text:
The "lost noble grape," Carmenere is at home in the Colchagua Valley. The winery's tasting note: "Veramonte´s Carmenere features intense red and black fruit aromas, juicy ripe fruit flavors and mature tannins with balanced acidity. Oak aging gives the wine complexity, a hint of toast and a soft, elegant finish. Enjoy pastas, strong cheeses and lamb." With 7% Merlot and 4% Cabernet Franc.
Below is a listing of the wine review scores we frequently reference in our notes. While these sources remain an excellent source of information, we always recommend that you trust your own palate. Scores in these publications often represent the personal taste of an individual critic, which may not always coincide with your own. We have a huge team of excellent wine professionals that can match wines you've had in the past with new arrivals that meet your flavor profile.
The item you have chosen is not in stock in our retail stores or within our main warehouse.
2014 Valentin Bianchi "Elsa Bianchi" Malbec Mendoza $9.99
2012 Bodegas Sur de los Andes Malbec Reserva Mendoza $9.99
2012 Santa Ema Cabernet Sauvignon Reserve Maipo Valley $13.99
2013 Pascual Toso Malbec Maipú $11.99
2011 Clos des Fous "Grillos Cantores" Cabernet Sauvignon Alto Cachapoal $14.99


text:
Snow Plow Driver Shows Off Amazing Spin Moves
The weather outside may be frightful, but the TV coverage is so delightful.
The weather outside may be frightful, but the TV coverage is so delightful.
During a live report, you never know what can happen. This video is proof of that.
What do Tom Petty and Sam Smith have in common? According to a handful of listeners their songs 'I Won't Back Down' and 'Stay With Me,' respectively, sound eerily similar.
A Lubbock missing person case has taken a strange turn. The family of 19-year-old Texas Tech student Gordon Fang-Wong is heading to California, where they believe he could be involved with a cult.
Awards season continued last night (Jan. 25) with the 2015 Screen Actors Guild Awards! And, because we can't resist a red carpet, we gathered up some of the night's most buzz-worthy looks for you!
Last night I received an email from the Amarillo Police Department stating that the eastbound lanes of 34th from Soncy to Brennan Ave. would be closed until 9am on Tuesday, due to repair of pot holes.
Check out these 10 sites where you can file your federal tax returns for free!
The case against Paul John Martinez, in the 251st District Court in Randall County ended with a guilty verdict. Martinez was originally arrested on October 22, 2013 on charges of possession of child pornography.
I remember a time when Amarillo was considered a safe place to live. We didn't have a huge crime rate and it was rare to hear about a murder or a robbery. Now it is an everyday occurrence, and frankly that is a scary thing.
Amarillo Crime Stoppers needs your help in locating this week's fugitive of the week Ky Nguyen.
Expensive gifts, dinners or trips don't necessarily mean the most for Valentine's Day. They just cost the most.

subset: commoncrawl


text:
FederalNewsRadio.com - Purpose of Comments statement Click to show
Hubbard Radio, LLC encourages site users to express their opinions by posting comments. Our goal is to maintain a civil dialogue in which readers feel comfortable. At times, the comment boards following articles, blog posts and other content can descend to personal attacks. Please do not engage in such behavior here. We encourage your thoughtful comments which:
- Have a positive and constructive tone
- Are on topic, clear and to-the-point
- Are respectful toward others and their opinions
Hubbard Radio, LLC reserves the right to remove comments which do not conform to these criteria.
You are reading comments on the story:
2012 Defense bill amendment capping contractor pay advances
An amendment to the 2012 Defense Authorization Bill, passed by the Senate last week, caps taxpayer-funded compensation for all contracting employees at $400,000. Senate sponsors of the measure say the measure is designed to head off burgeoning defense contractor salaries. Federal employee unions have applauded the bill, but it has drawn the ire of industry groups. The Senate version of the defense bill, which contains controversial provisions dealing with military detainees, must still be reconciled with a House version before becoming law.


text:
Group ExCom and Volunteers
All activities of the Mineral King Group are led and run by volunteers. The Executive Committee ("ExCom") is our main volunteer committee, elected from the membership of the group, with additional "ex officio" members who volunteeer to be active. The "Ex Comm" meets on the fourth Tuesday of each month. Meetings are open to all Sierra Club members.
Many volunteer positions in the Group do not require being an elected member of the Executive Committee. Volunteers are needed to help with conservation action, outings, newsletter, and other activities! To get involved, call Membership Chair Bev at 559-624-0199, e-mail: firstname.lastname@example.com.
Mineral King Group Executive Committee 2013 members:
Officer | Name | Phone | Term Ends 12/31 | |
Chair / Chapter Delegate Alternate / Wetlands | John Kamansky | 559-686-6418 | email@example.com | 2015 |
Vice-Chair / Bears & Water Issues | Richard Garcia | (559) 624-0199 | firstname.lastname@example.com | 2014 |
Secretary / Outings Chair | Joanne Dudley | 559-733-2078 | firstname.lastname@example.com | 2015 |
Treasurer | Janet Wood | 559-697-3525 | email@example.com | 2015 |
Conservation Co-Chair | Roy Kendall | 559-901-1974 | email@example.com | 2015 |
Conservation Co-Chair/ Chapter Delegate Alternate | Brian Newton | 559-627-3571 | firstname.lastname@example.com | 2014 |
Chapter Delegate | Bev Garcia | 559-624-0199 | firstname.lastname@example.com | 2015 |
ExCom Member | Dave Keller | 559-688-4813 | email@example.com | 2014 |
Ex Com Member | Alan George | 559-732-9751 | no email | 2014 |
Conservation Committee
The Sierra Club is unique in its ability to stand up to environmental threats on many fronts at once because of our active presence in Washington D.C., Sacramento, and here locally in Kings and Tulare Counties. Our region has many local examples of threats to the environment emanating from the Bush White House which directly impact us here by reducing air quality, increasing urban sprawl, destroying forest and watershed protection, and polluting our water. The Sierra Club supports sustainable agriculture, clean water, pure air, protection of wild lands and forests, and "smart growth." We need your help on these efforts! Contact one of the following to get involved!
Conservation Co-Chairs: Mary Moy and Brian Newton
- Agriculture/CAFO: Mary Moy
- Air Quality:
- Contained Animal Feeding: Mary Moy
- Energy:
- Global Warming: Harold Wood
- Mining: Mary Moy
- National Forests:
- Endangered Species/ Parks/Refuges: Brian Newton (559) 627-3571
- Political:
- Programs/Publicity:
- Sprawl: vacant
- Transportation: John Kamansky
- Water: Richard Garcia
- Wetlands: John Kamansky
- Wilderness:
Outings Co-Chairs: Joanne Dudley & Dave Keller
Newsletter/Web: Harold Wood
Join Sierra Club Online today!
e-mail a friend about this page
Back to
Mineral King Group Home Page.
Sierra Club California
Back to
Sierra Club chapters.
Back to
Sierra Club home page.
If you have problems or comments concerning the Kern-Kaweah Chapter website, please send e-mail to: email@example.com


text:
50th Wedding Anniversary Question A man and his wife were celebrating their 50th wedding anniversary. The... 2/11/2013 11:42:49 AM 15,517 33
Asking 100 Girls For Sex (Social Experiment) There is a saying that goes that if you ask enough girls (in this case 100)... 7/29/2013 2:12:07 PM 23,039 40
Asking Guys For Sex (Social Experiment) If the roles were reversed and a girl asked a bunch of guys to have sex,... 7/29/2013 2:12:20 PM 23,860 46
Bus Driver Freaks Out on Passenger Don't ask this bus driver any questions unless you want to get your ass... 4/17/2013 10:14:48 AM 28,979 66
Funny Questions If you take an Oriental person and spin him around several times, does he... 9/8/2005 12:00:00 AM 59,744 1
Jay Leno Questions Paris Hilton Here is a good interview of Paris Hilton. You wont beleive what she says! 6/29/2007 12:00:00 AM 216,257 355
Jeopardy Contestant Fails at Sports Question Clearly not a sports fan. 3/30/2014 11:33:57 AM 12,070 14
Miss Utah Question and Answer Epic Fail Miss Utah choked on stage. "We need to create education better..." 6/18/2013 10:58:01 AM 18,705 22
New to our Country... It was the first day of a school in USA and a new Indian student named... 9/7/2010 12:00:00 AM 3,735 1


text:
1969 BUICK RIVIERA MAGNIFICENT CONDITION! ONLY 46,588 ACTUAL MILES! ALL ORIGINAL! JUST 2-OWNER'S FROM NEW! TEXAS CAR (DRY CLIMATE)! FULLY DOCUMENTED WITH MILEAGE LOG! ORIGINAL OWNER'S MANUAL! STUNNING COLOR COMBINATION! FACTORY AIR CONDITIONING! BUCKET... More
1939 Buick 41 Special, Always in family won in Ivory Soap jingel contest, only one known to exist out of sixty cars won. Authentication... More
1965 Buick Special -New Convertible Top, New Interior, Very Nice Paint Job, has 100,000 miles and runs very well. Real Nice Driver Car. Automatic,... More
1940 Buick 41C Phaeton Beautiful convertible in Marron finish with dual side mounts. Black leather interior (original door panels) and brand new Haartz cloth top. Recent... More
Motorcar Studio is pleased to offer this exceptionally well-preserved 1933 Buick Series 50 Model 57 Sedan. The car is nearly all original and has... More
This Beautiful Right Hand Drive Sedan is ready for a nice Sunday Cruise.... More
1938 Buick Roadmaster phaeton, original family owned car since new, chauffeur driven, low mileage, low production, only 350 made, dual cowl, always garaged since... More
This rare 1940 Buick is a frame-off, well-documented older restoration just out of long-term storage. The car is straight and solid with no known... More
This is a factory Cortez Gold GS that is extremely solid and straight. It is equipped with Power Steering, Power Brakes, Factory Air Conditioning,... More
A real collector car , a 1969 Buick GS convertible.Once own and restored by Hockey all star Selanne . Car that runs and drive... More
1976 Buick Century Station Wagon used in 2013 HBO movie "Behind the Candelabra" starring Michael Douglas and Matt Damon, Same California owner since 1976... More
1955 Buick Super 2dr Convertible 8-cyl. 322cid/236hp 4bbl. Red with red vinyl interior and white convertible top. Fresh restoration. Starts and drives good. Very... More
Big Buicks have been overlooked for too long, but this 1963 Buick Wildcat convertible is looking to change all that. Handsome and restrained looks,... More
NEW PRICE! Clean California Riv with minor upgrades to give it an awesome look. Factory A/C, great color combination, and recent paint. These cars... More
This Buick is a very well done custom built Skylark. The car has beautiful two-tone metallic paint that is carried inside the car... More


text:
Big laughs planned for Saturday in Parkers Prairie
Next weekend, the Parkers Prairie Event Center will feature four of the most popular comedians around.
Not only are these comedians - Darlene Westgor, Dwight York, Jeff Bergs and Rox Tarrant - popular in Minnesota, but also on television and in clubs across the country.
Producer and comedienne Rox Tarrant is excited to bring this high-energy comedy show featuring national-level talent to the new Event Center in Parkers Prairie.
Are you a newspaper subscriber but you don't have a Digital Access account yet? https://secure.forumcomm.com/?publisher_ID=40&event=subscriber/lookup.
You will need your subscription account number and phone number. Not sure if you have an account? Email us at firstname.lastname@example.com and we can help you.


