Optimization of Formulation Variables to Increase Antigen Entrapment in PLGA Particles

Efficient antigen entrapment is a key factor in preparation of poly (lactide-co-glycolide) acid (PLGA) vaccine formulations when the antigen is of short supply. This study presents a systematic approach in the testing of formulation variables with the objective to increase antigen entrapment in particles when the antigen stock concentration was low. Some of the experimental variables tested were poly (vinyl) alcohol (PVA) concentration in the inner (W₁) and outer (W₂) aqueous phase, W₁/oil (O) phase ratio and choice of organic solvent. The double emulsion solvent evaporation technique was applied to prepare PLGA particles with sonication as the emulsifying force. To measure antigen entrapment efficiency, the antigen (bovine serum albumin, BSA) was isotope labeled with ¹²⁵iodine (¹²⁵I). Our results demonstrated that a low PVA concentration in the inner aqueous (W₁) phase was beneficial to achieve a high encapsulation efficiency of antigen. On the contrary, in the outer aqueous (W₂) phase, a high PVA concentration favored antigen entrapment. We also demonstrated that decreasing the W₁ to O/polymer ratio contributed to increased entrapment efficiency. Testing different organic solvents (ethyl acetate, dichloromethane and chloroform), either alone or in combination, revealed that using chloroform as solvent resulted in the highest encapsulation of antigen and the highest production yield. Some of the results presented in this work are in disagreement with well-established formulation variables from previous studies.     

Effective production of fluorescent nanodiamonds containing negatively-charged nitrogen-vacancy centers by ion irradiation

Fluorescence from negatively-charged nitrogen-vacancy centers (NV⁻s) in diamonds has unique optical properties with none of the undesirable effects such as photo-bleaching and photo-blinking. In addition, the spin-dependent fluorescence intensity of NV⁻s allows us to perform optically detected magnetic resonance (ODMR) investigation for evaluating the presence of NV⁻s and for the electronic local environment. In this work, we irradiated H⁺, He⁺, Li⁺ and N⁺ ions to nanodiamonds with a median size of 26 nm at various irradiation energies and doses for improving the NV⁻ concentration. ODMR observations of the nanodiamonds showed that ion irradiation increased the number of nanodiamonds containing NV⁻s up to 200 ppm, whereas without ion irradiation, only few NV⁻s were found. The number of nanodiamonds containing NV⁻s at various ion irradiation doses was not monotonous, but had maxima at certain irradiation doses. These results suggest a competition in two opponent roles of vacancies, effective for pairing with nitrogen atoms and as defects for developing damage in crystalline. We also found that sharp and strong ODMR signals were obtained from nanodiamonds irradiated at the optimal condition for the highest yield of NV⁻s. We concluded that He⁺ ion irradiations with 60 or 80 keV at a dose of 1 × 10¹³ ions cm^–2 are the conditions required for the most efficient production of a high quantity of nanodiamonds containing NV⁻s.     

Column Chromatography Using Nano-Sorbent: A Viable Approach Towards Post-Processing Concentration of 125I for Medical Applications

Successful use of ion-exchange chromatographic technique using nano-zirconia (t-ZrO₂), to concentrate ¹²⁵I solution obtained from wet chemical distillation method to levels required for preparation of ¹²⁵I brachytherapy sources, is reported. A careful scrutiny of the sorption parameters of t-ZrO₂ was carried out and implemented to arrive at conditions resulting in optimum retention of ¹²⁵I from its solution of low radioactive concentration on a tiny column containing t-ZrO₂, followed by its elution in a small volume of eluent. The developed procedure was validated using samples containing varying amounts of ¹²⁵I activity in the feed and the concentration factors (CF) of ¹²⁵I > 5 could be obtained. The overall recovery of ¹²⁵I was >90% with appropriate radiochemical purity. The compatibility of the product in the preparation of ¹²⁵I brachytherapy sources was found to be satisfactory.     

The effect of a short term saturated fat diet on the apoprotein composition and radioiodination properties of rat very low density lipoproteins

The effect of a saturated fat diet on the apoprotein composition and radioiodination properties of plasma very low density lipoprotein (VLDL) was studied in rats. After feeding the diet for 10 days, the proportion of¹²⁵I attached to VLDL lipid decreased from 50% (control animals) to 8%, the remainder (92%) being bound to the apoportein components. The decreased lipid labelling was associated with proportional changes in the fatty acid composition of serum and VLDL lipids, the most notable change being a reduction in linoleic acid (30–8%) content which occurred in all the major lipid classes of both serum and VLDL. Analysis of VLDL after radioiodination showed that most of the radioactivity incorporated into the lipid moiety was associated with phospholipid. The proportion of¹²⁵I bound to phospholipid decreased after feeding rats a saturated diet. The proportion of soluble (small molecular weight peptides and arginine rich peptide) to insoluble (B apoprotein) did not alter during the saturated fatty acid dietary regime and no differences in the distribution of soluble proteins were observed. It is concluded that feeding a saturated fat diet to rats for 10 days significantly improved¹²⁵I labelling of the apoprotein moiety while apparently not inducing changes in apoprotein composition.     

