Incubation Of Serum Research Articles

Immunoregulation induced by autologous serum collected after acute exercise in obese men: a randomized cross-over trial

In this study, we evaluated the effects of autologous serum collected after two types of exercise on the in vitro inflammatory profile and T cell phenotype of resting peripheral blood mononuclear cells (PBMCs) in obese men. Serum samples and PBMCs were obtained from eight obese men who performed two exercise bouts—high intensity interval exercise (HIIE) and exhaustive exercise session to voluntary fatigue—in a randomized cross-over trial. Pre-exercise PBMCs were incubated with 50% autologous serum (collected before and after each exercise bout) for 4 h. In vitro experiments revealed that post-HIIE serum reduced the histone H4 acetylation status and NF-κB content of PBMCs and suppressed the production of both TNF-α and IL-6 by PBMCs, while increasing IL-10 production. Post-exhaustive exercise serum induced histone H4 hyperacetylation and mitochondrial depolarization in lymphocytes and increased TNF-α production. In vitro post-HIIE serum incubation resulted in an increase in the frequencies of CD4 + CTLA-4 + and CD4 + CD25+ T cells expressing CD39 and CD73. Post-exhaustive exercise serum decreased the frequency of CD4 + CD25 + CD73+ T cells but increased CD4 + CD25-CD39 + T cell frequency. Both post-exercise serums increased the proportions of CD4 + PD-1 + and CD8 + PD-1+ T cells. Blood serum factors released during exercise altered the immune response and T cell phenotype. The type of exercise impacted the immunomodulatory activity of the post-exercise serum on PBMCs.

Biotin-Labelled Clavulanic Acid to Identify Proteins Target for Haptenation in Serum: Implications in Allergy Studies.

Clavulanic acid (CLV) and amoxicillin, frequently administered in combination, can be independently involved in allergic reactions. Protein haptenation with β-lactams is considered necessary to activate the immune system. The aim of this study was to assess the suitability of biotinylated analogues of CLV as probes to study protein haptenation by this β-lactam. Two synthetic approaches afforded the labeling of CLV through esterification of its carboxylic group with a biotin moiety, via either direct binding (CLV-B) or tetraethylenglycol linker (CLV-TEG-B). The second analogue offered advantages as solubility in aqueous solution and potential lower steric hindrance for both intended interactions, with the protein and with avidin. NMR reactivity studies showed that both CLV and CLV-TEG-B reacts through β-lactam ring opening by aliphatic amino nitrogen, however with different stability of resulting conjugates. Unlike CLV conjugates, that promoted the decomposition of clavulanate fragment, the conjugates obtained with the CLV-TEG-B remained linked, as a whole structure including biotin, to nucleophile and showed a better stability. This was a desired key feature to allow CLV-TEG-B conjugated protein detection at great sensitivity. We have used biotin detection and mass spectrometry (MS) to detect the haptenation of human serum albumin (HSA) and human serum proteins. MS of conjugates showed that HSA could be modified by CLV-TEG-B. Remarkably, HSA preincubation with CLV excess only reduced moderately the incorporation of CLV-TEG-B, which could be attributed to different protein interferences. The CLV-TEG-B fragment with opened β-lactam was detected bound to the 404–430HSA peptide of the treated protein. Incubation of human serum with CLV-TEG-B resulted in the haptenation of several proteins that were identified by 2D-electrophoresis and peptide mass fingerprinting as HSA, haptoglobin, and heavy and light chains of immunoglobulins. Taken together, our results show that tagged-CLV keeps some of the CLV features. Moreover, although we observe a different behavior in the conjugate stability and in the site of protein modification, the similar reactivity indicates that it could constitute a valuable tool to identify protein targets for haptenation by CLV with high sensitivity to get insights into the activation of the immune system by CLV and mechanisms involved in β-lactams allergy.

Shape and Size Tuning of BiIII-Centered Polyoxopalladates: High Resolution 209Bi NMR and 205/206Bi Radiolabeling for Potential Pharmaceutical Applications.

