assays. Other potential applications of this approach are discussed.

Direct Determination of Coagulation Factor IIa and Plasmin Activities for Monitoring of Thrombotic State.

Current laboratory examinations for hypercoagulable diseases focus on the biomarker content of the activated coagulation cascade and fibrinolytic system. Direct detection of physiologically important protease activities in blood remains a challenge. This study aims to develop a general approach that enables the determination of activities of crucial coagulation factors and plasmin in blood. This assay is based on the proteolytic activation of an engineered zymogen of l-phenylalanine oxidase (proPAO), for which the specific blood protease cleavage sites were engineered between the inhibitory and activity domains of proPAO. Specific cleavage of the recombinant proenzyme leads to the activation of proPAO, followed by oxidation and oxygenation of l-phenylalanine, resulting in an increase of chromogenic production when coupled with the Trinder reaction. We applied this method to determine the activities of both coagulation factor IIa and plasmin in their physiologically relevant basal state and fully activated state in sodium citrate-anticoagulated plasma respectively. Factor IIa and plasmin activities could be dynamically monitored in patients with thrombotic disease who were taking oral anticoagulants and used for assessing the hypercoagulable state in pregnant women. The high specificity, sensitivity, and stability of this novel assay not only makes it useful for determining clinically important protease activities in human blood and diagnosing thrombotic diseases but also provides a new way to monitor the effectiveness and safety of anticoagulant drugs.

Rapid bioluminescence assay for monitoring rat CES1 activity and its alteration by traditional Chinese medicines

In traditional Chinese medicine herbs (TCM), including Radix Salviae Miltiorrhizae (Danshen), Radix Puerariae Lobatae (Gegen), Radix Angelicae Sinensis (Danggui), and Rhizoma Chuanxiong (Chuanxiong) are widely used for the prevention and treatment of cardiovascular diseases and also often co-administered with Western drugs as a part of integrative medicine practice. Carboxylesterase 1 (CES1) plays a pivotal role in the metabolisms of pro-drugs. Since (S)-2-(2-(6-dimethylamino)-benzothiazole)-4,5-dihydro-thiazole-4-carboxylate (NLMe) has recently been identified by us as a selective CES1 bioluminescent sensor, we developed a rapid method using this substrate for the direct measurement of CES1 activity in rats. This bioluminescence assay was applied to determine CES1 activity in rat tissues after a two-week oral administration of each of the four herbs noted above. The results demonstrated the presence of CES1 enzyme in rat blood and all tested tissues with much higher enzyme activity in the blood, liver, kidney and heart than that in the small intestine, spleen, lung, pancreas, brain and stomach. In addition, the four herbs showed tissue-specific effects on rat CES1 expression. Based on the CES1 biodistribution and its changes after treatment in rats, the possibility that Danshen, Gegen and Danggui might alter CES1 activities in human blood and kidney should be considered. In summary, a selective and sensitive bioluminescence assay was developed to rapidly evaluate CES1 activity and the effects of orally administered TCMs in rats.

High ErbB3 activating activity in human blood is not due to circulating neuregulin-1 beta

Neuregulin-1β (NRG-1) is a membrane-bound or secreted growth and differentiation factor that mediates its action by binding to ErbB receptors. Circulating levels of NRG-1 are characterized by large inter-individual variability with the range of absolute values covering two orders of magnitude, from hundreds to tens of thousands of picograms per milliliter of blood. NRG-1 signaling via ErbB receptors contributes to the cell survival and downregulation of the inflammatory response. A higher level of circulating NRG-1 may indicate increased shedding of membrane-bound NRG-1, which in turn can contribute to better protection against cardiovascular stress or injury. However, it is unknown whether circulating NRG-1 can induce activation of ErbB receptors.In the current study, we performed an analysis of circulating NRG-1 functional activity using a cell-based ELISA measuring phosphorylation of ErbB3 induced by blood plasma obtained from healthy donors. We found high levels of ErbB3 activating activity in human plasma. No correlations were found between the levels of circulating NRG-1 and plasma ErbB3 activating activity. To determine the direct effect of circulating NRG-1, we incubated plasma with neutralizing antibody, which prevented the stimulatory effect of recombinant NRG-1 on activation of ErbB3. No effect of the neutralizing antibody was found on plasma-induced phosphorylation of ErbB3. We also found that a significant portion of circulating NRG-1 is comprised of full-length NRG-1 associated with large extracellular vesicles. Our results demonstrate that circulating NRG-1 does not contribute to plasma-induced ErbB3 activating activity and emphasizes the importance of functional testing of NRG-1 proteins in biological samples.

