Below 10 years mixed development (entire) Gold: Plaza Arkadia
As you meander around the open-concept Plaza Arkadia in Desa ParkCity, Kepong, Kuala Lumpur, you can’t help but feel like you have been transported to somewhere in Europe, albeit on a summer day. British colonial-style architecture with a modern contemporary twist embellish the pedestrian-friendly paved streets and courtyards flanked by retail stores, offices and Small-office Home-office (SoHo) units.
According to its developer ParkCity Holdings, Plaza Arkadia has been designed to redefine mixed-use developments in Malaysia and to walk the talk of sustainable living in Desa ParkCity, which many considers one of the most liveable communities in KL.
“ParkCity has reinvented the standard shopoffice concept by redefining it as a hybrid mixed-use property packaged in a contemporary design inspired by British colonial architecture. It caters not only to its existing residents within Desa ParkCity but also those from the nearby neighbourhoods,” says ParkCity group CEO Datuk Joseph Lau.
Situated on an 11.32-acre freehold site, Plaza Arkadia has a net lettable area of over 860,000 sq ft, complemented by 1,980 car park bays. It consists of 211 shops, 139 offices and 167 SoHos — all which were fully sold within 15 months after launch. As at December 2019, the retail and office units have achieved at least 90% overall occupancy rate, while the SoHos have an occupancy rate of 63%.
Unlike traditional shophouses, even the units on the higher floors here receive their fair share of visibility and accessibility. This ingenuity is achieved by building a link bridge on the first floor, which connects all of the eight blocks comprising four to five levels. Thus, all retail shops, no matter which floor they are on, receive equal exposure as those on the ground floors. This is further enhanced by the landscape planning around the open-air courtyards, which have been crafted so that the upper floors also enjoy an immersive open-air experience.
“We have created a completely new mixed-use district that uses traditional urban elements of arcade, park, streets and plazas; while also making it reflect a very modern style of living by using modern and traditional architectural vocabularies. In short, while Plaza Arkadia seamlessly inherits the ParkCity DNA of walkability, sustainability and sense of community, it also marks a new brand for ParkCity.
“We think Plaza Arkadia will set a precedent in urban development, where people can emotionally relate to its familiar urban elements. It is a destination that people will return to again and again because of the quality of the place and the rich experience it evokes,” Lau notes.
In remaining steadfast to its commitment to sustainability, the developer has incorporated green features to ease sustainable management and maintenance into the project right from its blueprint. These included water-resistant glass to prolong cleaning cycles and reduce the frequency of cleaning; energy-saving light bulbs and sensor lift lighting and escalators; low volatile organic compound (VOC) paints which emit vapour or gas that helps freshen the air; as well as rainwater harvesting system for all the vegetation at Plaza Arkadia. Notably, even excess water is channelled to the main lake at The Central Park of Desa ParkCity.
In recognition of its sustainable and management-friendly approach to property management, Plaza Arkadia has been bestowed the Gold in the below 10 years mixed development (entire) category of EdgeProp Malaysia’s Best Managed and Sustainable Property Awards 2020. Notably, ParkCity’s The Central Park at Desa ParkCity also won Gold in the 10 years and above specialised category of the same Awards.
A strong collection rate
The Joint Management Body (JMB) of Plaza Arkadia appointed ParkCity Management Sdn Bhd, a wholly-owned subsidiary of the developer, to manage Plaza Arkadia since its completion at end-2016. The management team is closely supported by ParkCity’s M&E team and consultants.
Its strong collection rate has been maintained at 95% with no write-off debts since the handover of the properties in the project.
Like most mixed developments, cleaning and electricity formed the bulk of the monthly expenditure. Substantial resources and funds are also allocated to security, which is a priority for an open-concept commercial lifestyle project. In line with its aim to optimise cost, long-term security features have been included in the project’s design and planning. For instance, the landscaping surrounding Plaza Arkadia acts as a passive perimeter protection to enhance security and deter crime. Where there are plants, they do not block the shopfronts’ visibility.
Besides that, roads serving Plaza Arkadia come with speed bumps to control safe driving while ample street lights are installed. On top of that, the project is equipped with an integrated security system comprising CCTVs, two security control rooms, patrolling at the premises, security checkpoints and help buttons at the carpark.
Although the commercial project has been planned to be away from the township’s housing precincts, it remains in walkable distance to the residents. In the township’s masterplan drawn up some 20 years ago, it was already decided that Plaza Arkadia was to have its own access road to avoid disruption to the residences.
“Winning this award will definitely authenticate Plaza Arkadia’s ideals, design philosophy and architecture. This will contribute to setting a new benchmark for real estate industry in Malaysia. This award will also raise the profile and upgrade the status of Plaza Arkadia as well as Desa ParkCity.
“This award is a recognition of the effort, aspiration and the vision of the company and more importantly, the support of our buyers over the years. ParkCity and the JMB would like to thank all our staff, committee members, buyers and business associates for supporting and making Plaza Arkadia a success,” says Lau.
Get the full results of EdgeProp Malaysia’s Best Managed & Sustainable Property Awards 2020 here: https://www.edgeprop.my/awards2020
View the virtual awards ceremony here: https://buff.ly/2YFNcVg


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Closes in 11 days
“Boroboro Dorodoro” — muddiness and rattiness — don’t come to mind as you stand outside Watari-um’s (www.watarium.co.jp ) clean postmodernist facade, but that’s what you’ll find inside. The exhibition aims to present “the return of Japanese subculture” through Taylor McKimens and Misaki Kawai, two young street artists currently based in Brooklyn.
The two’s works are fun and playful. McKimens, who is interested in the “overlooked, nondramatic decay that constantly surrounds us,” presents grotesque yet compelling domestic scenes. Through paintings and an inventive, life-size paper installation, he creates a festering world where chewed gum fills dirty living rooms and men sit in backyards with just their underwear and plastic bowls to cover themselves. Gross, yes, but McKimens’s choice of pastel colors, cartoon-like drawing style and his eye for detail — note the bits of fly in the fly swatter — create repellent works that don’t repel.
Kawai’s “Space House” is equally as detailed, though to a more cutesy effect. The giant installation is of connected space ships that appear to be the rooms of a house. Homespun dolls representing the artist and her friends occupy rooms with magazines and posters, glasses of wine and a Jacuzzi. Made out of junk and other cheap materials, its childishness — think sophisticated elementary school art — possesses its own charm.
Though this amusing show stands above the usual work produced by the manga-inspired generation, these young artist’s voices still need strengthening. Though their works are not mass manufactured comics, they are are not quite unique.
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domain: artistic


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Prep: 20 min. Cook: 1 hour
20 servings (5 quarts)
Updated: Oct. 26, 2023
2-1/2 pounds ground beef2 cups chopped onion1 cup chopped celery3 tablespoons chili powder1 tablespoon ground cumin1 teaspoon pepper1 can (4 ounces) chopped green chiles1 garlic clove, minced1 can (46 ounces) tomato juice4 cups V8 juice1 can (28 ounces) diced tomatoes, undrained2 cans (16 ounces each) kidney beans, rinsed and drained2 cans (16 ounces each) pink beans or pinto beans, rinsed and drainedOptional: Sour cream, cubed avocado, shredded cheddar cheese and sliced jalapeno pepper
In a large Dutch oven, cook beef over medium heat until no longer pink; drain. Continue to cook until beef is browned, 4-5 minutes longer. Add onion and celery; cook until tender. Stir in chili powder, cumin and pepper; cook 1 minute. Add green chiles and garlic; cook 1 minute longer.
Stir in juices and tomatoes. Bring to a boil. Reduce heat; simmer, uncovered, for 20 minutes. Add beans and simmer 20 minutes longer or until thickened to desired consistency. If desired, serve with sour cream, avocado, cheese and jalapenos. Freeze option: Freeze cooled chili in freezer containers. To use, partially thaw in refrigerator overnight. Heat through in a saucepan, stirring occasionally; add a little water if necessary.
This recipe was tested with Goya Pink Beans (Habichuelas Rosadas). Pinto beans may be substituted if pink beans are difficult to find.
1 cup: 225 calories, 7g fat (3g saturated fat), 35mg cholesterol, 595mg sodium, 23g carbohydrate (7g sugars, 6g fiber), 17g protein.

domain: food


Are you interested in improving cities and empowering your community? The Sociology Major provides an excellent foundation for urban careers! Our urban courses feature experiential learning opportunities like walking tours, visits with local organizations and change makers, and problem-solving class projects that improve our campus. If you’re interested in cities, you may enjoy working for city government in areas such as housing, transportation, or urban planning. Our alumni work in urban environmental sustainability, housing assistance, transportation planning, refugee assistance, urban planning, and in community organizing. This interest track works well with double major or minor in Urban Studies.
If you’re interested in an urban career, check out the following classes as you complete your Sociology Major and talk with Nancy Fischer (firstname.lastname@example.org).
- City Life: Intro. to Urban Sociology (COM 111)
- Race, Class, and Gender (SOC 265)
- Disaster and Resilience (SOC 380)
- City and Regional Planning (SOC 381)
- Internship in Urban-Related Organizations (SOC 399)
- Intersections of Race and Immigration (SOC390)
- Another Future is Possible: Sustainable Solutions in Amsterdam and Rotterdam (SOC 295)
- Social Justice in Urban America (POL 122)
- History of Architecture after 1750 (ART 244)
- U.S. Environmental History (HIS 316)
- Designed Environment (ART 249/349)
- Geographic Information Systems (GIS) classes (Macalester, St. Thomas)
- Study abroad opportunities in Copenhagen, Denmark at the Danish Institute for Study Abroad or in Berlin, Germany with IES.


