Advances in nanomedicine for the treatment of ankylosing spondylitis | IJN – Dove Medical Press

Yanhai Xi,1,* Tingwang Jiang,2,* Birendra Chaurasiya,3 Yanyan Zhou,1 Jiangmin Yu,1 Jiankun Wen,1 Yan Shen,3 Xiaojian Ye,1 Thomas J Webster4

1Department of Spine Surgery, Changzheng Hospital, Second Military Medical University, Shanghai, Peoples Republic of China; 2Department of Immunology and Microbiology, Institution of Laboratory Medicine of Changshu, Changshu, Jiangsu 215500, Peoples Republic of China; 3Department of Pharmaceutics, Center for Research Development and Evaluation of Pharmaceutical Excipients and Generic Drugs, China Pharmaceutical University, Nanjing, Peoples Republic of China; 4Department of Chemical Engineering, Northeastern University, Boston, MA, USA

*These authors contributed equally to this work

Correspondence: Xiaojian YeDepartment of Spine Surgery, Changzheng Hospital, Second Military Medical University, 360 Huntington Avenue, Shanghai, MA 200003, Peoples Republic of ChinaTel +86 1 381 734 6934Email xjyespine@smmu.edu.cn

Thomas J WebsterDepartment of Chemical Engineering, Northeastern University, Boston, MA 02115, USATel +1 617 373 6585Email th.webster@neu.edu

Abstract: Ankylosing spondylitis (AS) is a complex disease characterized by inflammation and ankylosis primarily at the cartilagebone interface. The disease is more common in young males and risk factors include both genetic and environmental. While the pathogenesis of AS is not completely understood, it is thought to be an immune-mediated disease involving inflammatory cellular infiltrates, and human leukocyte antigen-B27. Currently, there is no specific diagnostic technique available for this disease; therefore conventional diagnostic approaches such as clinical symptoms, laboratory tests and imaging techniques are used. There are various review papers that have been published on conventional treatment approaches, and in this review work, we focus on the more promising nanomedicine-based treatment modalities to move this field forward.

Keywords: ankylosing spondylitis, pathogenesis, genetic factors, environmental factors, treatment approaches

This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License.By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.

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Market Share in Nanotechnology and Nanomedicine for Treatment of Viral Infections Could Reach Tens of Billion Dollars – P&T Community

PALM BEACH, Florida, Oct. 29, 2019 /PRNewswire/ -- There is much activity in the nanotechnology & nanomedicine markets as new treatments and human clinical trials are growing at a record pace. And these new treatment are hope they could be key to combating viral infections such as, bacteria, viruses, fungi,HBV, hepatitis C, Influenza, HSV, Human papillomavirus and parasites account for approximately 15million deaths worldwide, with acute respiratory infections and human immunodeficiency virus (HIV) being the leading causes. The National Institutes for Health NCBI Center for Biotechnology Information (NCBI) reports: "Infectious diseases are the leading cause of mortality worldwide, with viruses in particular making global impact on healthcare and socioeconomic development. In addition, the rapid development of drug resistance to currently available therapies and adverse side effects due to prolonged use is a serious public health concern. The development of novel treatment strategies is therefore required. The interaction of nanostructures with microorganisms is fast-revolutionizing the biomedical field by offering advantages in both diagnostic and therapeutic applications. Nanoparticles offer unique physical properties that have associated benefits for drug delivery." Mentioned in today's commentary includes: TG Therapeutics, Inc. (NASDAQ: TGTX), NanoViricides, Inc. (NYSE: NNVC), Matinas BioPharma Holdings, Inc. (NYSE: MTNB), Clovis Oncology, Inc. (NASDAQ: CLVS), Pfizer Inc. (NYSE: PFE).

Nanoparticle-based delivery systems present new opportunities to overcome challenges associated with conventional drug therapies and have therefore attracted enormous interest in the treatment of viral infections. Nanomaterials can be engineered to incorporate conventional antiviral properties with those modifications that are unique to nanosystems (ultra small and controllable size, large surface area to volume ratio, and the ability to tailor the surface with the possibility of multi-functionalization). This is undoubtedly a promising tool for biomedical research and clinical use.

One of the more active companies in the industry includes NanoViricides, Inc. (NYSE American: NNVC). NanoViricides,a leader in nanomedicines with novel platform technology to treat difficult and life-threatening viral diseases, on October 9, 2019 announced that it has initiated bio-analytical studies as part of the required IND-enabling preclinical safety and toxicology studies of NV-HHV-101, moving towards human clinical trials.

The Company has contracted NorthEast BioLab, Hamden CT, to conduct the bio-analytical studies and facilitate the toxicokinetic analyses. These studies and analyses are part of the required general safety and toxicology studies that will go into an Investigational New Drug (IND) Application to the US FDA.

NorthEast BioLab has already performed the bio-analytical assay development and validation and is in the process of determining the concentrations of NV-HHV-101 in blood samples from the general safety and toxicology studies that are required for IND.

The company feels that the market size for its immediate target drugs in the HerpeCide program is variously estimated into Billions to tens of Billions of Dollars. The Company believes that its dermal topical cream for the treatment of shingles rash will be its first drug heading into clinical trials. The Company believes that additional topical treatment candidates in the HerpeCide program, namely, HSV-1 "cold sores" treatment, and HSV-2 "genital ulcers" treatment are expected to follow the shingles candidate into IND-enabling development and then into human clinical trials.

NanoViricidesalso released a report this past August that its first drug candidate, NV-HHV-101, is on track with required preclinical GLP Safety and Toxicology studies moving towards human clinical trials. The Company reports that NV-HHV-101 has been found to be safe and well tolerated in the clinical observation portion of the GLP Safety/Toxicology study of NV-HHV-101 as a dermal treatment.

The Company has previously found that NV-HHV-101 was safe and well tolerated in non-GLP safety/toxicology studies. The GLP studies are an expanded version of the non-GLP studies, with extended treatment, larger number of subjects, and stringent operational requirements as specified by the current Good Laboratory Practices guidelines for such studies.

Additional studies required for the Safety and Toxicology datasets for filing an IND are in progress.

The Company anticipates advancing NV-HHV-101 into human clinical trials for topical dermal treatment of the shingles rash as the initial indication, assuming that these studies are successful. The Company also continues to evaluate this broad-spectrum drug candidate as well as certain variations based on the same candidate, for the treatment of other herpesviruses, namely HSV-1 cold sores and HSV-2 genital herpes. The market size for its immediate target drugs in the HerpeCide program is variously estimated into billions to tens of billions of dollars. The Company believes that its dermal topical cream for the treatment of shingles rash will be its first drug heading into clinical trials. The Company believes that additional topical treatment candidates in the HerpeCide program, namely, HSV-1 "cold sores" treatment, and HSV-2 "genital ulcers" treatment are expected to follow the shingles candidate into IND-enabling development and then into human clinical trials.

In addition, the Company also recently announced that its first clinical drug candidate, NV-HHV-101, for the treatment of the Shingles virus (aka VZV), is on track with required preclinical GLP Safety and Toxicology studies moving towards human clinical trials. The Company has reported that NV-HHV-101 has been found to be safe and well tolerated in the clinical observation portion of the GLP Safety/Toxicology study of NV-HHV-101 as a dermal treatment.

NanoViricides' current programs target a potential market opportunity of over $20 Billion. Investors are urged to view an informative video interview with Anil R. Diwan, PhD, President and Executive Chairman, who was interviewed by broadcast journalist Christine Corrado of Proactive Investors, a leading multi-media news organization, investor portal and events management business with offices in New York, Sydney, Toronto, Frankfurt, and London. Click here to access the video interview.

In other biotech news in the markets this week:

TG Therapeutics, Inc.(NASDAQ: TGTX), a biopharmaceutical company developing medicines for patients with B-cell mediated diseases, announced that the follicular lymphoma (FL) cohort of the UNITY-NHL Phase 2b pivotal trial evaluating single agent umbralisib, the Company's novel, once daily, PI3K delta inhibitor, met the primary endpoint of overall response rate (ORR) as determined by Independent Review Committee (IRC) for all treated patients (n=118) who have received at least two prior lines of therapy including an anti-CD20 monoclonal antibody and an alkylating agent. The results met the Company's prespecified ORR target of 40-50%. Importantly, umbralisib monotherapy appeared to be well tolerated with a safety profile consistent with previous reports.

The Company plans to present the data at a future medical conference as well as discuss the data with the U.S. Food and Drug Administration (FDA).

Matinas BioPharma Holdings, Inc.(NYSE AMER: MTNB), a clinical stage biopharmaceutical company, this month announced that it has initiated its Phase 2 EnACT clinical study, which will explore the use of MAT2203 for both induction and maintenance therapy in HIV-patients with cryptococcal meningitis, a life-threatening fungal infection most commonly observed in immunocompromised individuals.

"We are extremely pleased to advance clinical development of MAT2203 for the treatment of cryptococcal meningitis," commented Theresa Matkovits, Ph.D., Chief Development Officer of Matinas.

Clovis Oncology, Inc.(NASDAQ: CLVS) this month announced that the National Institute for Health and Care Excellence (NICE) has recommended that women with relapsed ovarian cancer in England have access to rucaparib through the Cancer Drugs Fund (CDF).1 Rucaparib is available for use within the CDF as an option for the maintenance treatment of relapsed, platinum-sensitive high-grade epithelial ovarian, fallopian tube or primary peritoneal cancer that has responded to platinum-based chemotherapy in adults, based on the conditions outlined in the managed access agreement.

