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The following handle holds various files of this Leiden University dissertation:
Author: Kleinovink, E.W.J.
Title: The use of light in cancer immunotherapy Issue Date: 2018-04-19
Near-infrared labeled, ovalbumin loaded polymeric nanoparticles based on a hydrophilic polyester as model vaccine: In vivo tracking and evaluation of antigen-specific CD8+ T
cell immune response
Sima Rahimian*, Jan Willem Kleinovink*, Marieke F. Fransen, Laura Mezzanotte, Henrik Gold, Patrick Wisse, Hermen Overkleeft, Maryam Amidi, Wim Jiskoot, Clemens W. Löwik, Ferry Ossendorp and Wim E. Hennink
* Shared first authorship
Biomaterials (2015) Jan;37:469-77
Particulate antigen delivery systems aimed at the induction of antigen- specific T cells form a promising approach in immunotherapy to replace pharmacokinetically unfavorable soluble antigen formulations. In this study, we developed a delivery system using the model protein antigen ovalbumin (OVA) encapsulated in nanoparticles based on the hydrophilic polyester poly(lactide-co- hydroxymethylglycolic acid) (pLHMGA). Spherical nanoparticles with size 300-400 nm were prepared and characterized and showed a strong ability to deliver antigen to dendritic cells for cross-presentation to antigen-specific T cells in vitro. Using near-infrared (NIR) fluorescent dyes covalently linked to both the nanoparticle and the encapsulated OVA antigen, we tracked the fate of this formulation in mice.
We observed that the antigen and the nanoparticles are efficiently co-transported from the injection site to the draining lymph nodes, in a more gradual and durable manner than soluble OVA protein. OVA-loaded pLHMGA nanoparticles efficiently induced antigen cross-presentation to OVA-specific CD8 T cells in the lymph nodes, superior to soluble OVA vaccination. Together, these data show the potential of pLHMGA nanoparticles as attractive antigen delivery vehicles.
Immunotherapy aims at the induction or enhancement of cellular immune responses against the target of interest, and has the advantage of a high efficacy and less side effects based on antigen-specificity (1). One of the challenging aspects in the development of effective strategies for immunotherapy is to secure efficient antigen presentation by dendritic cells (DCs) in a targeted and sustained manner to subsequently result in activation of antigen-specific T cells (2). Dendritic cells are professional antigen-presenting cells (APCs) and have a pivotal role in the initiation and orchestration of T cell immune responses (3-6). Immature DCs constantly engulf and process soluble and particulate antigens, as well as necrotic and apoptotic cells via various mechanisms (7). Upon maturation, DCs migrate to draining lymph nodes and present the processed protein antigens in the form of linear peptide epitopes to CD4 and CD8 T cells through major histocompatibility complex (MHC) class I and class II molecules to initiate a proper immune response against the antigens (6,8,9).
The crucial role of DCs in adaptive immunity makes them an important target for the development of antigen vaccines. The weak immunogenicity of soluble protein antigens has led to the development of carrier systems that aim for DC targeting and intracellular antigen delivery (2,10-17). Particulate delivery systems have several advantages such as protection of antigen against enzymatic degradation and enhanced uptake by DCs, the possibility of co-delivery of antigen and adjuvants to DCs, and the possibility of introducing targeting ligands on the surface of the particle as well as prolonged antigen delivery for sustained antigen-presentation by DCs (2,3,7,15,18-22). Especially biodegradable polymeric nanoparticles (NPs) have been used extensively as antigen delivery vehicles (3,15,19,23). In particular, particles based on poly(lactic-coglycolic acid) (pLGA), a biodegradable polyester, have been widely investigated for diverse pharmaceutical applications including protein and antigen delivery (24-29). Several studies have shown the efficacy of antigen-loaded pLGA particulate systems in the induction of immune responses both in vitro and in animal models (18,30,31). pLGA particles are far more efficient in generating long-lasting CD8 T cell immunity than soluble antigen and antigen in incomplete Freund’s adjuvant (IFA) (32). Nevertheless, pLGA systems have several drawbacks as peptide/protein-releasing formulations. Upon degradation, acidification inside the particles results in aggregation and denaturation of the loaded peptide/protein (33). Moreover, peptide/protein encapsulated in pLGA are prone to chemical modifications such as acylation (34,35). Altogether these drawbacks lead to incomplete release (36) of the content from the particles and possible undesired immunogenicity and other adverse reactions (37). To overcome pLGA pitfalls, functionalized polyesters such as poly(lactic-co-glycolic-hydroxymethyl glycolic
acid) (pLGHMGA) and poly(lactic-co-hydroxymethyl glycolic acid) (pLHMGA) have been developed. These polymers are more hydrophilic than pLGA and therefore degrade faster, do not show a pH drop inside the degrading particles (38) and cause much lower acylation. Moreover, they show complete and faster release in comparison to pLGA particles (39-41). The fate of vaccines after administration has been investigated in several in vivo imaging studies (42-46). Fluorescence imaging has received considerable attention during the past few years because of its advantages such as the ease of translation from in vitro imaging studies and being non-invasive, fast and cost-effective (47-51). Several in vivo near-infrared (NIR) fluorescent dyes have been developed with excitation/emission maxima between 700 and 900 nm to minimize tissue absorbance and light scattering, resulting in increased depth of penetration, low fluorescence background, and high intensity signals (45). These dyes can be modified or conjugated to antigens or NPs/polymers (50). A considerable number of vaccine tracking studies with NIR fluorescent dyes focuses on tracking of DCs rather than the vaccine. Moreover, the fluorescent dye is often encapsulated in the particles which brings the risk of dye leakage from the particles (46,52), or is attached to other contrast agents such as superparamagnetic iron oxide particles (53,54). The use of NIR fluorescent dyes coupled both to the antigen of interest and to the delivery system may therefore be preferred. In this study, we evaluate the feasibility of polymeric pLHMGA nanoparticles as a protein antigen delivery system. The particulate antigen showed a superior ability to induce antigen cross-presentation to antigen-specific T cells compared to soluble antigen, both in vitro and in vivo. Using NIR dyes covalently linked to both the nanoparticle and the model protein antigen, we visualized the delivery system and its content in real-time, showing gradual relocation from the injection site to the draining lymph nodes. This shows the potential of pLHMGA nanoparticles as an efficient antigen delivery system for the induction of antigen-specific cellular immune responses and suggests its experimental application in cancer research.
Materials and Methods
Ovalbumin Low EndoTM (OVA, batch X2N13844) was purchased from Worthington, USA. IRDye800CW and IRDye680RD N-hydroxysuccinimide ester (NHS ester) were purchased from LI-COR Biosciences, USA. Alexa647-labeled ovalbumin was obtained from Molecular probes, The Netherlands. 5-Hexyn-1-ol, polyvinyl alcohol (PVA; Mw 30000-70000; 88% hydrolyzed), palladium on carbon (Pd/C; 10
% wt. loading dry support), tin(II) 2-ethylhexanoate (SnOct2), copper(I) acetate, dimethyl sulfoxide (DMSO) and tris((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)
amine (TBTA) were obtained from Sigma-Aldrich, USA. Benzyl alcohol, sodium dihydrogen phosphate (NaH2PO4) and disodium hydrogen phosphate (Na2HPO4) were obtained from Merck, Germany. D,l-Lactide was from Purac, the Netherlands.
N,N-dimethylformamide (DMF), chloroform, dichloromethane (DCM), n-propanol, methanol, tetrahydrofuran (THF) and toluene were from Biosolve, the Netherlands.
Sodium azide (NaN3, 99%), sodium hydroxide (NaOH) and sodium dodecyl sulfate 20% (SDS) were purchased from Fluka, the Netherlands. Bicinchoninic acid assay (Micro BCA) reagents were obtained from Thermo Scientific (USA). Pyrogen-free water was purchased from Carl Roth (Germany). Phosphate buffered saline (1.8 mM NaH2PO4, 8.7 mM Na2HPO4, 163.9 mM Na+, 140.3 mM Cl-) (PBS) was obtained from B Braun, Germany. Toluene and DMF were dried on 3Å molecular sieves prior to use. Chemicals were used as received without further purification, unless otherwise stated. Azide-functionalized water-soluble near-infrared fluorescent dye 10 (azide-NIR10) was prepared as described elsewhere (55). (Experimental details are provided in supporting information).
Synthesis of copolymers of 3S-(benzyloxymethyl)-6S-methyl-1,4-dioxane-2,5- dione with d,l-lactide (pLHMGA).
3S-(benzyloxymethyl)-6S-methyl-1,4-dioxane-2,5-dione (benzyloxymethylmethyl- glycolide) (BMMG) was synthesized as described before (56). A copolymer of d,l-lactide and BMMG (50:50 molar ratio) was synthesized via ring opening polymeriztion at 130 °C using benzyl alcohol as initiator with a 100/1 monomer- to-initiator molar ratio and stannous octoate (SnOct2) as catalyst (Figure 1) (39,56).
In a typical procedure, vacuum dried monomers (BMMG (2840 mg, 11.35 mmol) and d,l-lactide (1630 mg, 11.35 mmol)) were introduced into a dry schlenk tube under a nitrogen atmosphere. Next, benzyl alcohol (24.5 mg, 0.226 mmol; 223.1 µL from a 109.8 mg/mL stock solution in toluene) and Sn(Oct)2 (45.9 mg, 0.113 mmol;
409.8 µL from a 112 mg/mL stock solution in toluene) were added. Toluene was evaporated under vacuum for 2 hours, the tube was closed and immersed into a preheated 130°C oil bath for 16 hours while stirring. Next, the obtained polymer was dissolved in 10 mL chloroform and subsequently precipitated in 500 ml cold methanol to remove the unreacted monomers and to precipitate the polymer, which was collected by filtration and vacuum dried to yield 4.5 g (90%) poly (d,l-lactic-co- benzyloxymethylglycolic acid) (pLBMGA). Subsequently, pLBMGA was dissolved in 500 mL THF, and 5 g of 10 % w/w Pd/C catalyst was added. The flask was filled with hydrogen in three consecutive steps of subsequent evacuation and refilling with H2 and the reaction mixture was stirred for 16 hours under a H2 pressure. Next, Pd/C was removed using a glass filter and THF was removed by evaporation. The obtained polymer was then dissolved in 5 mL chloroform and precipitated in 200 mL n-propanol and vacuum dried after filtration to yield 3 g of pLHMGA (84%).
Synthesis of hexyn-poly(d,l lactide)(hexyn-pDLLA) to couple to azide-NIR10 via click chemistry
Hexyn-pDLLA was synthesized via ring opening polymerization similar to the method described in section 2.2.1. Briefly, vacuum dried d,l lactide (396 mg, 2.75 mmol) was transferred into a dry schlenk tube under a nitrogen atmosphere. Next, the initiator, 5-hexyn-1-ol (monomer-to-initiator ratio was 70 to 1, 3.85, 0.039 mmol;
75.5 µL from a 51 mg/mL stock solution in toluene and Sn(Oct)2 (7.9 mg, 0.020 mmol; 71.2 µL from a 112 mg/mL stock solution in toluene) were introduced into the schlenk tube. The tube was evacuated for 2 h, sealed, and immersed in an oil bath thermostated at 130 °C. The polymerization was performed for 16 hours and the formed polymer was purified by dissolution in chloroform and precipitation in n-propanol followed by filtration and vacuum drying to yield 370 mg of hexyn- pDLLA (Yield: 92 %).
Conjugation of azide-NIR10 to hexyn-pDLLA by click chemistry
Conjugation of the azide-NIR10 to hexyn-pDLLA was performed using copper catalyzed azide-alkyne cycloaddition (57-60). In a typical procedure, 30.4 mg hexyn- pDLLA (1.84 µmole) and 2 mg azide-NIR10 (1.84 µmole) were transferred into a vial. Then copper (I) acetate (0.2 mole per mole of polymer) and TBTA (0.05 mole per mole of polymer) were added to the vial from their stock solution in dried DMF and the volume was adjusted with DMF to 250 µL. The system was flushed with nitrogen for 20 minutes and the reaction was performed at 40 °C overnight.
The obtained conjugate (NIR10-pDLLA) was purified by precipitation in water. The obtained precipitate was washed with water and freeze dried at -50 °C and 0.7 mbar overnight (Chris Alpha 1-2 freeze-drier, Germany). The conjugation efficiency was calculated using GPC analysis by dividing the area under the chromatogram of the conjugated polymer using UV detection by the area under the curve of reaction mixture (Yield: 76%).
NMR Spectroscopy: A Gemini 300 MHz spectrometer (Varian Associates Inc., USA) was used to conduct 1H-NMR measurements at 298 K. Chemical shifts are reported in ppm with reference to the solvent peak (δ) 7.26 ppm for CDCl3. Gel Permeation Chromatography (GPC): Molecular weight and molecular weight distribution (polydispersity index (PDI); weight average molecular weight/
number average molecular weight) of the synthesized pLHMGA was measured by using a Waters Alliance system (Waters, USA) with a Waters 2695 separating module equipped with Waters 2414 refractive index detector. Fifty µL of 5 mg/
mL sample was injected onto two PL-gel 5 μm mixed-D columns fitted with a guard column (Polymer Laboratories, Mw range 0.2-400 kDa). Polystyrene
standards were used to calibrate the columns and AR grade THF was used as eluent at a flow rate of 1 mL/min and a run time of 30 minutes. For hexyn-pDLLA and NIR10-pDLLA, DMF containing LiCl 10 mM was used as eluent and a Waters 2478 UV detector was connected to the system and the UV absorbance of the dye was measured at 700 nm. Polyethylene glycol (PEG) standards were used to calibrate the column. The flow rate was set at 0.7 mL/min with a run time of 40 minutes. The results were analyzed using Empower software v. 2 (Waters, USA).