text:
Timmons Group Announces New Associates
Richmond, VA – January 29, 2014 – Timmons Group is very pleased to announce the following new Associates of the firm.
Lu Gay Lanier has been with Timmons Group for over 10 years and directs our emerging landscape architecture practice. She has assembled a talented team of specialists and created a thriving operation. Lu Gay and her team deliver exceptional projects to their clients, both internal and external. As an environmental scientist and licensed landscape architect with a specialty in comprehensive stormwater management, she has managed award-winning projects from large conservation areas to Federal headquarters and data centers, to corporate headquarters. From site layouts, design and preservation to stormwater harvesting and xeriscaping, Lu Gay integrates economic, environmental, community involvement and social responsibility into each design project.
David Saunders has been with Timmons Group for 4 years is a licensed professional engineer in numerous states as well as a designated Design-Build professional. David is a Senior Project Manager in the utilities practice focusing on major design-build projects. His extensive experience includes the design of water and sewer force mains and gravity sewers, water and wastewater pump stations, storage tanks, and water and wastewater treatment plants. Over the last four years, he has been instrumental in winning and completing numerous municipal and private utilities projects, including several large design-build projects.
Shawn Smith has been with Timmons Group for 17 years. Shawn has helped build and grow the firm's water and wastewater utilities and residential land development practices. A licensed professional engineer with extensive expertise in the design of sewer and water line extensions, sewer force mains, hydrologic and hydraulic analyses, subdivision layout and design, mixed-use development and stormwater management, Shawn currently oversees multiple residential land development projects. One of these is the Magnolia Green project in Chesterfield County, Virginia.
Vince Doherty joined Timmons Group nearly two years ago and is the firm's first Chief Financial Officer. He is a Certified Public Accountant and received his Bachelor's and Master of Business Administration from the University of Richmond. With over 19 years of experience working for both Fortune 500 companies and launching a development stage enterprise, Vince's expertise in accounting, finance and management is an asset to the firm.
"I am excited to announce the naming of these individuals to our management team," said Brian Bortell, President and Chief Executive Officer of Timmons Group. "Their collective capabilities and dedication to client and staff collaboration will be invaluable as we continue to expand our growth and geographic reach."
About Timmons Group
Timmons Group is a multi-disciplined engineering and technology firm recognized for nearly twenty years as one of Engineering News Record's (ENR) Top 500 Design Firms in the country. The firm provides economic development, civil engineering, environmental, geotechnical engineering, GIS/geospatial technology, landscape architecture and surveying services to a diverse client base. Headquartered in Richmond, Virginia, Timmons Group has regional offices throughout Virginia and North Carolina. For more information, visit www.timmons.com.
More User Press Releases
- Timmons Group Expands in Northern Virginia
- Timmons Group Announces Upcoming Webinar on Agile Development
- Raleigh-based Mitchell Environmental Merges with Timmons Group
- Slover Memorial Library Opens in Norfolk
- Timmons Group Welcomes Tim Sheldon to Their GIS Transportation Team
- Timmons Group's Geospatial Division Grows with Addition of Mark Cave
- Timmons Group Awarded Oregon Zoo's Elephant Lands Application
- Level 1 Risk Application Monitors Forestry and Community Safety
- Upcoming Webinar: Cityworks PLL Best Practices and Lessons Learned
- Timmons Group's Landscape Architects Win Design Competition
This press release is licensed under a Creative Commons Attribution 3.0 Unported License. Read full copyright information here.