"Ovacome welcomes the availability of rucaparib via the CDF as an option for maintenance treatment of platinum-sensitive relapsed high grade serous epithelial ovarian cancer regardless of BRCA status or line of treatment in the relapsed maintenance setting," said Victoria Clare, CEO of Ovacome, a United Kingdom ovarian cancer charity focused on providing support to anyone affected by ovarian cancer.

Pfizer Inc. (NYSE: PFE) reported financial results for third-quarter 2019 and updated certain components of its 2019 financial guidance.Third-Quarter 2019 Revenues of $12.7 Billion, Reflecting 3% Operational Decline; Excluding the Impact from Consumer Healthcare, Third-Quarter 2019 Revenues were Flat Operationally - See the full financial reporting at: https://finance.yahoo.com/news/pfizer-reports-third-quarter-2019-104500229.html

DISCLAIMER:FN Media Group LLC (FNM), which owns and operates Financialnewsmedia.com and MarketNewsUpdates.com, is a third- party publisher and news dissemination service provider, which disseminates electronic information through multiple online media channels.FNM is NOT affiliated in any manner with any company mentioned herein.FNM and its affiliated companies are a news dissemination solutions provider and are NOT a registered broker/dealer/analyst/adviser, holds no investment licenses and may NOT sell, offer to sell or offer to buy any security. FNM's market updates, news alerts and corporate profiles are NOT a solicitation or recommendation to buy, sell or hold securities. The material in this release is intended to be strictly informational and is NEVER to be construed or interpreted as research material. All readers are strongly urged to perform research and due diligence on their own and consult a licensed financial professional before considering any level of investing in stocks.All material included herein is republished content and details which were previously disseminated by the companies mentioned in this release.FNM is not liable for any investment decisions by its readers or subscribers.Investors are cautioned that they may lose all or a portion of their investment when investing in stocks.For current services performed FNM was compensated twenty five hundred dollars for news coverage of current press release issued by NanoViricides, Inc.by a non-affiliated third party.FNM HOLDS NO SHARES OF ANY COMPANY NAMED IN THIS RELEASE.

This release contains "forward-looking statements" within the meaning of Section 27A of the Securities Act of 1933, as amended, and Section 21E the Securities Exchange Act of 1934, as amended and such forward-looking statements are made pursuant to the safe harbor provisions of the Private Securities Litigation Reform Act of 1995. "Forward-looking statements" describe future expectations, plans, results, or strategies and are generally preceded by words such as "may", "future", "plan" or "planned", "will" or "should", "expected," "anticipates", "draft", "eventually" or "projected". You are cautioned that such statements are subject to a multitude of risks and uncertainties that could cause future circumstances, events, or results to differ materially from those projected in the forward-looking statements, including the risks that actual results may differ materially from those projected in the forward-looking statements as a result of various factors, and other risks identified in a company's annual report on Form 10-K or 10-KSB and other filings made by such company with the Securities and Exchange Commission. You should consider these factors in evaluating the forward-looking statements included herein, and not place undue reliance on such statements. The forward-looking statements in this release are made as of the date hereof and FNM undertakes no obligation to update such statements.

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Market Share in Nanotechnology and Nanomedicine for Treatment of Viral Infections Could Reach Tens of Billion Dollars - P&T Community

A highly biocompatible and intravenous injectable hafnium oxide nanocrystal synthesized to enhance radiaotherapy efficacy for cancer treatment -…

On October 11, the research team led by ZHOU Min from the Zhejiang University Institute of Translational Medicine published a research article entitled Gram-scale synthesis of highly biocompatible and intravenous injectable hafnium oxide nanocrystal with enhanced radiotherapy efficacy for cancer theranostic in the journal of Biomaterials.

Bio-inorganic nanomaterials are one of the hottest research topics in biomaterials, but materials that can be successfully applied to clinical trials are far and few between. The Hafnium Oxide (HfO2) based nanoagent has been employed in clinical trials for radiosensitized tumor therapy. However, the current clinically used HfO2 nanoparticles rely heavily on intratumoral injection, thus considerably hindering therapeutic effects.

To address this challenge, ZHOU Min et al. developed an intravenously injectable and clearable HfO2 nano-assembly (NA) which can be produced on a large scale. This NA can not only enhance radiotherapeutic effects, but it can also be removed from the body effectively, thereby reducing its side effect.

Schematic illustration of the synthesis process of HfO2 nanocrystal assemblies and enhanced radiotherapy performance against tumor.

ZHOU Min et al. found that this new type of HfO2 radiotherapy sensitizer can not only destroy breast cancer tumor cells efficiently upon X-ray radiation but also significantly inhibit the growth of tumor stem cells. CT and in vivo fluorescence imaging demonstrate its efficient tumor-homing ability and exhibit remarkable radiotherapeutic sensitization effects in a 4T1 breast tumor model after intravenous injection. More importantly, this HfO2 NA has low toxicity even at high concentrations. Moreover, it can be degraded and excreted effectively within a reasonable period in a living body, thus able to eschew long-term toxicity.

This work provides a new approach to promoting radiotherapeutic efficiency and safety. At present, this research is confined to small animals. However, it is expected that the HfO2 NA will be tested on large animals, thus attesting to the feasibility of clinical translation.

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A highly biocompatible and intravenous injectable hafnium oxide nanocrystal synthesized to enhance radiaotherapy efficacy for cancer treatment -...

Doheny and UCLA Stein Eye Institutes Welcome Kaustabh Ghosh, PhD, to the Scientific and Clinical Research Faculty – PRNewswire

"I am tremendously proud and honored to begin this position at Doheny-UCLA," says Dr. Ghosh. "I entered the field of biomedical research from an engineering background, which perhaps gave me a new perspective to see things differently. As a biomedical engineer, it allowed me to think about diseases in a way that a typical biomedical researcher and clinician may not."

Dr. Ghosh states that also as a vascular biologist, one such perspective he was able to successfully introduce was the importance of "stiffness" of blood vessels in disease pathogenesis.

"Doheny will be the ideal place for me to realize the true translational potential for my work as it offers strength and resources in ophthalmic imaging," shares Dr. Ghosh. "Doheny also provides the perfect balance between basic science and clinical research."

He adds, "I look forward to developing strong, collaborative relationships with members of Doheny-UCLA engineering, biomedical sciences and clinical infrastructure. Our goal will be to discover effective treatment strategies from a multidisciplinary approach especially in the area of investigating the role of chronic vascular inflammation, a major determinant of various debilitating conditions including macular degeneration and diabetic retinopathy."

Dr. Ghosh was most recently Associate Professor of Bioengineering at University of California, Riverside (UCR) as well as Participating Faculty in the Division of Biomedical Sciences, Stem Cell Center and the Program in Cell, Molecular and Developmental Biology. The Ghosh Research Group at UCR focused on leveraging the principles of mechanobiology to examine and treat inflammationmediated vascular degeneration associated with diabetic retinopathy and agerelated macular degeneration, the leading causes of vision loss in the diabetic and aging population. In 2016, these studies were supported by two R01 grants from the National Eye Institute (NEI), and a macular degeneration grant from the BrightFocus Foundation. Dr. Ghosh has received numerous awards during his research career, including the Hellman Fellowship and the NIH Postdoctoral Training Grant, and has published 24 peer-reviewed papers in highly-regarded journals that include PNAS, The FASEB Journal, Science, and Nano Letters, among others.

In 2011, prior to joining UCR, Dr. Ghosh was a postdoctoral fellow in the laboratory of Donald Ingber, MD, PhD, part of the Vascular Biology Program at Boston Children's Hospital and Harvard Medical School. In 2006, Dr. Ghosh received his PhD in Biomedical Engineering from Stony Brook University, New York. He obtained his undergraduate degree in Chemical Engineering from National Institute of Technology, Warangal, India in 2001.

Dr. Ghosh's dedication to collaborative research and team building is evident in his numerous and illustrious achievements. His distinguished scientific leadership demonstrates an excellence that will contribute greatly to Doheny Eye Institute's research programs.

About Doheny Eye InstituteFor over 70 years, Doheny Eye Institute has been at the forefront of vision science. From seeking new ways to free blockages that prevent fluid drainage in glaucoma, to replacing retinal cells in age-related macular degeneration, to providing colleagues worldwide with standardized analyses of anatomical changes in the eyes of patients, Doheny clinicianscientists and researchers are changing how people see and also how they think about the future of vision. Please visit doheny.org for more information.

Doheny Eye Institute and UCLA Stein Eye Institute have joined forces to offer the best inpatient care, vision research and education. This affiliation combines the strength, reputation and distinction of two of the nation's top eye institutions to advance vision research, education and patient care in Southern California.

CONTACT INFORMATIONMedia Contact:Matthew RabinDirect: (323) 342-7101Email: mrabin@doheny.org

SOURCE Doheny Eye Institute

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NanoRobotics Market Fastest hit at a CAGR of 12.5% and is expected To Reach $11.88 Billion by 2019-2026: Bruker, JEOL, Thermo Fisher Scientific,…

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NanoRobotics Market Fastest hit at a CAGR of 12.5% and is expected To Reach $11.88 Billion by 2019-2026: Bruker, JEOL, Thermo Fisher Scientific, Ginkgo Bioworks, Oxford Instruments, EV Group, Imina Technologies, Klocke Nanotechnik, Xidex, Synthace| Orian Research

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NanoRobotics Market Fastest hit at a CAGR of 12.5% and is expected To Reach $11.88 Billion by 2019-2026: Bruker, JEOL, Thermo Fisher Scientific,...

SUNY Potsdam chemistry professor awarded grant from National Institutes of Health – NNY360

POTSDAM Dr. Fadi Bou-Abdallah, a professor in the Department of Chemistry at SUNY Potsdam, has received a grant award from the National Institutes of Health for $414,047.