Differential Scanning Calorimetry (DSC): The thermal properties of the polymers were established by using differential scanning calorimetry (Discovery DSC, TA instrument, USA). Two to five mg of polymer sample was loaded into an aluminum pan which was subsequently heated from room temperature to 120 °C, with a heating rate of 5 °C/min, followed by cooling down to −50 °C. Thereafter, the sample was heated to 120 °C with a temperature modulation of ±1 °C and a ramping rate of 2 °C/min. The glass transition temperature (Tg) was recorded as the midpoint of the heat capacity change in the second heating run.
Labeling OVA with NIR fluorescent dyes
OVA was labeled with either IRDye800CW (IR800) or IRDye680RD (IR680) by coupling the NHS ester of the dyes to the protein. In a typical procedure, 20 mg of OVA was dissolved in 1 mL PBS (B Braun, Germany) pH 7.4 and the pH was adjusted to 8.5 by adding 0.1 mL of K2HPO4 1 M pH 9. The IRDye680RD NHS ester was dissolved in DMSO (4 mg/mL) and 0.25 mL of the stock solution containing 1 mg of the dye was added to the OVA solution at a 2:1 molar ratio. The reaction was carried out at room temperature for two hours. The unreacted dye was subsequently removed with pyrogen-free water by using Zeba™ spin desalting columns equilibrated with pyrogen-free water in two consecutive steps and IR680-OVA was collected after freeze drying (yield: 85%). IR800CW was conjugated and purified by using the same procedure with a dye/OVA feed molar ratio of 1.7 to 1 (yield: 79%).
Characterization of labeled-OVA
IR680-OVA was characterized by GPC using a Waters UPLC Acquity system equipped with a fluorescence detector. Two channels were used: channel 1 for detection of the protein (excitation at 280 nm and emission at 340 nm) and channel 2 for detection of the IR680 signal (excitation at 672 nm and emission at 694 nm). Seven and a half µL of IR680-OVA (10 µg/mL in PBS) was injected onto a BEH450 SEC 2.5 µm column (Waters, USA). PBS was used as elution buffer and the flow rate was set at 0.25 mL/
min. To calculate the dye/OVA molar ratio, the UV spectrum of the freeze-dried labeled OVA was recorded (Shimadzu 2450, Japan) and the absorbance values at 280 nm (A280) and 672 nm (A672) were used to calculate the degree of conjugation according to the following formula: (Dye/OVA) = (A672/εDye) / ((A280 – 0.07*A672)/
εOVA)). Dye/OVA is the molar ratio of IR680 to OVA and the molar extinction coefficient of IR680 (εDye) is 165,000 M–1 cm–1 (according to the manufacturer) and the molar extinction coefficient of OVA (εOVA) is 30590 M–1 cm–1 (61). IR800-OVA was characterized similarly by using the corresponding wavelengths suitable for IR800.
Preparation of nanoparticles
OVA loaded nanoparticles (NPs) were prepared by using a double emulsion solvent evaporation method essentially as described before (62). In brief, 50 μL of 30 mg/
mL OVA solution in pyrogen-free water was emulsified by sonication (30 s, 20 W-ultrasonic homogenizer (Labsonic P, B. Braun Biotech, Germany) in a solution of 50 mg of pLHMGA in 1 mL of DCM to obtain a water -in-oil emulsion (W1/O). Next, 2 mL of PVA 1% w/v solution in pyrogen-free water (filtered through a 0.2 μm filter) was added to this first emulsion and a water-in-oil-in-water emulsion was formed by a second round of sonication for 30 seconds. The double emulsion (W1/O/W2) was then added dropwise to 25 mL of PVA 0.3% w/v in pyrogen-free water at 40
°C while stirring for rapid removal of DCM. After 1 hour, the particles were collected by centrifugation for 30 min at 20,000 g, washed with PBS and consecutively with water, resuspended in pyrogen-free water, and freeze-dried overnight. Dual labeled NPs containing NIR10-labeled pLHMGA and IR680-OVA were prepared using the same method by addition of 4 mg of NIR10-pDLLA to 50 mg of pLHMGA and using IR680-OVA (20 mg/ml) as internal water phase. For preparation of empty NPs, NPs loaded with IR800-OVA or Alexa647-OVA, 50 µl pyrogen-free, 50 µl of 20 mg/mL IR800-OVA or Alexa647-OVA was used as internal water phase.
Nanoparticle characterization: Size and morphology analysis
Dynamic light scattering: The size of NPs was measured with dynamic light scattering (DLS) on an ALV CGS-3 system (Malvern Instruments, Malvern, UK) equipped with a JDS Uniphase 22 mW He–Ne laser operating at 632.8 nm, an optical fiber-based detector, and a digital LV/LSE-5003 correlator.
Freeze dried NPs were suspended in deionized water (RI = 1.332 and viscosity 0.8898 cP) and measurements were done at 25 °C at an angle of 90°.
Transmission electron microscopy (TEM): The size and morphology of NPs were analyzed by using transmission electron microscopy (TEM, FEI Tecnai T10). A droplet of nanoparticle suspension in water was placed on a carbon coated copper grid and the sample was stained with 2% uranyl acetate. NPs were visualized with 7-73 k fold magnification and analyzed by MeasureIT software.
Nanoparticle characterization: OVA content and loading efficiency of NPs
OVA loading efficiency of the NPs was determined by measuring the protein content of digested NPs essentially as described before (63). In short, about 5 mg of particles (accurately weighed) was dissolved in 0.5 mL of DMSO followed by addition of
2.5 mL of 50 mM NaOH containing 0.5% (w/v) SDS. After complete degradation of the particles, the resulting solution was analyzed by using the Micro BCA™
Protein Assay Kit (Thermo Scientific), a calorimetric method that quantifies protein concentration based on peptide bond and specific amino acid residues. Calibration was done by using 2-40 µg/mL of OVA solution in DMSO:NaOH 50 mM/SDS 0.5%
(1:5). Loading efficiency (LE%) is defined as the amount of OVA encapsulated in the nanoparticles divided by the amount of protein added × 100%. Loading percentage (L%) is reported as the amount of OVA entrapped in the particles per total dry mass of the nanoparticles × 100%. In case of dual labeled NPs (containing IR680-OVA) or IR800-OVA NPs, the degraded particle solution was analyzed for its OVA content based on the NIR label by using an Odyssey™ scanner (LI-COR Biosciences, USA) at the 700 nm channel for IR680-OVA and the 800 nm channel for IR800-OVA and the calibration was done by using IR680-OVA or IR800-OVA.
Nanoparticle characterization: In vitro release of OVA from NPs
About 5 mg of freeze-dried dual labeled NPs (containing IR680-OVA) was accurately weighed and suspended in 5 mL phosphate buffered saline (pH 7.4, 49 mM NaH2PO4, 99 mM Na2HPO4, 6 mM NaCl and 0.05% (w/v) NaN3). The particle suspension was incubated at 37 °C under mild agitation. At different time points, 300 µl was removed and centrifuged at 24000 g for 15 minutes. The amount of protein released and present in the supernatant was quantified by using an Odyssey™ scanner using the 700 nm channel for visualization of IR680-OVA. Calibration was done by using IR680-OVA in PBS.
Mice and cells
WT C57BL/6 (CD45.2/Thy1.2; H-2Kb) mice were obtained from Charles River Laboratories (France). Albino BL/6 mice (B6(Cg)-Tyrc-2J/J tyrosinase-deficient immunocompetent BL/6 mice) were bred in the animal breeding facility of the Leiden University Medical Center, the Netherlands. All experiments were approved by the animal experimental committee of Leiden University, the Netherlands. D1 cells are a dendritic cell line of BL/6 background (64), and B3Z is a CD8+ T cell hybridoma specific for the SIINFEKL epitope from OVA and carrying the lacZ (E.coli β-galactosidase) reporter gene induced by NFAT (nuclear factor of activated T-cells) (65). Antigen- specific activation of B3Z cells leads to T cell receptor-mediated triggering of NFAT, and the subsequent production of β-galactosidase can be quantified by addition of CPRG, a substrate for the β-galactosidase, as a measure of antigen cross-presentation (66). Cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, Lonza) containing 8% fetal calf serum (FCS, Greiner), supplemented with 2 mM GlutaMax (Gibco) and 80 IU/mL sodium-penicillin G (Astellas, the Netherlands) for D1 cells, or supplemented with 100 IU/mL penicillin/streptomycin (Gibco), 2 mM glutamin
(Gibco), 25 µM 2-mercaptoethanol and 500 µg/mL Hygromycin B (AG Scientific) for B3Z cells. OT-I*Thy1.1 mice are TCR-transgenic mice on B6 background whose CD8+ T cells recognize the OVA-epitope SIINFEKL presented in H2-Kb, crossed to Thy1.1 mice to obtain a congenic marker for distinction from endogenous CD8+ T cells in transfer experiments. Naïve OT-I CD8+ T cells were obtained by creating single-cell suspensions from spleens and lymph nodes of OT-I*Thy1.1 mice and subsequent purification of CD8+ cells.
Dendritic Cell viability assay
D1 dendritic cells (50,000 cells/well in 96-well plates) were incubated with dispersions of unlabeled and labeled NPs (7.8-1000 µg/ml) overnight at 37 °C, and cell viability was measured by flow cytometry using the DNA-binding fluorescent dye 7-amino-actinomycin-D (7-AAD) to stain dead cells.
Analysis of cellular uptake
D1 cells (100,000 cells/well in 96-well plates) were incubated with Alexa 647- OVA nanoparticles (final concentration of OVA in medium: 6 µg/ml) overnight.
D1 cells were subsequently transferred onto a confocal slide without washing or fixation. The intracellular localization of NPs was studied by confocal laser scanning microscopy imaging using inverted Leica SP2 confocal microscope and the samples were analyzed using the corresponding Leica confocal software in DIC mode.
In vitro antigen cross-presentation assay
In vitro cross-presentation of OVA by DCs was studied by incubating titrated amounts of OVA encapsulated in NPs or in soluble form with 50,000 D1 cells (100 µl of 0.2- 25 µg OVA/mL in PBS corresponding to 0.12 to 1.5 mg/ml of NPs) for two hours at 37°C followed by addition of 50,000 B3Z cells and incubation overnight at 37°C in 96-well plates. The cells were then incubated with a lysis buffer containing the CPRG substrate for lacZ (PBS +1% 9 mg/mL CPRG + 0.9% 1M MgCl2 + 0.125% NP40 + 0.71% 14.3M β-mercaptoethanol) at 37 °C until the color reaction had progressed sufficiently for readout in a plate reader measuring the optical density at 590 nm.
The direct MHC-binding minimal epitope SIINFEKL (100 ng/mL in PBS) was used as a positive control, and unstimulated D1 cells were negative controls.
In vivo tracking of labeled NPs
Albino BL/6 mice were vaccinated subcutaneously with 50 µg OVA encapsulated in dual labeled NPs dispersed in 100 µl PBS or 50 µg/100 µl PBS IR800-OVA. Vaccine kinetics were then studied at several time points after administration by using the IVIS spectrum preclinical in vivo imaging system (PerkinElmer). Kinetics were measured by quantifying the fluorescent intensity in pre-set regions of interest (ROI) at the injection site and the two vaccine-draining inguinal lymph nodes, expressed
as the total radiant efficiency in (p/s)/(µW/cm2). The fluorescent signal in the injection site at each time point is presented as the as percentage of the maximum recorded value, to show percentual decrease in time. In the lymph nodes the data is presented as the signal to background ratio and it is calculated by dividing the fluorescence signal of the lymph nodes by the background signal at each time point (67).
In vivo antigen cross-presentation
To establish antigen-presentation in vivo, BL/6 mice were vaccinated with OVA encapsulated in NPs or in its soluble form. Mice were injected subcutaneously with 200 µL of a 2 mg/mL suspension of NPs in PBS containing 50 µg of encapsulated OVA or 50 µg of soluble OVA. One day after vaccination, 500,000 OVA-specific OT-I CD8+ T cells were injected intravenously in the tail vein in 200 µL PBS. Mice were sacrificed 4 days after T cell transfer to analyze the expansion of the transferred OVA-specific T cells caused by presentation of the OVA antigen. Vaccine-draining inguinal lymph nodes and the spleen were taken out, mashed on 70 µm cell strainers (Becton Dickinson) and the cells were stained with fluorescently labeled antibodies for CD8β and the OT-I congenic marker CD90.1 (Thy1.1), and analyzed by flow cytometry. T cell expansion expressed as the number of transferred (OT-I) T cells as a percentage from total CD8+ T cells (68).
GraphPad Prism software was used for statistical analysis. Two groups were comparing by unpaired two-tailed Student’s t-test with alpha=0.05. Statistical significance was considered when p < 0.05.
Results and Discussion
Synthesis and characterization of pLHMGA
A random copolymer of BMMG and d,l-lactide was synthesized by using BnOH and SnOct2 as initiator and catalyst, respectively, via ring opening polymerization in the melt (Figure 1) and the polymer was obtained in high yield (~100%). The protective benzyl groups were subsequently removed overnight by hydrogenation to yield pLHMGA (83%). The structures of the synthesized polymers were confirmed by 1H NMR spectroscopy and this analysis showed that the molar ratio of BMMG and d,l-lactide in pLBMGA (protected polymer) was 48/52, which is close to feed ratio (50/50). Both pLBMGA and pLHMGA were amorphous with Tg values of 39
°C and 57 °C, respectively, which were similar to values reported earlier (39) and confirmed the random structure of the polymers. GPC analysis showed that the number average molecular weight of pLHMGA (16.2 kDa) was close to the aimed
molecular weight based on monomer-to-initiator molar ratio (15.8 kDa) and the PDI was 1.6. Based on previous studies using the same polymers, in our study we chose a relatively low molecular weight polymer in order to achieve small nanoparticles with high loading and fast release.
Figure 1. Synthesis of poly(lactic-co-hydroxymethylglycolic acid) (pLHMGA) in two steps. Synthesis of poly(lactic- co-benzyloxymethylglycolic acid) (pLBMGA) via ring opening polymerization of BMMG and D,L-lactide, followed by removing the benzyl group by hydrogenation to yield pLHMGA.
Synthesis and characterization of hexyn-pDLLA and NIR10-pDLLA