Synthesis and Characterization of a Specific Iodine-125-Labeled TRPC5 Radioligand

The nonselective Ca²⁺-permeable transient receptor potential channel subfamily member 5 (TRPC5) belongs to the transient receptor potential canonical (TRPC) superfamily and is widely expressed in the brain. Compelling evidence reveals that TRPC5 plays crucial roles in depression and other psychiatric disorders. To develop a TRPC5 radioligand, following up on our previous effort, we synthesized the iodine compound TZ66127 and its iodine-125-labeled counterpart [¹²⁵I]TZ66127. The synthesis of TZ66127 was achieved by replacing chloride with iodide in the structure of HC608, and the [¹²⁵I]TZ66127 was radiosynthesized using its corresponding tributylstannylated precursor. We established a stable human TRPC5-overexpressed HEK293-hTRPC5 cell line and performed Ca²⁺ imaging and a cell-binding assay study of TZ66127; these indicated that TZ66127 had good inhibition activity for TRPC5, and the inhibitory efficiency of TZ66127 toward TRPC5 presented in a dose-dependent manner. An in vitro autoradiography and immunohistochemistry study of rat brain sections suggested that [¹²⁵I]TZ66127 had binding specificity toward TRPC5. Altogether, [¹²⁵I]TZ66127 has high potential to serve as a radioligand for screening the binding activity of other new compounds toward TRPC5. The availability of [¹²⁵I]TZ66127 might facilitate the development of therapeutic drugs and PET imaging agents that target TRPC5.     

Electrochemical immunoassay for CA125 based on cellulose acetate stabilized antigen/colloidal gold nanoparticles membrane

A novel separation-free electrochemical immunosensor for carcinoma antigen-125 (CA125) was proposed based on the immobilization of CA125 antigen on colloidal gold nanoparticles that was stabilized with cellulose acetate membrane on a glassy carbon electrode. A competitive immunoassay format was employed to detect CA125 antigen with horseradish peroxidase (HRP) labeled CA125 antibody as tracer, o-phenylenediamine and hydrogen peroxide as enzyme substrates. After the immunosensor was incubated with a mixture of HRP labeled CA125 antibody and CA125 sample at 35 °C for 50 min, the amperometric response decreased with an increasing CA125 concentration in the sample solution. The decreased percentage of the electrocatalytic current was proportional to CA125 concentration ranging from 0 to 30 U ml⁻¹ with a detection limit of 1.73 U ml⁻¹ (S/N = 3). The proposed immunosensor showed good stability, acceptable accuracy, and would be applicable to clinical immunoassay of CA125.     

Infrared absorption study of surface functional groups providing chemical modification of nanodiamonds by divalent copper ion complexes

Infrared (IR) absorption data evidence the presence of multiple carboxyl and anhydride groups together with hydroxyl and C(sp³)-H groups in the dried copper-modified detonation nanodiamonds (DND). Insignificant differences in IR spectra of initially purified and copper-modified nanodiamonds indicate that in the ion exchange process Cu²⁺ ions substitute protons just in a small fraction of the entire amount of carboxyl groups on the DND surface. Surface anhydride groups appear as the result of mutual conversion of the neighboring carboxyl groups into the anhydride ones. The observation of specific shape of IR absorption spectrum of DND is a good practical rule for primary selection of detonation nanodiamonds with oxygen-containing functional groups well suitable for targeted surface modification by double-charged metal ions.     

Thermochemistry of nanodiamond terminated by oxygen containing functional groups

The standard enthalpies of formation at 25 °C of nanodiamonds terminated by oxygen containing functional groups have been investigated by high-temperature oxidation calorimetry. Depending on the amount of oxygen containing functional groups, the nanodiamonds (plus oxygen and hydrogen as represented in the surface functional groups) can be up to 52 kJ mol⁻¹ more stable in enthalpy than graphite, which means that less heat is evolved during oxidation of nanodiamonds terminated by oxygen containing functional groups, since their surface carbon is already partially oxidized. The stability of the nanodiamonds terminated by oxygen containing functional groups increases (enthalpy of formation becomes more negative) with increasing surface area within the studied range, reflecting the dominant effect of higher content of surface functional groups over the destabilizing effect of higher surface-to-volume ratio typical for nanoparticles.     