The NIH Academic Research Enhancement Award grant will fund Bou-Abdallahs project, titled Effect of Ferritin Subunit Composition on Iron Core Formation, Morphology and Iron Mobilization: Physical Characterization and Physiological Relevance, for the next three years (from 2019 to 2022).

Through this research, Dr. Bou-Abdallah seeks to understand how ferritin, the major iron storage protein in mammals, plays a crucial role in iron mineralization and housekeeping.

Improper iron mineralization has been suggested to contribute to the progression of diseases, and a number of studies have found abnormal brain iron and ferritin levels in neurodegenerative disorders, such as Parkinsons, Alzheimers disease and Friedreichs ataxia, Bou-Abdallah said.

These abnormalities have been discussed in terms of a loss of ferritins ability to maintain iron homeostasis, leading to ferritin and iron deposits in brain tissues. The proposed research will help to define the molecular origins of these debilitating diseases, and provide fundamental insights into the biochemical processes responsible for iron-related disorders and neurodegenerative diseases, Bou-Abdallah said. The results would also facilitate exploitation of ferritin as a nanotemplate, for uses in nanochemistry, nanobiology and nanomedicine.

It is a great honor to be recognized by NIH for the wonderful science we do at SUNY Potsdam with our undergraduates. This grant award would not have been possible without our students dedication and hard work, and represents a culmination of years of excellence in undergraduate teaching and research, Bou-Abdallah said. Support of undergraduate research at small institutions like ours is extremely important. The majority of students who receive bachelors degrees in our department go on to earn doctorates and/or medical degrees. Undergraduate research is a fundamental part of their education at SUNY Potsdam, and one of the high-impact learning practices they encounter here.

Bou-Abdallah plans to work with SUNY Potsdam undergraduates over the next three academic years and summers to complete the project. The students will be involved in all aspects of this research and be exposed to interdisciplinary research at the interface of chemistry, biology and materials science. They will also get a chance to travel to the University of Pennsylvania and work with experts in the fields of biosciences and materials science, and use a powerful scanning transmission electron microscope that provides images down to the atomic level, the first of its kind in the United States.

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SUNY Potsdam chemistry professor awarded grant from National Institutes of Health - NNY360

Improving 131I Radioiodine Therapy By Hybrid Polymer-Grafted Gold Nano | IJN – Dove Medical Press

Marine Le Goas,1 Marie Paquet,25 Aurlie Paquirissamy,1 Julien Guglielmi,24 Cathy Compin,24 Juliette Thariat,6 Georges Vassaux,24 Valrie Geertsen,1 Olivier Humbert,25 Jean-Philippe Renault,1 Graldine Carrot,1 Thierry Pourcher,24 Batrice Cambien24

1NIMBE, Commissariat lEnergie Atomique, Centre National Recherche Scientifique UMR 3685, Universit Paris-Saclay, Gif-sur-Yvette, France; 2Laboratory Transporter in Imaging and Radiotherapy in Oncology (TIRO), Institut de Biosciences et Biotechnologies dAix-Marseille (BIAM), Commissariat lEnergie Atomique, Nice, France; 3Laboratory Transporter in Imaging and Radiotherapy in Oncology (TIRO), University Nice Sophia Antipolis, Nice, France; 4Laboratory Transporter in Imaging and Radiotherapy in Oncology (TIRO), University Cte dAzur, Nice, France; 5Nuclear Medicine Department, Centre Antoine Lacassagne, Nice, France; 6Department of Radiation Oncology, Centre Franois Baclesse, Universit de Normandie, Caen, France

Correspondence: Batrice CambienLaboratory Transporter in Imaging and Radiotherapy in Oncology (TIRO), University Nice Sophia Antipolis, 28 Avenue Valombrose, Nice Cedex 2 06107, FranceTel +33 493 377 715Email cambien@unice.fr

Background: Human trials combining external radiotherapy (RT) and metallic nanoparticles are currently underway in cancer patients. For internal RT, in which a radioisotope such as radioiodine is systemically administered into patients, there is also a need for enhancing treatment efficacy, decreasing radiation-induced side effects and overcoming radio-resistance. However, if strategies vectorising radioiodine through nanocarriers have been documented, sensitizing the neoplasm through the use of nanotherapeutics easily translatable to the clinic in combination with the standard systemic radioiodine treatment has not been assessed yet.Method and materials: The present study explored the potential of hybrid poly(methacrylic acid)-grafted gold nanoparticles to improve the performances of systemic 131I-mediated RT on cancer cells and in tumor-bearing mice. Such nanoparticles were chosen based on their ability previously described by our group to safely withstand irradiation doses while exhibiting good biocompatibility and enhanced cellular uptake.Results: In vitro clonogenic assays performed on melanoma and colorectal cancer cells showed that poly(methacrylic acid)-grafted gold nanoparticles (PMAA-AuNPs) could efficiently lead to a marked tumor cell mortality when combined to a low activity of radioiodine, which alone appeared to be essentially ineffective on tumor cells. In vivo, tumor enrichment with PMAA-AuNPs significantly enhanced the killing potential of a systemic radioiodine treatment.Conclusion: This is the first report of a simple and reliable nanomedicine-based approach to reduce the dose of radioiodine required to reach curability. In addition, these results open up novel perspectives for using high-Z metallic NPs in additional molecular radiation therapy demonstrating heterogeneous dose distributions.

Keywords: internal radioisotope therapy, radioiodine, polymer-grafted gold nanoparticles, melanoma, colorectal cancer, radio-enhancement

This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License.By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.

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Improving 131I Radioiodine Therapy By Hybrid Polymer-Grafted Gold Nano | IJN - Dove Medical Press

Nanoparticles moving through airway mucus may predict severity of COPD – News-Medical.net

In a proof-of-concept experiment, researchers from Johns Hopkins Medicine say they have successfully used microscopic man-made particles to predict the severity of patients' chronic obstructive pulmonary disease (COPD) by measuring how quickly the particles move through mucus samples. The technique, say the researchers, could eventually help doctors deliver more effective treatments sooner.

"If further studies verify our findings, nanoparticles could establish early which patients with COPD are more likely to need more intensive interventions to avoid adverse outcomes," says Enid Neptune, M.D., associate professor of medicine at the Johns Hopkins University School of Medicine, who partnered with Justin Hanes, Ph.D., and Jung Soo Suk, Ph.D., from the Center for Nanomedicine at the Wilmer Eye Institute at Johns Hopkins.

A report on the research was published Aug. 1 in the European Respiratory Journal.

According to the World Health Organization, 65 million people have moderate to severe COPD, making it the fifth leading cause of death worldwide. Linked to smoking and pollution, COPD describes a group of incurable, chronic inflammatory, lung-damaging processes marked by coughing, breathlessness and excessive production of spit and mucus. Currently, the best diagnostic tool for COPD is spirometry, which measures how deeply a person can move air out through their lungs after taking a deep breath.

"Spirometry gives us a good picture of a person's current lung function, but tells us very little about how their disease will progress," says Neptune, which is why finding a predictive biomarker is so important.

In search of one, the research team focused on mucus, the slimy lubricant secreted by tissues that line airways, intestines and other body passages, forming protective barriers and -- in the lungs -- sweeping away potentially harmful particles inhaled with the air we breathe. Mucus has a structure similar to a water-filled sponge, with many tiny interconnected pores.

Hanes and Suk had previously engineered nanoparticles that don't stick to the mucus and discovered that these "muco-inert" particles were capable of navigating through the maze of mucus pores covering the airways. "The diffusion of muco-inert particles in airway mucus is primarily hindered by mucus' structure, and thus the mucus pore size can be estimated based on how fast the particles travel within the mucus samples collected from patients," says Suk.

That suggested to us that the structure of the mucus in people with COPD might be distinct and affect the way foreign particles or even pathogens move."

Enid Neptune, M.D., associate professor of medicine, Johns Hopkins University School of Medicine

To test this, the researchers next collected mucus samples from 33 patients who were active or former smokers: Seven of the smokers had no history of COPD, 18 had mild to moderate COPD, and eight had severe COPD as determined by lung function.

The team then added nanoparticles labeled with a fluorescent marker that could be traced by an ultrasensitive camera into the mucus and observed their movement, watching for the speed at which the nanoparticles diffused through the sample.

The researchers found that in mucus collected from patients with COPD, the nanoparticles moved significantly slower compared with patients without COPD, indicating that their mucus had a more restricted structure, which is harder for the nanoparticles to navigate. The effect was even more pronounced when observing the nanoparticle behavior in mucus samples from patients with severe COPD, indicating that the pore size within the mucus shrinks as COPD progresses.

"If this restricted structure impacts the ability of immune cells to engage infectious pathogens in the lungs, it could potentially serve as a predictor for COPD flares, a critical risk factor in COPD progression," says Neptune.

The researchers state that if future, larger studies validate their findings, the structure of mucus could provide new insight into how COPD progresses, as well as improve therapy.

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Nanoparticles moving through airway mucus may predict severity of COPD - News-Medical.net

MagForce AG to host Lunch Symposium on Local Therapies in Malignant Gliomas during the 19th European Congress of Neurosurgery (EANS2019) – BioSpace

Berlin, Germany and Nevada, USA, September 19, 2019 - MagForce AG (Frankfurt, Scale, XETRA: MF6, ISIN: DE000A0HGQF5), a leading medical device company in the field of nanomedicine focused on oncology, is pleased to announce that it will host a scientific lunch symposium titled "Local Therapies in Malignant Gliomas - update and new perspectives" during the 19th European Congress of Neurosurgery in Dublin, Ireland.