An alkyne-bearing homopolymer of d,l lactide (hexyn-pDLLA) was synthesized by using 1-hexyn-6-ol as initiator and stannous octoate as catalyst. The alkyne functionality in this polymer provides the possibility to conjugate azide-bearing molecules via click chemistry. The obtained polymer was amorphous with a Tg of 50 °C, similar to values reported in the literature (69). GPC analysis showed that the number average molecular weight of hexyn-pDLLA (11.0 kDa) was close to that of the aimed molecular weight based on monomer-to-initiator ratio (10.1 kDa). To obtain a NIR fluorescent-labeled polymer, the azide-NIR10 was coupled to hexyn- pDLLA by click reaction. Analysis of the reaction mixture after 16 hours by GPC with UV detection confirmed the presence of labeled polymer at a retention time of 20 minutes corresponding to that of the polymer, while a peak was also observed at 23.5 min, which corresponds to the retention time of the free dye. From the chromatogram a coupling efficiency of 85% was calculated. After purification, GPC analysis showed that the polymer contained 8% of free dye. GPC analysis of the freeze dried particles containing NIR10-pDLLA showed that only a negligible amount of free dye was present in the particles indicating that most of the free dye was removed during particle preparation process.
Characterization of IR680-OVA and IR800-OVA
In order to prepare a traceable OVA for in vivo imaging, the NHS ester of IRdye680RD was conjugated to OVA and the obtained IR680-OVA was analyzed by GPC. Overlapping peaks corresponding to OVA (excitation 280 and emission 340 nm) and IR680 (excitation 672 and emission 694 nm) in the GPC chromatogram of the purified IR680-OVA confirmed that the IR680 was indeed conjugated to OVA.
No additional peaks (fluorescence detection) were observed, indicating that the
BMMG d,l Lactide pLHMGA
free dye was completely removed by purification. Conjugation efficiency calculated by UV measurements showed an average molar dye/protein ratio of 1.7. IR800-OVA was successfully prepared and purified as described for IR680-OVA. The average molar dye/protein ratio was 1.0.
Table 1. Characteristics of NPs. Average ± SD (n=3) of representative data.
Size (nm) (DLS) PDI (DLS) Loading (%) Loading efficiency (%)
Empty NPs 364 ± 40 0.22 ± 0.08 - -
OVA NPs 340 ± 4 0.19 ± 0.04 1.71 ± 0.01 57.4 ± 0.4 Dual labeled NPs 293 ± 3 0.15 ± 0.02 1.37 ± 0.01 69.0 ± 0.4 Alexa-OVa NPs 384 ± 20 0.33 ± 0.05 1.15 ± 0.02 57.6 ± 0.4
Preparation and characterization of empty and OVA loaded NPs
Empty and OVA-loaded NPs particles were prepared by a double emulsion- solvent evaporation method, which is a well-known procedure and has been used frequently for encapsulation of hydrophilic drugs as well as biotherapeutics (70-73).
Empty and OVA loaded NPs with NIR fluorescent dyes on the polymer and/or the protein antigen had comparable characteristics in terms of morphology and size.
DLS analysis showed that the mean hydrodynamic diameter of the NPs ranged from 290-370 nm with a PDI of 0.16 - 0.27 (Table 1). The particle preparation procedure was optimized in order to obtain nanoparticles with a size of around 300 nm because nanoparticles of 300 nm were most efficient in activation of dendritic cells and inducing a systemic immune response as compared to bigger particles (74). Analysis of freeze-dried samples with TEM showed spherical NPs with a size ranging from 150-250 nm (Figure 2), which is smaller than the sizes obtained with DLS. This is due to the fact that DLS reports the hydrodynamic diameter of the particles based on scattering intensity which is proportional to the 6th power of the particle diameter, and therefore in polydisperse samples, relatively low scattering intensities of small particles are overshadowed by high scattering intensities of big particles, resulting in a skewed average size (75,76). The loading efficiency of unlabeled as well as labeled OVA in NPs was about 60%. The in vitro release of OVA from pLHMGA particles was studied by using dual labeled NPs (containing IR680-OVA).
Figure 2. TEM images of (a) empty NPs, (b) unlabeled OVA NPs and (c) dual-labeled NPs. Bar scale = 500 nm.
Figure 3 shows that the particles had a low burst release of about 5%, followed by sustained release up to 70% for 30 days. These data are in line with previous studies of pLHMGA particles with the same copolymer composition and loaded with bovine serum albumin (77). Considering slight variations in the loading efficiency of the nanoparticles and in order to allow a fair comparison in functional experiments, a fixed amount of OVA was used in various in vitro and in vivo studies.
Figure 3. In vitro release of IR680-OVA from pLHMGA NPs. Dual labeled NPs were dispersed in PBS (1 mg/mL) and kept at 37 °C under mild agitation. Following a low burst release of about 5%, IR680-OVA was release in a sustained manner in 30 days up to 70%.
Figure 4. In vitro protein cross-presentation of OVA- encapsulated NPs. DCs were incubated with 0.2-25 µg OVA/mL encapsulated in NPs or in soluble form, and co-cultured with OVA-specific B3Z hybridoma T cells overnight. Optical density (O.D.) at 590 nm is a measure of OVA cross-presentation by DCs to B3Z cells. The SIINFEKL peptide positive control was used at 100 ng/mL. n=3, representative graph of 5 independent experiments.
Cytocompatibility of empty and OVA loaded NPs
To assess the cytocompatibility of NPs, dendritic cells were incubated with empty and OVA NPs as well as dual labeled NPs for 16 hours at 37 °C. Dead cells were quantified by using 7-AAD, a DNA binding fluorescent dye which stains cells that have lost membrane integrity (78). Dendritic cells incubated with LPS and soluble OVA corresponding to the amounts of OVA in the loaded nanoparticles were used as controls. Cell viability was about 90% for dendritic cells incubated with empty and OVA-loaded nanoparticles (concentration of NPs ranging from 7.8-1000 µg/mL, data not shown), showing the cytocompatibility of these particles.
Antigen cross-presentation in vitro
D1 dendritic cells were incubated with (Alexa 647-OVA)-loaded NPs overnight and visualized by confocal microscopy to verify that OVA NPs are efficiently taken up by dendritic cells (data not shown). Subsequently, we wanted to test whether the uptake of OVA NPs by DCs leads to processing and cross-presentation of the OVA antigen to OVA-specific T cells. To this end, DCs were incubated with OVA NPs and co-cultured with OVA-specific B3Z hybridoma T cells. Figure 4 shows that OVA NPs induced antigen-specific T cell activation in a dose-dependent manner, while soluble OVA failed to activate T cells even at high doses. Since this assay is based on
OVA release (%)
00 10 20 30
Antigen cross-presentation (O.D. 590 nm)
OVA µg/mL 100
SIINFEKL (positive control) OVA NPs
Unstimulated (negative control)
the immunological principle of antigen-specificity of T cells, the T cell activation is the result of uptake, processing and cross-presentation of the antigen by DCs to T cells. Furthermore, the dual labeled NPs were equally efficient in T cell activation as the unlabeled NPs (data not shown), showing that labeling did not affect the ability of NPs to induce in vitro cross-presentation.
Figure 5. In vivo tracking of NPs and OVA antigen. Quantification of the fluorescent signal of IR680 and NIR10 in dual labeled NPs (containing NIR10 dye on the polymer and IR680 on the OVA), and IR800 signal of soluble antigen (IR800-OVA in PBS) after s.c. injection of 50 µg OVA in NPs or in soluble form. (A) Fluorescent signal at the injection site expressed as a percentage of maximum signal. (B) Fluorescent signal in the draining lymph nodes expressed as signal to background ratio. Statistical significance is indicated by asterisks comparing IR680 signal in NPs vs soluble IR800-OVA. Statistical analysis was performed by unpaired Student’s t-test, * = p < 0.05, ** = p < 0.01. n=mice per group.
In vivo tracking of labeled NPs
As our aim was to develop an antigen-delivery system that ensures a prolonged supply of antigen to the immune system, we visualized the presence of OVA NPs in the injection site and in the draining lymph nodes, where immune responses are initiated. The tail-base injection site was chosen as it drains specifically to the inguinal lymph nodes which are located at sufficient distance in the mouse to be able to distinguish the injection site from the lymph nodes, and to visualize and quantify their respective signals accurately. Using a dual-color in vivo imaging setup based on two distinct NIR fluorescent dyes, we were able to visualize the antigen and the delivery vehicle simultaneously. The NIR fluorescent dye IR680 (excitation 675 nm, emission 720 nm) was coupled to the OVA antigen and the dye NIR10 (excitation 745 nm and emission 840 nm) was coupled to the polymer to track the delivery vehicle. Figure 5a shows the presence of the double-labeled OVA NPs at the injection site in time, compared to a soluble formulation of NIR-labeled OVA protein in PBS. As expected, the vast majority of soluble OVA antigen leaves the injection site within the first day, leading to a short peak presence in the lymph nodes immediately after injection that rapidly decreases (Figure 5b). In contrast, OVA encapsulated in NPs was cleared from the injection site more gradually,
Fluorescence (signal-to-background ratio)
1 Days after injection
0 2 3 4 5 6 7
0 1 2 3 4 5
Days after injection 14
0 2 4 6 8 10 12 16 18 20
Fluorescence (% of max)
0 20 40 60 80 100
Soluble OVA in PBS
OVA signal from dual-labeled NPs NP signal from dual-labeled NPs
Lymph nodes Injection site
corresponding with gradual accumulation in the draining lymph nodes and remaining there for several days. Figure 6 shows examples of the presence of NP OVA at the injection site and in the draining lymph nodes. Interestingly, the signals from the NP vehicle and its OVA content show similar patterns in the injection site and lymph nodes, suggesting that the OVA antigen arrives in the lymph nodes in encapsulated form. The increasing fluorescent signal of encapsulated IR680-OVA in the first day after injection may be caused by initial quenching of the signal, for example by the hydrophobic environment in the NPs or by self-aggregation and stacking of the dye at high concentrations (79). Hydration of the NPs and a decreased stacking of NPs at the injection site during the first day would relieve the IR680 dye from both these quenching effects.
Figure 6. In vivo visualization of OVA NPs in the injection site and draining lymph nodes. Presence of OVA NPs in (A) the injection site and (B) the right inguinal draining lymph node, based on quantification of the fluorescent signal of NIR10 coupled to the NP polymer. Repeated measurements in time plotted on the same scale of fluorescence. The lymph node (LN) is indicated by an arrow. All images are overlays of grayscale photographs with fluorescence intensity measurements indicated on the color scale.
In vivo antigen cross-presentation
The efficient and prolonged delivery of antigen to the draining lymph nodes by OVA NPs led us to test their ability to induce antigen cross-presentation in vivo. To test this hypothesis, we administered OVA NPs to mice, followed after one day by
x1081 2 3 4 5
Fluorescence (Radiant Efficiency)
3 hours 6 hours 1 day 4 days 9 days 13 days
Time after injection
Fluorescence (Radiant Efficiency)
x108 0.5 1.0 1.5 2.0
6 hours 1 day
an adoptive transfer of OVA-specific CD8+ T cells (OT-I cells). By transferring these high numbers of OT-I T cells, we increase the precursor frequency of OVA-specific T cells to artificially high levels, which allows us to detect subtle effects in terms of cross-presentation of OVA antigen. Four days after T cell transfer, the proliferation of these transferred OT-I T cells as a result of in vivo cross-presentation of the OVA antigen was assessed in the vaccine-draining lymph nodes and the spleen (Figure 7). A strong expansion of OT-I cells was found in the lymph nodes and spleen of mice treated with OVA NPs, which proved far superior to the administration of soluble OVA. The absence of T cell proliferation after injection of equal amounts of empty NPs shows that the delivery system itself plays no role and emphasizes the antigen- specific nature of T cell activation. These results correspond with the superior ability of OVA NPs over soluble OVA to induce in vitro antigen cross-presentation to OVA- specific CD8+ T cells (Figure 4).
Figure 7. In vivo cross-presentation of OVA antigen to OVA-specific CD8 T cells.
Expansion of TCR-transgenic OVA-specific CD8 T cells (OT-I) in the draining lymph nodes and spleen, 4 days after transfer into recipient mice. One day before the OT-I transfer, mice were treated subcutaneously with 50 µg OVA either in soluble form or in 400 µg of NPs or with 400 µg of empty NPs. In vivo expansion of the transferred OT-I T cells was quantified by ex vivo flow cytometry and expressed as the percentage of total CD8 T cells. n
= 3-4 mice per group, representative of 2 independent experiments. Student’s t-test showed significant difference in OT-I T cell expansion between mice treated with OVA NPs versus soluble OVA, ** = p < 0.01, *** = p < 0.001.
We developed an antigen delivery system based on pLHMGA nanoparticles encapsulating the model protein antigen OVA. The nanoparticles showed sustained release of antigen in vitro and were able to induce in vitro antigen cross-presentation by DCs to antigen-specific T cells. Near-infrared fluorescence imaging showed that the antigen is efficiently transported from the injection site to the draining lymph nodes, both in encapsulated and in free form. In these lymph nodes, the OVA NPs exhibited a strong capacity to induce antigen-specific CD8+ T cell proliferation through cross-presentation of the antigen. In conclusion, pLHMGA nanoparticles are attractive vehicles for protein antigen delivery for effective stimulation of the cellular immune system.