text:
Nanobody-albumin nanoparticles (NANAPs) for the delivery of a multikinase inhibitor 17864 to EGFR overexpressing tumor cells.
ABSTRACT A novel, EGFR-targeted nanomedicine has been developed in the current study. Glutaraldehyde crosslinked albumin nanoparticles with a size of approximately 100nm were loaded with the multikinase inhibitor 17864-L(x) -a platinum-bound sunitinib analogue- which couples the drug to methionine residues of albumin and is released in a reductive environment. Albumin nanoparticles were surface-coated with bifunctional polyethylenglycol 3500 (PEG) and a nanobody -the single variable domain of an antibody- (Ega1) against the epidermal growth factor receptor (EGFR). EGa1-PEG functionalized nanoparticles showed a 40-fold higher binding to EGFR-positive 14C squamous head and neck cancer cells in comparison to PEGylated nanoparticles. 17864-L(x) loaded EGa1-PEG nanoparticles were internalized by clathrin-mediated endocytosis and ultimately digested in lysosomes. The intracellular routing of EGa1 targeted nanoparticles lead to successful release of the kinase inhibitor in the cell and inhibition of proliferation whereas the non-targeted formulations had no antiproliferative effects on 14C cells. The drug loaded targeted nanoparticles were as effective as the free drug in vitro. These results demonstrate that multikinase inhibitor loaded nanoparticles are interesting nanomedicines for the treatment of EGFR-positive cancers.
Article: Interconnected hyaluronic acid derivative-based nanoparticles for anticancer drug delivery.
Ju-Hwan Park, Hyun-Jong Cho, Ubonvan Termsarasab, Jae-Young Lee, Seung-Hak Ko, Jae-Seong Shim, In-Soo Yoon, Dae-Duk Kim[Show abstract] [Hide abstract]
ABSTRACT: Doxorubicin (DOX)-loaded nanoparticles (NPs) based on interconnected hyaluronic acid-ceramide (HACE) structure were fabricated and their anti-tumor efficacy was evaluated in vitro. Interconnected HACE was synthesized by cross-linking HACE with adipic acid dihydrazide (ADH) and its synthesis was identified by (1)H NMR analysis. DOX-loaded NPs with <200nm mean diameter, negative zeta potential, and spherical shape were prepared. Interconnected HACE-based NPs increased drug-loading capacity and in vitro drug release, compared to HACE-based NPs. DOX release was dependent on the environmental pH, implying the feasibility of enhancing drug release in tumor region and endosomal compartments. Synthesized interconnected HACE did not show cytotoxic effect up to 1000μg/ml concentration in NIH3T3 and MDA-MB-231 cells. In cellular uptake studies using confocal laser scanning microscopy (CLSM) and flow cytometry in MDA-MB-231 cells, higher uptake of DOX was observed in the interconnected HACE-based NPs than HACE NPs. In vitro anti-tumor efficacy was assessed by MTS-based assay, in which cytotoxic effect of DOX-loaded interconnected HACE NPs was higher than that of DOX-loaded HACE NPs. Thus, these results suggest the feasibility of interconnected HACE-based NPs to be used for efficient tumor-targeted delivery of anticancer drugs.Colloids and surfaces B: Biointerfaces 06/2014; · 4.29 Impact Factor - SourceAvailable from: Christophe A SerraYasser Shahzad, Ikram Ullah Khan, Talib Hussain, Alamgeer, Christophe A Serra, Syed A ARizvi, Minja Gerber, Jeanetta du Plessis[Show abstract] [Hide abstract]
ABSTRACT: Proteins are posed as the natural counterpart of the synthetic polymers for the development of drug delivery systems and few of them, have been regarded safe for drug delivery purposes by the United States Food and Drug Administration (FDA). Serum albumin is the most abundant protein in human blood. Interest in the exploration of pharmaceutical applications of albumin-based drug delivery carriers, especially for the delivery of chemotherapeutic agents, has increased in recent years. Albumin has several advantages over synthetic polymers, as it is biocompatible, biodegradable, has low cytotoxicity and has an excellent binding capacity with various drugs. Micro- and nano-carriers not only protect active pharmaceutical ingredients against degradation, but also offer a prolonged release of drugs in a controlled fashion. Since existing tumour chemotherapeutic agents neither target tumour cells, nor are they specific to tumour cells, a slow release of drugs from carriers would be beneficial in targeting carcinogenic cells intracellularly. This article aims at providing an overview of pharmaceutical applications of albumin as a drug delivery carrier in tumour chemotherapy.Current Cancer Drug Targets 10/2014; 14(8):752. · 3.58 Impact Factor - SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Head-and-neck cancer is a major form of the disease worldwide. Treatment consists of surgery, radiation therapy and chemotherapy, but these have not resulted in improved survival rates over the past few decades. Versatile nanoparticles, with selective tumor targeting, are considered to have the potential to improve these poor outcomes. Application of nanoparticle-based targeted therapeutics has extended into many areas, including gene silencing, chemotherapeutic drug delivery, radiosensitization, photothermal therapy, and has shown much promise. In this review, we discuss recent advances in the field of nanoparticle-mediated targeted therapeutics for head-and-neck cancer, with an emphasis on the description of targeting points, including future perspectives.International journal of medical sciences. 12(2):187-200.
Page 1
Nanobody-albumin nanoparticles (NANAPs) for the delivery of a multikinase
inhibitor 17864 to EGFR overexpressing tumor cells
Isil Altintasa, Raimond Heukersb, Roy van der Meela, Marie Lacombec, Maryam Amidia,
Paul M.P. van Bergen en Henegouwenb, Wim E. Henninka, Raymond M. Schiffelersa,d, Robbert J. Koka,⁎
aDepartment of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands
bCell Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands
cLinxis, Breda, The Netherlands
dLaboratory Clinical Chemistry and Haematology, UMC Utrecht, Utrecht, The Netherlands
a b s t r a c t a r t i c l ei n f o
Article history:
Received 3 September 2012
Accepted 10 November 2012
Available online 16 November 2012
Drug targeting
Coordination chemistry
Kinase inhibitor
Clathrin-mediated endocytosis
Engineered antibodies
A novel, EGFR-targeted nanomedicine has been developed in the current study. Glutaraldehyde crosslinked
albumin nanoparticles with a size of approximately 100 nm were loaded with the multikinase inhibitor
17864-Lx—a platinum-bound sunitinib analogue—which couples the drug to methionine residues of albumin
and is released in a reductive environment. Albumin nanoparticles were surface-coated with bifunctional
polyethylene glycol 3500 (PEG) and a nanobody—the single variable domain of an antibody—(Ega1) against
the epidermal growth factor receptor (EGFR).
EGa1-PEG functionalized nanoparticles showed a 40-fold higher binding to EGFR-positive 14C squamous head
and neck cancer cells in comparison to PEGylated nanoparticles. 17864-Lxloaded EGa1-PEG nanoparticles
were internalized by clathrin-mediated endocytosis and ultimately digested in lysosomes. The intracellular
routing of EGa1 targeted nanoparticles leads to a successful release of the kinase inhibitor in the cell and inhibi-
tion of proliferation whereas the non-targeted formulations had no antiproliferative effects on 14C cells. The
drug loaded targeted nanoparticles were as effective as the free drug in vitro. These results demonstrate that
multikinase inhibitor loaded nanoparticles are interesting nanomedicines for the treatment of EGFR-positive
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
One of the improvements of nanomedicine formulations is to in-
crease the therapeutic index of highly potent drugs that often show
severe side effects. Targeted delivery of these drugs to tumor tissues
can increase antitumor efficacy and reduce toxicity by avoidance of
normal tissues. Well known examples of nanoparticulate carriers
are liposomes [1,2], polymeric nanoparticles [3] and micelles [4]. Re-
cently, albumin nanoparticles have gained attention [5–10] especially
after the success of albumin-based formulations such as Abraxane®
[11]. These formulations take advantage of the properties of albumin,
which is nature's own carrier for binding and transporting drugs
within the body [12]. In recent years, albumin nanoparticles have
been further functionalized by coupling tumor-directed monoclonal
antibodies (i.e., trastuzumab, cetuximab, antiCD3) [13–19] and
other targeting ligands (i.e. folate, RGD, apolipoproteins) [20–25]
onto their surface in order to increase their uptake by tumor cells.
In the present study, a novel type of decorated nanobody-albumin
are functionalized with anti-EGFR nanobody (EGa1) attached to the dis-
tal end of surface coated PEG chains (Fig. 1). Nanobodies are low molec-
ular weight proteins (~15 kDa) that are derived from heavy chain-only
antibodies as found in camels, llamas and dromedaries [26]. The antigen
recognition domain of this type of antibodies is expressed in a single
chain, in contrast to conventional antibodies which encode the antigen
recognition loops in two separate heavy and light chains. Consequently,
the recombinant production of nanobodies is easier than that of conven-
such as high affinity to the target and the absence of an Fc domain
[27–29]. Particularly in the case of antibody coupled nanoparticles, the
presence of an Fc domain could lead to the recognition of the
nanoparticles by the immune system, resulting in their fast clearance
from the blood circulation [30]. Furthermore, compared to monoclonal
antibodies, the single domain format of nanobodies facilitates easy selec-
tion from phage libraries [31]. As a result, nanobodies have been rapidly
developed for many applications, i.e., imaging, cancer immunotherapy,
blocking blood clotting and resins for immuno-affinity purification [32].
In the present study, we explored the loading of a kinase inhibitor
to albumin nanoparticles via chemical conjugation to the albumin
backbone. The multikinase inhibitor 17864 used in this study targets
several growth-factor signaling pathways and is structurally closely
Journal of Controlled Release 165 (2013) 110–118
⁎ Corresponding author. Tel.: +31 620275995; fax: +31 30 251789.
E-mail address: firstname.lastname@example.com (R.J. Kok).
0168-3659/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
Contents lists available at SciVerse ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Page 2
related to sunitinib [33]. Multikinase inhibitors inhibit the activity of
several receptor tyrosine kinases by competitive inhibition with
ATP, and thus are able to intervene in proliferation and survival of
tumor cells and angiogenesis. Although the activity versus multiple
molecular targets is attractive from efficacy point-of-view, this is
also associated with lack of selectivity and hence greater risk for
side effects [34]. Therefore, to enhance the accumulation of 17864
in tumor cells and prevent the exposure of healthy cells to the inhib-
itor, in the present study we developed a targeted nanomedicine that
can guide the multikinase inhibitor to tumor cells.
17864 can be coupled to albumin nanoparticles by the use of a
platinum based linker that coordinates the drug at its pyridyl moiety
and albumin at methionine and cysteine residues. As such, the pres-
ently developed NANAPs that can accumulate in EGFR-expressing
tumor cells and upon intracellular degradation will release the con-
jugated multikinase inhibitor into the reductive environment of the
cytosol, where the 17864 is released from the platinum linker [35].
Both 17864 and the 17864-Lx adducts inhibit several important
tumor associated pathways like PDGFR and c-Kit [33]. The present
study explores the potential of 17864-albumin nanoparticles as
EGFR-directed targeted nanomedicine. The enzymatic degradability of
the developed nanoparticles and cellular uptake and intracellular
routing, as well as antiproliferative activity on EGFR positive human
head and neck squamous cell carcinoma (HNSCC) UM-SCC-14C (14C)
are studied.
2. Materials and methods
2.1. Chemicals
Albumin,human fraction V powder and glutaraldehyde, 8% aqueous
solution, grade I, were purchased from Sigma Aldrich. NHS-PEG3500-
maleimide was obtained from Jenkem Technology (Beijing, China).
Sunitinib-malate was purchased from LC Laboratories, Germany.
17864 was obtained from Vichem chemie (Budapest, Hungary) and
linked to Lxas described previously [33]. Rhodamine-Lxwas obtained
from Kreatech (Amsterdam, The Netherlands). Trypsin (5 mg/ml)
from porcine supplemented with EDTA and fetal calf serum was
obtained from PAA, GE Healthcare.
2.2. Preparation of nanoparticles
according to Langer et al., with some modifications [36]. Briefly,
50 mg of HSA was dissolved in 1 ml deionized water, pH was adjust-
ed to 8.3 by addition of 0.1 M NaOH and the solution was filtered
through a 0.2 μm filter. Nanoparticles were formed by adding 4 ml
of 92% ethanol to 1 ml of the HSA solution drop wise under constant
stirring at 450 rpm. Immediately after ethanol addition, the formed
particles were crosslinked with 8% glutaraldehyde (GA) and reacted
overnight at room temperature (RT) under stirring. 18, 29 or 59 μl
8% glutaraldehyde (corresponding with 18:1, 30:1, 60:1 GA:HSA
mol/mol ratios) was added to obtain low, medium and high
crosslinked nanoparticles, respectively. The nanoparticles were
purified by two-step centrifugation, once at 1000 g for 5 min and
once at 35,000 g three times for 1 h at 4 °C in deionized water.
After centrifugation, the nanoparticles were sonicated for 4 min and
stored in deionized water at 4 °C. The yield of nanoparticle prepara-
tions was calculated from the dry weights of the nanoparticle suspen-
sions after evaporation of the carrier solvent from 100 μl aliquots in a
thermographic analyzer (TGA Q50™ instrument, USA). Aliquots of
the suspensions corresponding to 6 μg albumin were loaded onto an
SDS gel (4–12% crosslinked, Invitrogen, Breda, Netherlands) under
reducing conditions to check for the possible presence of albumin
and soluble multimers by Coomassie blue staining.
werepreparedbya desolvationtechnique
2.3. Loading of kinase inhibitors into albumin nanoparticles
Loading of sunitinib into albumin nanoparticles with different
cross-link densities is described in the supplemental information.
Coupling of 17864-Lxwas performed according to Harmsen et al.
[33] with minor modifications. In brief, 12 mg of low crosslinked
nanoparticles (0.07 mg/ml in water) was incubated with 1.2 mg of
17864-Lx (10:1 mol/mol, 17864-Lx:HSA) in 20 mM tricine/NaNO3
17864 (entrapped in the core)
anti-EGFR nanobody
Fig. 1. Schematic representation of targeted nanobody-albumin nanoparticles (NANAPs) which consist of a glutaraldehyde crosslinked albumin core that has been surface-modified
with EGa1-PEG tethers. The multikinase inhibitor 17864 has been loaded into the core of the nanoparticles and is retained into the particles via Lx-based platinum coordinative
linkage. 17864 is an analogue of sunitinib.
I. Altintas et al. / Journal of Controlled Release 165 (2013) 110–118
Page 3
pH 8.5 buffer overnight at 37 °C. To prevent flocculation, SDS
was added (final concentration 0.008% (v/v)). 17864-Lx loaded
nanoparticles were purified by centrifugation and stored as described
above. The 17864 content of the nanoparticles was determined after
cleavage of the 17864-Lxcoordinative bond by an overnight incuba-
tion with 0.5 M KSCN at 80 °C, followed by extraction with ACN
(2:1, v/v) [37]. Released 17864 was determined with ultra-high per-
formance liquid chromatography (Acquity UPLC®) on an Acquity
UPLC® BEH C18 1.7 μm, 2.1×50 mm column (Waters, Ireland).
17864 was eluted with a water/ACN/TFA gradient starting from
100% eluent A (95% H2O, 5% ACN, 0.1% TFA) and going up to 100%
eluent B (5% H2O, 95% ACN, 0.1% TFA) in 7 min at a flow rate of
1 ml/min. 17864 was detected at 425 nm and eluted at 2.2 min.
17864-Lxwas injected as a reference and eluted at 2.7 min.
2.4. Preparation of labeled nanoparticles for imaging
For cellular uptake and intracellular routing, three types of fluo-
rescently labeled nanoparticles were prepared. Rhodamine was
linked to the nanoparticles via the same linkage approach as 17864,
usinga rhodamine-Lxadduct(Rho-Lx). Indetail, 12 mgof nanoparticles
(3 mg/ml) was labeled with 144 μg of Rho-Lx(from a 4 mg/ml water
stock), (1:1, mol/mol, Rho-Lx:HSA). Alexa-488 labeled nanoparticles
were prepared by reacting 10 mg of nanoparticles (3 mg/ml) with
33.9 nmol of Alexa488-NHS (Invitrogen, 0.2:1 mol/mol, Alexa488-NHS:
HSA) in sodium bicarbonate pH 8.4 for 2 h at RT. For lysosomal degrada-
tion studies, nanoparticles were labeled with the near-infrared dye
IRDye800(Westburg, Leusden, The Netherlands). 18 mg of nanoparticles
(3 mg/ml) was labeled with 85.5 nmol of IRDye800CW NHS
(0.3:1 mol/mol, IRDye800:HSA) in 0.1 M phosphate buffer (PB),
pH 8 for 2 h at RT. Labeled nanoparticles were purified as described
in Section 2.2 and resuspended in 0.1 M PB pH 7.4 prior to use.
2.5. Preparation of NANAPs
Production of the anti-EGFR-1 nanobody (EGa1) was done as de-
scribed by Roovers et al., with minor modifications [31]. Briefly,
Escherichia coli BL21-CodonPlus(DE3)-RIL cells (Agilent Technologies,
Inc., Santa Clara, CA, USA) transformed with pET28-nanobody
plasmid were grown overnight at 37 °C in 2× TY medium containing
2% glucose and supplemented with 100 μg/ml kanamycin and chlor-
amphenicol. Next, 400 ml of 2× TY (tryptone/yeast extract) medium
containing 0.2% glucose and 100 μg/ml kanamycin and chloramphen-
icol was inoculated with bacteria from an overnight culture with
an OD600 of 0.1. This bacterial culture was incubated at 37 °C at
250 rpm until it reached an OD600 of 0.6. Subsequently, 0.5 mM
IPTG was added to the bacterial culture to induce EGa1 expression
and the culture was further incubated at 25 °C for 6 h. Cells were