We have discovered five bismuth(III)-containing polyoxopalladates (POPs) which were fully characterized by solution and solid-state physicochemical techniques: the cube-shaped [BiPd12O32(AsPh)8]5- (BiPd12AsL), [BiPd12O32(AsC6H4N3)8]5- (BiPd12AsLN), and [BiPd12O32(AsC6H4COO)8]13- (BiPd12AsLC) as well as the star-shaped [BiPd15O40(PO)10H6]11- (BiPd15P) and [BiPd15O40(PPh)10]7- (BiPd15PL), respectively. The organically modified capping groups phenylarsonate, p-azidophenylarsonate, and p-carboxyphenylarsonate were chosen as the azido (-N3) and carboxyl (-COOH) groups open up opportunities to covalently conjugate (via click reaction, amide coupling, etc.) with targeting vectors. The synthesis of p-azidophenylarsonate is reported here for the first time. The effects of the BiIII template and the organoarsonate vs -posphonate capping groups on the resulting POP shape (cube vs star) are discussed. The 209Bi NMR (I = 9/2) spectra of BiPd12AsL, BiPd12AsLN, and BiPd12AsLC revealed narrow peaks (ν1/2 ∼ 200 Hz) at 5470 ppm with a longitudinal relaxation time in the millisecond range (at 8.46 T). The absence of a quadrupolar relaxation contribution could be attributed to the allocation of BiIII in the highly symmetrical cuboid POP host cage. Similar peaks were absent in the 209Bi-NMR spectra of the star-shaped POPs BiPd15P and BiPd15PL due to the less symmetric coordination environment around the central BiIII ion. Further, 205/206Bi-radiolabeled POPs have been synthesized by incorporating a 205/206BiIII ion in the center of the POP structures. Carrier-free 205/206Bi radioisotopes (as surrogates of α-emitting 213Bi) were incorporated into the POP host-cage for the preparation of 205/206BiPd12AsL, 205/206BiPd12AsLN, 205/206BiPd12AsLC, and 205/206BiPd15PL, respectively. The radiometal incorporation was complete (>99% radiochemical yield) in 10 min according to radio-thin-layer chromatography. The 205/206BiPd12AsL polyanion was purified by solid-phase extraction. The incubation in rat serum showed the formation of a 205/206BiPd12AsL-protein aggregate.

The serological responses to acute exercise in humans reduce cancer cell growth in vitro: A systematic review and meta-analysis.

We systematically reviewed and meta‐analyzed the effects of acute exercise‐conditioned serum on cancer cell growth in vitro. Five literature databases were systematically searched for studies that measured cancer cell growth after exposure to human sera obtained before and immediately after an acute bout of exercise. Standardized mean differences (SMDs) with 95% confidence intervals (CIs) were pooled using a three‐level random‐effects model. Meta‐regressions were also performed with participant age and disease status, exercise type, cell line TP53 status, and serum incubation time entered as covariates. Seven studies met the inclusion criteria encompassing a total of 21 effect estimates and 98 participants. Exercise‐conditioned serum significantly reduced cancer cell growth compared with preexercise serum (SMD = −1.23, 95% CI: −1.96 to −0.50; p = .002; I 2 = 75.1%). The weighted mean reduction as a percentage of preexercise values was 8.6%. The overall treatment effect and magnitude of heterogeneity were not statistically influenced by any covariate. There were concerns regarding the risk of bias within individual studies and Egger's test of the intercept showed evidence of small study effects (β = −3.6, p = .004). These findings provide in vitro evidence that the transient serological responses to acute exercises reduce cancer cell growth, although many questions remain regarding the underlying mechanistic pathways and potential effect modifiers. To strengthen this evidence‐base, future studies should seek to reduce the risk of bias by using more rigorous experimental designs, and consider using 3D cell culture models to better replicate in vivo tumor conditions. PROSPERO registration: CRD42020161333.