10 20 30 40
OT-I (% of total CD8 T cells)
40 60 80 100
OT-I (% of total CD8 T cells)
NPs Soluble NPs OVA OVA Empty NPs Soluble NPs
OVA OVA Empty
We thank Ana Luisa Silva, Paul Werkhoven, Nataschja Ho and Joep van den Dikkenberg for technical assistance and useful suggestions. This research was performed within the framework of the Cancer Vaccine Tracking project (#03O- 302), Center for Translational Molecular Medicine (CTMM).
1. Silva JM, Videira M, Gaspar R, Preat V, Florindo HF. Immune system targeting by biodegradable nanoparticles for cancer vaccines. J Control Release 2013;168:179e99.
2. Reddy ST, Swartz MA, Hubbell JA. Targeting dendritic cells with biomaterials: developing the next generation of vaccines. Trends Immunol 2006;27:573e9.
3. Akagi T, Baba M, Akashi M. Biodegradable nanoparticles as vaccine adjuvants and delivery systems: regulation of immune responses by nanoparticle-based vaccine. Polym Nanomedicine 2012;247:31e64.
4. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, et al. Immunobiology of dendritic cells. Annu Rev Immunol 2000;18:767e811.
5. Banchereau J, Palucka A. Dendritic cells as therapeutic vaccines against cancer. Nat Rev Immunol 2005;5:296e306.
6. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392:245e52.
7. Lanzavecchia A. Mechanisms of antigen uptake for presentation. Curr Opin Immunol 1996;8:348e54.
8. Vyas JM, Van der Veen AG, Ploegh HL. The known unknowns of antigen processing and presentation. Nat Rev Immunol 2008;8:607e18.
9. Schuurhuis D, Laban S, Toes R, Ricciardi-Castagnoli P, Kleijmeer M, van der Voort E, et al. Immature dendritic cells acquire CD8(+) cytotoxic T lymphocyte priming capacity upon activation by T helper cell-independent or –dependent stimuli. J Exp Med 2000;192:145e50.
10. Elamanchili P, Diwan M, Cao M, Samuel J. Characterization of poly(D,L-lacticco- glycolic acid) based nanoparticulate system for enhanced delivery of antigens to dendritic cells. Vaccine 2004;22:2406e12.
11. Elamanchili P, Lutsiak CME, Hamdy S, Diwan M, Samuel J. “Pathogenmimicking” nanoparticles for vaccine delivery to dendritic cells. J Immunother 2007;30:378e95.
12. Gregory AE, Titball R, Williamson D. Vaccine delivery using nanoparticles. Front Cell Infect Microbiol 2013;3.
13. Bolhassani A, Safaiyan S, Rafati S. Improvement of different vaccine delivery systems for cancer therapy. Mol Cancer 2011;10(3).
14. Joshi MD, Unger WJ, Storm G, van Kooyk Y, Mastrobattista E. Targeting tumor antigens to dendritic cells using particulate carriers. J Control Release 2012;161:25e37.
15. Krishnamachari Y, Geary SM, Lemke CD, Salem AK. Nanoparticle delivery systems in cancer vaccines. Pharm Res 2011;28:215e36.
16. Mahapatro A, Singh DK. Biodegradable nanoparticles are excellent vehicle for site directed in-vivo delivery of drugs and vaccines. J Nanobiotechnol 2011;9:55.
17. Park YM, Lee SJ, Kim YS, Lee MH, Cha GS, Jung ID, et al. Nanoparticle-based vaccine delivery for cancer immunotherapy. Immune Netw 2013;13:177e83.
18. Zhang Z, Tongchusak S, Mizukami Y, Kang YJ, Ioji T, Touma M, et al. Induction of anti-tumor cytotoxic T cell responses through PLGA-nanoparticle mediated antigen delivery. Biomaterials 2011;32:3666e78.
19. Hamdy S, Haddadi A, Hung RW, Lavasanifar A. Targeting dendritic cells with nano-particulate PLGA cancer vaccine formulations. Adv Drug Deliv Rev 2011;63:943e55.
20. Demento SL, Cui W, Criscione JM, Stern E, Tulipan J, Kaech SM, et al. Role of sustained antigen release from nanoparticle vaccines in shaping the T cell memory phenotype. Biomaterials 2012;33:4957e64.
21. Melief CJM. Cancer immunotherapy by dendritic cells. Immunity 2008;29:372e83.
22. van Montfoort N, Camps MG, Khan S, Filippov DV, Weterings JJ, Griffith JM, et al. Antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-priming capacity. Proc Natl Acad Sci USA 2009;106:6730e5.
23. Ma W, Chen M, Kaushal S, McElroy M, Zhang Y, Ozkan C, et al. PLGA nanoparticle-mediated delivery of tumor antigenic peptides elicits effective immune responses. Int J Nanomedicine 2012;7:1475e87.
24. Ye M, Kim S, Park K. Issues in long-term protein delivery using biodegradable microparticles. J Control Release 2010;146:241e60.
25. Giteau A, Venier-Julienne MC, Aubert-Pouessel A, Benoit JP. How to achieve sustained and complete protein release from PLGA-based microparticles? Int J Pharm 2008;350:14e26.
26. Sinha V, Trehan A. Biodegradable microspheres for protein delivery. J Control Release 2003;90:261e80.
27. van de Weert M, Hennink WE, Jiskoot W. Protein instability in poly(lactic-coglycolic acid) microparticles. Pharm Res 2000;17:1159e67.
28. Brannon-Peppas L, Blanchette J. Nanoparticle and targeted systems for cancer therapy. Adv Drug Deliv Rev 2004;56:1649e59.
29. Jain R. The manufacturing techniques of various drug loaded biodegradable poly(lactide-co- glycolide) (PLGA) devices. Biomaterials 2000;21:2475e90.
30. O’Hagan D, Singh M, Gupta R. Poly(lactide-co-glycolide) microparticles for the development of single-dose controlled-release vaccines. Adv Drug Deliv Rev 1998;32:225e46.
31. Rosalia RA, Silva AL, Camps M, Allam A, Jiskoot W, van der Burg SH, et al. Efficient ex vivo induction of T cells with potent anti-tumor activity by protein antigen encapsulated in nanoparticles. Cancer Immunol Immunother 2013;62:1161e73.
32. Mueller M, Schlosser E, Gander B, Groettrup M. Tumor eradication by immunotherapy with biodegradable PLGA microspheres-an alternative to incomplete Freund’s adjuvant. Int J Cancer 2011;129:407e16.
33. Ding AG, Schwendeman SP. Acidic microclimate pH distribution in PLGA microspheres monitored by confocal laser scanning microscopy. Pharm Res 2008;25:2041e52.
34. Sophocleous AM, Zhang Y, Schwendeman SP. A new class of inhibitors of peptide sorption and acylation in PLGA. J Control Release 2009;137:179e84.
35. Houchin ML, Topp EM. Chemical degradation of peptides and proteins in PLGA: a review of reactions and mechanisms. J Pharm Sci 2008;97:2395e404.
36. Kim HK, Park TG. Microencapsulation of human growth hormone within biodegradable polyester microspheres: protein aggregation stability and incomplete release mechanism. Biotechnol Bioeng 1999;65:659e67.
37. Hermeling S, Crommelin DJA, Schellekens H, Jiskoot W. Structure-immunogenicity relationships of therapeutic proteins. Pharm Res 2004;21:897e903.
38. Liu Y, Ghassemi AH, Hennink WE, Schwendeman SP. The microclimate pH in poly(D,L-lactide-co- hydroxymethyl glycolide) microspheres during biodegradation. Biomaterials 2012;33:7584e93.
39. Ghassemi AH, van Steenbergen MJ, Talsma H, van Nostrum CF, Jiskoot W, Crommelin DJA, et al. Preparation and characterization of protein loaded microspheres based on a hydroxylated aliphatic polyester, poly(lactic-cohydroxymethyl glycolic acid). J Control Release 2009;138:57e63.
40. Ghassemi AH, van Steenbergen MJ, Barendregt A, Talsma H, Kok RJ, van Nostrum CF, et al. afs controlled release of octreotide and assessment of peptide acylation from poly(D,L-lactide-co-
hydroxymethyl glycolide) compared to PLGA microspheres. Pharm Res 2012;29:110e20.
41. Samadi N, van Nostrum CF, Vermonden T, Amidi M, Hennink WE. Mechanistic studies on the degradation and protein release characteristics of poly(lactic co-glycolic-co-hydroxymethylglycolic acid) nanospheres. Biomacromolecules 2013;14:1044e53.
42. Srinivas M, Cruz LJ, Bonetto F, Heerschap A, Figdor CG, de Vries IJM. Customizable, multi- functional fluorocarbon nanoparticles for quantitative in vivo imaging using F-19 MRI and optical imaging. Biomaterials 2010;31: 7070e7.
43. Verdijk P, Scheenen TWJ, Lesterhuis WJ, Gambarota G, Veltien AA, Walczak P, et al. Sensitivity of magnetic resonance imaging of dendritic cells for in vivo tracking of cellular cancer vaccines. Int J Cancer 2007;120:978e84.
44. Long CM, van Laarhoven HWM, Bulte JWM, Levitsky HI. Magnetovaccination as a novel method to assess and quantify dendritic cell tumor antigen capture and delivery to lymph nodes. Cancer Res 2009;69:3180e7.
45. Yang Z, Zheng S, Harrison WJ, Harder J, Wen X, Gelovani JG, et al. Longcirculating near-infrared fluorescence core-cross-linked polymeric micelles: synthesis, characterization, and dual nuclear/
optical imaging. Biomacromolecules 2007;8:3422e8.
46. Heo MB, Lim YT. Programmed nanoparticles for combined immunomodulation, antigen presentation and tracking of immunotherapeutic cells. Biomaterials 2014;35:590e600.
47. Christian NA, Benencia F, Milone MC, Li G, Frail PR, Therien MJ, et al. In Vivo dendritic cell tracking using fluorescence lifetime imaging and near-infraredemissive polymersomes. Mol Imaging Biol 2009;11:167e77.
48. Frangioni J. In vivo near-infrared fluorescence imaging. Curr Opin Chem Biol 2003;7:626e34.
49. Escobedo JO, Rusin O, Lim S, Strongin RM. NIR dyes for bioimaging applications. Curr Opin Chem Biol 2010;14:64e70.
50. Key J, Leary JF. Nanoparticles for multimodal in vivo imaging in nanomedicine. Int J Nanomedicine 2014;9:711e26.
51. Rao J, Dragulescu-Andrasi A, Yao H, Yao H. Fluorescence imaging in vivo: recent advances. Curr Opin Biotechnol 2007;18:17e25.
52. Mou Y, Hou Y, Chen B, Hua Z, Zhang Y, Xie H, et al. In vivo migration of dendritic cells labeled with synthetic superparamagnetic iron oxide. Int J Nanomedicine 2011;6:2633e40.
53. Su H, Mou Y, An Y, Han W, Huang X, Xia G, et al. The migration of synthetic magnetic nanoparticle labeled dendritic cells into lymph nodes with optical imaging. Int J Nanomedicine 2013;8:3737e44.
54. Chen YC, Wen S, Shang SA, Cui Y, Luo B, Teng GJ. Magnetic resonance and near-infrared imaging using a novel dual-modality nano-probe for dendritic cell tracking in vivo. Cythotherapy 2014;16:699e710.
55. Lee H, Mason JC, Achilefu S. Heptamethine cyanine dyes with a robust CeC bond at the central position of the chromophore. J Org Chem 2006;71:7862e5.
56. Leemhuis M, van Nostrum C, Kruijtzer J, Zhong Z, ten Breteler M, Dijkstra P, et al. Functionalized poly(alpha-hydroxy acid)s via ring-opening polymerization: toward hydrophilic polyesters with pendant hydroxyl groups. Macromolecules 2006;39:3500e8.
57. Kolb H, Finn M, Sharpless K. Click chemistry: diverse chemical function from a few good reactions.
Angew Chemie Int Ed 2001;40:2004.
58. van Dijk M, Rijkers DTS, Liskamp RMJ, van Nostrum CF, Hennink WE. Synthesis and applications of biomedical and pharmaceutical polymers via click chemistry methodologies. Bioconjug Chem 2009;20:2001e16.
59. Moses JE, Moorhouse AD. The growing applications of click chemistry. Chem Soc Rev 2007;36:1249e62.
60. Binder WH, Sachsenhofer R. ‘Click’ chemistry in polymer and material science: an update.
Macromol Rapid Commun 2008;29:952e81.
61. Gill SC, von Hippel PH. Calculation of protein extinction coefficients from amino acid sequence
data. Anal Biochem 1989;182:319e26.
62. Silva AL, Rosalia RA, Sazak A, Carstens MG, Ossendorp F, Oostendorp J, et al. Optimization of encapsulation of a synthetic long peptide in PLGA nanoparticles: low-burst release is crucial for efficient CD8(þ) T cell activation. Eur J Pharm Biopharm 2013;83:338e45.
63. Sah H. A new strategy to determine the actual protein content of poly(lactideco-glycolide) microspheres. J Pharm Sci 1997;86:1315e8.
64. Winzler C, Rovere P, Rescigno M, Granucci F, Penna G, Adorini L, et al. Maturation stages of mouse dendritic cells in growth factor-dependent longterm cultures. J Exp Med 1997;185:317e28.
65. Karttunen J, Sanderson S, Shastri N. Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens. PNAS 1992;89:6020e4.
66. Shastri N, Gonzalez F. Endogenous generation and presentation of the ovalbumin peptide/k(b) complex to T-cells. J Immunol 1993;150:2724e36.
67. Filonov GS, Piatkevich KD, Ting L, Zhang J, Kim K, Verkhusha VV. Bright and stable near-infrared fluorescent protein for in vivo imaging. Nat Biotechnol 2011;29:757e61.
68. Hogquist KA, Jameson SC, Heath WR, Howard JL, Bevan MJ, Carbone FR. T cell receptor antagonist peptides induce positive selection. Cell 1994;76:17e27.
69. Perego G, Cella GD, Bastioli C. Effect of molecular weight and crystallinity on poly(lactic acid) mechanical properties. J Appl Polym Sci 1996;59:37e43.
70. Cohen S, Yoshioka T, Lucarelli M, Hwang L, Langer R. Controlled delivery systems for proteins based on poly(lactic glycolic acid) microspheres. Pharm Res 1991;8:713e20.
71. Mundargi RC, Babu VR, Rangaswamy V, Patel P, Aminabhavi TM. Nano/micro technologies for delivering macromolecular therapeutics using poly(D,L-lactide-co-glycolide) and its derivatives. J Control Release 2008;125:193e209.
72. Zambaux M, Bonneaux F, Gref R, Maincent P, Dellacherie E, Alonso M, et al. Influence of experimental parameters on the characteristics of poly(lactic acid) nanoparticles prepared by a double emulsion method. J Control Release 1998;50:31e40.
73. Couvreur P, Puisieux F. Nanoparticles and microparticles for the delivery of polypeptides and proteins. Adv Drug Deliv Rev 1993;10:141e62.
74. Joshi VB, Geary SM, Salem AK. Biodegradable particles as vaccine delivery systems: size matters.
Aaps J 2013;15:85e94.
75. Dhawan A, Sharma V. Toxicity assessment of nanomaterials: methods and challenges. Anal Bioanal Chem 2010;398:589e605.
76. Patterson JP, Robin MP, Chassenieux C, Colombani O, O’Reilly RK. The analysis of solution self- assembled polymeric nanomaterials. Chem Soc Rev 2014;43:2412e25.
77. Ghassemi AH, van Steenbergen MJ, Talsma H, van Nostrum CF, Crommelin DJA, Hennink WE.
Hydrophilic polyester microspheres: effect of molecular weight and copolymer composition on release of BSA. Pharm Res 2010;27:2008e17.
78. Zembruski NCL, Stache V, Haefeli WE, Weiss J. 7-Aminoactinomycin D for apoptosis staining in flow cytometry. Anal Biochem 2012;429:79e81.
79. Zhegalova NG, He S, Zhou H, Kim DM, Berezin MY. Minimization of selfquenching fluorescence on dyes conjugated to biomolecules with multiple labeling sites via asymmetrically charged NIR fluorophores. Contrast Media Mol Imaging 2014;9:355e62.