spun down at 4500 rpm and at 4 °C for 15 min and the pellet was
resuspended in 6.4 ml (per 400 ml culture) ice cold TES (200 mM
Tris-HCl, 0.5 mM EDTA, 500 mM sucrose, pH 8). To this suspension
10 ml of diluted ice cold TES (1:3 in water) was added and incubated
on ice for 30 min. The bacteria were spun down at 4 °C for 15 min at
4500 rpm and the supernatant was collected. The pellet was
resuspended in 10 ml ice cold TES, 120 μl 1 M magnesium sulfate
was added and the mixture was incubated on ice for 30 min. The
suspension was spun down at 4 °C for 15 min at 4500 rpm and the
supernatant was added to the previously collected supernatant.
Purification was performed on an ÄKTA purifier 10 (GE 7 Healthcare
Europe GmbH, Munich, Germany) using a HisTrap™ Column (GE
Healthcare) according to the manufacturer's protocol. Nanobodies
were eluted with an imidazole gradient (10–500 mM in 50 mM sodi-
um phosphate, 300 mM NaCl, pH 8 buffer) and the collected fractions
were dialyzed overnight against PBS pH 7.4 and stored at 4 °C.
This procedure yielded 10 mg EGa1/1L bacterial culture, as was
determined by a Micro BCA protein assay (Pierce Biotechnology,
Rockford, IL, USA).
EGa1 nanobody (18.3 kDa) was modified with an 8-fold molar ex-
cess of N-succinimidyl-S-acetylthioacetate (SATA) (Pierce Biotech-
nology, Rockford, IL, USA) as described in [38]. The introduced
sulfhydryl groups were deacetylated for 30 min at RT by a deprotection
buffer (0.5 M Hepes, 0.5 M hydroxylamine-HCl, 0.25 mM EDTA, pH 7)
prior to addition of SATA-EGa1 to freshly prepared maleimidiyl-PEG
nanoparticles. 12 mg of drug-loaded or empty nanoparticles
(3 mg/ml) was reacted with 6.3 mg of NHS-PEG3500-maleimide
(10:1, mol/mol, PEG:HSA) in 0.1 M phosphate buffer, pH 8 for 1 h
at RT on a roller bench. PEGylated nanoparticles were isolated by
centrifugation and resuspended in 0.1 M PB, pH 7 and immediately
reacted with SATA-modified EGa1 (46 μg EGa1/mg nanoparticle,
0.2 nmol EGa1/nmol albumin) for 2 h at RT. The depletion of the
free EGa1 in the coupling reaction was confirmed by loading an ali-
quot of the reaction mixture to a gel after 2 h. Control batches of
PEGylated nanoparticles not-bearing EGa1 were prepared by overnight
quenching of maleimidyl groups in 0.1 M sodium carbonate buffer,
pH 9. Final products were centrifuged, washed, and resuspended in
0.1 MPB,pH 7priortouse.CouplingofEGa1tonanoparticleswasstud-
ied by dot-blot immunodetection of EGa1 by spotting 2 μg of NANAPs
onto a nitrocellulose membrane, followed by anti-EGa1 western blot-
ting. The membrane was blocked with 5% BSA in Tris-buffered saline
with 0.1% Tween-20, pH 7.5 (TBS-T) at 4 °C overnight and incubated
with rabbit polyclonal anti-VHH serum (antiserum raised to llama
heavy chain only variable domain, diluted 1:5000 in 5% BSA in TBS-T)
for 2 h and subsequently with goat anti-rabbit peroxidase-conjugated
secondary antibody (1:1000 dilution in 5% BSA TBS-T, Thermo Fisher
Scientific, Rockford, IL, USA) for 1 h at RT. The signal was detected by
using the SuperSignal West Femto Maximum Sensitivity Substrate
(Thermo Fisher Scientific, Perbio Science Nerderland B.V) and chemilu-
tories, Inc, USA).
2.6. Characterization of NANAPs
Size distribution and polydispersity of the NANAPs were deter-
mined by dynamic light scattering (DLS) on a Malvern ALV CGS-3
(Malvern Instruments, Malvern, UK) containing a He\Ne laser source
(λ=632.8 nm, 22 mW output power) under an angle of 90°. The zeta
potential of the nanoparticles was determined using a Malvern
zetasizer Nano-Z (Malvern Instruments, Malvern, UK). The measure-
ments were performed in 5 mM phosphate buffer, pH 7.4 at 25 °C.
The morphology and size of the nanoparticles were visualized by
transmission electron microscopy (TEM). Briefly, samples (0.1 mg/ml
in water) were soaked onto carbon-coated copper grids for 2 min and
excess liquid was removed by a filter paper. The grids were negatively
stained with 2% uranyl acetate (Merck) for 45 s and dried for 10 min
at RT before acquisition of TEM images (Tecnai 10, Philips, The
2.7. Release of 17864 from NANAPs
Release studies with 17864-loaded NANAPs (0.7 mg/ml) were
conducted in PBS, 5 mM DTT in PBS and PBS supplemented with
10% fetal calf serum. At indicated time points samples were collected
and diluted with 2 volumes of ACN to extract the released drug from
the nanoparticles. Samples were spun down and the supernatants
were analyzed by UPLC as described in Section 2.3. Measurements
were performed in triplicate.
2.8. Cell experiments
Cell experiments were performed with the EGFR-positive head and
neck squamous cell carcinoma cell line UM-SCC-14C (abbreviated as
I. Altintas et al. / Journal of Controlled Release 165 (2013) 110–118
Page 4
tured in Dulbecco's Modified Eagle's Medium (DMEM, PAA, Pasching,
Austria) containing 3.7 g/l sodium bicarbonate, 4.5 g/l L-glucose,
2 mM L-glutamine and supplemented with 5% (v/v) fetal calf serum,
penicillin (100 IU/ml), streptomycin (100 μg/ml), and amphotericin B
(0.25 μg/ml) at 37 °C in a humidified atmosphere containing 5% CO2.
The tests for mycoplasma infection were performed every 3 months
and the cells were found to be mycoplasma-free.
2.9. Binding and intracellular routing of NANAPs
Cellular binding of NANAPs was studied using Rho-Lx labeled
nanoparticles. 14C cells were trypsinized and seeded at 50,000 cells/
well in round bottom 96-well plates (Becton & Dickinson, Mountain
View, CA, USA) on ice to prevent adherence. Next, the cells were incu-
bated at 4 °C with Rho-Lxlabeled nanoparticles at concentrations
ranging from 0.0625 to 2 mg/ml in culture medium. Incubations
were performed for 1 h in culture medium in the dark after which
the cells were washed three times with 0.3% BSA in PBS and fixed
with 10% formalin. The mean fluorescence intensity was determined
with a BD FACSCanto II and analyzed with BD FACSDiva™ software
(Becton & Dickinson, USA). Each experiment was performed in tripli-
cate. To demonstrate the involvement of EGa1/EGFR-1 in the interac-
tion of nanoparticles with the cells, incubations were performed in
the presence of an excess of free (100 μg) EGa1 nanobody (corre-
sponding to 22-fold molar excess for 1 mg/ml Ega1-PEG-NP).
Internalization of NANAPs was studied with Alexa-488 labeled
nanoparticles. 14C cells were seeded at 50,000 cells/well in 12 well
plates and adhered overnight at 37 °C. 14C cells were pre-incubated
with 2% of either 1-butanol or 2-propanol in serum free culture medi-
um for 1 min. Subsequently, Alexa-488-conjugated nanoparticles
(0.5 mg/ml) were allowed to internalize for 30 min in complete cul-
ture medium containing 2% of either 1-butanol or 2-propanol at
37 °C. After incubation, cells were put on ice and washed with ice-cold
PBS to stop the internalization. Surface bound nanoparticles were re-
moved by an acid wash (250 mM NaCl, 100 mM glycine, pH 2.5),
performed two times for 10 min on ice followed by fixation of cells
with 4% PFA (paraformaldehyde). Formalin-induced auto-fluorescence
was quenched with 100 mM glycine in PBS for 15 min. Nuclei were
stained with DAPI (Roche), 1 μg/ml in PBS for 5 min at RT. Images
were taken with a wide field Olympus AX70 microscope, attached to a
CCD camera (Nikon DXM1200) using a 60× objective oil immersion
(NA 1.25/PlanFl).
Lysosomal colocalization of NANAPs was studied with Alexa-488
labeled nanoparticles. 14C cells were seeded at 50,000 cells/well in
12 well plates containing uncoated 12 mm glass cover slips and
were allowed to adhere overnight. Cells were incubated with
Alexa-488 conjugated nanoparticle formulations (0.5 mg/ml) in cul-
ture medium for 2 h at 37 °C. As a positive control for lysosomal up-
take, Alexa-488 conjugated EGF (Invitrogen, 1 ng/ml) was allowed to
internalize for 15 min and was chased for an additional 45 min at
37 °C. Late endosomes and lysosomes were stained by adding
Lysotracker-Red DND-99 (Invitrogen, 65 nM in culture medium
containing 2% Marvel) 90 min before fixation. Subsequently, cells
were washed with PBS and fixed with 4% PFA for 30 min on ice and
autofluorescence was quenched with 100 mM glycine for 15 min.
Nuclei were stained with DAPI. Images were taken with a Zeiss
Axiovert 200M confocal microscope equipped with a 63× oil immer-
sion objective (NA 1.4).
Lysosomal degradation of NANAPs was studied with IRdye800la-
beled nanoparticles. After cellular internalization and digestion of
these NPs, the IRDye800metabolites will not diffuse out of the cells
due to the hydrophilic and charged nature of this label [39]. 14C
cells were seeded at 300,000 cells/well in 12-well plates (Becton &
Dickinson, Mountain View, CA, USA) and were allowed to adhere
overnight. Cells were pulsed with 0.5 mg/ml IRdye800labeled NPs,
PEG-NPs and EGa1-PEG-NPs for 2 h at 37 °C. The medium was re-
placed by fresh incubation medium supplemented with or without
10 μM of chloroquine (CQ) and cells were incubated for an additional
48 h at 37 °C. Cells were washed with PBS and lysed in 40 μl of re-
ducing sample buffer (50 mM Tris-HCl,
100 mM DTT, 2% SDS and0.01%bromophenolblue).Highandlowmo-
lecular weight IRDye800
peptidic fragments were separated by
SDS-PAGE and quantified using an Odyssey Infrared Imager ® (LI-COR
pH 6.8, 10% glycerol,
2.10. Inhibition of tumor cell proliferation
Antiproliferative effects of 17864 loaded NANAPs were deter-
mined by a BrdU assay performed according to the supplier's instruc-
tion (Roche Diagnostics, Mannheim, Germany). 14C cells were seeded
(4000 cells/well) in 96-well plates and allowed to adhere overnight.
Cells were incubated with nanoparticles diluted in culture medium
to concentrations ranging from 25 to 800 μg/ml (albumin concentra-
tion), corresponding to 3–100 μM conjugated 17864. In addition, cells
wereincubatedwithsolutionsof2.5–60 μMoffree17864and17864-Lx
for comparison withthe nanoparticle formulations. After incubationfor
48 h, the medium was replaced with BrdU (10 μM) containing com-
plete medium and cells were incubated overnight at 37 °C. BrdU incor-
poration into the DNA was determined according to the supplier's
protocol and measured on a SPECTROstarNano(BMG Labtech, Germany)
at 450 nm with a reference wavelength of 690 nm. Cell proliferation is
expressed relative to control cells not treated with nanoparticles.
3. Results and discussion
3.1. Preparation of albumin nanoparticles
The nanoparticles were prepared by ethanol desolvation of albu-
min followed by crosslinking with GA [17,18,36]. As shown in
Table 1, a subsequent two-step centrifugation yielded well-defined
100 nm nanoparticles with a narrow size-distribution. At pH 7.4 the
nanoparticles have a negative zeta-potential (as expected; the pI of
the native albumin, i.e. 4.5 [40]). The zeta-potentials became more
negative with increasing GA crosslinking, which is in agreement
with the depletion of primary amino groups upon increased GA
crosslinking. The yield of the nanoparticles can be increased by in-
creasing the ethanol volume fraction, however this also leads to the
unwanted formation of bigger aggregates.
3.2. Loading of multikinase inhibitors and preparation of NANAPs
Nanoparticles loaded with the clinically approved multikinase in-
hibitor sunitinib were prepared by either pre-loading or post-loading
of the drug into albumin nanoparticles (see supplemental informa-
tion). In vitro release studies, however, demonstrated a very rapid re-
lease of sunitinib from the nanoparticles, independent of the crosslink
density, in the presence of 1% or 4% albumin in the release buffer
(100% of the loaded amount was released in less than 30 min most
likely due to interaction with soluble albumin; data not shown).
Therefore, the studies were continued with the 17864-Lxcompound
which was conjugated to the albumin nanoparticles via a coordinative
Table 1
Characteristics of empty albumin nanoparticles.
Crosslink density Low MediumHigh
Size (nm)
Zeta potential (mV)
Yield (%)
PDI: polydispersity index (n=3).
I. Altintas et al. / Journal of Controlled Release 165 (2013) 110–118
Page 5
linkage [35]. Only low cross-linked nanoparticles were used for the fol-
lowing studies, since a better degradability and PEGylation efficiency
were anticipated, as compared to higher crosslinked nanoparticles.
Upon coupling of 17864-Lx, size, zeta potential and PDI of drug loaded
nanoparticles were similar as those of unloaded nanoparticles (see
Tables 1 and 2).
17864-loaded nanoparticles were surface modified with the bifunc-
tional NHS-PEG3500-maleimide polymer and finally with SATA-EGa1
to yield NANAPs. PEGylation shielded the surface of the nanoparticles,
as intended, since surface charge was slightly decreased from −26 to
−21 mV after PEGylation (Table 2). Importantly, the coupling of EGa1
to NANAPs was confirmed by a nanobody dot-blot analysis (Fig. 2A).
There was no free nanobody detectable 2 h after the coupling reaction
with EGa1 observed by running an aliquot of the reaction mixture on
a gel (results not shown). The final drug loaded NANAPs had a size of
138 nm, a narrow size distribution (PDI=0.13) and slightly negative
zeta potential of −21 mV. The size and morphology of the NANAPs
were visualized by TEM (Fig. 2B), confirming their spherical shape.
The observed size of the NANAPs (~100 nm) was in agreement with
the DLS measurements.
3.3. 17864 release and degradation of NANAPs
The 17864-Lx kinase inhibitor was conjugated to the albumin
nanoparticles via a Pt(II) coordinative bond at sulfur containing side
chain residues as methionine or cysteine. Previous studies with a
drug-Lxconjugate have shown that the Lxlinker is stable in plasma
or serum [41], but that drug can be released via competitive displace-
ment with thiol containing molecules [35]. As shown in Fig. 3, less
than 10% of the bound 17864 was released within 48 h in PBS or
serum supplemented PBS. In the presence of DTT, however, the re-
lease of 17864 increased to 55% in 48 h. Of note, only the free
17864 was detected and not the 17864-Lxdrug-platinum adduct.
These results are in agreement with previous data obtained from Lx
release [35]. Total release of the drug was obtained by competitive
displacement with an excess of a platinum ligand at higher tempera-
tures (see Section 2.3).
One of the advantages of albumin nanoparticles over polymeric
particles is their susceptibility of the core material to enzymatic di-
gestion. Modification of the albumin by cross-linking and attachment
of drug and PEG may however hamper its biodegradability. The influ-
ence of extent of crosslinking on degradability was studied by using
trypsin as a model enzyme. As expected and also observed by Langer
et al. [42] an increased crosslink density caused a decrease in degra-
dation rate (supplemental Fig. 1). In addition, PEGylation and drug
coupling both reduced the degradation rate of NPs (see supplemental
Fig. 2A). However, degradation was still observed for the three types
of nanoparticles of different crosslink density as was also confirmed
by SDS-PAGE analysis (supplemental Fig. 2B) of the tryptic digests
obtained after 6 h. Intact nanoparticles (lanes 1, 3, 5) were not able
to enter the polyacrylamide gel and were retained in the slots at the
top part of the gel. All digested samples (lanes 2, 4, 6) showed small
peptidic fragments, indicating that nanoparticles had been degraded
to fragments smaller than 10 kDa.
3.4. Binding and intracellular routing of NANAPs
The binding of NANAPs by EGFR expressing 14C tumor cells was in-
vestigated using Rho-Lxlabeled nanoparticles. Fig. 4A shows that after
incubation for 1 h at 4 °C, NPs bound non-specifically to 14C cells,
Table 2
Properties of nanoparticles after drug loading and surface functionalization.
Samples EE%LC%LR (drug:
PDIZeta pot.
72±54.0±0.37.2±0.5:1 100±8 0.05±0.02
EE: encapsulation efficiency, LC: loading capacity, LR: loading ratio (mol/mol), PDI:
polydispersity index, Zeta pot: zeta potential.
Fig. 2. A) Dot blot analysis of free EGa1, nanoparticles (NP), PEGylated nanoparticles (PEG-NP), EGa1 coupled PEGylated nanoparticles (EGa1-PEG-NP) and B) Transmission electron
microscopy image of 17864 loaded EGa1-PEG-NP. Size bar of image B is 100 nm.
Fig. 3. The release of 17864 from NANAPs in PBS, PBS with 10% serum and 5 mM DTT at
pH 7.4, 37 °C.
I. Altintas et al. / Journal of Controlled Release 165 (2013) 110–118
Page 6
however after PEGylation this non-specific binding was completely
prevented. Importantly, when the PEG-NPs were functionalized with
the targeting ligand EGa1, the binding increased 5-fold in comparison
to bare NPs. Moreover, at the highest dose EGa1-PEG-NPs bound over
40-fold more efficiently than PEGylated NPs without EGa1, clearly
showing the potential of EGFR directed targeting for this type of
head-and-neck carcinoma. To further demonstrate the interaction of
EGa1-decorated nanoparticles with the target EGFR receptor, the bind-
ing of these nanoparticles was studied after preincubation of the cells
with an excess of free EGa1. Fig. 4B shows that binding of the EGa1
nanoparticles was indeed strongly inhibited by free nanobody, demon-
strating the specificity of the interaction.
To study the uptake as well as the pathway of endocytosis and lyso-
somal colocalizations of the nanoparticles, 14C cells were incubated
with Alexa-488 labeled nanoparticles at 37 °C. To allow visualization
of internalized nanoparticles only, i.e. without interference of surface
located nanoparticles, the cells were stripped by a mild acid-wash pro-
cedure after the incubations. Activated EGF receptors are internalized
into cells by both clathrin-mediated (CME) (at physiological plasma
concentration of EGF; 1 ng/ml) and non-clathrin-mediated endocytosis
(high EGF concentration) [43]. We investigated which of these traf-
ficking routes is involved in the uptake of the EGFR targeted
nanoparticles by studying their
1-butanol. Phospholipids play an important role in CME, especially
during the formation of the clathrin coated vesicles that bud off