Bifunctional Therapeutic Peptides for Targeting Malignant B Cells and Hepatocytes: Proof of Concept in Chronic Lymphocytic Leukemia

AbstractProtein–protein interactions are well recognized as therapeutic targets and therefore interfering peptides (IP) that block these interactions are receiving increasing attention. Four different tumor‐penetrating peptides (TPPs) (iRGD, RPARPAR, Linear TT1 (LinTT1), and cyclic TT1 (TT1)) are associated to an IP that blocks the interaction between the protein phosphatase PP2A and its binding protein SET, generating new bifunctional peptides able to intracellularly target the PP2A/SET interaction in malignant B cells and tumoral hepatocytes. The TPPs are able to penetrate into B cells of patients suffering chronic lymphocytic leukemia (CLL) and into tumoral hepatocytes but not into B cells from healthy donors and healthy hepatocytes. The association of cargo does not affect the penetration of the TPPs in CLL B cells. All the bifunctional peptides induce apoptosis in human CLL B cells and tumoral hepatocytes, and stability tests reveal that iRGD‐IP, RPARPAR‐IP, and TT1‐IP are stable after 24 h incubation in human serum. The iRGD associated with the IP significantly increases the survival of mice bearing xenograft models of CLL without any symptom of toxicity, suggesting that the bifunctional peptides may have a therapeutic application for selective tumoral targeting of PP2A/SET interaction, which is deregulated in several cancers, including CLL.

Serum markers for immunological response to metal alloys of endoprostheses

A study of solid-phase structures of blood serum using wedge-shaped and marginal dehydration methods (Litos system technology) was conducted in order to find out the causes of an inflammatory reaction followed by fibrosis in the second operated joint in a patient with bilateral knee arthritis. The study was aimed at identifying specific morphological markers that characterize the bodys response to the endoprosthesis material. Its solid-phase structures indicated the activation of a hyperergic reaction with daily incubation of blood serum with an alloy of titanium, aluminum, and vanadium. On the contrary, the immunological activity of blood serum can be suppressed and the structures present in it can be transformed into amorphous detritus with the incubation of an alloy of cobalt, chromium, and molybdenum. It was observed from the study that the nature of the immunological reaction of a sensitized organism depends on the type of metals that are part of the endoprosthesis. The immune response causes inflammation of the periarticular tissue, followed by its fibrosation and the formation of a scar demarcation shell that separates the periarticular tissue from the endoprosthesis and performs the function of an immunological barrier on the alloy of titanium, aluminum, and vanadium. On the other hand, an immunological reaction causes the destruction of inflamed periarticular tissue, followed by gradual destruction of the articular bag on the alloy of cobalt, chromium, and molybdenum.

An untargeted metabolomics strategy to measure differences in metabolite uptake and excretion by mammalian cell lines

IntroductionIt is widely but erroneously believed that drugs get into cells by passing through the phospholipid bilayer portion of the plasma and other membranes. Much evidence shows, however, that this is not the case, and that drugs cross biomembranes by hitchhiking on transporters for other natural molecules to which these drugs are structurally similar. Untargeted metabolomics can provide a method for determining the differential uptake of such metabolites.ObjectivesBlood serum contains many thousands of molecules and provides a convenient source of biologically relevant metabolites. Our objective was to detect and identify metabolites present in serum, but to also establish a method capable of measure their uptake and secretion by different cell lines.MethodsWe develop an untargeted LC-MS/MS method to detect a broad range of compounds present in human serum. We apply this to the analysis of the time course of the uptake and secretion of metabolites in serum by several human cell lines, by analysing changes in the serum that represents the extracellular phase (the ‘exometabolome’ or metabolic footprint).ResultsOur method measures some 4000–5000 metabolic features in both positive and negative electrospray ionisation modes. We show that the metabolic footprints of different cell lines differ greatly from each other.ConclusionOur new, 15-min untargeted metabolome method allows for the robust and convenient measurement of differences in the uptake of serum compounds by cell lines following incubation in serum. This will enable future research to study these differences in multiple cell lines that will relate this to transporter expression, thereby advancing our knowledge of transporter substrates, both natural and xenobiotic compounds.