Dual-Response Detection of Oxidized Glutathione, Ascorbic Acid, and Cell Imaging Based on pH/Redox Dual-Sensitive Fluorescent Carbon Dots.

The pH/redox dual-sensitive fluorescent carbon dots (pHRCDs) with the fluorescence quantum yield of 16.97% were synthesized by the pyrolysis of l-glutamic acid (l-glu) and dopamine (DA). Compared with the quantum dot (QD)–dopamine conjugate, when the pH value of the solution was changed from neutral to alkaline, the pHRCDs exhibited unique optical phenomenon including red-shift of fluorescence peak and the fluorescence intensity first decreasing from pH 7 to 10 and then increasing from pH 10 to 13. The pHRCDs could be developed for a discriminative and highly sensitive dual-response fluorescent probe for the detection of oxidized glutathione (GSSG) and ascorbic acid (AA) activity in human blood. Under the optimized experimental conditions, the dual-response fluorescent probe can detect GSSG and AA in the linear range of 1.2–3.6 and 27–35 μM with the detection limits of 0.1 and 3.1 μM, respectively. In addition, the pHRCDs demonstrated low cytotoxicity and good biocompatibility, which can be well applied to in vitro cell imaging, and the pHRCDs/GSH fluorescence system has been successfully developed for the detection of AA in real samples.

Poly(D,L-Lactic Acid) Nanoparticle Size Reduction Increases Its Immunotoxicity.

Polylactic acid (PLA), a biodegradable and biocompatible polymer produced from renewable resources, has been widely used as a nanoparticulate platform for antigen and drug delivery. Despite generally regarded as safe, its immunotoxicological profile, when used as a polymeric nanoparticle (NP), is not well-documented. Thus, this study intends to address this gap, by evaluating the toxicity of two different sized PLA NPs (PLAA NPs and PLAB NPs), produced by two nanoprecipitation methods and extensively characterized regarding their physicochemical properties in in vitro experimental conditions. After production, PLAA NPs mean diameter (187.9 ± 36.9 nm) was superior to PLAB NPs (109.1 ± 10.4 nm). Interestingly, when in RPMI medium, both presented similar mean size (around 100 nm) and neutral zeta potential, possibly explaining the similarity between their cytotoxicity profile in PBMCs. On the other hand, in DMEM medium, PLAA NPs presented smaller mean diameter (75.3 ± 9.8 nm) when compared to PLAB NPs (161.9 ± 8.2 nm), which may explain its higher toxicity in RAW 264.7. Likewise, PLAA NPs induced a higher dose-dependent ROS production. Irrespective of size differences, none of the PLA NPs presented an inflammatory potential (NO production) or a hemolytic activity in human blood. The results herein presented suggest the hypothesis, to be tested in the future, that PLA NPs presenting a smaller sized population possess increased cytotoxicity. Furthermore, this study emphasizes the importance of interpreting results based on adequate physicochemical characterization of nanoformulations in biological medium. As observed, small differences in size triggered by the dispersion in cell culture medium can have repercussions on toxicity, and if not correctly evaluated can lead to misinterpretations, and subsequent ambiguous conclusions.

The Antistaphylococcal Lysin, CF-301, Activates Key Host Factors in Human Blood To Potentiate Methicillin-Resistant Staphylococcus aureus Bacteriolysis.