from the cell membrane. While the vesicles are forming, adaptor
proteins involved in clathrin-mediated endocytosis interact with
the phospholipid, phosphoinositide-4,5-biphosphate (PIP2) in the
membrane [44]. 1-Butanol, inhibits the formation of PIP2, resulting
in the removal of PIP2 from the membrane, consequently blocking
CME [45]. EGa1-PEG-NPs were internalized in the presence of
2-propanol (which does not interfere with CME) as shown in
Fig. 5A. In contrast, EGa1-PEG-NPs were not internalized in the presence
EGa1 were internalized via clathrin-independent mechanisms, as addi-
To investigate whether the internalized NPs were transported to
lysosomes, co-localization of Alexa-488 nanoparticles was studied
with lysotracker-red. EGF was used as a positive control for lysosomal
uptake in the presence of
Fig. 4. Fluorescence activated cell sorting analysis of binding of Rho-Lxlabeled
A) EGa1-PEG-NP, PEG-NP, NP and B) EGa1-PEG-NP with and without free EGa1
(100 μg) at 4 °C for 1 h on 14C cells. MFI (mean fluorescence intensity).
Inhibition of CME
EGa1 - PEG NP
Fig. 5. A) Internalization of nanoparticle formulations by 14C cells pre-incubated with either 2-propanol or B) 1-butanol for 30 min at 37 °C. Nanoparticles were labeled with Alexa-488
(green) and the nucleus was stained with DAPI (blue). Arrow 1a: EGa1-PEG-NPs are taken up. Arrow 2a: NPs are taken up. Arrow 1b: EGa1-PEG-NP uptake is clathrin-dependent. Arrow
2b: NP uptake is clathrin-independent.
I. Altintas et al. / Journal of Controlled Release 165 (2013) 110–118
Page 7
targeting. Both EGF and EGa1-PEG-NPs accumulated in the lysosomal
compartment, as demonstrated by co-localization of the green la-
beled nanoparticle with the red lysotracker, resulting in yellow
perinuclear staining (Fig. 6). In contrast, NPs were internalized via a
different type of endocytic vesicles and did not co-localize with
3.5. Lysosomal uptake and degradation of NANAPs
Lysosomal degradation of the particles after their uptake by 14C
cells was studied with IRDye800labeled nanoparticles. IRDye800re-
mains entrapped inside the cells due to the hydrophilic and charged
nature of this label [39]. 14C cells that were incubated with
nanoparticles for 24 h were lysed and the cell lysates were loaded
onto a gel. SDS-PAGE analysis showed the intracellular degradation
of the internalized EGa1-PEG-NPs, PEG-NPs and NPs into small frag-
ments (Fig. 7A). PEG-NPs showed a low extent of internalization
resulting in only minor degradation products. The degradation of
NPs was 5 times less than the EGa1-PEG-NPs (Fig. 7B). To demon-
strate that this degradation occurs in lysosomes, chloroquine was
added which impairs endocytosis, exocytosis and lysosomal degrada-
tion.Asa weakbaseitcanenterlowpHendosomalandlysosomalcom-
partments. Its accumulation results in an increase in pH and osmotic
pressure thereby inhibiting the lysosomal degradation and endosomal
trafficking inside the cells [46]. EGa1-PEG-NPs and NPs that were local-
ized in lysosomes were affected from CQ treatment similarly. As such,
the inhibition of lysosomal activity resulted in a 10-fold decrease in
degradation in the presence of CQ (Fig. 7B).
3.6. Inhibition of tumor cell proliferation
17864 inhibits multiple tyrosine kinases involved in tumor cell pro-
liferation. The anti-proliferation activity of 17864-loaded nanoparticles
was studied using a BrdU proliferation assay, after 48 h of incubation
with 14C cells. NANAPs efficiently inhibited tumor cell proliferation,
with an IC50of 40 μM (Fig. 8A), which is 2.8 times higher than the
IC50of free 17864 and 17864-Lx(Fig. 8B, 14 μM for both compounds).
As discussed in previous sections, EGa1-decorated nanoparticles enter
cells via CME and require lysosomal processing before 17864 can be re-
leased. In contrast, small molecule inhibitors diffuse across the cellular
membranes without prior activation steps, which often render small
molecules more effective than their nanomedicine counterpart in ex-
periments with in vitro cultured cells [47]. The in vitro experiments in
accumulation and activation of albumin nanoparticles. Non-targeted
PEGylated nanoparticles with 17864 did not inhibit proliferation of
14C tumor cells, which can be ascribed to the very low cellular uptake
of these particles. But even bare nanoparticles loaded with 17864,
which are internalized and degraded to some extent, were not capable
of reaching the IC50at the tested concentration range (Fig. 8C). EGa1
nanobody in itself is a blocker of EGFR-1 phosphorylation and may
therefore inhibit proliferation of EGFR-positive 14C cells. Previous
studies with EGa1-decorated liposomes have shown that such
nanoparticles, due to multimerization of the nanobody, are even stron-
ger inhibitors of EGFR phosphorylation than free nanobody [38]. How-
ever, the inhibitory effect of multivalent EGa1-liposomes is not
sufficient to completely prevent tumor cell proliferation and only
resulted in significant growth inhibition of 14C cells when the lipo-
somes were loaded with an antiproliferative drug [48]. Our results are
in line with such observations, as we only see profound inhibitory
merged image
merged image
merged image
merged image
Fig. 6. Co-localization of EGF, NPs, PEG-NPs and EGa1-PEG-NPs (green) with
Lysotracker Red (lysosome marker) on 14C cells after 24 h. Nuclei were stained with
DAPI (blue). Boxed areas were enlarged.
I. Altintas et al. / Journal of Controlled Release 165 (2013) 110–118
Page 8
effects of the combined coupling of EGa1 and 17864 to albumin
nanoparticles (Fig 8D).
4. Conclusion
with the multikinase inhibitor 17864 conjugated to the albumin back-
bone via a platinum based linker (Lx). PEGylation of nanoparticles suc-
cessfully inhibited the aspecific binding of the nanoparticles to cells.
Further functionalization with EGa1 resulted in nanoparticles that
were internalized in an EGFR-dependent manner. The pathway of
uptake and compartmentalization in the lysosomes was crucial for the
intracellular release of 17864 from Lx. The tunable characteristics of
the NANAPs make them promising new materials favorable for in vivo
use as targeted nanomedicines in the treatment EGFR positive cancers.
We thank Öznur Akbal from Hacettepe University for her assistance
in the experiments and Vichem Chemie for the production of 17864.
This project is supported by the Utrecht University Focus and Massa
program Nanobullets: The next generation in drug targeting.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://
log(conc 17864)
log(conc 17864)
-6.0 -5.5-5.0-4.5 -4.0
log(conc 17864)
% BrdU incorporation
% BrdU incorporation
% BrdU incorporation
0.0 0.20.4 0.60.8
% BrdU incorporation
mg/ml NP
Fig. 8. Inhibition of 14C cell proliferation with different types of NPs and drug treatments
after 48 h incubation at 37 °C. A) Incubation with 17864-Lxloaded EGa1-PEG-NPs (0.025–
0.8 mg/ml NP; equivalent to 3–100 μM 17864); B) Incubation with free 17864 and
17864-Lx;(equivalent to 1–40 μM 17864); C) Incubation with 17864-Lxloaded PEG-NP
and NPs (0.025–0.8 mg/ml NP; equivalent to 3–100 μM 17864); and D) Incubation with
EGa1-PEG-NPs not loaded with 17864-Lx(0.025–0.8 mg/ml NP).
Free dye
(degraded NP)
Fig. 7. Quantification of lysosomal degradation of NANAPS with and without chloro-
quine treatment. Cells were lysed and the lysates were loaded on an SDS-PAGE gel to
visualize the IRdye800after 24 h incubation with 14C cells at 37 °C. Band intensities
of degraded nanoparticles were quantified (n=3).
I. Altintas et al. / Journal of Controlled Release 165 (2013) 110–118
Page 9
[1] A. Abu Lila, T. Ishida, H. Kiwada, Targeting anticancer drugs to tumor vasculature
using cationic liposomes, Pharm. Res. 27 (2010) 1171–1183.
[2] F. Yang, C. Jin, Y. Jiang, J. Li, Y. Di, Q. Ni, D. Fu, Liposome based delivery systems in
pancreatic cancer treatment: from bench to bedside, Cancer Treat. Rev. 37 (2011)
[3] S. Parveen, S.K. Sahoo, Polymeric nanoparticles for cancer therapy, J. Drug Target.
16 (2008) 108–123.
[4] U. Kedar, P. Phutane, S. Shidhaye, V. Kadam, Advances in polymeric micelles for
drug delivery and tumor targeting, Nanomedicine: NBM 6 (2010) 714–729.
[5] S. Dreis, F. Rothweiler, M. Michaelis, J. Cinatl Jr., J. Kreuter, K. Langer, Preparation,
characterisation and maintenance of drug efficacy of doxorubicin-loaded human
serum albumin (HSA) nanoparticles, Int. J. Pharm. 341 (2007) 207–214.
[6] H. Jeong, M. Huh, S. Lee, H. Koo, I. Kwon, S. Jeong, K. Kim, Photosensitizer-conjugate
6 (2011) 230–239.
[7] S. Rhaese, H. von Briesen, H. Rübsamen-Waigmann, J. Kreuter, K. Langer, Human
serum albumin–polyethylenimine nanoparticles for gene delivery, J. Control. Re-
lease 92 (2003) 199–208.
[8] A.P. Retnakumari, P.L. Hanumanthu, G.L. Malarvizhi, R. Prabhu, N. Sidharthan,
M.V. Thampi, D. Menon, U. Mony, K. Menon, P. Keechilat, S. Nair, M. Koyakutty,
Rationally designed aberrant kinase-targeted endogenous protein nanomedicine
against oncogene mutated/amplified refractory chronic myeloid leukemia, Mol.
Pharm. (2012), http://dx.doi.org/10.1021/mp300172e.
[9] K. Ulbrich, M. Michaelis, F. Rothweiler, T. Knobloch, P. Sithisarn, J. Cinatl, J. Kreuter,
Interaction of folate-conjugated human serum albumin (HSA) nanoparticles with
tumour cells, Int. J. Pharm. 406 (2011) 128–134.
[10] F. Kratz, Albumin as a drug carrier: design of prodrugs, drug conjugates and
nanoparticles, J. Control. Release 132 (2008) 171–183.
[11] F. Petrelli, K. Borgonovo, S. Barni, Targeted delivery for breast cancer therapy: the
history of nanoparticle-albumin-bound paclitaxel, Expert. Opin. Pharmacother.
11 (2010) 1413–1432.
[12] F. Kratz, B. Elsadek, Clinical impact of serum proteins on drug delivery, J. Control.
Release 161 (2012) 429–445.
[13] K. Löw, M. Wacker, S. Wagner, K. Langer, H. von Briesen, Targeted human serum
albumin nanoparticles for specific uptake in EGFR-expressing colon carcinoma
cells, Nanomedicine: NBM. 7 (2011) 454–463.
[14] M.Anhorn,S.Wagner,J.Kreuter,K.Langer,H.vonBriesen,SpecifictargetingofHER2
overexpressing breast cancer cells with doxorubicin-loaded trastuzumab-modified
human serum albumin nanoparticles, Bioconjug. Chem. 19 (2008) 2321–2331.
[15] I. Steinhauser, B. Spänkuch, K. Strebhardt, K. Langer, Trastuzumab-modified
nanoparticles: optimisation of preparation and uptake in cancer cells, Biomate-
rials 27 (2006) 4975–4983.
[16] I.M. Steinhauser, K. Langer, K.M. Strebhardt, B. Spänkuch, Effect of trastuzumab-
modified antisense oligonucleotide-loaded human serum albumin nanoparticles
prepared by heat denaturation, Biomaterials 29 (2008) 4022–4028.
[17] H. Wartlick, K. Michaelis, S. Balthasar, K. Strebhardt, J. Kreuter, K. Langer, Highly
specific HER2-mediated cellular uptake of antibody-modified nanoparticles in tu-
mour cells, J. Drug Target. 12 (2004) 461–471.
[18] S. Wagner, F. Rothweiler, M.G. Anhorn, D. Sauer, I. Riemann, E.C. Weiss, A.
Katsen-Globa, M. Michaelis, J. Cinatl Jr., D. Schwartz, J. Kreuter, H. von Briesen,
K. Langer, Enhanced drug targeting by attachment of an anti αv integrin antibody
to doxorubicin loaded human serum albumin nanoparticles, Biomaterials 31
(2010) 2388–2398.
[19] N. Dinauer, S. Balthasar, C. Weber, J. Kreuter, K. Langer, H. von Briesen, Selective
targeting of antibody-conjugated nanoparticles to leukemic cells and primary
T-lymphocytes, Biomaterials 26 (2005) 5898–5906.
[20] L. Zhang, S. Hou, S. Mao, D. Wei, X. Song, Y. Lu, Uptake of folate-conjugated albu-
min nanoparticles to the SKOV3 cells, Int. J. Pharm. 287 (2004) 155–162.
[21] D. Zhao, X. Zhao, Y. Zu, J. Li, Y. Zhang, R. Jiang, Z. Zhang, Preparation, characteriza-
tion and in vitro targeted delivery of folate-decorated paclitaxel-loaded bovine
serum albumin nanoparticles, Int. J. Nanomedicine 20 (2010) 669–677.
[22] Q.Li,C.Liu,X.Zhao,Y.Zu,Y.Wang,B.Zhang,D.Zhao,Q.Zhao,L.Su,Y.Gao,B.Sun,Prep-
aration, characterization and targeting of micronized 10-hydroxycamptothecin-loaded
folate-conjugated human serum albumin nanoparticles to cancer cells, Int. J.
Nanomedicine 6 (2011) 397–405.
[23] J. Kreuter, T. Hekmatara, S. Dreis, T. Vogel, S. Gelperina, K. Langer, Covalent attach-
ment of apolipoprotein A-I and apolipoprotein B-100 to albumin nanoparticles
enables drug transport into the brain, J. Control. Release 118 (2007) 54–58.
[24] P.K. Dubey, D. Singodia, R.K. Verma, S.P. Vyas, RGD modified albumin nanospheres
for tumour vasculature targeting, J. Pharm. Pharmacol. 63 (2011) 33–40.
[25] X. Qu, C. Yang, J. Zhang, N. Ding, Y. Lu, L. Huang, G. Xiang, In vitro evaluation of a
folate-bovine serum albumin-doxorubicin conjugate, J. Drug Target. 18 (2010)
[26] S. Muyldermans, M. Lauwereys, Unique single-domain antigen binding fragments
derived from naturally occurring camel heavy-chain antibodies, J. Mol. Recognit.
12 (1999) 131–140.
[27] V. Joosten, M.S. Roelofs, N. van den Dries, T. Goosen, C.T. Verrips, C.A.M.J.J. van den
Hondel, B.C. Lokman, Production of bifunctional proteins by Aspergillus awamori:
llama variable heavy chain antibody fragment (VHH) R9 coupled to Arthromyces
ramosus peroxidase (ARP), J. Biotechnol. 120 (2005) 347–359.
[28] P.A. Barthelemy, H. Raab, B.A. Appleton, C.J. Bond, P. Wu, C. Wiesmann, S.S. Sidhu,
Comprehensive analysis of the factors contributing to the stability and solubility
of autonomous human VH domains, J. Biol. Chem. 283 (2008) 3639–3654.
[29] I. Vaneycken, J. Govaert, C. Vincke, V. Caveliers, T. Lahoutte, P. De Baetselier, G.
Raes, A. Bossuyt, S. Muyldermans, N. Devoogdt, In vitro analysis and in vivo
tumor targeting of a humanized, grafted nanobody in mice using pinhole
SPECT/Micro-CT, J. Nucl. Med. 51 (2010) 1099–1106.
[30] A.M. Eisenbeis, S.J. Grau, Monoclonal antibodies and Fc fragments for treating
solid tumors, Biologics 6 (2012) 13–20.
[31] R. Roovers, P. Henderikx, W. Helfrich, E. van der Linden, A. Reurs, A. de Bruïne, J.
Arends, L. de Leij, H. Hoogenboom, High-affinity recombinant phage antibodies to
the pan-carcinoma marker epithelial glycoprotein-2 for tumour targeting,Br. J.Can-
cer 78 (1998) 1407–1416.
[32] F. Rahbarizadeh, D. Ahmadvand, Z. Sharifzadeh, Nanobody; an old concept and
new vehicle for immunotargeting, Immunol. Invest. 40 (2011) 299–338.
[33] S. Harmsen, M.E.M. Dolman, Z. Nemes, M. Lacombe, B. Szokol, J. Pató, G. Kéri,
L. Orfi, G. Storm, W.E. Hennink, R.J. Kok, Development of a cell-selective and
intrinsically active multikinase inhibitor bioconjugate, Bioconjug. Chem. 22
(2011) 540–545.
[34] D. Berz, H. Wanebo, Targeting the growth factors and angiogenesis pathways:
small molecules in solid tumors, J. Surg. Oncol. 103 (2011) 574–586.
[35] K.Temming, M. Lacombe, P.van der Hoeven, J.Prakash,T.Gonzalo,E.Dijkers,L. Orfi,
G. Kéri, K. Poelstra, G. Molema, R.J. Kok, Delivery of the p38 MAPkinase inhibitor
SB202190 to angiogenic endothelial cells: development of novel RGD-equipped
and PEGylated drug-albumin conjugates using platinum(II)-based drug linker tech-
nology, Bioconjug. Chem. 17 (2006) 1246–1255.
[36] K. Langer, S. Balthasar, V. Vogel, N. Dinauer, H. von Briesen, D. Schubert, Optimi-
zation of the preparation process for human serum albumin (HSA) nanoparticles,
Int. J. Pharm. 257 (2003) 169–180.
[37] M.M. Fretz, M.E.M. Dolman, M. Lacombe, J. Prakash, T.Q. Nguyen, R. Goldschmeding,
J. Pato, G. Storm, W.E. Hennink, R.J. Kok, Intervention in growth factor activated sig-
nalingpathways by renally targeted kinase inhibitors, J.Control. Release 132 (2008)
[38] S. Oliveira, R.M. Schiffelers, J. van der Veeken, R. van der Meel, R. Vongpromek,
P.M.P. van Bergen en Henegouwen, G. Storm, R.C. Roovers, Downregulation of
EGFR by a novel multivalent nanobody-liposome platform, J. Control. Release
145 (2010) 165–175.
[39] S. Oliveira, R. Cohen, M. Stigter-van Walsum, G.A. van Dongen, S.G. Elias, P.J. van
Diest, W. Mali, P.M.P. van Bergen en Henegouwen, A novel method to quantify
IRDye800CW fluorescent antibody probes ex vivo in tissue distribution studies,
EJNMMI Research 2 (2012), http://dx.doi.org/10.1186/2191-219X-2-50.
[40] A.O. Elzoghby, W.M. Samy, N.A. Elgindy, Albumin-based nanoparticles as potential
controlled release drug delivery systems, J. Control. Release 157 (2012) 168–182.
[41] J. Prakash, M. Sandovici, V. Saluja, M. Lacombe, R.Q.J. Schaapveld, M.H. de Borst, H.
van Goor, R.H. Henning, J.H. Proost, F. Moolenaar, G. Këri, D.K.F. Meijer, K. Poelstra,
R.J. Kok, Intracellular delivery of the p38 mitogen-activated protein kinase inhibitor
in renal tubular cells: a novel strategy to treat renal fibrosis, JPET 319 (2006) 8–19.
[42] K. Langer, M.G. Anhorn, I. Steinhauser, S. Dreis, D. Celebi, N. Schrickel, S. Faust, V.
Vogel, Human serum albumin (HSA) nanoparticles: reproducibility of prepara-
tion process and kinetics of enzymatic degradation, Int. J. Pharm. 347 (2008)
[43] A. Sorkin, L.K. Goh, Endocytosis and intracellular trafficking of ErbBs, Exp. Cell Res.
314 (2008) 3093–3106.
[44] E. Boucrot, S. Saffarian, R. Massol, T. Kirchhausen, M. Ehrlich, Role of lipids and
actin in the formation of clathrin-coated pits, Exp. Cell Res. 312 (2006)
[45] C. Antonescu, G. Danuser, S. Schmid, Phosphatidic acid plays a regulatory role in
clathrin-mediated endocytosis, Mol. Biol. Cell 21 (2010) 2944–2952.
[46] J.L. Ambroso, C. Harris, Chloroquine accumulation and alterations of proteolysis
and pinocytosis in the rat conceptus in vitro, Biochem. Pharmacol. 47 (1994)
[47] M. Talelli, M. Iman, A.K. Varkouhi, C.J.F. Rijcken, R.M. Schiffelers, T. Etrych, K.
Ulbrich, C.F. van Nostrum, T. Lammers, G. Storm, W.E. Hennink, Core-crosslinked
polymeric micelles with controlled release of covalently entrapped doxorubicin,
Biomaterials 31 (2010) 7797–7804.
[48] R. van der Meel, S. Oliveira, I. Altintas, R. Haselberg, J. van der Veeken, R.C. Roovers,
P.M.P. van Bergen en Henegouwen, G. Storm, W.E. Hennink, R.M. Schiffelers, R.J.
Kok, Tumor-targeted nanobullets: anti-EGFR nanobody-liposomes loaded with
anti-IGF-1R kinase inhibitor for cancer treatment, J. Control. Release 159 (2012)
I. Altintas et al. / Journal of Controlled Release 165 (2013) 110–118