Brucine PEGylated nanoemulsion: In vitro and in vivo evaluation

Cancer represents one of the most life threatening disease worldwide. Poly ethylene glycol nanoemulsion (PEG-NE), a new drug delivery system (DDS) is widely utilized for encapsulating water insoluble drugs and delivering it into cancerous tissue following its intravenous injection. Brucine (BRU) is an effective anti-carcinogenic agent toward different cancer cell lines; however, its low solubility represents a great obstacle in its formulation. The objective of this investigation was to develop and evaluate BRU-PEG-NE. Physicochemical evaluation including, viscosity, particle size and entrapment efficiency investigations were performed. Quantitative and qualitative estimation of total serum protein adsorbed onto the surface of BRU-NE were performed. in vitro release studies of BRU-NE with and without serum incubation were assayed. Formulations were tested for their cytotoxic activity using MTT assay. Finally, in-vivo anticancer effect was evaluated in tumor-inoculated mice. Homogenous NEs were obtained with particle size less than 140 nm and viscosity less than 3.3 cP for BRU-PEG-NE. Total serum protein adsorbed was less than 17.33 ± 0.76 μg/μmol total lipid for BRU-PEG-NE. in vitro drug release was less than 65 % over 24 h for PEG-NE. As well, PEG-NEs could achieve significant inhibition for the viability of cancer cells. Finally, significant reduction in tumor growth rate and mortality rate for BRU PEG-NE formulations were obtained. The obtained results suggested that BRU PEG NE could be considered as a potential mean for cancer therapy.

Antibodies From Children With PANDAS Bind Specifically to Striatal Cholinergic Interneurons and Alter Their Activity.

Pediatric obsessive-compulsive disorder (OCD) sometimes appears rapidly, even overnight, often after an infection. Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections, or PANDAS, describes such a situation after infection with Streptococcus pyogenes. PANDAS may result from induced autoimmunity against brain antigens, although this remains unproven. Pilot work suggests that IgG antibodies from children with PANDAS bind to cholinergic interneurons (CINs) in the striatum. CIN deficiency has been independently associated with tics in humans and with repetitive behavioral pathology in mice, making it a plausible locus of pathology. The authors sought to replicate and extend earlier work and to investigate the cellular effects of PANDAS antibodies on cholinergic interneurons. Binding of IgG to specific neurons in human and mouse brain slices was evaluated ex vivo after incubation with serum from 27 children with rigorously characterized PANDAS, both at baseline and after intravenous immunoglobulin (IVIG) treatment, and 23 matched control subjects. Binding was correlated with symptom measures. Neural activity after serum incubation was assessed in mouse slices using molecular markers and electrophysiological recording. IgG from children with PANDAS bound to CINs, but not to several other neuron types, more than IgG from control subjects, in three independent cohorts of patients. Post-IVIG serum had reduced IgG binding to CINs, and this reduction correlated with symptom improvement. Baseline PANDAS sera decreased activity of striatal CINs, but not of parvalbumin-expressing GABAergic interneurons, and altered their electrophysiological responses, in acute mouse brain slices. Post-IVIG PANDAS sera and IgG-depleted baseline sera did not alter the activity of striatal CINs. These findings provide strong evidence for striatal CINs as a critical cellular target that may contribute to pathophysiology in children with rapid-onset OCD symptoms, and perhaps in other conditions.

A complement-mediated rat xenotransfusion model of platelet refractoriness

BackgroundPlatelet refractoriness remains a challenging clinical dilemma although significant advancements have been made in identifying human leukocyte antigen (HLA) matched or HLA compatible units. Antiplatelet antibodies are the major risk factor for immune-mediated platelet refractoriness, yet the role of antibody-initiated complement-mediated platelet destruction remains poorly understood. Study Design and MethodsHuman complement-mediated opsonization and killing of platelets was assayed ex vivo using antibody-sensitized human platelets incubated with complement-sufficient human sera. A new animal model of platelet refractoriness utilizing Wistar rats transfused with human platelets is described. ResultsHuman platelets sensitized with anti-platelet antibodies were rapidly opsonized with iC3b upon incubation in human sera. This opsonization could be completely blocked with a classical pathway complement inhibitor, PA-dPEG24. Complement activation decreased platelet viability, which was also reversible with complement inhibitor PA-dPEG24. A new rat model of platelet refractoriness was developed that demonstrated some platelet removal from the blood stream was complement mediated. ConclusionsComplement activation initiated by anti-platelet antibodies leads to complement opsonization and decreased platelet viability. A new rat model of platelet refractoriness was developed that adds a new tool for elucidating the mechanisms of platelet refractoriness.