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Bigger is always better, right? We all know that is the case with Mob Wive'sBig Ang! Now, the over-the-top Ang is following the lead of many reality stars before her. That's right–Big Ang has some big news of the alcoholic variety!
The Drunken Monkey owner has come out with her own line of vino. Ang's first offering treats us to a chardonnay, cabernet sauvignon, and prosecco. Mob Wives creator Jennifer Graziano recently tweeted a picture of wine line, sharing, "They're here!!!! Everyone stay tuned for info on where to buy!! @biggangVH1 @ManiacManagemnt."
Good ol' Wacky Jackie is up to her antics yet again, and this time she's channeling her inner E.L. James! Basketball Wives: LA star Jackie Christie recently launched her new book Sexual Relations: A His & Her Guide to Greater Intimacy, but she's not planning to stop there!
No ma'am. Jackie has bigger plans than reality television and kinky tomes. She's moving on to film, y'all! Just wait until you hear about Jackie's latest project!
Run, Bruce Jenner! Run as fast as you can! We all know you can do it, and you've got the medal to prove it. Do not pass go, do not collect $200! I can't even. I mean, I may very well be at a loss for words, and y'all know that is never the case!
On the heels of their separation announcement, Kris Jenner is taking every opportunity she can to share her relief over being rid of husband Bruce. It's really quite sad and disrespectful, but then again, that's par for the course with Kris, isn't it? People has the details of the pimpmomager's cover story for New You magazine, and in it, Kris describes what she would want for her one do-over. Hint: it has nothing to do with Bruce.
Kris reveals, "The one regret, if I had to do it over, would be divorcing Robert Kardashian. But then there wouldn't have been Kendall and Kylie, so that's the way I look at it. Everything happens for a reason, and we learn from our mistakes. I thought I was so smart when I was young; I was fearless." I am really holding back here, and it's harder than expected.
In her Bravo blog this week, Caroline dishes on just how over she is of the bitter battle of the sister-in-laws and once again (enough already!) clarifies her comments about a cheating Al. I know she annoys the sanity out of most folks, but you have to admit, she's usually right. I personally don't think Caroline perceives herself as the matriarch or the all-knowing, I just think she's the only woman on RHONJ with one iota of common sense.
Don't lie. Y'all have missed Somaya Reece since she departed from Love & Hip Hop. I know I have, and I certainly didn't have to refresh my memory as to what she looks like or the drama she caused during her tenure on VH1. Clearly it wasn't much or she'd likely still be on it!
I have to commend her from taking a break from scripted cat fights, girl drama, and drink tossing that has become synonymous with all VH1 reality shows. But it begs the question…what is Somaya doing now?
The singer and former reality star recently checked in with the folks of The Jasmine Brand. While Somaya is no longer gracing the small screen on L&HH, she's mastered the art of reality television stardom: do everything! Whether it's schlep a cocktail with your name on it or taking credit for some ghostwriter penning your life story, do it! I kid. Kind of.
Not surprisingly, Bravo's favorite potty-mouthed Polish beauty has her take on what happened, and she's never one to hold back. Joanna remains vehemently loyal to Lea and is quick to belittle her fiance's feelings in one breath and then praise him with the next. She's exhausting, no?
Last night was the premiere of Oxygen's Preachers of L.A. To be honest, I am not really sure what to think. One guy looks like Billy Dee Williams and used to fancy the angel dust. I swear another one of the ministers is twins with D.J. Drew from Love & Hip Hop. They all drive ridiculously expensive luxury sports cars, and they appear to have more money than God. Pun clearly intended.
I am extremely intrigued by the entire premise of the show. It's likely going to be very controversial, and I apologize in advance if I offend anyone with my attempts to recap–and snark–on a show about men of the cloth.
We are first introduced to Bishop Dietrick Haddon. He describes himself as "a preacher, a pastor, a gospel entertainer, and a Grammy nominee." Impressive! He was called to minister at a young age, and he preached his first sermon at age eleven.
Dietrick married young and went through a contentious divorce. In the midst of the dramatic split, Dietrick relocated from Detroit to Los Angeles where he met Dominique. Soon after they started dating, Dominique became pregnant. Not yet divorced and fathering a child out of wedlock, Dietrick believed he had two strikes against him. Now engaged to Dominique, he is working on making his choir an international success.

domain: celebrity


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Charleston's, West Virginia life in video
On our site, you will find the only video portal of the city of Charleston, which contains the most exciting and interesting videos of the region. Participate in the life of the project, be aware of all the events !!!
Best videos of Charleston online
Charleston is a city in West Virginia, United States. It is the most populous municipality designated as a “city” in West Virginia, and the third most populous of its kind in the United States.
Charleston is a city with character. Beautiful, modern, hardworking, West Virginia city! Charleston residents from all walks of life share their vision of one of West Virginia's largest cities, its history, and future.
The video library about Charleston contains:
- Charleston's history and facts;
- the most interesting and vibrant sports and cultural events;
- large-scale construction projects;
- interesting events;
- about life in West Virginia (Charleston, United States);
- beautiful views of the city.
All videos posted on the site are of high quality, taken by amateurs or professional videographers.
Watch the best Charleston videos online right now on our website.
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The desired video about the city of Charleston is very easy and simple to find. The information is well structured by month and date. In addition, the desired video can be found by the hashtag entered in the search. You can also watch videos on specific topics.
Watch videos about the city of Charleston, download and save or distribute information on social networks.
The last photos of Charleston
The latest news in Charleston
- U.S. Attorney Thompson Invites Media to Human Trafficking Training Workshop February 8, 2023, 1:20 PM United States Attorney Will Thompson of the Southern District of West Virginia invites the media to ...
- Concord, WVSOM professors finalists for annual honor February 8, 2023, 1:56 AM charleston, w,va. – Two professors, one at Concord University and the other at the West Virginia Sch...
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- IT Information Technology Specialist (IT Specialist) Department Of The Treasury
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Charleston, United States - the big trip
If you want to get to know Charleston as a tourist, start with the main video on this site: Presentation of Charleston City. It contains the main attractions of Charleston, panoramic beautiful views - all this is in the video about Charleston. Also, pay attention to Charleston's video review and video news. Plunge into the past. We have a lot of videos on our site for those who are interested in the history of the city. In a word, in this video section, the city of Charleston will open up for you from a new side. You will make the city picturesque and vibrant, business and formal, forward-looking and modern. We wish you a pleasant viewing!

domain: geography


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But these tutoring sessions at St. Margaret’s in Aigburth got him up to speed, and with his dad Paul, his mom Avril and his uncle Barry, all former bodybuilders, it didn’t take long for 24-year-old Marcel to be on his way to much bigger things.
When Marcel Vormawah Jonas started going to the school gym at noon, he had no more ambition than to improve the physique of an average teenager.
Marcel Vormawah Jonas from Wavertree continued a family tradition and won the Mr. Britain Bodybuilding title
He has just received the trophy in the beginner’s class, a reward for almost six months of careful, strict diet and exercise, during which he ate seven strictly controlled meals and six liters of bottled water a day and spent up to three hours a day in the gym.
Now, almost a decade later, Marcel is twice the man he once was – and the proud bearer of the NABBA (National Amateur Body Building Association) Mr. Britain title.
It was a sacrifice, but it paid off, and for Marcel, it was his first UK title since his rise from the junior classes.
By the day of the competition he had lost his muscles and his body fat had dropped below 5%.
“It hasn’t been pleasant these last few weeks,” he admits. “I woke up three or four times a night, it was so hard to sleep, and I pretty much locked myself in my room when I wasn’t in the gym. I would just sit there and pass the time between meals.
His career as a serious bodybuilder began at the age of 15 when, inspired by the fitness of his family, he decided to start training regularly.
“I’ve always been quite athletic and keen on sports, but back then I only had an average physique,” he recalls. “I started going to the school gym with a group of friends because that was something you could do over lunch. St. Margaret’s Sports Center wasn’t a big, fancy gym with lots of machines, but there was enough and you could train every part of your body.
“At the time, he suggested attending NABBA for under-18s, and that gave me something to work towards.
“I did it four times a week and went to family gymnastics on the weekends – the training station in Wavertree – and I noticed quite a bit of improvement. But then I remember one day, when I was about 16 years old, I went to our gym and I got dizzy and my uncle sat me down and explained to me that I had to eat right and in the right amounts if I was going to exercise. I followed that advice and over the next year I put about one stone on the board.
Marcel did his first 10 week intensive competition diet when he was 17 years old, then he entered and won the North West and British under 18s classes. He was then invited to Greece where he won the Mr. Universe title.
The following year he finished second in the British finals, so he took a break to improve before returning to compete again and win in the under 21s class.
“But after about a year it was so far that I could imagine going backwards, so I knew I had to get back in because I hated looking like that.
Once he had reengaged himself, Marcel started to work on concentrated weights, cardio and diet from January 1st this year, targeting the trophies of the NABBA North West and the British Novice class.
At 20, he decided to take a year off because, as he admits, he felt that his competition regime meant that he was giving up parties with his friends….

domain: sports


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Game Preview: Pelicans vs. Knicks - February 19, 2014
After a memorable 2014 NBA All-Star weekend in New Orleans, the second time the city has hosted over the past six years, the Pelicans return to the hardwood Wednesday, when they host the New York Knicks. New Orleans will try to pick up from where it left off prior to the All-Star break, when the Pelicans put together a 3-1 homestand that included wins over Chicago, Atlanta and Minnesota from Feb. 1-7. Dating back to late January, the Pelicans have won four of their last five games in the Smoothie King Center, with the lone defeat in that span coming against defending Western Conference champion San Antonio.
New Orleans will also try to continue to build on its rampant success this season against the Eastern Conference. Prior to All-Star, the Pelicans were 14-7 against the East, including at least one win over 10 of its 15 clubs. New Orleans has already swept two-game series from Chicago, Detroit, Cleveland and Philadelphia.
The Pelicans are seeking another two-game series sweep Wednesday against the Knicks, having prevailed in New York City on Dec. 1 by a 103-99 margin. New Orleans won in the Big Apple despite a first-half broken left hand injury suffered by Anthony Davis, who exited the game after playing just 10 minutes. Off the bench, Ryan Anderson picked up the slack with one of the best games of his NBA career, a 31-point outing that including 7-for-11 three-point shooting. Also in a reserve role, Tyreke Evans piled up 24 points. Evans continues to spark the Pelicans off the bench, while Anderson has been sidelined since Jan. 3 with a herniated disc, the result of an accidental collision with Boston’s Gerald Wallace.
New York, which begins its second half with a Tuesday game at Memphis, is trying to move into the top eight of the standings in the East and return to the playoffs. The Knicks reached the second round of the postseason in 2013, but were eliminated by the Pacers in six games.
Tyreke Evans vs. J.R. Smith

domain: news


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Has anyone been to Wonton House in Santa Clara (Chinese name for the restaurant is "Chez Chicken/House of Chicken")?
I happened to drive past the restaurant on my way to Tofu House further down El Camino. I noticed a lot of customers and it was during lunch hour.
Was wondering if they serve wonton noodles as the name suggested and how the food is compared to e.g. Fat Wong's in Millbrae?