Cost-effective disposable thiourea film modified copper electrode for capacitive immunosensor

Cost-effective disposable electrodes were fabricated from copper clad laminate, usually used for printed circuit board (PCB) in electronic industries, by using dry film photoresist. Electro-oxidation (anodisation) was employed to obtain a good formation of thiourea film on the electrode surface. The affinity binding pair of carcinoembryonic antigen (CEA) and anti-carcinoembryonic antigen (anti-CEA) was used as a model system. Anti-CEA was immobilized on thiourea film via covalent coupling. This modified electrode was incorporated with a capacitive system for CEA analysis. This capacitive immunosensor provided a linear range between 0.01 and 10 ng ml⁻¹ with a detection limit of 10 pg ml⁻¹. When applied to analyze CEA in serum samples, the results agreed well with the enzyme linked fluorescent assay (ELFA) technique (P > 0.05). The proposed strategy for the preparation of disposable modified copper electrode is very cost effective and simple. Moreover, it provides good reproducibility. This technique can easily be applied to immobilize other biological sensing elements for biosensors development.     

The effects of surface oxidation on luminescence of nano diamonds

In this work we quantify and characterise the effects of air-oxidation on nitrogen-vacancy defect luminescence in both high-temperature-high-pressure and detonation synthesized nanodiamonds using Raman and luminescence spectroscopies. We find that oxidation treatments result in an increased nitrogen-vacancy centre excited state lifetime from 13 ns to 25 ns and in 5-nm diamonds the intensity of this luminescence increases by at least 5-fold. At the same time, in 5-nm diamonds, short lived surface-defect related luminescence is reduced by 10-fold. Furthermore we find that air oxidation reduces the sp² and disordered carbon fraction of nanodiamonds by up to 5-fold in 5-nm nanodiamonds. Based on these results, the authors suggest that the disordered-carbon and graphite shell of 5-nm nanodiamonds quenches nitrogen-vacancy luminescence, and that this quenching can be partially reduced by surface oxidation. These findings provide useful insights into the role of the graphite and disordered carbon shell in quenching luminescence, and have implications for the applicability of 5-nm nanodiamonds to bio- and quantum physics applications.     

In vitro cadmium removal from human serum by Cibacron Blue F3GA–thionein‐complex conjugated affinity membranes

A new membrane affinity biosorbent carrying thionein has been developed for selective removal of cadmium ions from human serum. Microporous poly(2‐hydroxyethyl methacrylate) (pHEMA) membranes were prepared by photopolymerization of HEMA. The pseudo dye ligand Cibacron Blue F3GA (CB) was covalently immobilized on the pHEMA membranes. Then, the cysteine‐rich metallopeptide thionein was conjugated onto the CB‐immobilized membrane. The maximum amounts of CB immobilized and thionein conjugated on the membranes were 1.07 µmol cm⁻² and 0.92 µmol cm⁻², respectively. The hydrophilic pHEMA membrane had a swelling ratio of 58% (w/w) with a contact angle of 45.8 °. CB‐immobilized and CB‐immobilized–thionein‐conjugated membranes were used in the Cd(II) removal studies. Cd(II) ion adsorption appeared to reach equilibrium within 30 min and to follow a typical Langmuir adsorption isotherm. The maximum capacity (q _m) of the CB‐immobilized membranes was 0.203 (µmol Cd(II)) cm⁻² membrane and increased to 1.48 (µmol Cd(II)) cm⁻² upon CB–thionein‐complex conjugation. The pHEMA membranes retained their cadmium adsorption capacity even after 10 cycles of repeated use. © 2000 Society of Chemical Industry     

Deposition of detonation nanodiamonds by Langmuir–Blodgett technique

Langmuir–Blodgett technique, which classically allows preparing monomolecular films, was here used to deposit a film of detonation nanodiamond particles.We proceeded to the functionalization of the nanodiamond (nD) particles so as to obtain hydrophobic nanodiamonds. Cetyltrimethylammonium chloride (C₁₉H₄₂ClN) (CTAC) was used for this purpose in order to form an ionic complex ND–COO^?(NH₃)⁺–R with the functional groups of the nanodiamonds.Compressions with various strengths (10 to 30 mN/m) were performed on the Langmuir–Blodgett device in order to prepare different types of deposits on mica substrates. Atomic Force Microscopy was used to characterize these deposits. It was shown that compressions with low intensity result in discontinuous distributions of the particles on the surface. Conversely, very dense and continuous deposits were observed for higher compression strengths. By optimizing the nD/CTAC ratio in the suspension, a very regular deposit with a monoparticle height was obtained.     