Bacteriophage-derived lysins are cell-wall-hydrolytic enzymes that represent a potential new class of antibacterial therapeutics in development to address burgeoning antimicrobial resistance. CF-301, the lead compound in this class, is in clinical development as an adjunctive treatment to potentially improve clinical cure rates of Staphylococcus aureus bacteremia and infective endocarditis (IE) when used in addition to antibiotics. In order to profile the activity of CF-301 in a clinically relevant milieu, we assessed its in vitro activity in human blood versus in a conventional testing medium (cation-adjusted Mueller-Hinton broth [caMHB]). CF-301 exhibited substantially greater potency (32 to ≥100-fold) in human blood versus caMHB in three standard microbiologic testing formats (e.g., broth dilution MICs, checkerboard synergy, and time-kill assays). We demonstrated that CF-301 acted synergistically with two key human blood factors, human serum lysozyme (HuLYZ) and human serum albumin (HSA), which normally have no nascent antistaphylococcal activity, against a prototypic methicillin-resistant S. aureus (MRSA) strain (MW2). Similar in vitro enhancement of CF-301 activity was also observed in rabbit, horse, and dog (but not rat or mouse) blood. Two well-established MRSA IE models in rabbit and rat were used to validate these findings in vivo by demonstrating comparable synergistic efficacy with standard-of-care anti-MRSA antibiotics at >100-fold lower lysin doses in the rabbit than in the rat model. The unique properties of CF-301 that enable bactericidal potentiation of antimicrobial activity via activation of "latent" host factors in human blood may have important therapeutic implications for durable improvements in clinical outcomes of serious antibiotic-resistant staphylococcal infections.

Microbubbles, Nanodroplets and Gas-Stabilizing Solid Particles for Ultrasound-Mediated Extravasation of Unencapsulated Drugs: An Exposure Parameter Optimization Study

Ultrasound-induced cavitation has been proposed as a strategy to tackle the challenge of inadequate extravasation, penetration and distribution of therapeutics into tumours. Here, the ability of microbubbles, droplets and solid gas-trapping particles to facilitate mass transport and extravasation of a model therapeutic agent following ultrasound-induced cavitation is investigated. Significant extravasation and penetration depths on the order of millimetres are achieved with all three agents, including the range of pressures and frequencies achievable with existing clinical ultrasound systems. Deeper but highly directional extravasation was achieved with frequencies of 1.6 and 3.3 MHz compared with 0.5 MHz. Increased extravasation was observed with increasing pulse length and exposure time, while an inverse relationship is observed with pulse repetition frequency. No significant cell death or any haemolytic activity in human blood was observed at clinically relevant concentrations for any of the agents. Overall, solid gas-trapping nanoparticles were found to enable the most extensive extravasation for the lowest input acoustic energy, followed by microbubbles and then droplets. The ability of these agents to produce sustained inertial cavitation activity whilst being small enough to follow the drug out of the circulation and into diseased tissue, combined with a good safety profile and the possibility of real-time monitoring, offers considerable potential for enhanced drug delivery of unmodified drugs in oncological and other biomedical applications.

Gas-Stabilizing Gold Nanocones for Acoustically Mediated Drug Delivery.

The efficient penetration of drugs into tumors is a major challenge that remains unmet. Reported herein is a strategy to promote extravasation and enhanced penetration using inertial cavitation initiated by focused ultrasound and cone-shaped gold nanoparticles that entrap gas nanobubbles. The cones are capable of initiating inertial cavitation under pressures and frequencies achievable with existing clinical ultrasound systems and of promoting extravasation and delivery of a model large therapeutic molecule in an in vitro tissue mimicking flow phantom, achieving penetration depths in excess of 2 mm. Ease of functionalization and intrinsic imaging capabilities provide gold with significant advantages as a material for biomedical applications. The cones show neither cytotoxicity in Michigan Cancer Foundation (MCF)-7 cells nor hemolytic activity in human blood at clinically relevant concentrations and are found to be colloidally stable for at least 5 d at 37 °C and several months at 4 °C.