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School: Georgia Southern University
Course: Anatomy and Physiology 1
Professor: Diana sturgess
Term: Fall 2016
Tags: epithelial tissues, homeostasis, cells, and chemical
Name: Test 1 Study Guide
Description: This covers the entire test 1.
Also review the notes for Week 1 and each assignment from Topics 1-4.
Epithelial Tissue WILL be on the test.

domain: biology


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Research insight into mycotoxin mitigation in dairy cows
Scientists renowned for their research on mycotoxin risk management in livestock shared insights on how mycotoxins affect the bottom line of dairy cow operations during the Selko Feed Additives global webinar "Multi- pronged Approach to Mycotoxin Risk Management in Dairy Cows," on 27 October 2022. Prof. Antonio Gallo, Animal Nutrition Department at Università Cattolica del Sacro Cuore, Italy and Dr. Swamy Haladi, Global Programme Manager, Mycotoxin Risk Management at Selko, shared how dairy cows are exposed to higher number of mycotoxins as compared to monogastric animals, the challenges faced in diagnosing mycotoxicosis, the economic impact of mycotoxins and the latest research on the practical strategies for mitigating mycotoxin risk in dairy cows.
Extreme Conditions Elevating Mycotoxin Risk
Both geopolitical and climate factors are elevating mycotoxin risk for livestock production. Beyond a reduced grain supply resulting from the Russia-Ukraine conflict, widespread droughts have lowered grain production in many regions. Lower grain supplies have led to concerns about moulds and grain quality, triggering import alerts.
Dispelling ruminant myths
Ruminants are often thought to be less susceptible to mycotoxins than monogastric animals. The rationale is that the rumen helps break down contaminants in the rumen. While the rumen protects against some contaminants, it does not defend against all mycotoxins. For some mycotoxins, the mycotoxin degradation in the rumen can lead to more toxic metabolites than the parent mycotoxin. The complex dairy cow diet presents more opportunity for contamination with multiple types of mycotoxins.
Diagnosing mycotoxin risk is challenging in dairy cows
Mycotoxins can cause acute or chronic mycotoxicosis in dairy cows. Some commonly recognized moulds that generate major mycotoxins include Aspergillus, Penicillium, Fusarium graminearum, Fusarium and Ergot. As dairy cows' diets are more diverse than many other livestock diets, detecting the presence of mycotoxins among multiple ingredients requires an extensive and sometimes impractical sampling processes. Testing for lesser-known emerging mycotoxins should be conducted along with the most common mycotoxins. Interactions between mycotoxins can lead to additive, synergistic, or antagonistic effects that make toxins' effects more damaging to animals.
Although there are specific signs of contamination – like levels of aflatoxin M1 in milk – a range of non-specific problems including undigested feed in the faeces, reduced feed intake or milk yield, poor reproductive performance, lameness, rumen acidosis and mastitis may suggest a mycotoxin issue. At the cellular level, increased permeability in the intestinal tract and elevated production of oxidants may also result from mycotoxins.
Research at Università Cattolica del Sacro Cuore also explored the cooccurrence of well-known and emerging mycotoxins in corn silage. The project found that many silage samples include concerning levels of multiple contaminants. Analysis of the milk generated by cows receiving diets with the contaminated silage found that the metabolic profile of the milk changed in correlation with silage quality. Contamination level appeared to alter milk composition and metabolite levels.
Managing mycotoxins with mitigation agents
Not all mycotoxins respond the same way to mitigants. For example, binding agents can bind aflatoxins, Ergot toxins, and endotoxins in the intestinal tract, but are less effective against T-2 toxin, ZEA and OTA, and do very little to bind DON and fumonisins. Thus, a strategy deploying multiple modes of action provides a more holistic approach to mitigating mycotoxin contamination in the ration (figure 1).
Figure 1 – Among the "Big 6" mycotoxins, not all respond to binding agents, and some, like Fumonisins, can be bound at acidic pH but not at alkaline pH.
Considering the mode of action, mycotoxin mitigation products target a range of mycotoxins by reducing their bioavailability and boosting animal responses. Blended feed additives can support mycotoxin adsorption, provide gut wall protection, and boost immune, and antioxidant systems.
Emerging mycotoxin research
As more attention is focusing on emerging mycotoxins, researchers are trying to study their negative effects on livestock. For the first time, Selko evaluated the binding efficacy of the TOXO product range on three of these emerging toxins – sterigmatocystin, enniatin B, and roquefortine C – at two different pH levels to mimic potential uptake by an animal's gastrointestinal tract. All three toxins displayed the potential for a high adsorption rate to TOXO products.
Figure 2 – Less may be known about emerging mycotoxins, but ongoing research is finding that they can be managed through the application of TOXO product range in the feed.
in vivo studies with mycotoxin mitigation products
Looking at the drought situation in many parts of the world this year, dairy producers will be challenged to supply the milk with below the regulatory levels of aflatoxin M1. Research examining a feed additive found that it helped lower aflatoxin transfer.
Figure 3 – Mycotoxin mitigation product (TOXO-MX) can help to reduce transfer of aflatoxin M1 into milk.
Another study examined a feed additive with multiple modes of action – TOXO-XXL –provided to cows ingesting feed with moderate levels of Fusarium mycotoxin contamination for 54 days. The diets included ingredients naturally contaminated with DON, ZEA, and Fumonisin B1 and B2. Overall, cows getting the feed additive had better milk yield and tended to have slightly better feed efficiency than cows receiving contaminated diets. Milk components were similar for all cows.
Figure 4 – Inclusion of mitigation product into mycotoxin-contaminated feed showed best production indices.
"Producers face a difficult year for sourcing quality feed ingredients, putting dairy cows at an increased risk of receiving diets with multiple mycotoxins" said Swamy Haladi. He said supporting cows' ruminal and intestinal health, immune function, and antioxidant status can help defend against these threats.
More research is needed to gauge the effect of mycotoxin contamination on elements like dry matter intake, feed digestibility, and milk quality, according to Professor Antonio Gallo. "A lot of regulated and emerging Fusarium toxins can contaminate the feed, in particular silage and bailage, because they are characterized by a complex microflora."
For more of the article, please click here.
Article made possible through the contribution of Selko


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Gina Harlow and the Cutthroats were icons in the hey-day (1970’s) of Punk and Nuevo Wavo, in New York City. Their shows at Max’s Kansas City, CBGB’s, Trax, Heat, Hurrah’s, and The Mudd Club were legendary. They shared the bill with The Heartbreakers at Max’s Kansas City, at SNAFU’s they shared the bill with the Johnny Average Band featuring Mick Ronson, they headlined the Beacon Theatre to a sold out audience, and performed at the original Copacabana several times.
Gina Harlow & the Cutthroats – Live on Stage became one of Billboard magazine’s “Recommended LP’s” when it debuted in 1978. The record was recorded in 1977 at Max’s Kansas City (the legendary New York City Punk/Rock club). The band consisted of Gina Harlow: Bass and Vocals; Jesse Davis: Guitar and Vocals; and Nick Fuse: Drums. It was released with no overdubs, preserving the raw and live sound of a new group in its infancy; it was recorded on their fourth performance.
The group was a classic in the Punk Rock genre all songs are original and reflect the social climate of the 70’s. They performed high-energy rock, the musical theme was a reflection of the era, served up with social, political and religious satire, and it was a
highly visual stage show.
The group disbanded in the 1980, Gina Harlow continued to perform in New York, until 1985. Then, she disappeared…it was reported that she lived in an undisclosed location, with wild and savage head-hunters, until 2011, when Elvis came to her in a dream…Well, she answered his call and she’s back, watch out, she’s wild.
In 2012, Gina Harlow ran for President, on the T&A Party, which she formed in response to the Tea Party/Republican Party.

domain: entertainment


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Finally got around to scanning my sketchbook, a couple of images were lost so i'll have to re-scan those later but here's what we have so far. I still can't work out how to re-size my blog layout in order to have the pictures in full view, in order to see the image in all it's glory simply click on it and you will be taken to the photobucket album.
Here are a couple of storyboards for the heavy lifting excercise.
Here is the storyboard for the emotion to emotion excercise.
Here are some of my character designs for the 11 second club project. I was hoping to create some characters that were crude but that still possessed a charm. I'm hoping to do some more detailed and coloured version in Illustrator (once I've gotten to grips with it) but in the meantime here's the concept art.
And here are some storyboards for a series of shorts that I'm planning. I realise the humor, like the characters, is crude and probably only funny to me, but it's my own brain child so I can do whatever the hell I like...


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Wiesner, D. (2001). The Three Pigs. New York, NY: Clarion Books.
The story of the “Three Little Pigs” begins off as expected with three pigs choosing to build their houses out of straw, sticks, and bricks. When a wolf comes along to blow down the house of straw, he blows the first pig out of the panel of the story. This first pig convinces the other two to follow him on an adventure outside of their own. They travel through “Hey Diddle Diddle” and a story about a dragon who must be slain before deciding to return to their own story and alter the ending.
This delightful re-imagining of a classic fable uses differences in illustration technique to show the difference between the three stories and the “real world” that immediately convey to the reader what is happening as the characters move between panels and different stories. We move from a comic style in the original three pigs story to a more realistic illustration, then “Hey Diddle Diddle” takes us to a colorful, bright, and whimsical world. Finally, the dragon’s story is black and white, the starkest difference from the real world portrayed in this picture book between stories.
The ending where the pigs turn their story upside down to get the ending they desire shows the reader in an obvious but delightful lesson—we are in control of our own stories. We write them ourselves and have the freedom to do what we wish with them, whether it is making friends outside our own “story,” defeating the bully, or escaping from what we’ve always known.
From Publisher’s Weekly: “ Wiesner’s (Tuesday) brilliant use of white space and perspective (as the pigs fly to the upper right-hand corner of a spread on their makeshift plane, or as one pig’s snout dominates a full page) evokes a feeling that the characters can navigate endless possibilities–and that the range of story itself is limitless.”
From School Library Journal: “Children will delight in the changing perspectives, the effect of the wolf’s folded-paper body, and the whole notion of the interrupted narrative.”
From Booklist: “Jon Scieszka’s The True Story of the Three Little Pigs (1989) turned the favorite porkers’ story upside-down by allowing the grossly misjudged wolf to tell his side of the story. Wiesner’s latest is a post-modern fantasy for young readers that takes Scieszka’s fragmentation a step further: it not only breaks apart and deliciously reinvents the pigs’ tale, it invites readers to step beyond the boundaries of story and picture book altogether.”
-Use in connection with other versions of The Three Little Pigs:
The Three Little Pigs by James Marshall
The True Story of the Three Little Pigs by Jon Sciezka
The Three Little Wolves and the Big Bad Pig by Eugene Trivizas
No Lie, Pigs (and Their Houses) Can Fly!: The Story of the Three Little Pigs As
Told By the Big Bad Wolf by Jessica Gunderson
The Three Ninja Pigs by Corey Rosen Schwartz
-Read with other fables:
The Ant and the Grasshopper by Rebecca and Ed Emberley
The Tortoise and the Hare by Jerry Pinkney
The Lion and the Mouse by Jerry Pinkney
The Wolf in Sheep’s Clothing by Mark White