A Dodecapeptide Selected by Phage Display as a Potential Theranostic Probe for Colon Cancers

AimColon cancer is one of the leading causes of cancer-related mortality. However, specific biomarkers for its diagnosis or treatment are not established well. MethodsWe developed a colon-cancer specific peptide probe using phage display libraries. We validated the specificity of this probe to colon cancer cells with immunohistochemical staining and FACS analysis using one normal cell and five colon cancer cell lines. ResultsThis peptide probe maintained binding affinity even after serum incubation. For therapeutic applications, this peptide probe was conjugated to hematoporphyrin, a photosensitizer, which showed a significantly enhanced cellular uptake and high photodynamic effect to kill tumor cells. As another application, we made a nanoparticle modified from the peptide probe. It efficiently delivered SN-38, an anticancer drug, into tumor cells, and its tumor-targeting ability was observed in vivo after intravenous injection to the same xenograft model. ConclusionThe noble dodecapeptide probe can be a promising candidate for both colon tumor diagnosis and targeted drug delivery.

Recombinant cold shock domain containing protein is a potential antigen to detect specific antibody during early and late infections of Haemonchus contortus in goat

BackgroundHaemonchus contortus (H. contortus) is one of the most important parasites that cause huge economic losses to small ruminant industry worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods for the identification of prepatent H. contortus infection are available to identify prepatent H. contortus infection properly. The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H. contortus protein (rHc-CS) during early and late infections of H. contortus in goat.ResultsPurified rHc-CS exhibited a clear band, with a molecular weight about 38 kDa. H. contortus eggs were not detected by fecal egg count technique from feces collected at 0 to 14 days post infection (D.P.I). However, eggs were detected at 21, 28 and 35 D.P.I. Hence, results of immunoblotting assay showed specific anti rHc-CS antibody detection in all goat sera collected at early stage (14 D.P.I) and late stage (21–103 D.P.I) of H. contortus infection. Furthermore, no cross reactivity was observed against Trichinella spiralis, Fasciola hepatica and Toxoplasma gondii or uninfected goats. Among several evaluated rHc-CS indirect-ELISA format variables, favorable antigen coating concentration was found 0.28 μg/well at 37 °C 1 h and overnight at 4 °C. Moreover, optimum dilution ratio of serum and rabbit anti-goat IgG was recorded as 1:100 and 1:4000, respectively. The best blocking buffer was 5% Bovine Serum Albumin (BSA) while the best time for blocking, serum incubation and TMB reaction were recorded as 60, 120 and 10 min, respectively. The cut-off value for positive and negative interpretation was determined as 0.352 (OD450). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded as 100%.ConclusionThese results validated that rHc-CS is a potential immunodiagnostic antigen to detect the specific antibodies during early and late H. contortus infections in goat.

Simple FRET Electrophoresis Method for Precise and Dynamic Evaluation of Serum siRNA Stability.

Small interfering RNAs (siRNAs) are potent therapeutic molecules, but despite recent progress, their administration in vivo remains challenging due to their low stability in the bloodstream. Thus, techniques for investigating the stability of siRNA are fundamental for the development of efficient siRNA delivery systems. We designed a simple FRET electrophoresis method to dynamically evaluate serum siRNA stability in parallel with its interaction with the serum components. Each strand of the siRNA was labeled with the fluorophore carboxyfluorescein (FAM) at the 5'-end and the quencher carboxytetramethylrhodamine (TAMRA) at the 3'-end. After incubation in serum, molecular stability was proportional to the FRET efficiency that could be quantified in-gel by ImageJ analysis. Compared to the usual gel-shift and other plate-based FRET assays, this method is more sensitive and allows investigation of the stability of serum siRNA and siRNA-based nanoparticles, as well as the extrapolation of degradation kinetics in parallel with interaction analysis.

Morphological and Spectroscopic Study of an Apatite Layer Induced by Fast-Set Versus Regular-Set EndoSequence Root Repair Materials.