Chaired by Prof. Dr. Walter Stummer, Director of the Department of Neurosurgery at the University Hospital of Mnster (UKM), Germany, the one-hour lunch symposium will feature two keynote speeches: After an introduction and overview on the current status of glioma treatments by Prof. Dr. Stummer, Ricardo Dez Valle, MD PHD, Head of the Department of Neurosurgery, Hospital Group Quirn Madrid, Spain, will give an update on local neurosurgical therapies. The symposium will be rounded off with a talk from Prof. Dr. Stummer on An Emerging Adjunct: NanoTherm NanoPaste Application. During the subsequent wrap-up and discussion, participants will have the opportunity to ask questions.

Symposium details:


Local Therapies in Malignant GliomasUpdate and new perspectivesNanoTherm - NanoPaste Application


Wednesday, September 25, 2019


1.15-2.15 p.m. local time/GMT


EcoCem meeting room, The Convention Centre Dublin


Prof. Dr. Walter Stummer (Chair)University Hospital of Mnster, GermanyIntroductionCurrent Status of Glioma Treatments

Ricardo Dez Valle, MD PHDHospital Group Quirn Madrid, SpainLocal Neurosurgical Therapies: An Update

Prof. Dr. Walter StummerUniversity Hospital of Mnster, GermanyAn Emerging Adjunct: NanoTherm NanoPaste Application

In addition to the lunch symposium, MagForce will host Meet the Expert sessions on Thursday, September 26th and Friday, September 27th, at 10 a.m. respectively, at the MagForce booth No. 35 located at Level 0 The Forum. On both days, Dr. Michael Schwake, Senior Physician in the Department of Neurosurgery at the University Hospital of Mnster and member of Prof. Dr. Stummers team, will be available to interested participants for questions as an expert in the use of the NanoTherm therapy system in clinical practice.

Prof. Dr. Stummer and his team at the University Hospital of Mnster have been treating brain tumor patients with MagForce's NanoTherm therapy since early 2015 and were the first to introduce, clinically, the new nanoparticles application technique called 'NanoPaste'. In previous clinical research, the UKM team demonstrated that a better applicability of heat-focussing nanoparticles around the resection rim after surgical removal of a brain tumor could boost the thermotherapy treatment for tumor ablation and as a radiosensitiser in glioma therapy. With the use of the new NanoPaste technique, the team was able to create sufficient nanoparticle concentrations to reach effective thermotherapy in the glioma resection cavity wall and its vicinity, allowing for more control of remaining tumor infiltration. Especially in comparison to stereotactic techniques with their associated imponderabilities in scarred and pretreated tissues, the 'NanoPaste' application was technically easy, controllable and quick to perform.

About NanoTherm TherapyMagForce's NanoTherm therapy provides a novel, nanotechnology-based approach for the treatment of solid tumors by introducing magnetic nanoparticles either directly into the tumor or into the resection cavity wall. These particles are subsequently heated by an alternating magnetic field that allows targeted treatment by irreparably destroying, or weakening, cancer cells making them more sensitive to concomitant radiotherapy or chemotherapy. Since the particles remain at the site of application due to their special coating, the surrounding healthy tissue is spared and repeated treatments as well as the integration into multimodal therapy concepts are made possible. MagForce AG was granted the European CE certificate ("European Certification") in 2011 and thus the official approval of NanoTherm therapy for the treatment of brain tumors in Germany and all member states of the European Union.

About EANS2019The European Association of Neurosurgical Societies (EANS) is both an independent federation of European national neurosurgical societies and a fast-developing association of individual neurosurgeons from around the world, aiming to enhance the quality of neurosurgical patient care through training, education and research. A key way in which the association fulfils this objective is by facilitating the exchange of scientific information at the highest level through the organisation of meetings, symposia and educational courses with the annual congress as its flagship event.

The overall scientific programme will take the familiar form that has been established over the last few years bringing together international experts to discuss and inform. To help develop best practice in all subspecialities under the headings of Technology, Techniques, Training and increasingly important Transparency. The four days of scientific discussions offer plenty of opportunities for meetings, socialising and hearing of the latest scientific developments.

For more information on the EANS2018 programme, please click here: https://eans2019.com/programme-and-abstracts/scientific-programme.html.

About MagForce AG and MagForce USA, Inc.MagForce AG, listed in the Scale segment of the Frankfurt Stock Exchange (MF6, ISIN: DE000A0HGQF5), together with its subsidiary MagForce USA, Inc. is a leading medical device company in the field of nanomedicine focused on oncology. The Group's proprietary NanoTherm therapy enables the targeted treatment of solid tumors through the intratumoral generation of heat via activation of superparamagnetic nanoparticles.

NanoTherm, NanoPlan, and NanoActivator are components of the therapy and have received EU-wide regulatory approval as medical devices for the treatment of brain tumors. MagForce, NanoTherm, NanoPlan, and NanoActivator are trademarks of MagForce AG in selected countries.

For more information, please visit: http://www.magforce.comGet to know our Technology: video (You Tube)Stay informed and subscribe to our mailing list

DisclaimerThis release may contain forward-looking statements and information which may be identified by formulations using terms such as "expects", "aims", "anticipates", "intends", "plans", "believes", "seeks", "estimates" or "will". Such forward-looking statements are based on our current expectations and certain assumptions, which may be subject to a variety of risks and uncertainties. The results actually achieved by MagForce AG may substantially differ from these forward-looking statements. MagForce AG assumes no obligation to update these forward-looking statements or to correct them in case of developments, which differ from those, anticipated.

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MagForce AG to host Lunch Symposium on Local Therapies in Malignant Gliomas during the 19th European Congress of Neurosurgery (EANS2019) - BioSpace

Synthetic networks with tunable responsiveness, biodegradation, and molecular recognition for precision medicine applications – Science Advances


In 2015, the Obama administration launched the precision medicine initiative (1). An emerging engineering challenge within precision medicine is the need for versatile platform technologies that can be tailored to individual patients or pathologies (1, 2). A common approach within the fields of biomaterials and nanotechnology has been to design highly tailored formulations that target specific cell and tissue characteristics of a single pathology. These formulations, which can be fabricated in a variety of supramolecular structures [i.e., linear polymer conjugates (3), gels (4, 5), and self-assembled materials (6, 7)], recognize hallmark overexpressed cellular markers for the purpose of disease targeting. Nanoparticle carriers for precision medicine applications are typically dynamic in nature, swelling and/or degrading in intracellular environments to deliver therapeutic payloads to the cytosol of target cells (8).

In cancer treatment, there is precedence that multiple therapeutics can act synergistically to target and kill tumors. Chemotherapeutic agents act through a variety of mechanisms, including, but not limited to, DNA intercalation, enzyme inhibition, and cell cycle arrest (9, 10). Targeted agents, such as monoclonal antibodies, alter cell signaling pathways and engage the immune system. Photothermal therapy leads to tumor reduction by increasing membrane fluidity (~43C) or ablating the cells (~50C) (11). All of these therapeutic modalities benefit from targeting strategies, which concentrate the therapeutic agent within the tumor.

Each therapeutic option has distinct potential to aid in an individual patients treatment regimen. But, there is also marked variability between patients, necessitating precise and tailored treatments specific for the genetic and biophysical properties of the individual pathology. Advancements in genomic and proteomic technologies have made the collection of these relevant individual data a reality. The major hurdles left to overcome include, first, establishing predictive models of patients response to treatment and, second, engineering highly tunable platform technologies that deliver multiple therapeutic modalities in a patient-specific manner. Our modular strategy addresses the latter challenge and could serve as a useful tool in future studies on the former.

Previous studies on nanoparticle development for cancer precision medicine have focused on highly specified platforms that efficiently target and kill a single tumor population. For example, Conde et al. (12) recently designed a composite platform composed of gold nanorods, gold nanoparticles, therapeutic antibodies, and small interfering RNA encapsulated within an adhesive hydrogel patch. This system targeted and killed colorectal cancer cells through multiple modalities (i.e., photothermal therapy, RNA interference, and targeted chemotherapy), increasing treatment efficacy in vitro and in vivo.

In another illustrative example, Liu et al. (13) constructed a hierarchical nanomaterial assembly that delivered a cytotoxic protein (ribonuclease A) and antibiotic (doxycycline). This platform targeted cancer stem cells within heterogeneous cancer populations. The targeted, dual therapy led to a significant reduction in tumor volume relative to both the controls and individually administered therapeutics. These are only two examples, out of many promising studies on cancer nanomedicines that have used multiple therapeutic modalities (1215). There is a need, therefore, for a readily modifiable platform that facilitates the rapid customization of cancer nanomedicines to individual patients pathologies.

We previously demonstrated the ability to tune the hydrodynamic diameter and magnitude of pH response of poly(acrylamide-co-methacrylic acid) [P(AAm-co-MAA)] nanogels by modulating the monomer feed, polymerization parameters, or purification strategy (16). In the present work, our base platform is this random P(AAm-co-MAA) copolymer cross-linked into a nanogel with either a nondegradable or a redox-labile cross-linker. We present a new, modular sequence of nanogel modifications with small molecules, peptides, or proteins; these nanogels are multifunctional and multiresponsive, exhibiting dynamic loading and release of therapeutic payloads, engaging in a bioactive manner with biological substrates, transducing external signals into therapeutic heating, and promoting cellular internalization.

To achieve modular functionalization while retaining the bioactivity of conjugated molecules, we rely on facile and biocompatible conjugation schemes. While there are numerous bioconjugation strategies documented in the literature (1719), we use carbodiimide-mediated coupling to attach diverse ligands to pendant carboxylic acid groups via a stable amide bond. In this coupling scheme, carboxylic acid groups are activated with a catalyst to form a reactive ester intermediate, which is highly reactive with primary amines in slightly acidic aqueous solutions. We can, therefore, couple any water-soluble, amine-terminated moleculeincluding, but not limited to, proteins, peptides, and small moleculesdirectly to our polymer backbone. The diversity of bifunctional linker molecules that are available commercially, such as poly(ethylene glycol) derivatives (20, 21), further diversifies the ligands that our platform can accommodate, including those with amine, carboxylic acid, hydroxyl, or sulfhydryl groups.