domain: chemistry


text:
Barrie Day Camp
June 20—Aug. 12
13500 Layhill Road,, Silver Spring, Md. 20906
Barrie School's outdoor summer day camp takes place at the school's 45-acre campus. Campers participate in a range of traditional activities, from athletics to the performing arts, and attend daily swim lessons. Speciality camps for riding, theater, nature, karate, digital video, sports, art, and counselors-in-training are also offered. Ages 4 to 14.

domain: artistic


text:
Photograph [Dug-outs in a wood]; [?]; [?]; CT80.1408d1
Photograph [Dug-outs in a wood]About this object
Photograph; a black and white photograph depicting dug-outs of sandbags in a wood. A soldier walks past in the foreground.
The original photograph has been digitised. A digital image and copy print are available for research access.
[?]Inscription and Marks
Handwritten on reverse: 'sent home by / W. Dunlop / Tahakopa'Format
From the collection of Owaka Museum Wahi Kahuika The Meeting Place "a rest on your journey"Object number

domain: library


text:
Celluloid, metal, paper
1/8 x 7/8 in. diameter
A beige pinback button printed with a state seal, crest, symbol or motto from U.S. states and territories; marked "Indiana", with a scene of a man swinging an axe at trees with mountains in the distance and a bull nearby. Part of a series of pre-World War I United States state buttons; some of the region names are no longer current. The reverse is printed "Sweet Caporal Cigarette", indicating that this button was part of a series of collectible premiums given by tobacco and gum manufacturers.
Gift of Bella C. Landauer
Due to ongoing research, information about this object is subject to change.


MLS® ID: 13523137
2504 Oakland Boulevard
Fort Worth, Texas, 76103 $350,000
Just off E. Lancaster which has numerous retail outlets, restaurants, auto repair companies and personal service providers. Churches, fast food restaurants, service stations, and grocery stores are all in the vicinity. A new library is being built just around the corner. Located on the public transit system service line.