This study aimed to evaluate the morphology and chemistry of an apatite layer induced by fast-set versus regular-set EndoSequence root repair materials using spectroscopic analysis. Holes of a 4 mm diameter were created in the root canal dentin, which were filled with the test material. Fetal calf serum was used as the incubation medium, and the samples incubated in deionized water were used as controls. The material-surface and material-dentin interfaces were analyzed after 28 days using Raman and infrared spectroscopy, scanning electron microscopy/energy dispersive X-ray, and X-ray diffraction. After incubation in fetal calf serum, both materials formed a uniform layer of calcium phosphate precipitate on their surfaces, with the dentinal interface. This precipitated layer was a combination of hydroxyapatite and calcite or aragonite, and had a high mineral maturity with the regular-set paste. However, its crystallinity index was high with the fast-set putty. Typically, both consistencies (putty and paste) of root repair material have an apatite formation ability when they are incubated in fetal calf serum. This property could be beneficial in improving their sealing ability for root canal dentin.

Bile salt-coating modulates the macrophage uptake of nanocores constituted by a zidovudine prodrug and enhances its nose-to-brain delivery

We have previously demonstrated that the ester conjugation of zidovudine (AZT) with ursodeoxycholic acid (UDCA) allows to obtain a prodrug (U-AZT) which eludes the active efflux transporters (AET). This allows the prodrug to more efficiently permeates and remains in murine macrophages than the parent compound. Here we demonstrate that U-AZT can be formulated, by a nanoprecipitation method, as nanoparticle cores coated by bile acid salt (taurocholate or ursodeoxycholate) corona, without any other excipients. The U-AZT nanoparticles appeared spherical with a mean diameter of ∼200 nm and a zeta potential of ∼−55 mV. During the incubation (5 h) in fetal bovine serum, the ursodeoxycholate-coated nanoparticle size did not change. Differently, taurocholate-coated particle size was firstly reduced and then increased up to 800 µm, thus suggesting the high aptitude of these nanoparticles to interact with serum proteins. The in vitro uptake of taurocholate coated particles by murine macrophages was strongly higher than that of ursodeoxycholate-coated particles or free U-AZT (∼500% and ∼7000%, respectively). AZT was also detected in macrophages following the prodrug uptake, with the greatest amounts observed after the taurocholate-coated nanoparticle incubation. As macrophages in the subarachnoid spaces of cerebrospinal fluid (CSF) constitute one of the most unreachable HIV sanctuaries in the body, we also tested the ability of taurocholate-coated nanoparticles (i.e., nanoparticles highly internalized by macrophages) to reach them after their nasal administration in the presence or absence of chitosan. The results indicate that chitosan allowed to obtain a relatively high uptake (up to 4 µg/ml) of U-AZT in CSF. Taking into account that chitosan may promote the direct brain nanoparticle uptake, these findings can be considered an initial step toward the in vivo targeting of the subarachnoid macrophages by U-AZT prodrug.

Paclitaxel-loaded sodium deoxycholate-stabilized zein nanoparticles: characterization and in vitro cytotoxicity

Paclitaxel (PTX) is one of the most successful antineoplastic drugs and is widely used for the treatment of many forms of advanced and refractory cancer. Unfortunately, various drawbacks including non-selective cytotoxicity, poor water solubility and low bioavailability limit its clinical use. The aim of this study was to characterize a novel colloidal system made up of the natural protein zein, that would be able to efficiently retain the anticancer compound and increase its in vitro pharmacological effects. In fact, zein has promising characteristics that render it a potential material to be used in drug delivery application. The influences of temperature, pH and serum incubation on the stability of these particles, entrapment efficiency of PTX and in vitro toxicity on different cancer cell lines were evaluated. The nanosystems containing PTX demonstrated suitable storage stability, and were not destabilized by temperatures of up to 50 °C, pH alterations, the freeze-drying process or serum proteins. The encapsulation of PTX did not destabilize the structure of the zein nanoparticles and a suitable drug entrapment efficiency resulted. PTX-loaded zein nanoparticles showed an increased toxicity on different cancer cell lines with respect to the free drug, confirming its potential application in preclinical and clinical investigations.