We hypothesized that a single platform, when modified in a modular manner with bioactive components, could respond dynamically to tumor physiological environments, partition and elute therapeutic agents in a controlled manner, transduce external signals for therapeutic heating, and target tumor populations. We believe that this platformwhich can be modified to achieve environmental responsiveness, therapeutic delivery, and molecular recognitionis an enabling technology for delivering personalized and calibrated combination therapies. A summary schematic for our platform, along with the reagents, chemical modifications, and therapeutic modalities explored, is given in Fig. 1. In this proof-of-concept study, we demonstrate how a single, biocompatible platform can be quickly and precisely modified for personalized and precision medicine applications. Furthermore, in addition to standard characterization techniques, we developed and applied two new experimental methods: a quartz crystal microbalance with dissipation (QCM-D) assay for nanogel degradation and a high-throughput cell imaging assay for determining the kinetics of nanogel uptake. These new techniques expand upon the fields repertoire of experimental methods for evaluating and comparing new nanoparticle systems for precision medicine.

Nanoscale networks of acrylamide (AAm) and methacrylic acid (MAA), cross-linked with methylenebisacrylamide (BIS) or its degradable disulfide analog [N,N-bis(acryloyl)cystamine], were synthesized by inverse emulsion polymerization and modified via carbodiimide chemistry with tyramine (Tyr), N,N-dimethylethylenediamine (DMED), proteins, or peptides. In an additional post-synthesis step, gold nanoparticles (AuNP) were precipitated within DMED-modified (DMOD) nanogels. Here, we document the synthesis and modification of this nanogel platform and demonstrate the impact of nanogels modification on their ability to respond to the pH environment, load and release a model cationic drug, target cells, act as a functional enzyme, and transduce green light for photothermal therapy. Because of its tunability and the variety of therapeutic modalities enabled, we believe that this platform is suitable for precision medicine applications. DTT, dithiothreitol; TMB, 3,3,5,5-tetramethylbenzidine.

Our base platform for small moleculemodified nanogels was an ionomer collapsepurified P(AAm-co-MAA) nanogel, synthesized as described by Zhong et al. (16). These nanogels had a swollen hydrodynamic diameter of 768 nm, were 63% acidic copolymer by mass, and exhibited pH-responsive expansion/syneresis behavior with a critical pH transition point of 4.8. Ionomer collapsepurified nanogels, as opposed to those purified by dialysis alone, were selected because the basic conditions (0.5 N sodium hydroxide) are known to induce hydrolysis of some of the nanogel acrylamide content to acrylic acid, providing additional acid groups for bioconjugation. The increased presence of carboxylic acids allowed us to couple a greater quantity of functional small molecules to each nanogel.

An additional degree of tunability was introduced to the P(AAm-co-MAA) nanogels by introducing a biodegradable cross-linker. N,N-bis(acryloyl)cystamine is a bisacrylamide analog that contains a labile disulfide linkage. It has been used previously as a component of digestible gels for drug and gene delivery applications (2224). We successfully cross-linked P(AAm-co-MAA) nanogels with N,N-bis(acryloyl)cystamine. These biodegradable nanogels were similar in hydrodynamic diameter, zeta potential, and pH-responsiveness to their nondegradable analogs (fig. S1).

The kinetics and mechanisms of biodegradation for these nanogels were quantified by optical and gravimetric analyses. Optical analysis was conducted via dynamic light scattering (DLS) with a fixed detector position and signal attenuation. Under these measurement conditions, the count rate is related to the decrease in the number of suspended nanoparticles (25). Simultaneously, the hydrodynamic diameter measurements collected provide inference to the mechanism of biodegradation (i.e., surface erosion and bulk degradation). We assessed biodegradation by DLS for degradable nanogels in the presence of 10 mM dithiothreitol (DTT) or glutathione in 1 phosphate-buffered saline (PBS) at pH 7.4. DTT is a common reducing agent used for biological applications. It has been used previously to degrade systems cross-linked with N,N-bis(acryloyl)cystamine and was selected to ensure that the nanogels degrade completely. Glutathione (10 mM) in 1 PBS was selected as a biologically relevant reducing condition, as it mimics the intracellular environment (26).

The chemical mechanism of nanogel degradation by each reducing agent, as well as a pictorial depiction of the biodegradation process, is given in Fig. 2A. DLS analysis confirmed that both DTT and glutathione were able to reduce the disulfide cross-linker and consequently degrade the nanogel network (Fig. 2B). In the presence of DTT, the nanogels degraded rapidly and were indistinguishable from a linear polymer solution of the same concentration (i.e., completely degraded) after 40 min. Nanogels degraded with reduced kinetics in a 10 mM glutathione solution. The normalized count rate declined by 72.05.8% after 50 min in glutathione solution, and the nanogels were indistinguishable (by DLS count rate) from linear polymer after 48 hours.

(A) N,N-bis(acryloyl)cystamine cross-linked nanogels degrade via reduction of the disulfide. The diagram demonstrates how, after an initial period of surface erosion, the nanogels experience bulk degradation, leading to simultaneous network swelling. (B) DLS analysis of nanogel degradation. While bisacrylamide cross-linked nanogels did not degrade under reducing conditions, those cross-linked with a disulfide cross-linker were digested by both reducing agents (n = 4, mean SD). (C) QCM analysis demonstrated the kinetic decomposition of nanogels under reducing conditions and flow. While the mass of nondegradable nanogels was relatively unaffected by reducing conditions, the mass of degradable gels declined rapidly (n = 3, mean SD). (D) Hydrodynamic diameter analysis by DLS supported the degradation mechanism of initial surface erosion followed by bulk degradation. While the normalized count rate declined steadily throughout the extended measurement, the hydrodynamic diameter decreased initially (surface erosion) and then increased for the remainder of the experiment (i.e., decrease in cross-links led to a reduction in the total number of nanoparticles but swelling of the remaining intact nanogels) (n = 3, mean SD).

In QCM-D experiments, the nanogels were covalently conjugated to a gold-coated quartz sensor, and the mass loss, under reducing conditions, was monitored by measuring the change in the quartz sensors fifth harmonic resonance frequency (27). Mass loss was normalized to the initial mass of coupled nanogels to determine a relative measure. It is noteworthy that because the nanogels are covalently conjugated to the quartz sensor, the mass loss will never reach 100%. Some linear polymer strands will remain immobilized on the sensor following complete degradation of the cross-links.

Under a steady flow of fresh 10 mM DTT (1 PBS, pH 7.4), the mass of nondegradable nanogels increased slightly. This increase was likely due to adsorption of DTT molecules. On the other hand, the relative mass of degradable nanogels decreased rapidly, reaching a degraded state in 15 min (Fig. 2C). As shown in fig. S2, in parallel with an increasing resonance frequency, the dissipation of sensors coated with degrading increased. This indicated that as the nanogels were degrading, they were simultaneously losing mass and imbibing water. This observation was consistent with our DLS measurements, which showed that the nanogels simultaneously degraded and swelled under reducing conditions (Fig. 2D).

Next, we explored the ability to modify the pendant acid groups on P(AAm-co-MAA) with amine-terminated small molecules (tyramine and N,N-dimethylethylenediamine) to add phenol or tertiary amine groups to the polymer backbone, respectively. Nanogels modified to different extents with tyramine (TMOD) or N,N-dimethylethylenediamine (DMOD) were characterized by Fourier transform infrared (FTIR spectroscopy), potentiometric titration, DLS, and zeta potential measurement (Fig. 3).

(A) FTIR spectra of TMOD and DMOD nanogels, as compared with the unmodified formulation. The peaks at 1700 and 1200 cm1 correspond to the carboxylic acid, at 1660 and 1590 cm1 correspond to the amide, and at 800 cm1 correspond to the aromatic groups, confirming the incorporation of each small-molecule ligand through covalent coupling. FTIR analysis of all formulations is presented in fig. S1. (B) Nanogel modification proceeded with approximately 60% efficiency when the ligand concentration did not exceed the carboxylic acid concentration (stoichiometric ratios less than 1). (C) Potentiometric titrations were used to quantify the carboxylic acid content of all formulations, elucidating the extent of small-molecule coupling. (D) Modified nanogels exhibited a pH-responsive zeta potential transition (anionic to cationic), whereas unmodified nanogels were anionic across all pH values tested (n = 3, mean SD). (E) Unmodified and TMOD nanogels exhibited a pH-responsive collapse with a critical transition point at pH ~ 4.8. DMOD nanogels did not undergo substantial pH-responsive swelling.

FTIR analysis showed that the small molecules were covalently bound to the nanogel network, as evidenced by the reduction in peaks corresponding with the carboxyl carbonyl (1700 cm1) and carbon-oxygen single bond (1200 cm1). A graphical depiction of select formulations is given in Fig. 3A, with the full analysis of all formulations given in fig. S3. From the FTIR analysis, it initially appeared that the DMOD reaction proceeded with greater efficiency, as the disappearance of carboxylic acid peaks was more pronounced. However, potentiometric titration revealed that the percent of modified methacrylic acid moieties trended with the stoichiometric ratio of ligand to carboxylic acid similarly for both ligands (Fig. 3, B and C). Therefore, the trends observed in the FTIR spectra are likely reflective of the location of nanogel modification (surface for DMOD and bulk for TMOD) rather than the efficiency of the reaction. The circled formulations (0.5 TMOD and 0.78 DMOD) in Fig. 3B, which achieved a high degree of efficient molecular coupling, were used in each of the following experiments.