What are the common solutions for DC power systems?
Issuing time:2024-02-27 18:05
DC power supply systems are widely used in various power plants, substations and other power system core hubs
Introduction to the composition and function of DC power supply system
The DC power supply system is widely used in various power plants, substations and other power system core hubs, and is composed of battery packs, charging devices, DC feeder screens, DC distribution cabinets, DC power monitoring devices, DC branch feeders, etc., to provide safe and reliable working power for relay protection devices, circuit breaker tripping, signal systems, DC chargers, UPS, communications and other subsystems.
As the core equipment of the substation, the DC power supply system of the substation supplies power to the control equipment of the substation, and if the power supply is stopped due to failure, it will directly affect the normal operation of the substation. At present, most of the chargers use high-frequency switching power supply type, and the battery has also been upgraded from the initial acid-proof lead-acid battery to the VRLA sealed lead-acid battery, and is equipped with a DC power supply microcomputer monitoring device.
The main functions of the battery pack in the operation of the DC power system:
1) When the mains power is interrupted or the DC charging equipment fails, the battery pack will supply power to the load separately to ensure that the power supply is not interrupted.
2) Under the short-circuit impact of the load or the initial impact of the normal load, the part exceeding the rated output of the charger shall be borne by the battery.
3) Smooth filtering. The battery pack has the same charging and discharging function as the capacitor, and the AC components are isolated, so that the AC components sent to the load are further reduced, and the voltage quality requirements of the load are guaranteed.
The contents of the daily regular inspection of the battery pack and the common faults:
1) The VRLA battery operates in the mode of float charge in normal operation, the single float charge voltage value should be 2.23-2.28V, and the balanced charge should be 2.3-2.35V, and the terminal voltage value, float charge current value, single battery voltage value, ground resistance and insulation state of the battery pack and DC bus should be monitored during operation.
2) Daily regular inspection of the battery pack: terminal voltage measurement, internal resistance test, dynamic discharge test, AC input power switching test of charging device.
3) The function of the safety valve of the VRLA battery: the VRLA battery is in a sealed state under normal charging, and a safety valve that can be automatically opened and closed is installed. When the internal pressure exceeds the specified opening threshold, the safety valve will be automatically opened, and it will be automatically closed when the pressure is lower than the closing threshold. The safety valve is equipped with an acid filtration device, which will not discharge harmful gases such as acid mist, and will not leak electrolyte, which can prevent the battery from exploding due to sparks.
4) Common faults of VRLA batteries: abnormal VRLA shells and abnormal discharge voltages of VRLA batteries.
Charging device protection function and operation monitoring
The charger should have over-voltage, over-current, under-voltage, DC insulation monitoring, AC loss of voltage, AC phase loss and other protection and sound and light alarm functions. The daily inspection of the charger is essential, such as: whether the three-phase AC input voltage is balanced or missing, whether the operating noise is abnormal, whether the protection signals are normal, whether the AC input voltage value, DC output voltage value, DC output current value, etc. are correct, and whether the insulation state to the ground is good. If the AC power supply is interrupted, the battery pack will supply power to the load uninterruptedly, if there is no automatic voltage regulation device, manual voltage regulation should be carried out to ensure that the bus voltage is stable, and the AC power supply is restored, and the charging device should be started manually or automatically immediately.
2. Routine test during DC power system overhaul
After the installation of the DC power supply system, the handover acceptance test should be carried out before it is put into operation, and the test project must meet the technical requirements, and the trial operation shall be normal for 72 hours. The main content of handover acceptance:
Insulation monitoring and signal alarm device
Withstand voltage and insulation test
Battery pack capacity test
Charging device current stabilization accuracy test
Voltage regulation accuracy test of charging device
Ripple coefficient test of charging device
DC bus continuous power supply test


text:
46,111 under bench storage Home Design Photos
Troy Dean Interiors
Mizner Park - Boca Residence
Bench at end. Black accent wall
Love the use of Dark Woods & Contrast plus Space & Possible Storage!
Nice room with storage...Storage behind bef...Wall storage...Where is the bench from
$1,200 Off Of Your Complete Remodel
Minimum $25,000 Project
Remodeling From Concept to Completion in Northern Virginia
Kitchen, Bath and Interior remodeling company featuring the latest bath and kitchen designs and the largest selection of flooring available in Northern Virginia. Showrooms in Sterling and Leesburg.
Signature Design & Cabinetry LLC
Rustic & Ritz
Love the bar fridge under the bench easy access. Also love the window open to the outdoors clear views
Incorporate the wine cooler and wine storage into the island for Mom's kitchen remodel.
Cooler with wine storage on side....fridge and wine storage
Michael K Chen Architecture
Cupboard under book shelf (folds up)
Blue storage - looks extremely multi-functional
Transformer bench...Hidden storage
Also need a place to sit, a chair instead of a space hogging bench.
good ideas for closet clothes shoes storage spaces
great basement storage, ceiling hung clothes hanger
coats w rod... maybe under stairs...Clothing storage...Retail storage
Kristi Spouse Interiors
Whidbey Island Beach House - Kitchen Remodel
I like the under counter storage
Island storage...great storage space....island as storage alternative
Parallel to the sea
!! How big does the space need to be to incorporate this base with the storage etc? What ar
What's in the bottom draw, it looks quite long. Is it storage, or a pull out bed?
Raised bed with desk and drawers under (vet)
Raised bed, storage drawers beneath, pull out desk
zones the (limited) space and the raised section can incorporate lots of storage, too. Design a mix of pull-out drawers of all shapes and sizes and perhaps
kids – they’re still children, after all. A platform bed with built-in storage will hopefully keep some of the mess at bay, and a slide-out desk is both
good idea FOR BED, WITH STORAGE
Michelle Walker architects
Paddington Butterfly House
Under-bench twin sinks
sink in the bench...Galley kitchen long bench...Window to bench height...Window at bench...Underbench sink colour of bench
Kress Jack At Home
Home office reading nook
Love the two benches, where can i buy them?
Cool little bench...Sofa bench

domain: design


text:
Preporučuju 23 lokalca ·
A short walk from our AIRBNB to this park will not disappoint. There is the Shinzen Friendship Garden that is a traditional northwestern American Japanese Garden. enjoy astroll, jog, run or biking on the many trails along the river. Gorgeous views for a picnic. . There are also special events held…
Large and sprawling park with a Fenced Dog Park, Picnic Tables/BBQ, Disc Golf Course, Jogging/Horseback Riding Trails, San Joaquin River Access, and Bird Watching.
360+ acres of wooded park. Feed ducks and geese, wander the Japanese Garden, play on the playgrounds, take in an outdoor show, jog, bike, even ride horses!
Pretty much everything is here for enjoyment (only 10 minutes away home), the best location compares Old town Clovis, Serria Vistas Mall, Fashion Fair mall, Fig Garden Village, Tower District...
Great and close to shopping! Great place for families, picnics, hiking, and biking.

domain: geography


•Dawson Competition Black Fixed Rear .225 Tall x .135 Notch Width
•Dawson Fiber Optic Front Sight .205 Tall x .105 Wide
•Easy to Install - Sight Punches, Allen Wrench and Instructions Included!
•Perfect Impact Policy Applies *See Details*
•CNC Machined From Bar Stock To Exacting Specifications
For years Bo-Mar rear sights set the standard of which all other rear sights were judged, so why not make a fixed rear that uses that knowledge. Dave Dawson set out to improve on that and make the best fixed rear sight on the planet for competition and target shooting. The blade is back angled 3 degrees to reduce glare. The deep notch provides more visual reference for faster precise windage alignment. This also gives the shooter the advantage of tracking the front sight better during recoil and placement on target, for those tight hardcover shots. The serrations on the black sights are designed to draw your eyes to the HiDef sight notch, providing a clear, crisp sight picture, yet snag free on outer profile. Chamfered to break all sharp edges and not rub you the wrong way. The competition black .135 notch rear sight with .105 wide fiber optic front provide Dave Dawson the best focus for his average eyes and is what is on all of his Glock's.
Don't Forget The Loctite 262 Threadlocker Works on front and rear Glock sights
POA-POI , installed without any issues, quality product at a fair price. Will recommend and would buy again.
I was looking for something to give me a leg up in a local stock class competition where you can only make open sight modifications to your pistol. After looking and many different brands I decided to go with Dawson and I’m so glad I did! It took just under a hour to fit these sights to my glock 19 gen 4, and they look fantastic. Plus out at 15 yards I’m hitting golf ball sized groups which you wouldn’t even be able to see with stock sights! For the price you can’t beat them and supper fast shipping! I’ll be buying again!
These are an absolute must have for any serious competition shooter. The narrow front sight allows for easy target acquisition and sight alignment on targets beyond 25 yards. The sights mount firmly and do not come loose. They are of high quality and do not damage easily.
I've shot Glock pistols for nearly 15 years. I've used all type of sights, from factory Glock plastics, several brands of night sights, and TFOs. For my night-stand pistol, I still like the TFOs. But for competition, nothing beats the Dawson Precision. The FO front helps with quick target acquisition and the black rear helps keep your focus on the front sight where it belongs. I am converting all of my competition Glocks over to the Dawson Precision sights.
The bright fiberoptic front sight is easy to pick up during Daytime and low light.
I have these sights on 1911's, G34, G27 and now the polymer 80 G19's. The wider blacked out rear notch helps with sight alignment for my aging eyes.
Jay in customer service is the man! I recieved ZERO static when I called to exchange my suppressor height for the competition sight set.
Customer service is really 1st class for a great product.
I put together a Glock 22 with 6" threaded barrel and added a sight block for USPSA Limited. I have us ed Dawson sights on 2 other competition guns, so I knew they wouldn't fail me. But I really had no idea if this would work. My worries were not warranted. Installed in minutes. Centered the sights. No further adjustments needed! This new rig is a 20 yard tack driver. Perfect! So not hesitate to use these sights. Thank you Dawson!
I have purchased two sets of these sights now, one set for a Glock 19 the other mounted on a 17, both guns are used in Steel
Challenge competition. The sight height and rear notch/front sight width are the perfect combination for all of the varied ranges of this game.
Dave Dawsons experience here is evident, his formula was spot on for both of these guns. At age 69. I can't even name all of the iron sights I've tried, to no avail, I thought that carry optics was my only recourse, these sights put me back in production class where I really want to be, in fact on very bright days with sun directly on the targets, these sights are much easier to pick up than any electronic dot sight that I own, and I own some very high end ones. I would highly recommend them and Dawson precision to anyone.
Great range sights. Love the black rear, no added distraction from the front sight. Easy & fast for me to get an acceptable sight picture & also accurate for bullseye. The rear sight notch width/front sight width combo seems perfect. They installed easy with no fitting or filing required. I suck much less using these sights.
These fixed sights are so much better than the original Glock 34 adjustable sights. Able to pick them up and get on target quicker. Easy swap out. Just doubled the fun factor! Would I recommend these sights? Yes! Worth every penny.