text:
The Lanzar Optidrive Series Amplifiers answer the call for immersive and powerful bass reproduction in any mobile environment. These amps are designed with the latest technology available and engineered to promote energy efficiency with a significant increase and better distribution of signal strength. Transform your vehicle's audio system with a serious improvement in power output capability with the Optidrive Series! These amps provide headroom for even the most demanding peaks and dynamic ranges found in today's audio files, have High-Efficiency Heavy-Aluminum Heat sinks, Nickel-Plated Power RCA and Speaker Connectors, Bass Boost Circuitry, Power and Protection LED Indicators, Variable High and Low-Pass Crossover Controls and plenty more to take your audio to the next level.
Additional Info
Additional Info
Manufacturer | Lanzar |
Model Number | OPTI1600X2 |
Length | 40.70 |
Width | 16.00 |
Height | 7.90 |
UPC Code | N/A |
Condition | New |
Color | No |
Size | N/A |


text:
Article Preview
Officers stopped shooting suspect on day of Ariz. massacre
Published 01/12/2011, Associated Press
TUCSON, Ariz. (AP) — The suspect in a deadly Arizona shooting ran a red light and was stopped by a wildlife officer less than three hours before the attack that wounded Rep. Gabrielle Giffords.
Word count: 81Log In
The full article is available to newspaper subscribers. If you are a subscriber please log in to continue reading.