domain: library


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A film fanatic from Orpington brushed shoulders with a star from the new Spider-Man movie this month.
Jamie Bebbington, 19, who goes to Ravensbourne College, had been invited by charity Into Film to a Q&A and talk at the British Film Institute (BFI) in London to discuss The Amazing Spider-Man 2.
At last Wednesday’s event he met actor Dane DeHaan, who plays the villainous Green Goblin, as well as director Marc Webb and producers Matthew Tolmac and Avi Arad.
The cinema fan was one of a lucky few to put a question to the Hollywood panel, which has the likes of X-Men, 500 Days of Summer, and Iron Man to its credit, and said he was happy with the industry insight he gained.
He said: “I got to ask Marc Webb about the characters and how he went about transferring them from the comic book universe, to the film.”
Alongside 100 others he was also handed a 30-minute sneak peek of the superhero picture, which is set for release today (April 16).
The event was organised, in conjunction with BFI Future Film, as part of Into Film’s ‘Behind-The-Scenes’, which enables young people from all backgrounds meet the stars and realise their dreams.
The Amazing Spider-Man 2, which also stars Andrew Garfield, Emma Stone, and Jamie Foxx, was released yesterday (April 16).
- WATCH: Shocking video of man hit by driving instructor's car in Woolwich
- No more happy endings after New Cross 'sex cinema' is shut down
- PICTURED: Sidcup road closed both ways due to sinkhole
- 'She must be avoided': Hilarious sign warns of Barnehurst's 'dumpy pervert'
- Chart topper Jamie Lawson tells us how a chance meeting with Ed Sheeran in Balham changed his life


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Funeral services for Frank Joseph Kida, Sr., 71, of North Judson, Indiana are Saturday, Nov. 29, 2014 at 10 a.m. CT at SS Cyril and Methodius Catholic Church in North Judson. Visitation is Friday, Nov 28, 2014 from 4 p.m. to 8 p.m. CT with a 7:30 p.m. CT Rosary service at the O’Donnell Funeral Home in North Judson.
Starke County had an October unemployment rate of 6.1 percent. The percentage fell two points in October. Starke County is ranked 15th in the state.
Money has been earmarked for the construction of a shell building in the Knox Industrial Park.
Starke County Economic Development Foundation Executive Director Charlie Weaver told the Knox City Council members last night that companies are looking for existing structures to move right into to start up their businesses. Weaver said companies don’t want to have to go through the process of building the site with infrastructure, permitting and zoning issues. They want to have a building that they can customize to their needs and begin their business right away.
William Jones pleaded guilty in a plea agreement with the state to a charge of domestic battery as a Class D felony. Judge Kim Hall accepted the plea agreement and Jones was sentenced to 18 months in the Indiana Department of Corrections with the entire sentence suspended to be served on probation. Jones must pay a $50 domestic violence fee along with court costs.
The employees in the government offices in Pulaski County are collecting food for the Pulaski County Human Services Food Pantry.
Auditor Shelia Garling asked the commissioners for permission to place boxes in the courthouse to collect food for those who are less fortunate this holiday season. A box is located in the courthouse and in the Pulaski County Justice Center.
If you have a donation you’d like to drop off, get those in today by the end of the day to help someone in need at either location. Non-perishable food items are welcome, as well as soaps, deodorant, shampoos, and diapers.
The Marshall County Metronet project is drawing to a close.
The commissioners last week approved a payment agreement between the county and the City of Plymouth for shared conduit installation. The county is to pay half of the cost of the installation and handholds on Lincolnway to State Road 17 on Pioneer Drive, Pioneer Drive to Overmyer Drive and three lateral connections to the three county buildings for a total cost of $37,513.76. The city will pay the same cost for those installation costs.
Pulaski County Community Development Commission Executive Director Nathan Origer brought forth the idea of a redevelopment commission to the county commissioners during their recent meeting.
Origer explained that there are opportunities the county could be missing out on without a redevelopment commission.
“It allows basically two important things that we cannot do right now,” said Origer. “If ever a town or the county wanted to buy real estate for a downtown revitalization effort because they have absentee owners who are not making buildings usable for potential tenants, the RDC could much more easily purchase that and put any work into it as necessary. They can sell it or lease it to tenants than a municipal entity on its own could. The other thing is setting up TIF Districts.”
Funeral services for Linda Prater Patrick, 95, of Knox, Indiana are Sunday, Nov. 30, 2014 at 1 p.m. CT at the Braman and Son Memorial Chapel in Knox. Visitation is Saturday, Nov. 29, 2014 from 4 p.m. to 7 p.m. CT at the funeral home.
Gary Chavez appeared for a status hearing in Starke Circuit Court on Monday afternoon with his attorneys, Leslie Baker and Dave Welter. Starke County Prosecutor Nicholas Bourff indicated that there were some discovery issues with several pieces of evidence. Dave Welter told the judge that some medical reports are also needed and Chavez may require another surgery.
A 12-member jury found Curtis DeVries not guilty on a charge of burglary, but he was found guilty on a lesser included offense of residential entry, a Class D felony. The jury also found DeVries guilty on a charge of criminal mischief as a Class A misdemeanor. The verdicts came in on Friday, Oct. 24.
Judge Kim Hall told DeVries Monday that he has a large drinking problem that he needs to get under control. Judge Hall stated that if he hadn’t have consumed such a large amount of alcohol on the night of this event, he wouldn’t be in court facing a sentence.
Hendron was charged with maintaining a common nuisance and possession of methamphetamine. Hendron took the stand where he explained that he had held at position at a North Judson business for three decades. He left his long-time employment to care for his mother who moved into his home after being diagnosed with Alzheimers. He said that he’s been working out of his home attempting to make ends meet. He accepted that fact that what he did was wrong and is ready to put this behind him.
The Knox City Council will meet tonight where more discussion will be held regarding a dumpster ordinance.
When the council members last met, City Attorney David Matsey issued policy copies from other municipalities and to choose specific options that would pertain to city. He noted that the main goal is to restrict the time in which dumpsters can be placed at a site where a project is in progress. More information will be sorted out during tonight’s meeting.
Pulaski County Community Development Commission Executive Director Nathan Origer gave the commissioners an update on his discussions surrounding the changes in the CAGIT tax with the local legislators.
Origer had approached the commissioners at their earlier November meeting for support in overturning legislation to free up money for the CEDIT fund for economic development. He believes that the special CAGIT tax should be used to help pay the jail lease instead of the CEDIT fund. Origer argued that the CEDIT fund should be used for economic development projects and not just the jail lease. To do this, Origer needs to approach local legislators to change legislation allowing language to change the law to reflect his request.
The Marshall County Commissioners recently discussed a Memorandum of Understanding to continue the Meals on Wheels program at the Marshall County Council on Aging.
County Attorney Jim Clevenger stated that the current agreement expires Dec. 31 of this year and it needed to be renewed. The agreement allows the meals to be cooked at the Marshall County Jail at a cost of $3.25 per person. The personnel at the Marshall County Council on Aging pick up the meals at the jail and deliver them to those in need.
The Pulaski County Sheriff’s Department investigated an accident on Thursday involving a semi-tractor trailer and a 14-year-old pedestrian on U.S. 421 just south of Gordon Street in Francesville.
The truck driver, 57-year-old Jerry E. Dehaven of Rossville, Indiana, was southbound on U.S. 421 when he observed a pedestrian walking along the road, according to a news release. Dehaven stated that the pedestrian turned and walked into the southbound lane and he attempted to miss the pedestrian. He reportedly told police that he was unable to miss the pedestrian and she was struck by the center of the tractor-trailer knocking her to the ground.
Pulaski County EMS Director Nikki Lowry told the commissioners Monday night that she has hired seven part-time personnel to help with the shortage of staff and training is ongoing this week.
While that helps cover shifts, Lowry explained that only one of those hired is a paramedic. All of the other new hires are basic life service trained only. One may move to a full-time position. She said at least one paramedic is needed per shift and there is one on each shift now.
The Marshall County Commissioners held a public hearing during their meeting Monday morning concerning an ordinance regulating building setbacks at Pretty Lake.
Plan Director Ralph Booker explained that the Pretty Lake Homeowners Association approved a change where new construction at the lake would need to be a 45 foot setback from an 789.0 elevation line. The plan commission also approved the ordinance with one commission member against.
Request for a recount in two races in the General Election have been filed in the Starke County Clerk’s office.
Clerk Evelyn Skronski told WKVI News that a recount has been requested in the County Council District 4 race between Republican Don White and Democrat Pamela Stalbaum. Stalbaum had received 707 votes and White had 705 votes. The second request has been filed in the race for Railroad Township Trustee. Republican Mandy Thompson won the seat by three votes over Democrat Roger Chaffins, Sr.
The North Judson-San Pierre School Board will discuss several items tonight when they meet in the central office at 7 p.m. CT.
The balanced calendar will be discussed. Superintendent Lynn Johnson has previously stated that the calendars need to be similar with Knox and Oregon-Davis to accommodate students in the JESSE and vocational cooperative programs. Corporations normally adopt school calendars by January and the school board could adopt a calendar tonight.
The Knox City Police Department is investigating another church burglary.
According to police, the United Methodist Church at 201 S. Shield Street was broken into Saturday night or early Sunday morning. Electronic equipment was stolen and the building suffered a lot of damage.
Knox officers believe that two suspects forced entry to building. The incident remains under investigation.


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SHARE YOUR STORY 故事
Home has a different meaning for all of us. Is it tied to a place, a time? Is it a scent, a sound, a story? Maybe it’s a single memory or an intangible feeling that hits as you enter a room. Answer these quick questions and your story may be published in our digital and physical exhibition spaces.
Note: All fields are mandatory, except for the video link.