Red PA Plastic Media Blasting PA40 For Plastic Sandblasting Surface Treatment
|Place of Origin:||Henan, China|
Payment & Shipping Terms:
|Minimum Order Quantity:||25kgs|
|Packaging Details:||25kgs paper bags+Pallet|
|Delivery Time:||5-8 working days|
|Payment Terms:||L/C, T/T, Western Union, MoneyGram|
|Supply Ability:||300MT Per Month|
|Material:||PA Polyamide Nylon 6||Molding Shrinkage:||1.2—1.5%|
|Shape:||Cylindrical||Specific Gravity:||1.14 G/cm3|
|Color:||White, Red||Tensile Force:||750 Kg / Cm2|
|Bending Stress:||1000 Kg / Cm2|
PA40 Plastic Media Blasting,
Surface Treatment Plastic Media Blasting,
PA40 plastic abrasive media
PA plastic media red color size PA40 for Plastic Sandblasting Surface Treatment
Our PA plastic media is produced from imported raw materials and by our patented technology by our independent research pf high-speed precision granulation. With uniform particles, wear-resistant and long-life antistatic. All products have passed SGS tested and ISO9001 certification.
PA plastic media mainly used for plastic products sandblasting surface treatment to remove spilled glue and deburring.
Remark: Dimension accuracy ±0.05mm
Special specification and color can be customized
- Physical Index:
|Color:||Translucent milky white or Red|
|Hardness:||Rockwell hardness HRC 120|
|Specific gravity:||1.14 g/cm3|
|Tensile force:||750 kg / cm2|
|Bending stress:||1000 kg / cm2|
|Water absorption rate 23 ℃:||1.8%|
|Melting point:||220 ℃|
- Main Features
1. PA plastic media is artificial abrasive, with cylindrical and hexahedral shapes;
2. PA plastic media has a certain degree of toughness, non-cutting, and will not cause damage to the product to be processed during use.
The material is soft and has a long service life.
3. PA plastic media is mainly used for sandblasting and shot blasting equipment to clean and remove burrs and burrs on the surface of the workpiece.
Widely used in sandblasting surface treatment:
1. Remove burrs of precision thermoplastic plastic parts such as mobile phone cases, connectors or thermosetting plastic parts such as transformer bakelite skeleton Bobbin;
2. Integrated circuit IC epoxy resin packaging, printed circuit board PCB drilling, SMD resin packaging of electronic components, such as spillage, flashing or burrs;
3. Homogenize plastic parts to eliminate bad marks and improve product quality;
4.Decontamination and paint removal of precision parts, used for aircraft, yacht, automobile and other vehicle maintenance.
Our PA plastic media has added appropriate amount of antistatic liquid in the raw materials formula, so it will prevent to generate static electricity in the process of using and recycling. It will bring you good using experience without any troubles.


text:
Easy Banana Bread Recipe Let s Mingle Blog from easy banana bread recipe 2 bananas , image source: letsmingleblog.com
- 2 cup purpose flour
- 1 tsp baking soda
- Pinch salt
- 1 cup sugar, or you can add a little bit more
- 1 egg
- 1/2 cup vegetables oil, you can use 1/2 cup melted butter instead
- 3 ripe banana (smash it)
- 2 tbsp almond milk, you can use any milk
- 1 tsp vanilla extract
- Handfuls slices almond or you can use any nuts
- Preheat the oven at 325 F. Greased the loaf pan or bundt pan. Mixed flour, baking soda, and salt aside.
- Mixed egg, sugar, and oil combined together. I used mixer to combined it.
- Add the dry ingredients into the liquid ingredients. Mixed well. It will be little bit dry, so please don’t freaking out.
- Add milk, vanilla extract, and smashes banana. Mixed together until smooth. Finally add half of almonds slices into it, mixed well with spatula.
- Sprinkles half of almond into the pan, pour the batter into it and top with the rest of the almond. Bake for 50-60 minutes. Check frequently. It’s done when the knife comes out clean from the middle of the loaf or bundt.
- Let the bread stand warm. Use sharp knife loosen the bread from sides and set aside. Enjoy!
Gallery of √ Easy Banana Bread Recipe 2 Bananas
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Banana Blueberry Bread Recipe from banana blueberry bread recipe , image source: recipeland.com Ingredients 1 1/4 cup granulated sugar 2 stick Butter, softened plus save wrappers 1 pints blueberries/blackberries 2 large eggs 3 1/2 bananas 1/2 cup buttermilk 2 tsp vanilla extract 2 1/2 cup all purpose flour 1 tsp baking soda 1 tsp salt Steps 1 hour 30 mins Preheat oven to 350. Mix […]
15 Easy & Healthy Apple Recipes from healthy oatmeal bread recipe , image source: www.runningwithspoons.com Ingredients 4 Medium Size Ripe Bananans 1 1/2 Cup Whole wheat Flour 1/2 Cup Quick Oats 3/4 Cup Sugar 1/2 Cup Canola oil 1 Tsp Vanilla Extract 1/4-1/2 Tsp Cinnamon Powder 1/4 Tsp Nutmeg Powder (Optional) 1 1/2 Tsp Baking Powder 1/2 Tsp Baking Soda Pinch Salt 1/2 Cup Shelled Walnuts 1/4 […]
EASY Banana Bread Recipe moist & delicious I Heart from homemade banana bread recipe , image source: www.iheartnaptime.net Ingredients 3 Very ripe bananas, peeled 1/3 cup Melted butter 1 cup Sugar 1 Egg,beaten 1 tsp Vanilla extract 1 tsp Baking soda 1 pinch Salt 1 1/2 cup All-purpose flour Steps Preheat the oven to 350°F (175°C), and butter a 4×8-inch loaf pan. In […]

domain: food


text:
I have been writing for many years, but only recently started sharing my work with the world. My first book was published in April 2017, and since then I have written three more books (two of which are available on Amazon).
My current project is a series of fantasy novels set in an original universe called The World of Aluvia. This is where you can find information about the series as well as links to purchase each book individually or all at once
Back to Main


text:
The majority of orthodontic patients are children and teenagers who are fitted with dental braces to correct misaligned teeth. Thankfully, the days of ‘train track’ braces are behind us and largely due to advances in dental technologies, attractive lightweight braces are commonly available.
Braces are discussed in more detail in our orthodontic treatments section.
What is the most common age for orthodontic treatment?
Most young people have treatment around the ages of 12 or 13. This is because their permanent teeth are breaking though which means that they are easier to move into the correct position. A brace will exert gradual pressure on these developing teeth, re-aligning them as they do so which means an improved facial structure and appearance.
This process varies from one child to another but in general, they require a brace at the start of their teenage years. However it is not uncommon for a child to need a brace at an earlier age.
Braces for young people
There are other types of braces which are suitable for young people and should also be considered.
NHS orthodontic treatment
Orthodontic treatment within the NHS is free to anyone under 18 as long as there is a clear, demonstrable need. The definition of ‘need’ is governed by the Index of Orthodontic Treatment Need (IOTN) which grades this according to the extent of the orthodontic problem.
This rating system is discussed in greater detail in our orthodontics and the NHS section.
Treatment is free to young people but there are limitations on this due to scarcity of NHS funding. And it does not include the newer forms of treatment such as transparent braces (e.g. Invisalign) or clear brackets (e.g. Damon).These are only available through private treatment. What about adults? Adults are not usually eligible for NHS treatment, but, there are exceptions to this. Find out more in our orthodontics and NHS section. Many young people benefit greatly from orthodontic treatment.

domain: medical


text:
Like many other philosophical writings, the Tao Te Ching is a timeless Chinese treasure. These ancient writings are credited to Lao Tzu, although some believe them to be authored by a number of persons writing under the Lao Tzu pen name. Nevertheless, this audio gives the listener a taste of the wisdom of age and the important values of human existence.
Free Audio Book
To stream audio: click the play button.
To download audio: right click the track and save to your device.
ThoughtAudio PDF Transcript: TA0010_TaoTeChing_LaoTzu.pdf

domain: literature


text:
2022 Sauvignon Blanc
94 Pts, Gold - 2023 Los Angeles International Wine Awards
92 Pts, Gold - 2023 Toast of the Coast Wine Competition
Product on Backorder
Bright, varietal and dry, this Sauvignon Blanc presents citrusy notes and aromas of grapefruit and peach. The finish is vibrant and persistent.
Two vineyards from the Lodi Appellation make up the grapes sourced for this wine. On the northeastern side of our property, you will find the Estate Sauvignon Blanc grapes in sandy loam soils. This certified and sustainable vineyard located in the Mokelumne River sub AVA is oriented at an angle to provide as much evenly balanced sunlight as possible. This site allows for the grapes to not be stressed and to fully mature, creating a fresh, citrus and fruit forward Sauvignon Blanc. The other grapes are sourced by another certified sustainable vineyard grown by Bruce Fry from Mohr-Fry Ranches, located in the Mokelumne river sub AVA as well. These vines are oriented east to west in flat sandy loam soil. This vineyard is not trellised and helps to capture the earthiness in this wine. It adds aromas like freshly cut grass and pyrazines like dried herbs and green pepper. These vineyards combined together help to round out the aromas and mouthfeel, leaving a more complex and interesting wine in the overall product.
These grapes were harvested primarily in two separate picks, about a week apart, in order to achieve different ripeness levels. The first pick captures more pyrazines, while the second pick captures more esters and thiols. All of the picks were hand-picked and gently whole cluster pressed to a temperature controlled stainless steel tank. The juice was racked, commercial yeast was added, and the juice was then fermented to dryness. After a long 24-day fermentation, the wine was racked again and held cold in tank until the early bottling in December to maintain freshness and liveliness.


July 18th, 2010
Near the end of the ride we had two accidents. Firstly, Penny hit a bollard in the middle of the trail (the kind that keep cars off the bike trails) and actually although she is fine now (ribs excepted) she hit it an awful crack! In the photos above you can see that her right pedal arm and her left pedal arm are no longer the same… having bent the right one inwards about an inch or more. Amazingly the only damage seems to be the bent arm; all other systems seem ok, at least for now.
And, to compound things, my velomobile was already limping home a bit. I had a drive chain skip on a VERY steep stretch and the drive chain had developed an odd click. About 300 meters from the end of our trip, on a reasonably steep stretch, the chain snapped.
So, all you bike wrench types, I need some advice.
#1 – Can Penny’s crank arm be straightened or do we need to replace the whole kit and caboodle? Would straightening it possibly weaken some already weakened metal further?
#2 – After some incredible gymnastics on my overturned Mango (below) I managed to replace and re-thread a new chain section onto the chain path of the velomobile (there has to be an easier way but that is another question). Now I am trying to close the quick-link EXCEPT the quick-link method assumes that I can tension the chain to get it to click closed. In a velomobile, I am jammed on my right ear into the cockpit on the wrong side of the seat to be able to get anywhere near the pedals… so I am not sure how to put tension on the chain sufficient to click the quick-link into place… if you get my drift?
Just because I blog about this stuff doesn’t mean I have any idea how to really do it! After fighting with the chain for about two hours yesterday, my body feels a bit like I was pushed through the keyhole backwards.


text: “San Francisco, 1947. Bartender Sammy “Two Toes” Tiffin and the rest of the Cookie’s Coffee Irregulars–a ragtag bunch of working mugs last seen in Noir–are on the hustle: they’re trying to open a driving school; shanghai an abusive Swedish stevedore; get Mable, the local madam, and her girls to a Christmas party at the State Hospital without alerting the overzealous head of the S. F. P. D. vice squad; all while Sammy’s girlfriend, Stilton (a. k. a. the Cheese), and her “Wendy the Welder” gal pals are using their wartime shipbuilding skills on a secret project that might be attracting the attention of some government Men in Black. And, oh yeah, someone is murdering the city’s drag kings and club owner Jimmy Vasco is sure she’s next on the list and wants Sammy to find the killer. Meanwhile, Eddie “Moo Shoes” Shu has been summoned by his Uncle Ho to help save his opium den from Squid Kid Tang, a vicious gangster who is determined to retrieve a priceless relic: an ancient statue of the powerful Rain Dragon that Ho stole from one of the fighting tongs forty years earlier. And if Eddie blows it, he just might call down the wrath of that powerful magical creature on all of Fog City.” — Provided by publisher. Unrated. Commercial audiobook. Bestseller. 2022.