text:
Politics pushes Japan towards 'fiscal cliff'
PUBLISHED: 24 Aug 2012 12:33:00 | UPDATED: 24 Aug 2012 12:39:06PRINT EDITION: 25 Aug 2012Japan is slowly edging toward its own version of a "fiscal cliff" as legislation needed to sell bonds for this year's budget languishes in a split parliament, suggesting the government could run out of money soon.
Already a subscriber? Please login here
Stay with the story for less than $1.90 a day
Read more
Topics | Politics /World Politics , Economy /Fiscal Policy |


text:
Every Type of Check Fraud You Have to Worry About
Checks distressingly easily give identity thieves a chance to take over your account. Learn how to avoid check washing and paperhanging, too.
Checks distressingly easily give identity thieves a chance to take over your account. Learn how to avoid check washing and paperhanging, too.
Aerial photography and mapping, crop monitoring and inspections of tall structures would be acceptable, but it's unclear if you could get a pizza delivered.
Kidde is recalling nearly 5 million disposable fire extinguishers because they may not discharge properly, posing a risk of injury.
Sites let us to easily budget and track our expenses. Like online banking and shopping, these sites vastly improve upon past methods. But are they safe?
There are tons of easy, no-tech ways to improve your home's security for free or next to nothing. Or go for security equipment or an alarm company.
The Sanus Simplicity TV mounts, sold for about $125 at Costco, have a nut that can come loose. One consumer was injured by a falling TV.
The FAA lifts its ban on U.S. flights in and out of Israel, which the agency had imposed out of concern for the risk of planes being hit by Hamas rockets.
Delta will extend its suspension of flights to Israel amid hostilities between Israel and the militant group Hamas, its chief executive said on CNBC.
GM has doubled to 1.6 million the number of small cars it is recalling to fix faulty ignition switches linked to multiple fatal crashes.
GM is recalling nearly 780,000 compact cars in North America because the engines can shut down unexpectedly, which has resulted in 22 crashes that have killed six people.
Toyota says it is recalling 1.9 million hybrid Prius cars globally for a software glitch that could cause the vehicle to stall.
Toyota has told North American dealers to stop selling six popular models with heated seats because the fabric doesn't meet flammability standards.
Subcompact cars fared poorly in new crash tests performed by an insurance industry group.
ETFs are the new way to invest in the market, but one recently launched fund tracks old reliable companies that have been around since your grandparents were buying stocks.
Combi USA is recalling more than 33,000 child car seats made before January 2013 because the harnesses aren't strong enough to meet federal safety standards.
General Motors' China joint venture will recall close to 1.5 million vehicles due to potential safety issues.
Honda tops the insurance industry's annual list of the safest new vehicles.
Federal bus safety regulators have shut down 52 companies in what they describe as a major nationwide crackdown on unsafe outfits.
We've all heard the Black Friday horror stories about out-of-control crowds, so retailers are taking to protect customers and employees during the shopping holiday.
The country's four biggest cellphone companies are set to begin their first joint advertising campaign against texting while driving.
Used in everything from laptops to electric cars, lithium-ion batteries nonetheless have an image problem following several high-profile incidents involving smoke and fire.
best smoke alarms, smoke and carbon monoxide detectors, dual alarm smoke detectors, smoke alarm tips, fire safety, first alert, carbon monoxide alarms
GM CEO Dan Akerson is charged up about the politically manufactured controversy surrounding the Chevy Volt. "We did not engineer the car to become a political punching bag," he said. Tough words -- but rescuing the Volt's reputation will be a tough fight.
We all know that buying a fake Fendi is "bad" to the degree that selling such goods is illegal, and they hurt the companies whose good are being copied, and the economy overall. But it turns out that knockoffs are problematic in many other ways as well: Buying them can put your health and your money at risk.
Last year, the federal government made substantial progress in the regulation of thousands of children's products, child safety advocacy group Kids...
Ford is expanding a recall of its popular F-Series truck line because of faulty airbags that can deploy unexpectedly and possibly lead to injuries, federal regulators said Thursday. The expanded action now includes some 1.2 million trucks from the 2004 through 2006 model years.
It's a giant see-through plastic ball you climb into to walk on water, but the U.S. Consumer Product Safety Commission warned consumers not to use...
A new consumer product safety information database is up and running where consumers can share information with others about product injuries or...
A software glitch in certain Buick Lacrosse sedans and Cadillac SRX crossovers could decrease a driver's visibility in fog or ice, prompting GM to recall 10,000 of the vehicles.
Toyota is recalling another 22,000 trucks and sport-utility vehicles, this time so that it can repair faulty tire pressure monitoring systems. Vehicles affected by the recall include the Toyota FJ Cruiser, Land Cruiser, Sequoia, Tacoma and Tundra from the 2008 through 2011 model years.


text:
Pronunciation: spericolato
Added on: 2011-02-14
Listened: 6 times
Pronunciation in Italian [it]
- Pronunciation by pillola (Female from Italy)
- Could you do it better? different accent? Pronounce spericolato in Italian
Accents & languages on maps
- Random word: trattenere il fiato
by Yandex Translate English translation:
spericolato = reckless
Not satisfied?
Modify word:
· Add languages
· Add categories
Report word:
Is there anything wrong with this word?
Please report it here.


text:
| |
COMMERCIAL DESCRIPTION An Ølfabrikken guest brew. For the third time we have invited one of our colleagues to come by and do any brew he or she would like. Gunnhild Kolvereid Brewmaster at Brewpub Copenhagen has made a sour brown ale brewed with a normal Belgian ale yeast as well as brettanomyces, pediococcus and lactic acid bacteria. The beer has been matured on french oak and sour cherries and bottle conditioned for six moths before release. It is bottled with brettanomyzes which provides the beer with a nice sparkle underscoring the refreshing sourness. Unpasteurized, unfiltered and bottle conditioned like all of our beers.
We Want To Hear From YouJoin us! RateBeer is made by beer enthusiasts for the craft beer community. Your basic membership is free and allows you to read all beer ratings. Click here to create your account... and give your opinion! Join Us » Page 1 2 3 4 5 6 7 |
about us About RateBeer FAQ Contact/Feedback New Beers |
add Advanced Search Add A Beer Add A Brewer Add A Place Events |
membership Log In Edit Personal Info Buy Premium Membership Your Messages |
the best RateBeer Best 100 Beer Club The Top 50 |


text:
Sleeps | 8 |
Bedrooms | 5 |
Bathrooms | 2 |
Property type | house |
Minimum Stay | nights |
Beautiful Home! Views of the bay and the dunes! Private beach access across the street. Home features Large dinning room seats 8. Two living rooms, 2 more dining areas, full kitchen washer and dryer, cable tv, large front porch, lounge chairs. Back deck, gas grill teak picinic table , views are amazing.
on bus line to Ptown.
parking for 8 cars
Great Location!: Private beach directly across the street ! Two large decks to enjoy the sunset and have a family cookout! or light the fire pit. The home boast two living rooms and two dining areas as well as a large dining room. Pefect vacation home for large or multi family gatherings. You would wish you could spend the whole summer here.
On bus line to Ptown!! no driving required.
No pets please and No smoking.
You must bring:: Linens and towels.
Cape Cod


text:
| |
RATINGS: 1053 WEIGHTED AVG: 3.7 SEASONAL: Autumn EST. CALORIES: 183 ABV: 6.1%
COMMERCIAL DESCRIPTION Brewing with fresh hops is a big endeavor, requiring four to five times the hop volume compared to using pelletized hops. Although the process is complex, it brings impressive results: Fresh Hop is an American-style pale ale with moderate hop bitterness marked by a unique and intensely grassy hop flavor and aroma. Fresh Hop is a superbly refreshing, medium bodied, light-copper colored pale ale weighing in at 55 IBUs and 6.1% ABV.
We Want To Hear From YouJoin us! RateBeer is made by beer enthusiasts for the craft beer community. Your basic membership is free and allows you to read all beer ratings. Click here to create your account... and give your opinion! Join Us » Page 1 « 95 96 97 98 99 100 101 » 106 |
More American Pale Ales By Style
Outland American Pale Ale 81 Ottos Mt. Nittany Pale Ale 83 Häffner Bräu Hopfenstopfer Citra Ale 91 Pit-Caribou LÉtoile du Brasseur Pale Ale Austraméricaine 91 Flat 12 Walkabout Pale Ale 97 Hailstorm Prairie Madness Pale Ale 67 Collective Arts Rhyme and Reason 99 Marshall Arrowhead Pale Ale 74 |
about us About RateBeer FAQ Contact/Feedback New Beers |
add Advanced Search Add A Beer Add A Brewer Add A Place Events |
membership Log In Edit Personal Info Buy Premium Membership Your Messages |
the best RateBeer Best 100 Beer Club The Top 50 |


text:
Ask Legal Question
Legal Answers
Search Past Legal Answers
Find an Attorney
Browse Legal Articles
Customer Support
About Us
Attorney Network
Attorney Search
9 Appeals and Writs Attorneys in Connecticut
Page 1 of 1
Emile Barton
Law Offices of Emile J. Barton
Stamford, CT
LawGuru Attorney
Frank McCoy
McCoy & McCoy, LLC.
Hartford, CT
LawGuru Attorney
Max Rosenberg
Rosenberg & Press L.L.C.
Stratford, CT
LawGuru Attorney
Nicholas Adamucci
Stamford, CT
LawGuru Attorney
Anthony Pantuso
The Quinn Law Firm, LLC
Milford, CT
LawGuru Attorney
Mark Dumas
Dumas Law Firm
Milford, CT
LawGuru Attorney
Michael Skiber
Michael E. Skiber, Esq., Attorney and Counselor at Law
Bridgeport, CT
LawGuru Attorney
Pamala Favreau
Law Office of Pamala J. Favreau
Vernon-rockville, CT
LawGuru Attorney
Peter Van Dyke
Eagan, Donohue, Van Dyke, & Falsey, LLP
West Hartford, CT
LawGuru Attorney
9 results / Page 1 of 1
Get Free Legal Advice
active attorneys ready to answer your legal questions today.
Featured Attorneys
Timothy McCormick
Libris Solutions - Dispute Resolution Services
San Francisco, CA
Michael E. Hendrickson
Attorney & Counsellor at Law
Barry Stein
De Cardenas, Freixas, Stein & Zachary
Miami, FL
Are you an Attorney?
Earn additional revenue and grow your business.
Join LawGuru Now
Find a Legal Form
Browse and download our attorney-prepared and up-to-date legal forms from
Find a Form