Influence of a Protein Corona on the Oral Pharmacokinetics of Testosterone Released from Mesoporous Silica

AbstractThe formation of a protein corona adsorbed on mesoporous silica particles with 2D and 3D pore structures after incubation in fetal bovine serum is investigated. The compositional properties of the resulting protein–particle complex is determined by using mass spectrometry as a function of serum incubation time. The effect of the adsorbed proteins on the drug release properties of the mesoporous particles when these are used to deliver the hormone TST is examined in dissolution media. Changes to the pharmacokinetic parameters are validated in vivo in a rat model. Small changes to the maximum plasma concentration, terminal half‐life, and area under the plasma concentration time curve are observed with pre‐incubation of a hard protein corona around the particles. This strategy is used to slow down the release of pharmaceutical drugs loaded onto mesoporous silica, and acts as a formulation strategy for future nanomedical applications involving advanced drug delivery.

Synthesis, characterization, and mechanistic studies of a gold nanoparticle-amphotericin B covalent conjugate with enhanced antileishmanial efficacy and reduced cytotoxicity.

BackgroundAmphotericin B (AmB) as a liposomal formulation of AmBisome is the first line of treatment for the disease, visceral leishmaniasis, caused by the parasite Leishmania donovani. However, nephrotoxicity is very common due to poor water solubility and aggregation of AmB. This study aimed to develop a water-soluble covalent conjugate of gold nanoparticle (GNP) with AmB for improved antileishmanial efficacy and reduced cytotoxicity.MethodsCitrate-reduced GNPs (~39 nm) were functionalized with lipoic acid (LA), and the product GNP-LA (GL ~46 nm) was covalently conjugated with AmB using carboxyl-to-amine coupling chemistry to produce GNP-LA-AmB (GL-AmB ~48 nm). The nanoparticles were characterized by dynamic light scattering, transmission electron microscopy (TEM), and spectroscopic (ultraviolet–visible and infrared) methods. Experiments on AmB uptake of macrophages, ergosterol depletion of drug-treated parasites, cytokine ELISA, fluorescence anisotropy, flow cytometry, and gene expression studies established efficacy of GL-AmB over standard AmB.ResultsInfrared spectroscopy confirmed the presence of a covalent amide bond in the conjugate. TEM images showed uniform size with smooth surfaces of GL-AmB nanoparticles. Efficiency of AmB conjugation was ~78%. Incubation in serum for 72 h showed <7% AmB release, indicating high stability of conjugate GL-AmB. GL-AmB with AmB equivalents showed ~5-fold enhanced antileishmanial activity compared with AmB against parasite-infected macrophages ex vivo. Macrophages treated with GL-AmB showed increased immunostimulatory Th1 (IL-12 and interferon-γ) response compared with standard AmB. In parallel, AmB uptake was ~5.5 and ~3.7-fold higher for GL-AmB-treated (P<0.001) macrophages within 1 and 2 h of treatment, respectively. The ergosterol content in GL-AmB-treated parasites was ~2-fold reduced compared with AmB-treated parasites. Moreover, GL-AmB was significantly less cytotoxic and hemolytic than AmB (P<0.01).ConclusionGNP-based delivery of AmB can be a better, cheaper, and safer alternative than available AmB formulations.

A Conversation Between Hedvig Hricak and Johannes Czernin.