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A painting can be a mirror to our soul and to pause and gaze at the reflection is not vanity.
Friday, October 2, 2009
Award for Tree of Life
This drawing entitled Tree of Life won an award at the Canton Fine Arts Exhibition last night. It was very exciting to win an award from a show with such good work on display. There were 147 submitting artists, with over 500 entries and 41 of those artists were chosen each displaying one piece. It's such a good feeling. Cheers


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1. Department of Pharmaceutical Analysis, School of Pharmacy, Fourth Military Medical University, Xi'an 710032, China;
2. Innovation experimental college, Northwest A&F University, Yangling 712100, China;
3. State Key Laboratory of Military Stomatology, Fourth Military Medical University, Xi'an 710032, China;
4. Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China;
5. Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M University Health Science Center, Kingsville, Texas 78363, United States.
* These authors contributed equally to this work.
Poly(β-L-malic acid) (PMLA), a natural aliphatic polyester, has been proven to be a promising carrier for anti-cancer drugs. In spite of excellent bio-compatibility, the application of PMLA as the drug carrier for cancer therapy is limited by its low cellular uptake efficiency. The strong negative charge of PMLA impedes its uptake by cancer cells because of the electrostatic repulsion. In this study, a dual pH-sensitive charge-reversal PMLA-based nanocomplex (PMLA-PEI-DOX-TAT@PEG-DMMA) was developed for effective tumor-targeted drug delivery, enhanced cellular uptake, and intracellular drug release. The prepared nanocomplex showed a negative surface charge at the physiological pH, which could protect the nanocomplex from the attack of plasma proteins and recognition by the reticuloendothelial system, so as to prolong its circulation time. While at the tumor extracellular pH 6.8, the DMMA was hydrolyzed, leading to the charge reversal and exposure of the TAT on the polymeric micelles, thus enhancing the cellular internalization. Then, the polymeric micelles underwent dissociation and drug release in response to the acidic pH in the lyso/endosomal compartments of the tumor cell. Both in vitro and in vivo efficacy studies indicated that the nanocomplex significantly inhibited the tumor growth while the treatment showed negligible systemic toxicity, suggesting that the developed dual pH-sensitive PMLA-based nanocomplex would be a promising drug delivery system for tumor-targeted drug delivery with enhanced anticancer activity.
Keywords: PMLA, pH-sensitive, charge-reversal, TAT, cancer therapy.
Cancer is one of the leading causes of death around the world. According to the 2016 report of the National Center for Health Statistics, about 16.8 million new cancer cases and nearly 0.6 million of cancer deaths are projected to occur in the United States . Chemotherapy is one of the most commonly used options for cancer treatment, while it may cause various undesired side effects mainly due to its low tumor targetability and off-tumor toxicity [2, 3]. Due to the unique characteristics, nanoscale drug delivery systems (NDDS) have received considerable attention in biomedical applications [4-6]. Nanoparticles of certain sizes (< 200 nm) prefer to accumulate in the tumor site through a passive targeting mechanism, i.e. the enhanced permeation and retention (EPR) effect, and decrease the side effects and increase the drug's efficacy [7, 8].
In order to compose proper nanoparticles, a variety of nanomaterials have been investigated [9-11]. Due to the excellent properties, the natural or synthetic polymer-based carriers are of particular interests . Among them, the PMLA emerging as a novel nanomaterial has been proved to be biodegradable, non-toxic and non-immunogenic [13, 14]. Besides, a great number of pendant carboxyl groups on PMLA provide the extra opportunities for further functionalization [14-17]. However, the strong negative charge of PMLA increases the electrostatic repulsion with the negatively charged cell membrane, leading to the low cellular uptake, and restricts its application as the drug carrier .
An ideal nanoparticle-based drug delivery system should possess the key characteristics, such as long circulation, high tumor accumulation and cellular uptake. PEGylation is one of the mostly used technologies to engineer the surface of the nanoparticles, to shield the undesired properties. The PEGylated nanoparticles usually have long blood circulation time due to the PEG's hindrance to the plasma proteins and the reticuloendothelial system (RES), which facilitates the EPR effect-mediated tumor targeting. However, PEGylation impedes the internalization of nanoparticles by cancer cells, and therefore is not beneficial for the cellular uptake [19-22]. In contrast, the positively charged nanoparticles effectively interact with the negatively charged cell membrane by the electrostatic attraction and trigger the efficient cell internalization [23, 24]. Nevertheless, the positive charge may cause nonspecific interactions of the nanoparticles with serum components and cell membrane of the normal cells, resulting in short circulation time and nonspecific distribution [25, 26].
Cell-penetrating peptides (CPPs), such as transactivator of transcription (TAT) peptide, were able to translocate across the cell membrane via various mechanisms. The CPP-conjugated nanoparticles have been extensively investigated as the intracellular drug delivery tools . However, the lack of selectivity of CPPs may cause undesired side effects, which is one of the major obstacles and remains unresolved . This dilemma is similar to that of positive surface charge of the nanoparticles. In response to these challenges, the stimuli-responsive NDDS has emerged as a smart tumor-targeted drug delivery system. Among various stimuli, pH is the most extensively exploited one for tumor-targeted drug delivery due to the pH difference between the normal tissues (pH 7.4) and tumor extracellular environment (pHe 6.8) [29, 30].
Here, a dual-pH sensitive and charge-reversal PMLA-based nanocomplex was prepared for effective drug delivery, enhanced cellular uptake, and intracellular drug release (Fig. 1). The PMLA was first modified with polyethylenimine (PEI) to allow the polymer to bear the positive charge (PMLA-PEI). Covalent conjugation of pH-sensitive cis-aconitic anhydride-modified doxorubicin (DOX-CA) with PMLA afforded the amphiphilic co-polymers that readily formed the polymeric micelles by the dialysis method (PMLA-PEI-DOX). The TAT peptide was conjugated to the PMLA (PMLA-PEI-DOX-TAT) to further increase the cellular uptake efficiency of polymeric micelles. Then, the 6-armed polyethylene glycol (PEG) decorated with 2,3-dimethylmaleic anhydride (DMMA) (PEG-DMMA), was used to 'wrap'/modify the polymeric micelles, to shield the positive charge and TAT peptide. The obtained dual pH-sensitive charge-reversal nanocomplex (PMLA-PEI-DOX-TAT@ PEG-DMMA) was a 'Trojan horse'-like system. In the blood (at normal pH), the PEGylated nanocomplex is negatively charged, which prolongs its circulation time and minimizes the rapid clearance of the nanocomplex from the body. However, the nanocomplex undergoes the PEG deshielding via the pH-sensitive hydrolysis of DMMA in the tumor microenvironment, leading to a quick conversion from the negative charge to the positive charge. The resultant positively charged and TAT exposed polymeric micelles would be efficiently internalized by the tumor cells. After endocytosis, the polymeric micelles could rapidly release the loaded drugs at the endosomal/lysosomal pH, to facilitate the drug action. All these make this dual-pH sensitive charge-reversal nanocomplex as a smart drug nanocarrier with enhanced tumor inhibition efficacy.
The entire synthetic routes for PMLA-PEI-DOX-TAT, PEG-DMMA and PEG-SA are illustrated in Fig. S1. Firstly, PMLA was modified by PEI, and the degree of substitution (DS) of PEI over PMLA was 0.4%. Then pH-sensitive doxorubicin prodrug (DOX-CA) was synthesized through the reaction between doxorubicin and cis-aconitic anhydride. After that, DOX-CA reacted with 1,4-diaminobutane to provide amino groups for the conjugation with PMLA, thus polymer PMLA-PEI-DOX was obtained. In order to conjugate TAT to the polymer, TAT-cysteine peptide firstly reacted with N-(2-aminoethyl)maleimide through maleimide-thiol covalent coupling, and since N-(2-aminoethyl) maleimide could provide the amino groups, then the polymer PMLA-PEI-DOX-TAT was obtained by the reaction between the amino groups and carboxyl groups in PMLA. The obtained PMLA-PEI-DOX-TAT was identified by 1H NMR. As shown in Fig. S2, the signals at 2.95 and 5.38 ppm were assigned to methylene and methane protons of PMLA respectively. The signals at 2.13-2.33 ppm were assigned to the protons of -NHCH2CH2- of PEI. The signals at 3.99, 4.58 and 7.93 ppm were assigned to the benzene, methyl and methane protons of DOX respectively. The 1H-NMR spectra indicated the successful synthesis of polymer PMLA-PEI-DOX-TAT.
Due to extreme sensitivity to tumor pHe, DMMA was used to design pH-sensitive deshielding PEG layer [31-33]. Non charge-reversal SA-conjugated PEG was chosen as a control. DMMA or SA was conjugated to 6NH2-PEG through the reaction between the amines in 6NH2-PEG and the anhydride in DMMA or SA, and the resultant PEG-DMMA and PEG-SA were also identified by 1H NMR. The signals at 3.09-3.84 ppm were assigned to methylene protons of PEG. While the signal at 1.97 ppm represented the methyl of DMMA, the signals of methyl of SA appeared at 2.49 and 2.46 ppm after the conjugation (Fig. S3 and S4). The 1H-NMR spectra indicated the successful synthesis of PEG-DMMA and PEG-SA. According to UV-vis absorbance at 481 nm, the DOX content in the polymer-drug conjugates was 17.71 wt.% for PMLA-PEI-DOX and 16.82 wt.% for PMLA-PEI-DOX-TAT (Table S1).
(a) Preparation of nanocomplex PMLA-PEI-DOX-TAT@PEG-DMMA. (b) Illustration of the dual pH-sensitive property, pH-sensitive charge-reversal and pH-sensitive drug release. (c) Schematic illustration of the dual pH-sensitive DOX-loading nanocomplex with the charge-conversional function for effective tumor-targeted drug delivery, enhanced cellular uptake, and intracellular drug release.