Potentiometric titration, pH-responsive zeta potential, and size measurements for TMOD, DMOD, and unmodified nanogels are also presented in Fig. 3 (C to E). As shown in the potentiometric titration analysis, unmodified nanogels were 63% polyacid [i.e., poly(acrylic acid) and poly(methacrylic acid)] by mass, as compared with 22 and 25% for the TMOD and DMOD nanogels, respectively. The reduction in acid content, because of modification, trended linearly with ligand concentration in the modification reaction at low extents of modification and plateaued at 69.83.7% modification. Full potentiometric titration analysis of all TMOD and DMOD formulations is presented in fig. S4.

All three formulations were anionic at pH values greater than five, as the carboxylic acid groups were predominantly deprotonated [pKa (where Ka is the acid dissociation constant), ~4.8] and held a negative charge. As the pH was reduced from 8 to 3, the TMOD and DMOD nanogels zeta potential was less negative than that of unmodified nanogels and became positive at pH 4.7. In this pH environment, as well as those more acidic, the carboxylic acid groups are protonated and therefore uncharged, whereas the tertiary amines contributed by N,N-dimethylethylenediamine and adsorbed sodium ions from the solution (5 mM sodium phosphate buffer) are positively charged. This pH-responsive ionization change for the modified nanogels is especially critical for environmentally responsive drug delivery, as will be shown in a later section.

The modified and unmodified nanogels hydrodynamic diameters, as measured by DLS, also changed in response to the pH environment. TMOD and unmodified nanogels exhibited similar pH-responsive collapses, with a critical pH transition of approximately 4.8. As the pH of the solution was dropped below 4, both the TMOD and DMOD nanogels aggregated. For the purpose of visualization, hydrodynamic diameter measurements from aggregated states were omitted from Fig. 3E. The full data are presented in fig. S5.

It is noteworthy that DMOD nanogels exhibited a nearly complete loss of pH-responsive swelling. This can be attributed to the fact that because of the modified networks amphoteric nature, it bears charge across all pH values. Its state of electrical neutrality at pH 4.7 is a result of balanced negatively and positively charged species, rather than a loss of ionization. On the contrary, the pH-responsive behavior of both the unmodified and TMOD nanogels suggests aggregation caused by a hydrophobic transition and loss of ionization. Taken in combination with the observed trends in zeta potential, this suggests that the TMOD nanogels negative-to-positive charge transition is a result of the association of ionic species, both salts from the buffer and additional tyramine molecules that were neither conjugated nor extracted during purification, rather than the network components themselves bearing a positive charge.

Suspensions of DMOD, TMOD, and unmodified nanogels were incubated separately with methylene blue in distilled water, and methylene blue loading was achieved through equilibrium partitioning. Methylene blue was selected as a model therapeutic agent because of its cationic nature, use as a photosensitizer, and similarity to the chemotherapeutic 5-fluorouracil. Methylene blue is a hydrophilic compound (logP=1.1), similar to 5-fluoruracil (logP=0.89). Methylene blueloaded nanogels were dialyzed against 1 PBS (of pH 4.5 or 7.4), which was exchanged regularly with fresh buffer to both simulate drug sequestration/metabolism and establish a semi-sink condition. The buffer condition (1 PBS at pH 7.4) was intended to simulate the pH environment in circulation, whereas the pH 4.5 condition was meant to emulate the environment of the late endosome, which nanocarriers will experience during lysosomal trafficking following cellular uptake. It is noteworthy that in the case of cancer drug delivery, the nanocarriers will experience a gradient of pH, decreasing from circulation through the endosomal pathway. The drug release environment was maintained at 37C, and methylene blue elution was monitored until complete release was achieved (28 hours).

Unmodified nanogels loaded significantly more methylene blue than their TMOD and DMOD derivatives (fig. S6). Prior to modifications, nanogels loaded methylene blue with 99.50.3% efficiency (equal mass ratio nanogels: methylene blue in ultrapure water). Increasing nanogel modification with tyramine or N,N-dimethylethylenediamine decreased the equilibrium partitioning of methylene blue. Specifically, TMOD and DMOD nanogels loaded methylene blue with 59.72.1% and 34.9 9.2% efficiency, respectively. This decrease in equilibrium partitioning, relative to unmodified control nanogels, is due to the hydrophobicity or cationic character that the respective ligands contribute. As a cationic and hydrophilic payload, methylene blue enages in complementary electrostatic interactions with deprotonated methacrylic acid groups. Furthermore, as methylene blue partitions preferentially in water over organic phases, we expected loading efficiency to correlate positively with nanogel hydrophilicity. In the preceding section, we showed that the extent of nanogel functionalization correlated with the amount tyramine or N,N-dimethylethylenediamine in the reaction solution. Furthermore, as each modification reaction depleted a pendant methacrylic acid group, there is a negative relationship between extent of nanogel modification and the available methacrylic acid groups to interact with methylene blue. Following modification with N,N-dimethylethylenediamine, the amphoteric nanogels lost pH-responsive swelling behavior. Their tertiary amine moieties, which are cationic, exerted a repulsive force on methylene blue. As a result of tyramine modification, the nanogels became more hydrophobic, similarly lowering the networks ability to partition methylene blue. These physicochemical characteristics of TMOD and DMOD nanogels are useful for responsive release behavior, but as they decrease the nanogelmethylene blue affinity, they decrease methylene blue loading efficiency.

A drug release experiment probed the ability of each modified or unmodified nanogel system to act as an intelligent drug delivery vehicle. In this experiment, nanogels loaded with methylene blue [nanogels (1 mg/ml), with corresponding loading described above] were placed in dialysis tubing [regenerated cellulose, molecular weight cutoff (MWCO), 12,000 to 14,000 kDa] and dialyzed against 1 PBS (pH 4.5 or 7.4). The dialysate was exchanged for fresh buffer every 2 hours to simulate drug metabolism. At each time point, a sample was taken from both within the dialysis tubing and outside it (i.e., the dialysate) to ensure precise measurement of the kinetic methylene blue release.

Unmodified nanogels exhibited sustained-release kinetics without a noticeable burst release or pH-responsiveness. In 2 hours, unmodified nanogels eluted 41.015.5% and 46.04.0% of their loaded methylene blue at pH 7.4 and 4.5, respectively (Fig. 4A). The unmodified nanogels consistency, in their rate of methylene blue elution between the two pH environments, is consistent with their continuously anionic zeta potential. While the pH 4.5 environment is below the unmodified nanogels pKa, sufficient acid moieties remained deprotonated to engage in electrostatic interactions with methylene blue and promote payload retention in a manner similar to the pH 7.4 condition.

(A) Methylene blue experienced complementary electrostatic interactions with unmodified nanogels at both pH 4.5 and 7.4, leading to sustained release in both conditions. (B) TMOD nanogels exhibited an initial burst release of methylene blue, where the quantity of that release was greater in acidic than neutral conditions. (C) DMOD nanogels exhibited a burst release of greater than 50% the loaded payload in each pH condition, with more rapid release in acidic than neutral conditions. (D) DMOD and TMOD nanogels exhibited similar methylene blue release behavior in acidic conditions, while unmodified gels exhibited a more sustained-release profile. (E) DMOD nanogels released methylene blue rapidly in 1 PBS (pH 7.4), while unmodified nanogels exhibited sustained-release and TMOD gels displayed intermediate behavior. The results in (D) and (E) indicated that the nanogels zeta potential is largely predictive for their release profile [all panels: n = 4, mean SD; *P < 0.05, **P < 0.01, and ***P < 0.001, two-way analysis of variance (ANOVA) with Tukey posttest].

On the other hand, TMOD nanogels exhibited an initial burst release, which varied significantly with the pH environment (78.59.6% and 43.7 18.9% of the loaded payload in the first 15 min, at pH 4.5 and 7.4, respectively; P < 0.05). After the burst release, TMOD nanogels gradually released methylene blue at pH 7.4 and rapidly released it at pH 4.5 (Fig. 4B). TMOD nanogels bear a cationic zeta potential in acidic buffers and further undergo a hydrophile-to-hydrophobe transition around the critical pH point (pH 4.8). These physical and chemical alterations, which were unique to the TMOD nanogels, explain their significant and unique pH-responsive methylene blue release profile. DMOD nanogels exhibited substantial burst release, followed by rapid methylene blue elution at both pH 4.5 and 7.4 (Fig. 4C). Methylene blue release was more rapid from DMOD nanogels at pH 4.5 than pH 7.4, which can be attributed to the transition from anionic to cationic zeta potential, as was shown previously.

Figure 4 (D and E) highlights the differences in pH-responsive methylene blue elution for the three formulations. All nanogel formulations eluted the entirety of the methylene blue payload within 28 hours. Unmodified nanogels exhibited a sustained-release profile in both pH conditions, demonstrating their use for controlled release but lack of responsive release. DMOD nanogels, conversely, released methylene blue rapidly in both pH environments, acting as neither a sustained-release depot nor a responsive delivery vehicle. TMOD nanogels acted as a pH-responsive delivery vehicle, responding to the acidic environment by rapidly releasing methylene blue. In the pH 4.5 environment, there were significant differences (P<0.001) between the relative elution of methylene blue from modified and unmodified nanogels. However, there were no differences between the methylene blue elution profiles of the DMOD and TMOD nanogels. This indicated that the release profile is driven primarily by the nanogels cationic zeta potential and not a hydrophile-to-hydrophobe transition (which was unique to TMOD). In the pH 7.4 environment, there were significant differences between the methylene blue elution profile of all three formulations.