3D-nanostructured scaffold electrodes based on single-walled carbon nanotubes and nanodiamonds for high performance biosensors

We report a novel strategy for controlled nanostructuring of electrodes surfaces using single-walled nanotubes and nanodiamonds in order to form highly porous structures for biosensors applications. These nanostructures were obtained by subsequent deposition of nanotubes and nanodiamonds followed by functionalization of this assembly with pyrenenitrilotriacteic acid (NTA) via dip coating. The electropolymerizaton of the pyrene derivative leads to a reinforcement of the structure and allows simultaneously the immobilization of biotin labeled bioreceptor units via coordinative affinity interactions. By using biotin modified glucose oxidase (B-GOx) as model bioreceptor, we could obtain impressive maximum current densities and sensitivities up to 465 μA cm⁻² and 85.78 mA M⁻¹ cm⁻², respectively.     

Effect of adsorbed metal ions and buffer nature on IgG separation from human plasma by column chromatography using an ion exchange resin,Amberlite IRC‐718

Fractionation of human plasma on ion exchanger resin was performed on Amberlite IRC‐718 saturated with metal ions. Depletion of human immunoglobulin G was carried out by column chromatography using Tris‐HCl, pH 7 at different concentrations. Results showed that, when Cu⁺² and Ni⁺² were adsorbed on the resin, one or two fractions of purified IgG were obtained, respectively. Whereas Fe⁺² and Zn⁺², both retain IgG and serum albumin or serum albumin alone. Furthermore, the Ni⁺²‐resin retention of serum proteins is too strong that the use of 700 mMTris‐HCl cannot liberate any other proteins than nonadsorbed serum albumin. In conclusion, this investigation demonstrates that immobilized metal ion affinity chromatography with Cu²⁺, Ni²⁺, and Fe²⁺ immobilized on Amberlite IRC‐718 has the potential to be developed as part of a process to purify IgG out of untreated human plasma as acceptable adsorption and elution levels of IgG could be achieved. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Prognostic Value of Tumor Markers,NSE, CA125 and SCC,in Operable NSCLC Patients

The aim of this study was to investigate the prognostic value of tumor markers in operable non-small cell lung cancer (NSCLC) patients. A total of 481 NSCLC patients were enrolled in the present study. High levels of neuron-specific enolase (NSE), carbohydrate antigen 125 (CA125) and squamous cell carcinoma antigen (SCC) were detected in 306 (63.6%), 89 (18.5%) and 125 (26.0%) patients, respectively. Seventy-eight of 481 patients died of disease progression, and the median disease-free survival (DFS) and overall survival (OS) were 16.0 and 21.0 months, respectively. The three-year DFS rate was 56.7%, and the OS rate was 75.3%. For serum NSE, the three-year cumulative DFS rate for the normal and elevated group was 67.7% and 51.8% (p = 0.007). The OS in patients with high and normal levels of NSE was 34.0 months and 48.0 months, respectively. The median DFS was 46.0 months versus 32.0 months (p = 0.001), and the OS was 48.0 months versus 44.0 months (p = 0.001) in patients with normal and high levels of CA125. For patients with squamous cell carcinoma, the overall survival was significantly shorter in patients with elevated levels of SCC (p = 0.041). In the multivariate analysis high levels of NSE, CA125 and clinical stage were significantly correlated with worse prognosis (p < 0.05). Patients with all three tumor markers elevated presented the worst prognosis (p < 0.05). In our analysis, high levels of preoperative serum NSE and CA125 are correlated with worse survival in operable NSCLC patients.     

Influence of HMX particle size on the synthesis of nanodiamonds in detonation waves

The effect of the particle size of HMX in alloys with TNT on the synthesis of nanodiamonds in a detonation wave was studied experimentally. Mixtures with a TNT content of 40 to 90% and the specific surface area of HMX varied in the range of 5–510 m²/kg were investigated. For all mixtures, an increase in the particle size of HMX was found to lead to an increase in the yield of nanodiamonds with the maximum yield shift toward alloys with increased TNT content. The results are explained using a model based on the absence of thermodynamic equilibrium between the components of the heterogeneous explosive during detonation. __________ Translated from Fizika Goreniya i Vzryva, Vol. 44, No. 2, pp. 79–84, March–April, 2008.     