The polymeric micelles were then constructed from the PMLA-PEI-DOX-TAT via self-assembly, followed by shielding with the pH-sensitive charge-reversal PEG-DMMA or non-charge-reversal PEG-SA through electrostatic adsorption. The four types of the polymeric micelles (PMLA-PEI-DOX and PMLA-PEI-DOX-TAT) or nanocomplexes (PMLA-PEI-DOX-TAT@PEG-SA and PMLA-PEI-DOX-TAT@PEG-DMMA) were prepared. In our previous study, the charge-conversional nanoconjugates based on PMLA were prepared for effective and specific anti-tumor drug delivery . Compared to the nanoconjugates, the nanocomplex was more stable and convenient, more importantly, possessed the stimuli-responsive properties, the pH-sensitive charge reversal and pH-sensitive drug release.
The dynamic light scattering (DLS) measurement showed that the average particle size of the DOX-loaded nanoparticles was 108.3 ± 5.5 nm for PMLA-PEI-DOX, 95.0 ± 5.7 nm for PMLA-PEI-DOX-TAT, 126.1 ± 3.5 nm for PMLA-PEI-DOX-TAT@PEG-SA, and 123.0 ± 2.2 nm for PMLA-PEI-DOX-TAT@PEG-DMMA, respectively (Fig. 2a, Fig. S5 and Table S1). Based on the TEM image, the PMLA-PEI-DOX-TAT@PEG-DMMA showed a spherical morphology with a size of 100.6 ± 3.1 nm that was smaller than DLS measurement due to the hydrodynamic radius in the aqueous buffer (Fig. 2a). The zeta potential of these micelles were +10.47 ± 0.51 mV, +23.51 ± 0.76 mV, -16.04 ± 0.34 mV and -16.33 ± 0.63 mV, respectively. The CMC value of PMLA-PEI-DOX-TAT was 5.222 mg/L, which was determined by fluorescent spectroscopy using pyrene as a probe (Fig. S5d). These results indicated that DOX-conjugated polymers were successfully self-assembled into the core-shell structured micelles in aqueous solution, in which DOX molecules were packed in the core through hydrophobic interaction, while PMLA-PEI acted as the shell. Moreover, the nanocomplex PMLA-PEI-DOX-TAT@PEG-DMMA exhibited excellent stability in different physiological solutions, including PBS (pH 7.4) and RPMI 1640 medium with 10% FBS (Fig. S6b).
(a) DLS and TEM results of the nanocomplex PMLA-PEI-DOX-TAT@PEG-DMMA, the scale bar is 200 nm. (b) Release profile of DOX from the nanocomplex PMLA-PEI-DOX-TAT@PEG-DMMA in PBS (pH 5.0, pH 6.8 or pH 7.4) at 37 ºC. (c) Zeta potential changes of PMLA-PEI-DOX-TAT@PEG-DMMA and PMLA-PEI-DOX-TAT@PEG-SA nanocomplexes when incubating at pH 7.4 and 6.8 for different time periods.
In our previous study, DOX was directly conjugated to PMLA via the amide bond and the formed nanoconjugates showed a pH-sensitive drug release pattern . Nevertheless, it's uncertain whether the released drug formulation was free DOX or not, the pH-sensitive pattern probably caused by the hydrolysis of PMLA at acid environment. Thus, in this work, DOX was conjugated to PMLA-PEI via a pH-sensitive maleic acid amide bond and ensured effective release of free DOX in response to the acidic endosomal/lysosomal pH. As shown in Fig. S7, free DOX exhibited its peak at an elution time of 2.82 min and DOX-CA exhibited its peak at an elution time at 1.15 min. Furthermore, the incubation of DOX-CA at pH 4.0 and pH 5.0 resulted in significant degradation, with a half-life of 3.5 h and 1 h respectively (Fig. S7b). The intensity of the peaks DOX-CA decreased while the intensity of the peaks of free DOX increased sharply at pH 5.0 and pH 4.0. In contrast, no degradation was observed and the maleic acid amide bonds in DOX-CA were very stable under pH 7.4 and pH 6.8 at 37 °C for 12 h. In addition, the incubation of PMLA-PEI-DOX-TAT at acidic environment revealed that the released drug formulation from the polymer was free DOX (Fig. S7c). These results indicated that the maleic acid amide bonds in DOX-CA could be cleaved under endo/lysosomes conditions of cancer cells, resulting in the release of free DOX, while remain stable under normal physiological conditions and in extracellular environment.
Release of DOX from the nanocomplex PMLA-PEI-DOX-TAT@PEG-DMMA was also measured in order to confirm the pH sensitivity of the nanocomplex. The in vitro DOX release profile of the nanocomplex was investigated at different pH values (Fig. 2b). The nanocomplex exhibited the relatively high stability at pH 7.4 with a no more than 20% DOX release after 72 h, while the drug release rate slightly increased at pH 6.8, which was caused by the hydrolysis of the DMMA. However, when the pH declined to 5, a rapid drug release was observed. The DOX release at pH 5.0 was in a biphasic manner, an initial rapid release in 12 h followed by a slow and continuous release for up to 72 h, and the cumulative release of DOX from the nanocomplex was over 90% after 3 days. This result was due to the quick cleavage of the acid-sensitive amide bond between DOX and PMLA at lower pH (the endosomal and lysosomal environments). This suggested that the nanocomplex PMLA-PEI-DOX-TAT@PEG-DMMA might hold the drugs under physiological condition and reduce the in vivo drug leakage, and release the drug in response to the endosomal pH (pH 5~6) or lysosomal pH (pH 4~5), to ensure the drug action in the intracellular compartment, i.e. cell nuclei.
DMMA was extremely sensitive to acidic tumor extracellular microenvironment (pHe, ~6.8), the negatively charged polymer PEG-DMMA could be hydrolyzed to become positively charged 6NH2-PEG, and then the PEG layer could detach from the positively charged PMLA-PEI-DOX-TAT due to the electrostatic repulsion. This property was confirmed by the zeta potential of PMLA-PEI-DOX-TAT@PEG-DMMA at the tumor extracellular pH 6.8 and normal physiological pH 7.4 (Fig. 2c). PMLA-PEI-DOX-TAT@PEG-SA was chosen as a control, because PEG-SA was stable under the acidity of the tumor. At pH 7.4, both PMLA-PEI-DOX-TAT@PEG-DMMA and PMLA-PEI-DOX-TAT@PEG-SA exhibited non-charge reversal characteristics with a consistent negative charge after incubation at 37 °C for 4 h, while at pH 6.8, PMLA-PEI-DOX-TAT@PEG-DMMA showed obvious charge-reversal property from the negative charge to positive charge (from -16.33 to +10.81 mV) after incubation at 37 °C for 4 h, and PMLA-PEI-DOX-TAT@PEG-SA did not exhibit the charge conversion.
It is believed that most of amide bonds only can be degraded at extreme pH values (pH < 1 or pH > 13), while maleic acid amide derivatives can be degraded at mild acidic pH values [34, 35]. In this study, we used two maleic acid amide derivatives, cis-aconitic acid amide and dimethylmaleamidic acid amide, to obtain pH-sensitive degradability, one for pH-sensitive drug release and the other one for pH-sensitive charge-reversal. cis-aconitic acid amide was used as pH-sensitive linker between DOX and PMLA since it contains two carboxylate groups, the β-carboxylate group for pH degradability to release free DOX and the other group for linking with PMLA. Dimethylmaleamidic acid amide was employed to design acidity-controlled charge-reversal deshielding systems due to its extreme sensitivity to tumor pHe. The difference of pH-sensitivity between two maleic acid amide derivatives was caused by the difference of their chemical structure, particularly the substituents of the cis-double bonds. cis-aconitic acid amide with one carboxyethyl and one hydrogen substituent showed appropriate degradability at the endosomal/lysosomal pH, while two methyl groups substituent on dimethylmaleamidic acid amide played a crucial role in the extreme pH-sensitivity (pHe).
Confocal laser scanning microscopy (CLSM) analysis and flow cytometry study were performed to investigate the effects of the pH-sensitive charge-reversal property on the cellular uptake of nanoparticles. The Cy3-labeled polymeric micelles (PMLA-PEI-DOX and PMLA-PEI-DOX-TAT) or nanocomplexes (PMLA-PEI-DOX-TAT@PEG-SA and PMLA-PEI-DOX-TAT@PEG-DMMA) were incubated with cancer cells for 4 hours at pH 7.4 or 6.8.
As shown in Fig. 3, in A549 cells, the PMLA-PEI-DOX-TAT group showed the strongest intracellular fluorescence of Cy3 at both pH 7.4 and 6.8, indicating the combination of positive charge and TAT could greatly enhance the cellular uptake. Compared with PMLA-PEI-DOX-TAT group, the PMLA-PEI-DOX group showed much lower intracellular fluorescence, suggesting that TAT was more efficient to induce the endocytosis than the PEI. At pH 7.4, both nanocomplexes (PMLA-PEI-DOX-TAT@PEG-SA and PMLA-PEI-DOX-TAT@PEG-DMMA) showed low intracellular fluorescence. The electrostatic repulsion between the negatively charged nanoparticles and cell membranes decreased the cell uptake. However, when the pH declined to 6.8, due to the exposure of positively charged TAT-conjugated polymeric micelles, the PMLA-PEI-DOX-TAT@PEG-DMMA group showed much higher intracellular fluorescence. In contrast, the intracellular fluorescence of PMLA-PEI-DOX-TAT@PEG-SA group was similar to that at pH 7.4. As shown in Fig. 4, at pH 7.4, the cellular uptake of the PMLA-PEI-DOX-TAT@PEG-SA and PMLA-PEI-DOX-TAT@PEG-DMMA was almost identical. PMLA-PEI-DOX showed a little bit higher cellular uptake due to the positive charge surface, and the uptake of PMLA-PEI-DOX-TAT was the highest. While at pH 6.8, the cellular uptake of PMLA-PEI-DOX-TAT@PEG-DMMA was greatly enhanced. All these results were consistent with CLSM analysis.
Confocal microscopic images of cellular internalization of Cy3-labeled nanoparticles by A549 cells incubated at pH 7.4 and 6.8 for 4 h. DAPI (blue) was used to stain cell nucleus. The scale bar is 100 μm.
(a) Flow cytometry analysis of cellular uptake of Cy3-labeled nanoparticles by A549 cells at pH 7.4 and pH 6.8 conditions for 4 h. (b) Quantitative analysis of the fluorescence intensity of Cy3-labeled nanoparticles in A549 cells.
To further confirm the cellular uptake results, in addition to the lung cancer cells A549, human hepatocellular carcinoma cells MHCC97-H were also used as the in vitro cancer models. In MHCC97-H cells, the PMLA-PEI-DOX-TAT group showed the highest cellular uptake at both pH 7.4 and 6.8 (Fig. S9 and S10), which was consistent with the results in the A549 cells. In addition, the nanocomplexes PMLA-PEI-DOX-TAT@PEG-SA and PMLA-PEI-DOX-TAT@PEG-DMMA displayed comparable intracellular fluorescence at pH 7.4, while the cellular uptake of the positively charged polymeric micelles PMLA-PEI-DOX was about 4-fold increased (Fig. S10), indicating that the positive charge could facilitate the interaction with negatively charged cell membrane to promote the cellular internalization. However, when the pH decreased from 7.4 to 6.8, the hydrolysis of DMMA caused the charge reversal of PEG layer, resulting in the exposure of the positively charged TAT-conjugated polymeric micelles (PMLA-PEI-DOX-TAT), so as to enhance the cell internalization. All these results indicate that the combination of the positive charge and TAT could greatly promote the cellular uptake of the nanoparticles and the cellular uptake of the nancomplex, PMLA-PEI-DOX-TAT@PEG-DMMA, was pH-dependent.