These results illustrated how modification of the acid moiety, through changing the nanogels environmentally responsive swelling and ionization, altered the systems use as a drug delivery vehicle. While unmodified nanogels were most advantageous for steadily delivering a hydrophilic, cationic payload to the surrounding environment, TMOD nanogels exhibited rapid pH-responsive delivery. This pH responsiveness could lead to triggered release in the acidic tumor or endosome microenvironments. Therefore, the identity and extent of nanogel surface modifications should be carefully tuned to yield combinations of sustained and responsive release for specific drug delivery applications.

Our original design goal was to construct a tunable nanoscale hydrogel platform that was cytocompatible and could be diversified in a modular manner with bioactive moieties. To assess cytotoxicity, we incubated nanogels with murine fibroblasts for 24 hours and measured the impact of nanomaterial exposure on the cells membrane integrity and metabolic activity.

Intact nanogels (degradable and nondegradable) exhibited limited toxicity to fibroblasts after 24 hours of incubation, while nanogels degraded by 10 mM glutathione in cell culture medium were nontoxic at concentrations up to 2 mg/ml (fig. S7A). Fibroblast membrane integrity was largely unaffected by 24-hour incubation with nondegradable, degradable, or degraded nanoparticles (fig. S7B), indicating that the reduction in metabolic activity observed in fig. S7A was not due to cell lysis. Modification of nanogels with tyramine or N,N-dimethylethylenediamine did not alter their cytotoxicity (fig. S7C), as measured by cell metabolic activity following 24-hour exposure to a dose of 2 mg/ml. Peptide incorporation (fig. S7D) at approximately 2 weight % (wt %) of the dry nanogel weight did not significantly affect nanogel cytotoxicity (fibroblasts, 24-hour exposure, 2 mg/ml dose), as peptide-modified nanogels did not alter the cells metabolic activity.

We then monitored the extent to which nanogel toxicity differed across different cell types (fibroblast, macrophage, and colon epithelial). These were selected as model cell systems for the different cell types that would experience a nanomaterial insult following injection. We recognized that each cell line would interact with the nanogels differently, altering the extent to which the material impairs the cell viability. No significant differences were observed in the cells viability, as determined by metabolic activity or membrane integrity, for degradable, nondegradable, or degraded nanogels at concentrations up to 2 mg/ml (fig. S8). It is noteworthy that we saw a nonstatistically significant trend in macrophage activity, where metabolic activity increased and membrane integrity decreased at the top concentration (2 mg/ml, 24 hours). This does indicate acute toxicity to macrophages at this dose.

We assessed the impact of the nanogels chemistry, through surface modification, on their uptake by different model cell lines. We selected fibroblasts, macrophages, and epithelial cells because they model components of the connective tissue, immune system, and tissues/organs, respectively. Furthermore, by selecting colon epithelial carcinoma (SW-48) cells as the epithelial model, we simultaneously probed the impact of surface modification on preferential uptake by human tumor cells.

Modified nanogels for uptake studies were prepared in the same manner as in previous modification efficiency, therapeutic efficacy, and cytotoxicity studies, except for the addition of a carboxylic acidreactive fluorophore in the modification solution. To make the nanogels fluorescent, we added 5-(aminoacetamido)fluorescein at 0.8 wt % of the dry polymer (for comparison, the tyramine or N,N-dimethylethylenediamine ligand was added simultaneously at 10 wt %) to the modification solution. This fluorophore was conjugated to all of the nanogel formulations, including the unmodified nanogels.

The fluorophore was successfully conjugated to unmodified, TMOD, and DMOD nanogels, although a decreased fluorophore coupling efficiency was observed for DMOD nanogels. We produced calibration curves for all modified nanogel formulations and normalized our subsequent image analyses to the relative slope for each formulation (correction factors: unmodified, 1.27; TMOD, 1; and DMOD, 5.44). We also validated that fluorophore conjugation did not significantly alter the nanogels cytotoxicity by conducting MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and LDH (lactate dehydrogenase) assays for nanogel exposure to each of the three cell lines at concentrations up to 2 mg/ml. No significant cytotoxicity was observed by either measure at concentrations up to 1 mg/ml (24-hour exposure, all three cell lines) (fig. S9). Consequently, the maximum nanoparticle dose for all uptake studies was maintained at 1 mg/ml.

Even at low doses (less than 40 g/ml, 24-hour exposure), murine macrophages imbibed substantial quantities of all three nanogel formulations (Fig. 5, A to C). On the other hand, fibroblasts exhibited limited uptake of unmodified and TMOD but took up DMOD nanogels. Human colon epithelial cells took up all three formulations, exhibiting no preference for unmodified or TMOD nanogels, but a 13.4-fold increase in uptake when exposed to DMOD nanogels (relative to unmodified nanogels, 250 g/ml, 24-hour exposure). Representative images, visualizing nanogel uptake by each of the three cell lines, are given in fig. S10.

The relative uptake was computed by normalizing the green fluorescent protein (GFP) (nanoparticle) signal to the slope of the calibration curve and then normalizing that value to the 4,6-diamidino-2-phenylindole (DAPI) (cell nucleus) signal. Note that the y axis quantities differ between plots, as the DMOD nanogels were uptaken in significantly greater quantity than TMOD or unmodified nanogels. (A to C) Relative uptake of unmodified, TMOD, or DMOD fluorescent nanogels by each cell line, as a function of dose (24-hour exposure). (D to F) Kinetic uptake of unmodified, TMOD, and DMOD nanogels (400 g/ml dose). Representative images for each plot are given in fig. S10 (all panels, n = 4, mean SEM).

These dose-response results demonstrated that, while nanogel modification with N,N-dimethylethylenediamine generally increased uptake, the extent to which uptake was enhanced differed between cell lines. Compared with unmodified nanogels, DMOD nanogels exhibited a 4.5-fold increase in uptake by macrophages, 11.6-fold by fibroblasts, and 17.0-fold by colon carcinoma cells (250 g/ml, 24-hour exposure, all differences significant at the P < 0.05 level). This result suggests that the cell-nanomaterial interactions, which promoted uptake and were imparted by the N,N-dimethylethylenediamine moiety, triggered varying degrees of response from different cells. Furthermore, TMOD nanogels were uptaken similarly to unmodified nanogels. Tyramine modification led to a 21% decrease in uptake by macrophages, 31% decrease by fibroblasts, and 3.8% increase by colon carcinoma cells, none of which were statistically significant. This confirmed that a surface modification that imparts environmental responsiveness or alters therapeutic partitioning does not necessarily also enhance cell uptake.

Image analysis revealed that the nanogels interacted with each cell line in a different spatiotemporal manner. Nanogels did not interact substantially with fibroblasts, and when they did, they colocalized primarily with the cell membrane. Macrophages rapidly internalized the nanogels, with images demonstrating cytosolic colocalization in as little as 30 min. In the case of colon epithelial cells, nanogels first associated with the cell membrane, which preceded uptake. DMOD nanogels associated with the colon epithelial cells membranes and were internalized more rapidly than unmodified and TMOD nanogels (fig. S10).

Kinetic analyses of nanogel uptake further clarified the differences in nanogel uptake within cell lines and between formulations (Fig. 5, D to F). For precision medicine applications, we want to ensure that target cells (i.e., colon cancer cells) internalize the nanomaterial prior to complete therapeutic elution or clearance by off-target cells (i.e., fibroblasts or macrophages). It is relevant to recall that, depending on the particular surface modification and pH environment, the majority of the loaded methylene blue was eluted in less than 4 hours. Therefore, a formulation that rapidly associates with, and facilitates uptake by, target cells will enhance cytosolic delivery of the payload.

Murine macrophages took up all three nanogel formulations with near zero-order kinetics for the first 6hours. On the other hand, colon carcinoma cells exhibited a rapid cell-nanoparticle association [i.e., a spike in the green fluorescent protein (GFP)/4,6-diamidino-2-phenylindole (DAPI) signal in the first 15 min to 2hours], followed by a plateau in the signal intensity. Nanogels did not associate with the membrane or cytosol of murine fibroblasts until 24 hours of exposure. We looked specifically at nanogel uptake within the first 2 hours of dosing, as this is when majority of the methylene blue elution occurred in our drug release studies (at pH 7.4, 41% released by unmodified, 64% released by TMOD, and 91% released by DMOD). To compare the nanogel uptake at 2hours across cell lines, we computed the relative uptake as the ratio of the 2- and 24-hour uptake (400 g/ml dose). A two-way analysis of variance (ANOVA) revealed that formulation accounted for only 11.1% of the total variation in relative uptake (not significant), whereas the cell line identity explained 38.6% of the total variation in relative uptake (significant at the P < 0.001 level). Consistent with the dose-response study presented above, DMOD uptake by colon cancer cells was 12.4 times greater than that of unmodified nanogels. Tyramine modification did not significantly affect the extent of nanogel uptake by any cell line.

The rapid association and uptake of DMOD nanogels by colon cancer cells is particularly interesting, as it suggests that this particular surface modification could enhance specific drug delivery to target tumor cells. However, as these experiments were conducted in homogeneous, static cell cultures, we are unable to conclude whether this preference for colon tumor cells would translate to in vitro coculture or in vivo models.