text:
Cool Comic Cover Gallery Archives - Page 2 of 2 - Comics Should Be Good! @ Comic Book Resources
Cool Comic Cover Gallery
I thought this might be a fun bit. I'll pick a cool comic cover artist, and then show a gallery of 10 cool comic covers by said artist. Should be a fun waste of time, no?
The first subject is Chris Bachalo!
Enjoy! Continue Reading »


text:
Access your personal favorites account here Log In or Register
The newest homes for sale and real estate listings in 50207 are displayed below. Browse through our real estate listings and homes for sale, view virtual tours and contact one of our experienced real estate agents to answer any questions you may have about moving to the New Sharon area.
New Sharon, IA 50207
$275,000 4 BR, 1 full BA, 2 ½BA, 1 ¾BA Offered By: Gatton Realty
$229,900 5 BR, 1 full BA, 1 ½BA Offered By: EXIT Huls Realty
$63,000 4 BR, 1 full BA Offered By: RE/MAX Pride, Inc
$61,500 2 BR, 1 full BA Offered By: SUMMIT REALTY
$30,000 Offered By: McGriff Realty
Create a personal scrapbook of favorite listings and services.
Search foreclosure, auction & bank owned properties for sale
New homes from America's top home builders


text:
A confirmation request has been sent to you on your email address. Check it right away.
Tell us your age and gender to get 100 Reward points
Hey ICC is looking for the cricketers of the Decade in every category.. and Jumbo is been nominated too. So follow the Link and VOTE for him..
Ever since you gave me the link, I have voted for our skipper as well as, I have asked others to do the same. He is a winner and nobody else deserves the award more than AK.
If you can vote for me at http://www.royalchallengers.com/cricket-online-contests/mighty14/application/Wizardprince
I will be delighted. You can vote for me after you have voted for AK, I can wait, but, you better not forget
I found a new poll recently regarding something like this
Anyway Jumbo is placed in India's All time XI by Cricinfo Jury!.. Thats wonderfull
+1 for anil
nice leader
good player
will always vote for him as he deserves every bit of it
ya definitely.
he is the best man ever likely to seen


text:
Ingredients & Description
Enjoy the total-body health benefits of Grape Complete by Country Life. This natural blend of grapes and pine bark work as an antioxidant to help protect the body's cells against damaging free radicals.Grape Complete helps to strengthen capillaries and veins to enforce their structural integrity. It also works to support healthy connective tissue throughout the body. Grape Complete is an Hypoallergenic/Vegetarian/Kosher supplement.
*Not intended to diagnose or treat diseases or ailments, and is not reviewed by the FDA.
Uses & Indications.


text:
Sears offers the Crane 32" Ceramic Tower Heater, model no. EE-8080, for $79.99. Coupon code "SEARS5OFF50" cuts it to $74.99. With free shipping, that's $27 below yesterday's expired mention (which included $20 in Kohl's Cash) and the lowest total price we could find by $38. It features two heat settings, oscillation, touchscreen LED display, overheat protection, and a sleep timer.
Of note, Shop Your Way Rewards members will earn a Sears credit with this purchase and may be eligible for additional credits and discounts.


text:
"Save the Whales" is the famous shout out that brought millions of people together and produced a worldwide ban on commercial whaling in 1986. Whales were and still are the most extreme example of the shocking exploitation and lack of protection of ocean life.
It was claimed that there used to be so many Right whales in Cape Cod Bay on the east coast of the USA, that locals could walk across their backs from one side of the bay to the other – a distance of 40km. It is estimated that there are 400 left in the area. The global movement that "saved the whales" is still needed today.
Activists have risked their lives in the Southern Ocean, putting themselves between the whales and the harpoons; campaigners in Japan have risked their freedom to expose the corruption of the whaling programme, people have marched, petitioned and demanded change; in 2013 one government took Japan to court over its continued whaling. Even cartoon characters want to save the whales – so why are they still at risk?
Not only do whales continue to be hunted by Japan, Norway and Iceland, they are also increasingly falling victim to ship strikes and being caught in nets as bycatch. Hundreds of thousands of whale and dolphins die each year in this way. Pollution, plastic trash in the ocean and overfishing are taking a heavy toll. The blubber in some whales caught in Arctic regions is so heavily contaminated with airborne chemicals and pesticides that it would class as toxic waste and numerous whales have been washed ashore having starved to death with plastic bags and sheeting stuck in their oesophagus or stomachs.
Whales are long-lived, from 70-150 years, and slow to reproduce. It makes them very vulnerable to population collapses. Blue whales – the biggest animal to have ever existed - have still not recovered from being hunted to the brink of extinction. Only one population, the East Pacific grey whale, is thought to have recovered to its pre-hunting levels, but the closely related West Pacific grey whale population is the most endangered in the world. It hovers on the edge of extinction with just over one hundred remaining. The north Atlantic right whales are endangered.
And even these estimates may not be reliable. Previous estimates of population sizes have had to be dramatically reduced by the International Whaling Commission (IWC), the organisation whose job it is to enforce the whaling ban and monitor populations.
Some measures are being taken to protect whales. The Southern Ocean Whale Sanctuary came as the result for great public demand for protected areas for the great whales. The Japanese whaling fleet continues to ignore the sanctuary status of the Southern Ocean, but a properly enforced ocean sanctuary could provide much needed protection for whales and countless other marine species.
Karli Thomas, Greenpeace oceans campaigner writes from the IWC meeting in Morocco... The town of Sidi R'bat on Morocco's Atlantic coast is where the biblical Jonah is said to have been vomited up by a whale. Less than 100km from...
John Frizell - Greenpeace International whales campaigner writes from the IWC meeting in Morocco, Greenpeace campaigners at international meetings usually find themselves crammed into chairs at the back trying to listen to the...
In response to discussions taking place today at International Whaling Commission meeting in Morocco that signal that there will be no deal, Greenpeace Japan Programme Director Junichi Sato issued the following statement:
As the International Whaling Commission (IWC) annual meeting begins in Morocco, there has been a flurry of media coverage over a possible 'deal' or 'compromise'. Often the details, and sometimes the central points, can get lost as things are...
Non-governmental organisations and their activists play an active and essential role in representative democracies. Like journalists and other watchdogs of society, they are part of the vanguard against tyranny and their service to the political...
46 - 50 of 190 results.
Tweets by @fishandship


text:
Simply Breathless Bridal
3 total reviews
- 1
Wedding: 6/11/2011
Gerri and Marie,
I want to thank you for doing my hair and make up as well as my bridal party. The both of you do such a wonderful job. I will recommend anyone to the both of you. Your price was very reasonalbe and you both all great people. Enjoy your summer and I hope to see you soon.
Thank you so much.
Wedding: 8/7/2010
Marie and Gerri did hair and makeup for me, both moms and the maid of honor. Everyone was happy with their looks and the styles lasted all day in the August heat! They were professional, friendly, and perfect with getting all of us ready on schedule.
Wedding: 9/24/2010
Marie and Gerri did my hair and makeup as well as most of my bridesmaids hair and make up and my mom's makeup. The timing that they came up with was perfect and allowed us all to be ready for pictures on time. Everyone's hair and makeup looked great and lasted through a long day. Marie and Gerri were super easy to get in touch with and were really responsive to my emails and requests. They were a pleasure to work with.
- 1


text:
HELLO MY DEAR THNX . I HOPE ALL IS WELL ,GOD BLASE U MY CONT- EMAIL [email protected] & ALL SO U VISIT MY FACEBOOK
Thanks I have recieve your text . I will do that as soon as i login to my facebook account
Hello my dear i am miss jane how are you i hope all is well with you my dear i sow your conact on facebook and i become intrerstaed in you please i will like you to contact me with my email address [email protected] so that i will send you my photo and tell you more about myself yours in love jane


text:
Considering Changing Your Blogging Software? WordPress Makes It Easy- They Import Your Posts and more
This is a little feature I wish I knew about when I started this blog. I did it all by hand! This is one of the reasons why I asked people to contact me and tell me about their favorite Wordpress resources. Below are your options when importing with WordPress: (From WordPress) Import If you have posts or comments […]
Make Your WordPress Blog Search Engine Friendly
If you are new to WordPress and you are concerned about SEO this is a MUST see Video by the Graywolf SEO Blog. He discusses a siloed blog. Very informative. I think a lot applies to web sites as well. Make Your WordPress Blog Search Engine Friendly
Negative SEO Article You Need To Read
Damaging a Competitor's Ranking is Possible By Ross Dunn (c) 2007 SiteProNews.com
No Matter Where You Are In Your Blogging, These Blogs Will Be Useful!
I truly believe that you always keep learning, even if you think your brain is fried! These blogs have some outstanding information that will help you with your blog! Blog Tutorials Performancing Read/WriteWeb Download Squad! Matt Cutts– of course he has to be listed as does… ProBlogger.net The Blog Columnist Work From Home Facts Dosh […]
Blog Tutorials Provides Great Info on Feedburner Pro For Free
Use FeedBurner Stats PRO and MyBrand For Free!
What Are Your Favorite Web Sites/Blogs For WordPress Information, Templates, Tips…?
I am looking to create a list. Please tell me your favorites and I will add your suggestions and your site/blog to the list. Thanks. Melissa- SEOAware.com
The Content of Your Web Site Has Several Jobs:
I cannot say any job is more important than the other because they are all very important. I am going to focus on two… ————————————————– 1 ). Please the Reader/Web Visitor When a web visitor comes to your site and they begin to scan your site your content needs to be easy-to-read, scannable and convincing! Easy-to-read- […]
Site Pro News pointed out some great articles today for bloggers, web users and SEOs
Internal Link Architecture Made Easy The Forgotten Fundamentals of SEO How Many Links Could a Link Lover Love? FBI to Notify Microsoft Windows Users Who Were Victims of Botnets Open standards for social networking
So you are an SEO…Are you tired? I am!
Well, anyone in this line of business is quite clear that this type of work can be exhausting. I know there are other jobs that take a ton of physical and mental strength, but I don't have experience with those jobs! I think that this particular job is exhausting because there is a lot at […]
The Top 10 Things I Have That Make My Home Office Very Efficient
My HP Pavilion Entertainment PC- Yes, an Entertainment PC that has the ability to run Dreamweaver, Photoshop, InDesign, Word, Email and have 10 windows open without a glitch. It never goes down, that makes my life much easier. I have had an HP (8 years ago), Compaq and a Dell- none have worked this well. […]


text:
Please confirm your Email address
Search Now for Low Fares
Fares from San Antonio, TX (SAT)
Last published fare: $249 RT Flight from San Antonio to Chicago (SAT-ORD)
United, American
Expired: Friday, 06 March 2015 See Details >
United, American
Seats are not available on all flights or all dates of travel. You may need to have flexible travel dates to find this fare. Fares for travel on holidays may be higher.
7 days/week, if available
Travel Starting:
Monday, 13 April 2015
Travel Ending:
Friday, 05 June 2015
More Details:
Non-Stop Flights, No min. stay
Last Updated:
Thursday, 05 March 2015
Our Notes:
CheapAir charges a $9.95 booking fee.
Find this Fare:
Dealhound Confirmed!
Found using flexible dates.
Sign up for alerts on this route:
There are no longer active fares from San Antonio, TX (SAT) to Chicago, IL (ORD) that meet our Dealhounds' standards.
To find the best price available now, search below to compare our airline partners.
Reviews for San Antonio to Chicago (SAT-ORD)
Flown this route before? Write a review
Flown this route before? Write a review
See What Our Dealhounds Have Found from Other Cities