In vitro antitumor activities of various DOX formulations were evaluated by Cell Counting Kit-8 (CCK-8) assay on A549 and MHCC97-H cells, and 50% inhibitory concentration (IC50) was determined. In A549 cells, as shown in Fig. 5 and Table S2, the pH value of the culturing media hardly affected the cytotoxicity of the free DOX, PMLA-PEI-DOX, PMLA-PEI-DOX-TAT and PMLA-PEI-DOX-TAT@PEG-SA. The PMLA-PEI-DOX-TAT@PEG-DMMA showed the comparable cytotoxicity with the PMLA-PEI-DOX-TAT@PEG-SA at pH 7.4, but had an 8.97-fold lower IC50 value at pH 6.8. Higher cytotoxicity was probably caused by higher cellular uptake. At both pH 7.4 and 6.8, the PMLA-PEI-DOX-TAT showed the lowest IC50 value, indicating the great cellular uptake efficacy caused by the combinatory effects of the positive charge and TAT. In MHCC97-H cells, at pH 7.4, no statistical difference was observed between the PMLA-PEI-DOX-TAT@PEG-SA and PMLA-PEI-DOX-TAT@PEG-DMMA (Fig. S11 and Table S3). However, the cell death increased significantly when the cancer cells were incubated with PMLA-PEI-DOX-TAT@PEG-DMMA at pH 6.8 due to the charge-reversal induced cellular internalization.
The cytotoxicity profiles of various DOX formulations indicated that the combination of the positive charge and TAT in the nanoparticles would have a synergistic effect on cytotoxicity. Besides, the cytotoxicity of polymers (PMLA-PEI, PMLA-PEI-TAT, PEG-DMMA and PEG-SA) without DOX conjugation was also evaluated. As shown in Fig. S11c, the polymers without DOX conjugation exhibited negligible cytotoxicity against A549 cells at various concentrations ranged from 0.1 to 1000 µg/mL for 48 h at pH 7.4, which indicated that the polymers were biocompatible and safe.
The antitumor activity of the nanocomplex, PMLA-PEI-DOX-TAT@PEG-DMMA, and other DOX formulation were measured in A549 xenograft mouse model. Firstly, in order to demonstrate the targeting effect of the nanoparticles, the bio-imaging study was conducted using an in vivo fluorescence imaging system. Fluorescent images of the treated mice were recorded at 24 h post injection using an IVIS® Spectrum Imaging System. As shown in Fig. 6, mice treated with Cy3-labled nanocomplexes, PMLA-PEI-DOX-TAT@PEG-SA and PMLA-PEI-DOX-TAT@PEG-DMMA, showed strong fluorescence signal at the tumor site, indicating a better tumor targeting efficiency. In contrast, almost no fluorescence was detected in the tumor after 24 h of treatment with Cy3-labled polymeric micelles PMLA-PEI-DOX and PMLA-PEI-DOX-TAT. The relatively stronger fluorescence was observed mainly in the liver and kidney, indicating that the positively charged nanoparticles, PMLA-PEI-DOX and PMLA-PEI-DOX-TAT, would be more likely to be captured by the liver and kidney rather than the tumor. Although the mice treated with the negatively charged and PEG-coated nanocomplexes showed some fluorescence in the liver and kidney, the fluorescence is remarkably low compared to the positively charged polymeric micelles. All these results suggest that the negative surface charge and PEG coating could reduce the nonspecific protein binding, avoid the opsonization by RES, and facilitate the ERP effect-mediated tumor accumulation.
As shown in Fig. 7a, the tumor volume of the saline-treated group rapidly increased over 21 days. The PMLA-PEI-DOX-TAT and PMLA-PEI-DOX@PEG-SA moderately inhibited tumor growth. In contrast, the PMLA-PEI-DOX@PEG-DMMA displayed the highest tumor inhibition effect. The growth of tumors on free DOX injected mice was slightly inhibited over 9 days. Moreover, we also measured the weight of each tumor mass at the end of the treatment (Fig. 7c), which confirmed that the tumor growth inhibition data. In addition, the H&E and TUNEL examination of tumor sections (Fig. 7d) showed that the PMLA-PEI-DOX-TAT@PEG-DMMA treated group resulted in significantly reduced proliferation and increased apoptosis compared that of the other groups.
Cytotoxicity of Free DOX and various nanoparticles against A549 cells at different concentrations of DOX under (a) pH 7.4 and (b) pH 6.8 for 48 h.
In vivo imaging of tumor bearing mice. Fluorescent signal captured by IVIS Lumina Imaging System in tumor bearing mice after injection with Cy3-labeled PMLA-PEI-DOX (a), PMLA-PEI-DOX-TAT (b), PMLA-PEI-DOX-TAT@PEG-SA (c) and PMLA-PEI-DOX-TAT@PEG-DMMA (d) for 24 h.
The antitumor effect of nanocomplex on nude mice bearing A549 cells subcutaneously. (a) The variation profiles of tumor volumes during injection. (b) The variation profiles of body weights during injection. (c) Dissected tumor tissues from the nude mice (Free DOX group, survived for 9 days and the weight of dissected tumor tissues. (d) H&E and TUNEL examination of the tumors from mice bearing A549 mammary tumors after 21-day injection. Data was given as mean ± SD (n=4). The scale bar is 100 μm.
Histopathological analysis. Images of tissue sections stained with Hematoxylin and Eosin after treatments with Saline, Free DOX, PMLA-PEI-DOX-TAT, PMLA-PEI-DOX-TAT@PEG-SA and PMLA-PEI-DOX-TAT@PEG-DMMA. The scale bar is 100 μm.
Body weight changes in all mice groups, as an indicator of systemic toxicity, were measured (Fig.7b). Serious systemic toxicity was observed in animals treated with free DOX (p<0.05). The death of mice was observed on the 9th after free DOX treatment. However, the body weight of other four groups was not obviously affected over the investigation period, indicating that the nanoparticles could reduce the side effect of drugs. In addition, the histological analysis of the main organs including heart, liver, spleen, lung, and kidney was shown in Fig. 8. There were no apparent damages in the detected organs from free DOX group, PMLA-PEI-DOX-TAT group, PMLA-PEI-DOX-TAT@PEG-SA group, PMLA-PEI-DOX-TAT@PEG-DMMA group, and control group. The results demonstrate that the nanocomplex PMLA-PEI-DOX-TAT@PEG-DMMA could improve the drug's anticancer activity and decrease its side effects.
In summary, a dual pH-sensitive charge-reversal nanocomplex PMLA-PEI-DOX-TAT@PEG-DMMA was successfully prepared for effective drug delivery, enhanced cellular uptake and intracellular drug release. At normal physiological condition (pH 7.4), the nanocomplex showed a negative surface charge. While at tumor extracellular microenvironment (pHe 6.8), the pH-sensitive charge-reversal PEG-DMMA layer would be taken off, resulting in the exposure of the positively charged TAT-conjugated polymeric micelles, so as to enhance cell internalization. After internalization, the polymeric micelles could facilitate the escape from endosome/lysosome of tumor cells, simultaneously trigger the drug release, and ensure the nuclear delivery of the anticancer drug. The nanocomplex showed high tumor accumulation and excellent tumor growth inhibition in both the cancer cells and xenograft. The study indicated that the nanocomplex, PMLA-PEI-DOX-TAT@PEG-DMMA, could be a promising nanocarrier for tumor-targeted drug delivery with enhanced anticancer activity.
TAT-cysteine peptide (TATp-Cys 12-mer: CysTyrGlyArgLysLysArgArgGlnArgArgArg; molecular mass 1662.99 Da with one reactive thiol group) was synthesized by KE BIOCHEM company (Shanghai, China). Polyethyleneimine (PEI with a MW of 1.8 kDa, branched) and Doxorubicin hydrochloride (DOX•HCl) were purchased from Sigma-Aldrich (Shanghai, China). 2,3-dimethylmaleic anhydride (DMMA), succinic anhydride (SA) and cis-aconitic anhydride (CA) were purchased from Alfa Aesar (Beijing, China). N-(2-aminoethyl)maleimide trifluoroacetic acid was purchased from Energy Chemical (Shanghai, China). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC•HCl) and N-hydroxysuccinimide (NHS) were purchased from TCI (Shanghai, China). 1,4-diaminobutane and N-(2-aminoethyl)maleimide trifluoroacetic acid were purchased from J&K SCIENTIFIC LTD (Shanghai, China). 6-armed polyethylene glycol (PEG) (Mw: 10 kDa) was purchased from Sunbio (Korea). Cy3-PEG2000-NH2 was purchased from Nanocs Inc. (New York, USA). HPLC-grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). All solvents were thoroughly dried and distilled before use. RPMI-1640 medium, Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone Cell Culture and Bioprocessing (Thermo Scientific, USA). EnoGeneCellTM Counting Kit-8 (CCK-8) and 4',6-diamidino-2-phenylindole (DAPI) were purchased from Beyotime Institute of Biotechnology (Shanghai, China).
PMLA (Mw: 7.89 kDa) was synthesized by ring-opening polymerization and then modified with PEI to obtain positive charge as described in our previous study . DOX-CA was synthesized by the ring-opening reaction between DOX and CA with triethylamine (TEA) as catalyst. In DMF (10 mL), DOX▪HCl (58 mg), CA (17.2 mg) and triethylamine (8.4 μL) were added. After reacting at room temperature under nitrogen atmosphere for 24 h, the mixture was added EDC▪HCl (40 mg) and NHS (25 mg) for another 4 h reaction. Then, the mixture was dropwise added into1,4-diaminobutane (0.11 mmol, 9.8 mg) solution for 12 h reaction in dark. After that, pH-sensitive DOX-CA was conjugated to PMLA-PEI via amide reaction, thus polymer PMLA-PEI-DOX was obtained. TAT-cysteine peptide (0.003mmol, 5 mg) and N-(2-aminoethyl)maleimide (0.003mmol, 0.765 mg) were dissolved in 4 mL DMSO, then the solution was stirred at room temperature for 12 h. Subsequently, the solution was added into the solution of polymer PMLA-PEI-DOX for 12 h reaction to obtain polymer PMLA-PEI-DOX-TAT. Finally, the mixture was dialyzed against water for 48 h by using a dialysis membrane with MWCO of 12 kDa. After being frozen dried, the obtained polymer PMLA-PEI-DOX-TAT was stored at -20 °C for further use. In addition, Cy3-labled polymers were synthesized by the reaction between the amino groups in Cy3-PEG-NH2 and carboxyl groups in PMLA.
In ~ pH 8.5 PBS (20 ml), 6NH2-PEG (50 mg, 10 kDa) was dissolved. Then, DMMA (152 mg) or SA (127 mg) was added slowly into the solution. After reacting at room temperature for 24 h, the mixture was dialyzed against water for 48 h by using a dialysis membrane with MWCO of 8 kDa. After being frozen dried, the obtained polymer PEG-DMMA or PEG-SA were stored at -20 °C for further use.