Preparation of Polyglycidylmethacrylate Macropore Beads and Application in Candida species 99–125 Lipase Immobilization

Polyglycidylmethacrylate beads with macroporous structure were synthesized by suspension polymerization and directly used for Candida species 99–125 lipase immobilization because of their rich surface epoxy groups. The properties of the synthesized polymer beads were characterized by scanning electron microscopy and FT‐IR spectroscopy. To increase the enzyme activity, divinyl benzene and toluene/heptane were used as cross‐linker and porogen, respectively, and the effects of cross‐linker content and porogen content on lipase immobilization were studied. After optimizing the synthesis conditions, the optimum lipase concentration for immobilization was determined as 10 mg mL^–1. The immobilized lipase showed a broader pH stability and higher temperature stability. The immobilized enzyme could be reused for ten batches with 62 % of residual activity.     

An efficient purification method for detonation nanodiamonds

This article reports the purification process of detonation soot to obtain pure nanodiamond powder. Nanodiamonds are synthesized by detonation using a high explosive mixture composed of trinitrotoluene and hexogen. The detonation of the charge leads to a powder containing nanodiamonds as well as metallic impurities and sp² carbon species. Further, to remove metallic particles, an unusual acidic treatment (hydrofluoric/nitric acids; i.e. fluorinated aqua regia) was set up. To eliminate sp² carbon species such as graphite and amorphous carbon, a thermal oxidation treatment was performed at 420 °C under air in a furnace during several hours. Transmission Electronic Microscopy, Raman spectroscopy, X-ray diffraction and Thermo-Gravimetric Analysis showed that this purification process is very efficient. From TGA measurements, a model of the carbon grain combustion was developed by considering graphitic shells surrounding the nanodiamond particles, and was used to demonstrate that the selective oxidation of graphite was experimentally realistic. Moreover, another model was set up from specific area measurements to evaluate the thickness of the functional groups surrounding the nanodiamonds after the oxidation of sp² carbonaceous species. The treatment described herein was achieved on several tens of grams of product and could be easily adapted to the industrial scale.     

Electrochemical sensing platform based on tris(2,2′-bipyridyl)cobalt(III) and multiwall carbon nanotubes–Nafion composite for immunoassay of carcinoma antigen-125

A new strategy for constructing a sensitive mediator-type electrochemical immunosensor for the detection of carcinoma antigen-125 (CA125) was developed. In this strategy, mediator tris(2,2′-bipyridyl)cobalt(III) (Co(bpy)₃³⁺) was incoporated into the multiwall carbon nanotubes–Nafion (MWNTs–Nafion) composite film via a simple ion-exchange route. Then, gold colloidal nanoparticles (nano-Au) were attached onto Co(bpy)₃³⁺/MWNTs–Nafion film through electrostatic interaction between negatively charged nano-Au and positively charged Co(bpy)₃³⁺. Finally, CA125 monoclonal antibody (anti-CA125), used as a model antibody, was assembled onto the surface of nano-Au to achieve an immunosensor for the determination of CA125 antigen. X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the assembly process of the modified electrode. The resulting immunosensor showed a high sensitivity, wide dynamic range consisting of two linear parts from 1.0 to 30 U mL⁻¹ and 30 to 150 U mL⁻¹ with a low detection limit of 0.36 U mL⁻¹ at 3 times the background noise. Moreover, it displayed good reproducibility and stability, and would be potentially attractive for clinical immunoassay of CA125. The integration of mediator Co(bpy)₃³⁺ and MWNTs–Nafion composite would offer potential promise for the fabrication of biosensors and biocatalysts.     

Cisplatin-Resistant CD44+ Lung Cancer Cells Are Sensitive to Auger Electrons

Cancer stem cells (CSCs) are resistant to conventional therapy and present a major clinical challenge since they are responsible for the relapse of many cancers, including non-small cell lung cancer (NSCLC). Hence, future successful therapy should also eradicate CSCs. Auger electrons have demonstrated promising therapeutic potential and can induce DNA damage while sparing surrounding cells. Here, we sort primary patient-derived NSCLC cells based on their expression of the CSC-marker CD44 and investigate the effects of cisplatin and a thymidine analog (deoxyuridine) labeled with an Auger electron emitter (¹²⁵I). We show that the CD44⁺ populations are more resistant to cisplatin than the CD44⁻ populations. Interestingly, incubation with the thymidine analog 5-[¹²⁵I]iodo-2′-deoxyuridine ([¹²⁵I]I-UdR) induces equal DNA damage, G2/M cell cycle arrest, and apoptosis in the CD44⁻ and CD44⁺ populations. Our results suggest that Auger electron emitters can also eradicate resistant lung cancer CD44⁺ populations.