1038 Introduction: Gold nanostar (AuNS) displays a high photothermal conversion efficiency and has been applied for in vitro and in vivo photothermal therapy (PTT) of various tumors. Single photon emission computed tomography (SPECT) and positron emission tomography (PET) provide a powerful tool for in vivo tracking of nanoparticles. In this study, we synthesized PEGylated AuNS (pAuNS) and its radioactive surrogate to evaluate its biological characteristics. The results of biodistribution and SPECT images may be adopted in designing the procedure of cancer treatment. The potential of pAuNS-mediated PTT was evaluated in a SKOV-3 human ovarian tumor-bearing mouse model. Methods: AuNS with a maximal plasma absorption peak at ~790 nm and an average size of 46.8±3.6 nm was synthesized by HEPES-reduction method, and then PEGylated to obtain pAuNS. The temperature rise of pAuNS in different buffers after 793 nm near infrared (NIR) laser irradiation was recorded with a thermal camera. DTPA-conjugated pAuNS was labeled with indium-111 to obtain 111In-DTPA-pAuNS with high radiolabeling efficiency (94.8±1.4%) and good in vitro stability (>95% after 72 h of incubation in serum). The biological characterizations of 111In-DTPA-pAuNS were conducted in mice bearing SKOV-3 human ovarian xenografts. The tumor gold concentration was estimated based on the results of biodistribution and validated by the inductively coupled plasma mass spectrometry (ICP-MS). The anti-tumor effect of pAuNS-mediated PTT was also evaluated in the same animal tumor model. Results: The heat generation after NIR irradiation (1.0 and 1.5 W/cm2) of pAuNS-containing normal saline and fetal bovine serum increased in a dose-dependent manner. MicroSPECT/CT images revealed that most of 111In-DTPA-pAuNS after intravenous injection retained in liver and spleen in the tumor-bearing mice. The gold content in tumor determined by 111In-radioactivity and ICP-MS was considerably correlated (R2 >0.7), indicated that 111In-DTPA-pAuNS is a suitable radioactive surrogate of pAuNS. At 24 h post-injection of 111In-DTPA-pAuNS, the tumor-to-muscle ratio reached plateau and was selected as the time point for pAuNS-mediated PTT. Comparing to NIR irradiation only, the tumor temperature rise was more remarkable, accompanied with a significantly inhibited tumor growth, in pAuNS-mediated PTT treatment group. No pathological changes or injuries were detected in main organs following pAuNS administration. Conclusions: We demonstrated the anti-tumor effect of pAuNS-mediated PTT in treating ovarian cancer. 111In-labeled pAuNS (i.e. 111In-DTPA-pAuNS) was successfully synthesized and used for investigating the biological behaviors of pAuNS, which hence facilitated the selection of the appropriate time point for pAuNS-mediated PTT.

Preparation and imaging of 18F-VUIIS1008 targeting TSPO in rheumatoid arthritis model

Objective To synthesize a novel 18F labeled probe targeting translocator protein (TSPO) ligand 2-(5, 7-diethyl-2-(4-(2-fluoroethoxy)phenyl)pyrazolo[1, 5-a]pyrimidin-3-yl)-N, N-diethylacetamide (VUIIS1008), and evaluate its biodistribution and imaging in rheumatoid arthritis (RA) model. Methods The tosylate substrate was labeled with 18F using a tosyloxy for fluorine nucleophilic aliphatic substitution to obtain 18F-VUIIS1008. The labeling efficiency, radiochemical purity, and stability in vitro were determined. In vitro cellular uptake and competitive binding assay were performed on RAW264.7 macrophage cells. Biodistribution and microPET/CT imaging were investigated on RA mice established by Complete Freund′s Adjuvant. Two-sample t test was used to analyze the data. Results 18F-VUIIS1008 was synthesized with the labeling yield up to (41.00±5.00)%, the radiochemical purity >98.00%, and the specific radioactivity >1.52×108 MBq/mmol. 18F-VUIIS1008 was highly stable with the radiochemical purity >98.00% at 4 h after incubation in mouse serum. In vitro, it also exhibited high specific TSPO binding in RAW264.7 macrophage cells. The uptake ratio was (14.00±0.30)% at 1 h after incubation, and decreased significantly ((4.00±0.70)%; t=12.894, P<0.05) after adding excessive unlabeled VUIIS1008. The half maximal inhibitory concentration (IC50) of 18F-VUIIS1008 binding to TSPO was 0.05 nmol/L in RAW264.7 macrophage cells. In vivo distribution results showed that the uptake of 18F-VUIIS1008 in the left arthritic ankles reached the peak value of (1.33±0.02) percentage activity of injection dose per gram of tissue (%ID/g) at 1 h after injection. The radioactivity ratio of left ankle arthritic tissue to blood (A/B) and to normal muscle (A/M) was 4.40±0.22 and 1.65±0.07 respectively. MicroPET/CT imaging demonstrated that 18F-VUIIS1008 could specifically target and retained in the inflammation site. Conclusion 18F-VUIIS1008 can be easily synthesized with high radiochemical purity and can clearly visualized in RA imaging with low background, suggesting its potential as a novel promising molecular probe targeting TSPO for RA PET imaging. Key words: Membrane transport proteins; Pyrazoles; Pyrimidines; Fluorine radioisotopes; Arthritis, rheumatoid; Positron-emission tomography; Tomography, X-ray computed; Mice