The synthesized polymers PMLA-PEI-DOX-TAT, PEG-DMMA and PEG-SA were confirmed by 1H NMR. NMR spectra were obtained by Varian 400 MHz (Bruker, Germany). Chemical shifts were expressed as parts per million (ppm). UV-Vis spectroscopy (Shimadzu, Japan) was used to quantify the amount of DOX conjugated on the polymers. Briefly, DOX-conjugated polymers were dissolved in deionized water and the absorbance of the solutions at 481 nm was measured. Using a calibration curve obtained by measuring the absorbance of different concentrations of free DOX in deionized water at 481 nm, and the DOX content in the polymer was calculated. The critical micelle concentration (CMC) of polymer was determined with fluorescent spectroscopy using pyrene as a probe. Polymer solution with a series of concentration range from 8×10-4 to 0.8 mg/mL and a solution of pyrene in acetone (1×10-5 mol/L) were prepared. Then, 1 mL pyrene solution was taken, after evaporation of the solvent, 10 mL polymer solution was added. The excitation wavelength was set at 335nm and the measured emission wavelengths were set at 373 and 383 nm.
The PMLA-PEI-DOX-TAT micelle was prepared as described below: PMLA-PEI-DOX-TAT (80 mg) was dissolved in DMSO (2 mL), and the solution in DMSO was added dropwise to the water under stirring. The mixed solution was dialyzed in 10 kDa dialysis bag with the water changing every 2 h for 24 h. Then polymeric micelles PMLA-PEI-DOX-TAT complexed with PEG-DMMA or PEG-SA was proceeded. PMLA-PEI-DOX-TAT (1.0 mL, 1 mg/mL in water) was added dropwise to PEG-DMMA or PEG-SA (2.0 mL, 1 mg/mL in water), respectively. The solution was stirred at room temperature overnight before being frozen dried, and the obtained EG-DMMA or PEG-SA complexed PMLA-PEI-DOX-TAT@PEG-DMMA or PMLA-PEI-DOX-TAT@PEG-SA was stored at -20 °C for further use. The particle size of polymeric micelles and nanocomplexes were measured by DelsaTM Nano C Particle Analyzer (BECKMAN COULTER, USA), and the morphology of polymeric micelles and nanocomplexes were observed via transmission electron microscopy (TEM).
Nanocomplex (PMLA-PEI-DOX-TAT@PEG-DMMA) (0.5 mL, 1.0 mg/mL) was incubated with phosphate-buffered saline (PBS) (pH 7.4, 0.5 mL), or Roswell Park Memorial Institute (RPMI) 1640 medium contained 10% FBS (0.5 mL). At designated time intervals, the solution of nanocomplex was used for DLS measurements.
The zeta potentials of nanocomplexes (PMLA-PEI-DOX-TAT@PEG-DMMA and PMLA-PEI-DOX-TAT@PEG-SA) were measured by photon correlation spectroscopy using DelsaTM Nano C Particle Analyzer (BECKMAN COULTER, USA) at 25 °C. Briefly, 2 mg of nanocomplexes was added into 2 mL PBS solution (phosphate buffered saline, pH 7.4 or pH 6.8). At designated time intervals, a certain volume of solution was withdrawn and used for zeta potential measurements.
DOX-CA was incubated with different pH values of phosphate-buffered saline (PBS) solution (pH 4.0, pH 5.0, pH 6.8 and pH 7.4). At designated time intervals, the solution was used for high performance liquid chromatography (HPLC) (Shimadzu 1630, Japan) measurements. HPLC analysis was performed on Agilent 1260 Infinity LC system (Agilent, Germany). Chromatographic separation was carried out at room temperature on a CAPCELL PAK C18 column (3.0 mm × 100 mm, 3 μm, SHISEIDO, Japan). The mobile phase consisted of solvent A (sodium dodecyl sulfate solution), solvent B (acetonitrile) and solvent C (methanol). The flow rate was 0.8 ml/min and the injection volume was 10 μL. The detection wavelength was set to 254 nm. Then, the release rate of DOX from the nanocomplex (PMLA-PEI-DOX-TAT@PEG-DMMA) was investigated by a dialysis method. Typically, 1 mL of nanocomplex solution (2 mg/mL) was dialyzed (MWCO: 3.5 kDa) against 20 mL PBS solution (pH 7.4, 6.8 or 5.0) at 37 °C. At given intervals, 100 μL of external buffer was collected and replaced by the same volume PBS solution. The concentration of DOX released from polymeric micelles was quantified by ultraviolet spectrophotometry (Shimadzu, Japan) at 481 nm.
The human lung epithelial tumor cell line A549 and human hepatocellular carcinoma cell line MHCC97-H were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. In all experiments, A549 cells and MHCC97-H cells were cultivated in RPMI-1640 and DMEM medium supplemented with 10% fetal bovine serum respectively at 37 °C in a humidified 5% CO2/95% air atmosphere.
A549 cells and MHCC97-H cells were seeded into 6-well plates at a density of 3×105 cells/well and incubated overnight. For cellular internalization observation, cells were incubated with the polymeric micelles (PMLA-PEI-DOX and PMLA-PEI-DOX-TAT) or nanocomplexes (PMLA-PEI-DOX-TAT@PEG-SA and PMLA-PEI-DOX-TAT@PEG-DMMA) at an equivalent Cy3 concentration of 0.25 μg/mL of fresh culture medium at pH 7.4 or 6.8, respectively. After incubation for 4 h, the cells were washed three times with PBS solution. The cells were then harvested by trypsinization and centrifuged at 1000 rpm for 5 min, resuspended with 500 μL PBS and analyzed by a FACScan instrument (Becton Dickinson, USA).
Confocal fluorescent microscopy was used to compare the cellular uptake of Cy3 labeled polymeric micelles or nanocomplexes. Similar to flow cytometry, A549 cells and MHCC97-H cells were seeded into glass-bottomed dishes at a density of 3×105 cells/well and incubated overnight. Then, the cells were treated with various Cy3 labeled polymeric micelles or nanocomplexes in fresh culture medium at pH 7.4 or 6.8, respectively. After 4 h incubation, the cells were washed three times with PBS solution to remove the remnant growth medium and fixed in 4% paraformaldehyde for 10 min, followed by cell nuclei staining with DAPI for 15 min. After the cells were washed with PBS solution, fluorescent images of cells were analyzed by using a FV1000 confocal microscope (Olympus, Japan).
The cytotoxicity of free DOX, polymeric micelles and nanocomplexes against A549 cells and MHCC97-H cells were investigated by using Cell Counting Kit-8 (CCK-8) assay. The cells were seeded into 96-well plates at a density of 1×104 cells per well and incubated overnight. Then the medium was replaced by free DOX, polymeric micelles or nanocomplexes in cell culture medium with different pH (6.8 or 7.4) and incubated for 48 h. After that, 10 μL CCK-8 solution was added to each well of the plate. Then, the plate was incubated for 2 h. Cell viability was determined by scanning with a microplate reader at 450 nm. The viability rate (VR) was calculated according to the manufacturer's instructions. Besides, the cytotoxicity assessment of polymers (PMLA-PEI, PMLA-PEI-TAT, PEG-DMMA and PEG-SA) without DOX conjugated was also conducted similarly to the above process, at pH 7.4 with various concentrations (0.1 to 1000 µg/mL).
Five-week-old BALB/c athymic nude mice (~ 20 g) were purchased from Peking University Health Science Center and feed at the condition of 25 °C and 55% of humidity. All animal experiments were approved by the Animal Research Committee of the Fourth Military Medical University and conducted in accordance with the international standards on animal welfare.
First, the tumors were built up by subcutaneous injection of A549 cells into the left axilla of each female mouse with a density of 5 × 106 cells in 100 μL PBS. After growing for 1 week, the tumors reached the size of about 6-8 mm. Then, the tumor-bearing mice were randomly divided into five groups (n = 4 for each group) and were treated by intravenous injection with saline, free DOX, PMLA-PEI-DOX-TAT, PMLA-PEI-DOX-TAT@PEG-SA and PMLA-PEI-DOX-TAT@PEG-DMMA, respectively. The injected amount for different polymeric micelle or nanocomplexes was 200 μL saline with DOX dose of 5 mg/kg body weight, and administered every 3 days. The tumor size and body weights were measured every day after the treatment.
The in vivo imaging system was used to study the biodistribution of Cy3-labeled polymeric micelle (PMLA-PEI-DOX-TAT) or nanocomplexes (PMLA-PEI-DOX-TAT@PEG-SA and PMLA-PEI-DOX-TAT@PEG-DMMA) in the A549 cell subcutaneous xenograft model in BALB/c athymic nude mice. The mice were observed at the 24 h after intravenous injection Cy3-labeled polymeric micelle or nanocomplexes at Cy3 dose of 0.1 mg/kg into the tumor-bearing mice.
Histology analysis was carried out at the 22nt day after the treatment. Typical heart, liver, spleen, lung, and kidney tissues of the mice in the control group and the best treatment group were isolated. Then, the organs were dehydrated using buffered formalin, ethanol with different concentrations, and xylene. After that, they were embedded in liquid paraffin. The sliced organs (3-5 mm) were stained with hematoxylin and eosin (H&E), and examined on a Leica SP8 microscope. In addition, Paraffin-embedded 5 µm thick tumor sections were obtained for the terminal transferase dUTP nick-end labeling (TUNEL) assay.
The mean values ± SD were determined for all the treatment groups. Each value is the mean of at least three repetitive experiments in each group. Statistical analysis was performed by Student's t-test. The differences between two groups was considered significant for *p<0.05, and very significant for **p<0.01, ***p<0.001.
PMLA: poly(β-L-malic acid); RES: reticuloendothelial system; NDDS: nanoscale drug delivery systems; EPR effect: enhanced permeation and retention effect; DMMA: 2,3-dimethylmaleic anhydride; SA: succinic anhydride; CA: cis-aconitic anhydride. TAT peptide: transactivator of transcription peptide; CPPs: cell-penetrating peptides; NMR: nuclear magnetic resonance; CMC: critical micelle concentration; HPLC: high performance liquid chromatography.
Supplementary figures and tables.
The authors would like to acknowledge support from the National Natural Science Foundation of China (Grant No. 81271687 and 81571786).
The authors have declared that no competing interest exists.
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Corresponding author: Hong Wu, Department of Pharmaceutical Analysis, School of Pharmacy, Fourth Military Medical University, Xi'an 710032, China. Tel: +86-29-84776823; E-mail: wuhongedu.cn.


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Pat Rawson shaped this one, nuff said.
6′-2″x 18.75″x 2 5/8″. Crisp clear Local motion logos top and bottom, Pat Rawson’s signature along with world class hawaiian spray artist Powers. The top of this board is a 9.5 and the bottom is a 7.5 Overall Condition 1-10: 8.5
Interested in this vintage board? Call 772-631-7106 or email us to make an offer.