text:
Lead you business, we solve the rest
We have an integrated system solutions for everything you need.
Our way of doing things
-> Operational load reduction
-> Information traceability
-> Supplier self-management
-> Improvement in the image of the company and service to suppliers
-> Permanent support through help desk, chat and call-center
-> Unique solution with finance, purchasing and tax modules.
-Peticiones de oferta
Solution of taxes
-Filing of Invoices
-Invoices in process

domain: economics


Providing you the best range of grid resistance starter with effective & timely delivery.
Grid Resistance Starter
Get Best Quote
✓Thanks for Contacting Us.
Approx. Rs 2 Lakh / PieceGet Latest Price
Product BrochureProduct Details:
Test And Reset Button
I Deal In
Country of Origin
Made in India
Grid Resistance Starter is for starting the Slip Ring Induction Motor. Metallic resistance elements made up of Stainless Steel (S.S.), either in the form of Punched Grid type or Coil type are used to provide the required resistance in the rotor circuit. These resistance elements are skillfully fabricated and designed for continuous rating to suit the motor parameters. The steps are selected adequately for precise speed control, to increases / decrease the speed linearly as to avoid large current kicks. Speed variation between each steps are restricted 1% to 2.5% (or as required by the customer). Different tapping from the resistance element through the power shorting contactors are taken to design these steps. With the help of PLC logic the power shorting contactors are turned off/on enabling to reach the desired speed. Cooling blowers are provided in the resistance chamber to remove the heat getting generated. It helps in controlling the change in resistivity of the element with temperature rise and increases the life of the resistance elements.


text:
Are you dreaming of an opulent escape to the picturesque South of France, where the sun-kissed shores and charming villages beckon you for an unforgettable holiday experience? Look no further! Qhome.fr offers a stunning collection of villas available for rent that will elevate your holiday to unparalleled levels of sophistication and luxury.
Why Choose a Villa?
When you opt for a villa rental in the South of France, you embark on a journey of pure indulgence and comfort. Imagine waking up to breathtaking views of the Mediterranean Sea, enjoying your morning coffee on a private terrace surrounded by lush gardens, and basking in the warmth of the sun as you relax by your own private pool.
Unmatched Elegance and Comfort
Qhome.fr specializes in curating luxurious villas that are meticulously designed to cater to the most discerning travelers. From exclusive properties nestled in the hills of Provence to seaside retreats along the French Riviera, each villa is a masterpiece of design and comfort.
Our dedicated team at Qhome.fr is committed to ensuring that your stay in the South of France is nothing short of perfection. From arranging private chefs to organizing luxury yacht rentals for exploring the azure waters of the Mediterranean, we go above and beyond to fulfill your every desire.
Explore the South of France
With a villa rental from Qhome.fr as your base, you can immerse yourself in the rich culture, gastronomy, and natural beauty that the South of France is renowned for. Indulge in Michelin-starred dining experiences, explore charming provincial towns, and unwind on pristine beaches that glisten under the Mediterranean sun.
Booking Your Dream Villa
Ready to embark on a journey of luxury and elegance in the South of France? Visit Qhome.fr today to browse our exclusive collection of villas for rent and start planning your unforgettable holiday in this paradise destination.
Start Your South of France Adventure Today!
Book your villa rental with Qhome.fr and immerse yourself in the luxe lifestyle of the South of France. Your dream holiday awaits!villa south of france rent

domain: travel


text:
SAN JOSE, Calif. — One month to the day after his injury, Avalanche captain Gabe Landeskog will play Tuesday against the San Jose Sharks and the player whose hit to the head kept him out of the lineup most of that time.
Landeskog suffered a concussion after a big hit from Sharks defenseman Brad Stuart in a Jan. 26 game at HP Pavilion. Landeskog returned to the lineup Saturday against Los Angeles and has one point in two games.
Those hoping for a royal rumble of revenge against Stuart from Landeskog will likely be disappointed.
“I’m treating it just like any other game,” Landeskog said. “It’s a game we need to win, and I’m just concerned with us getting two points.”
Landeskog played well against the Kings in a 4-1 Avs loss Saturday, but he struggled along with linemates Paul Stastny and David Jones in a 4-3 overtime loss to Anaheim on Sunday.
Landeskog did not record a point and had no shots on net. Stastny had an assist but only one shot on net, and Jones had no points and has only five this season.
Stastny has 10 points in 17 games and is a minus-7.
“I’m not going to single out anybody tonight,” Avs coach Joe Sacco said after Sunday’s loss, when the Stastny line struggled. “I thought our guys worked hard, and of course you’d like to get some production from your guys. Right now, our focus is on trying to gain points.”
After getting five points in a three-game span late last month and into February, Stastny has one goal and five points in his past nine games. One area of Stastny’s game that has been good lately, however: faceoffs. The center has been 55-percent or better in his past five games, including a 63 percent mark (10-for-16) against the Ducks Sunday.
Landeskog started the season on a line with Matt Duchene and P.A. Parenteau, but since he returned, he has been playing with Stastny and Jones. That may change Tuesday against the Sharks, with Jamie McGinn possibly going back to playing with Stastny and Jones.
Duchene, too, has struggled recently, with no points and a minus-4 in his past three games. In the past three, McGinn has the same statistics.
“We’ve got to find a way to get a win. We know we need to put points on the board, and it starts with me,” Landeskog said.
Footnotes. Entering Monday, things seemed quiet on the Ryan O’Reilly front. The center remains unsigned, and a trade seems likely. At Sunday’s game against Anaheim, scouts from several Eastern teams were in attendance — notable since the conferences don’t play each other in the regular season. … Defenseman Erik Johnson remains sidelined with a concussion and has not resumed skating.

domain: news


CPD investigate stabbing in Northeast Columbia
Columbia, S.C. (WOLO) – Update: Columbia Police Department says a female has been arrested in connection with the deadly Prescott Manor stabbing. 23 year-old, Zhane Pride, has been charged with murder and possession of a weapon during a violent crime.
Columbia Police Department say they are actively investigating a stabbing in Northeast Columbia on Wednesday afternoon (2/28).
According to officials, the stabbing occurred at 1601 Prescott Rd. (Prescott Manor).
Officials say a male victim was injured in the lower body inside an apartment this afternoon. He was taken to a local hospital, but officials say he was pronounced dead as a result of his injuries.
Investigators say they are currently speaking with a female who was initially detained.
Stay with ABC Columbia for more updates as they become available.


text:
|Title||:||The Supreme Court EBOOK|
|Format Type||:||PdF, ePub, Audiobook & Magazine|
The Supreme Court EBOOK Totally free Guides on the internet, wanting to download instantaneously in PDF, Epub and Kindle. All of our free ebooks are Lawfully Certified to your Peace of Mind.
NOTE : If you have a complaint the DMCA please send an email to our contact page.

domain: law


text:
SoftPro® In-Bed AFO
Ideal positioning AFO to treat mild plantarflexion (< 15 degree) contractures of the ankle / foot. This recumbent device comfortably maintains the ankle / foot in neutral while protecting the heel. The semi-rigid insert can accommodate a plantarflexion contracture of up to 15 degrees.
Orthotic therapy for mild contracture or abnormal tone and spasticity of the ankle and foot. The AFO is made of a flexible material, and will provide a gentle dorsi-flexion stretch of the ankle / foot. Ideal for bedbound patients. Adjustable toe post protects the toes; hip rotation bar can be used to treat internal / external rotation of the hip. The SoftPro® In-Bed AFO has a non-skid pad sewn into the bottom of the AFO cover to facilitate transfers without having to remove the AFO.
HCPCS – L4396


text:
The 4-star St. Anthony Hotel - A Wyndham Historic Hotel is located in old San Antonio, minutes away from Nightmare on Grayson, Southtown and Temple Beth-El. An example of European architecture, this hotel opened in 1909 and was fully restored in 2008.
The hotel is conveniently situated in a vibrant area, close to a river and gardens. The venue is set just 10 minutes from San Antonio center.
St. Anthony Hotel - A Wyndham Historic Hotel is set close to museums and theaters as well as San Antonio River, Henry B. Gonzalez Convention Center and Charline McCombs Empire Theatre.
The property enjoys a spectacular location within a 15-minute drive to San Antonio International airport.
Modern rooms feature a writing desk, LCD flat-screen TV, a minibar, individual climate control and a living area. Rooms offer a stunning view over the park. Bathrooms are fitted with bathrobes, free toiletries and slippers.
Eat & Drink
Guests can have breakfast in the restaurant every morning. At the club restaurant guests can enjoy American cuisine. Refreshing drinks are offered in the lobby bar.
A full breakfast is served and charges are applicable.
A solarium and a spa center are available on site. Such sports activities as volleyball, boating and snorkeling are provided on-site.
Wireless internet is available in public areas for free.
Wireless internet is available in the hotel rooms for additional charge
private parking is possible on site at USD 29 per day.


text:
Information on Clomid Side Effects
Many women who have difficulty getting pregnant discover they have menstrual irregularities. If you have been diagnosed with an ovulatory disorder, your doctor may prescribe Clomid, a fertility drug used to induce ovulation. Clomid, or clomiphene citrate, is a commonly prescribed drug that works by stimulating the body to produce more of the hormones that will cause your body to ovulate, or release an egg.
Any woman who is going through fertility treatments will most likely feel some sort of side effects. Although Clomid is widely used and most women go through a Clomid cycle without difficulties, there may be some minor changes that you will notice. In general, the most common side effects are similar to those you might feel in a heavy menstrual cycle:
mood swings, nausea, breast tenderness, abdominal pain or bloating, and headache or dizziness. Some women also experience vision problems such as blurring. However, if you begin taking Clomid and you experience any immediate or severe side effects, such as a serious rash or difficulty breathing, you should contact your health-care provider immediately.
Another commonly reported side effect of Clomid is a condition known as “hostile” cervical mucus. When you take Clomid, your body sometimes fails to produce fertile, sperm-friendly cervical mucus at the right time. Women who are taking Clomid sometimes find that during ovulation they are still producing thick cervical mucus, which can prevent sperm from traveling through the cervix.
If your health care provider determines that your cervical mucus is too thick, you can use a sperm-friendly lubricant such as PreSeed. You can also take guaifenesin, found in the over-the-counter cough syrup Robitussin, to thin the cervical mucus. Take two teaspoons of Robitussin, three times a day, starting five days before you expect to ovulate. Continue taking the Robitussin up until three days after you ovulate.
Beyond the immediate side effects, your doctor should explain to you some of the other possible risks involved in taking Clomid. For example, you should be aware that with Clomid, you have an approximately eight percent chance of having twins (compared to a 1-3 percent chance of conceiving twins naturally).
Clomid signals your body to produce more estrogen, which can sometimes result in the release of two eggs during a menstrual cycle . (That is why “Clomid twins” are almost always fraternal twins, or twins conceived from two different eggs.)
It’s also worth mentioning that in the past there have been suspicions about the increased incidence of ovarian cancer among women who took Clomid. However, recent research has shown no direct evidence of a link.
Aside from the physical effects, the time you spend taking Clomid may be a difficult time for you emotionally. The stress and demands of infertility treatments may lead you to feel lethargic or depressed. It is important to get good support during this period, whether it’s through a local support group or through the online community.
Most women respond to Clomid by at least the third menstrual cycle, or take the medication for no longer than six cycles. If you haven’t responded within this time, it’s generally safe to assume that you will need to pursue a different course of treatment. So no matter what side effects you may experience with Clomid, you can rest assured that they won’t last long. With luck, you will soon get pregnant and you can put your Clomid days behind you.


This Peugeot highwheel is now in the Velorama museum in Nijmegen, in the Netherlands. It is a rare machine with interesting details, like the unique ball bearings, detachable handlebars and rear spring support. This proves that Peugeot didn't just copy other bikes in its early years: they really were innovators.
This bike has a 51 inch driving wheel and the serial number 1968 is engraved in the right side of the neck and in the handlebars. This makes it an 1887-1888 model.
Interesting detail: the spring mentions 'Peugeot 1' - which was the most expensive Peugeot model at the time.
I added a picture from the book 'L'ere de Grand Bi' by Claude Reynaud, of an original Peugeot in the late 1880's. It is the same model.
Click here to enter my page with more info on Peugeot serial numbers.
Click a picture to enter Rober Sterba's (he sold the bicycle in 2013) site for more detailed pictures.