One advantageous therapeutic quality of DMOD nanogels was their ability to act as an intrinsic reducing agent and, subsequently, act as centers for gold nanoparticle precipitation. DMOD nanogels with the three highest degrees of modification (0.78:1 DMOD or greater) were able to reduce gold chloride successfully, forming nanogel-coated gold nanoparticles. Nanogels with lesser quantities of N,N-dimethylethylenediamine did not form gold nanoparticles. Analysis of the composite nanogels absorbance spectrum (Fig. 6A) revealed that the conjugates absorb visible light strongly, with a maximum absorbance wavelength of 536 nm. In the transmission electron microscopy (TEM) images presented here (Fig. 6B), the gold nanoparticles are visible as dark circular regions within the nanogel bulk. Some, but not all, of the nanogels contained gold nanoparticles after the precipitation reaction.

Gold nanoparticles were precipitated in DMOD nanogels. DMOD gels with a 0.39:1 ratio of N,N-dimethylethylenediamine:methacrylic acid or less were unable to facilitate gold nanomaterial formation. (A) Absorbance spectra of composite nanogels containing gold nanoparticles. (B) Transmission electron micrographs of gold nanomaterials within 3.1:1 DMOD nanogels. Arrows point to gold nanoparticles. (C) Proof of concept for the composite nanogels ability to transduce visible light (=532 nm) into heat. DMOD (3.1:1) nanogels with gold nanoparticles effectively and rapidly heated a 1 PBS suspension. (D) Concentration-dependent photothermal activity of 3.1:1 DMODgold nanoparticle composites (n = 4, mean SD).

DMOD nanogelgold nanoparticle composites (3.1:1) were suspended at various concentrations in 1 PBS and were irradiated with a 532-nm laser at 200 mW. Within 30 s, the PBS suspension reached an equilibrium temperature (Fig. 6C), while the heat rapidly dissipated when the laser was turned off. Nanogels alone, in the absence of precipitated gold nanoparticles, did not heat the surrounding medium when irradiated with the same laser, indicating that the gold nanomaterials were acting as a transducing element. The heat generated by laser irradiation increased with nanoparticle concentration, with a maximum heating of 10.30.20C by a nanoparticle-in-nanogel suspension (1 mg/ml) (Fig. 6D).

Next, we demonstrated the feasibility of peptide and protein coupling to the base nanogel platform. Peptides can be used to impart specific biological behaviors, including molecular recognition, cell targeting, cell penetration, and endosomal escape. Bioactive proteins can contribute enzymatic activity to the otherwise inert network or be used as a molecular recognition unit for targeting applications.

Two independent peptide conjugation reactions were explored: one for coupling cysteine-containing peptides via a thiol-maleimide reaction and a second for coupling the N-terminal amine or pendant lysine groups to carboxylic acids in the nanogel network. Five diverse, cysteine-containing peptide sequences were selected to sample a diverse array of peptide properties (two cationic, two anionic, and one electrically neutral at physiological pH, all water soluble). These peptides were previously identified by the authors as candidates for trypsin recognition in physiological fluids. In the present study, they were used as model oligopeptides to optimize a generalized nanogel-peptide conjugation strategy and conclude relationships between a peptides formal charge and its coupling efficiency. In a two-step conjugation schema (Fig. 7A), we first coupled a maleimide-terminated linker molecule to the nanogels via carbodiimide-mediated coupling (pH 4.5). After 2 hours, we adjusted the nanogel suspension pH to 7.0 to favor the thiol-maleimide click reaction with the cysteine-containing peptides, as opposed to any aminecarboxylic acid side reaction (i.e., those between the nanogels carboxylic acid and the peptides N terminus, or peptide dimerization via the C and N termini of multiple peptides).

(A) A thiol-maleimide click reaction effectively conjugated cysteine-containing peptides to the nanogel network. (B) A carboxylic acidamine reaction linked the peptides N terminus with the carboxylic acidcontaining nanogels. (C) Differential incorporation of diverse peptides was explained by their net charge at physiological pH. (D) Nanogel conjugation at 2 wt % did not significantly alter the nanogel diameter or zeta potential. (E) Peptide content in the final conjugate product can be readily tuned by altering the peptide feed concentration via reaction scheme (B). (F) Horseradish peroxidase (HRP) and wheat germ agglutinin (WGA) were incorporated into nanogels with 56.4 and 81% efficiency, respectively. (G) HRP retained 66.533% of its activity upon conjugation to the nanogel platform, as evidenced by the ability of HRP-nanogel conjugates to convert TMB substrate. (H) WGA-NP conjugates retained native WGA activity, as they bound and stained the cell membrane of L929 murine fibroblasts (blue, DAPI stain of nucleus; red, WGA-NP conjugates) (C to F, n = 3, mean SD; G and H, n = 3, representative data).

We were successful in conjugating all five peptides to the network, demonstrating the feasibility of conjugating diverse peptide ligands to the platform. Peptide content, within each nanogel network, was quantified with a Micro BCA colorimetric assay. Cationic (FAHWWC and HAHWEC) and electrically neutral (CDHFAI) peptides were incorporated with nearly complete efficiency (theoretically complete incorporation was 2% of the dry weight). On the other hand, anionic peptides were incorporated with lesser efficiency (43.78.5% and 50.98.6% for CDNWQY and ADCFLQ, respectively) (Fig. 7C). This highlighted the effect of peptide formal charge, which influences its equilibrium partitioning in the nanogel phase during the conjugation reaction, on efficient coupling. The extent of nanogel decoration with anionic peptides was increased by elevating the concentration of the anionic peptide in the coupling reaction, but is still significantly less efficient than the coupling of neutral and cationic peptides. Nanogel decoration with peptide, at 2 wt %, did not significantly alter the nanogels size or zeta potential (Fig. 7D).

In a separate bioconjugation schema, the peptides were linked directly to the nanogel network via a reaction between the peptide N terminus and pendant carboxylic acid groups (Fig. 7B). Again, conjugation of a cationic peptide (HAHWEC) was efficient, as the quantity of the peptide within the network was readily controlled by modulating the peptide concentration in the coupling reaction (Fig. 7E). Our model anionic peptide (CDNWQY) was incorporated into the nanogels, but with a lesser efficiency (62.715.0%, depending on the peptide concentration in the modification solution).

Wheat germ agglutinin (WGA) and horseradish peroxidase (HRP) were conjugated to nanogels via carbodiimide-mediated coupling, with 2 wt % protein in the modification reaction. These model proteins were selected, as they are commonly applied for immunohistochemistry and biosensing applications, respectively. As a result of selecting these two protein targets, we had methods for verifying the retention of protein activity following conjugation to the nanogels. Furthermore, the result is applicable to other proteins that have affinity for extracellular targets (similar to WGA) or catalyze small-molecule conversion (similar to HRP). Each protein was incorporated successfully (Fig. 7F) and retained its bioactivity after conjugation. HRP activity was quantified by the colorimetric determination of enzymatic conversion of 3,3,5,5-tetramethylbenzidine (TMB) substrate. Standard curves were generated for TMB conversion as a function of HRP concentration (free HRP or HRP bound covalently to nanogels). By comparing the conjugated HRP activity to that of free HRP at the same concentration, we determined that 66.533% of the HRP activity was conserved (Fig. 7G). This provided evidence that our nanogels acted as scaffolds for retaining and presenting bioactive HRP to the surrounding environment.

WGA activity was assessed by determining the effectiveness with which WGA-labeled nanogels labeled fibroblast cell membranes. Fibroblasts were selected because unlabeled nanogels neither associate with fibroblast cell membranes nor are uptaken by fibroblasts within 2 hours, as quantified in Fig. 6 and illustrated in fig. S10. Therefore, colocalization of nanogels with the fibroblast membranes, or uptake into the cytosol, is due to the membrane-targeting activity of WGA. As shown in Fig. 7H, the WGA-labeled nanogels (red) colocalize with the cell cytosol, indicating that the conjugated WGA facilitated cell-nanogel interactions and subsequent uptake.

Here, we documented a modular, tunable nanogel platform for therapeutic applications. P(AAm-co-MAA) nanogels were decorated with numerous amine-containing ligands (i.e., small molecules, peptides, and proteins) and retained the ligand bioactivity (i.e., intrinsic reducing ability, pH sensitivity, hydrophobicity, molecular recognition characteristics, and enzymatic activity). We tuned the extent of ligand decoration by modulating the characteristics of the modification reaction and yielded a range of therapeutic capabilities, including cell targeting, enhanced nanomaterial uptake, intelligent drug delivery, and photothermal therapy.

In its present form, unmodified P(AAm-co-MAA) nanogels are suitable for loading high weight fractions of hydrophilic, cationic therapeutics. A suitable initial chemotherapeutic agent will be 5-fluorouracil, which is used to treat a number of cancers including colorectal cancer. These unmodified nanogels exhibited sustained therapeutic delivery for greater than 6 hours. Tyramine-modified nanogels were responsive to the pH environment and, as a result, delivered methylene blue more rapidly in acidic than neutral buffer. N,N-dimethylethylenediaminemodified nanogels were amphoteric in nature, eluted methylene blue the most rapidly of the tested formulations, and increased nanogel uptake by colon cancer cells.

Gold nanoparticle precipiation enabled photothermal therapy. Following DMOD nanogel accumulation in tumor sites, excitation with a focused green laser would heat the tumor tissue. Previous studies using gold nanoparticles for photothermal therapy applications have demonstrated efficacious heating using green light (28, 29). However, our platform combines photothermal therapy and chemotherapeutic delivery in a new, modular manner. In the future, platform functionalization with targeting peptides, monoclonal antibodies, or other targeting molecules could further enhance nanogel targeting and cell uptake.

As presented in Introduction, research on treating cancer with multiple therapeutic modalities is increasing in prevalence (30, 31). This platform technology, with its highly tunable nature, is amenable to delivering multiple chemotherapeutics and facilitating combination therapies, each with precise targeting or environmental responsiveness. Calibrated combinations of modified and unmodified nanogels in a single regimen could produce new physical distributions and release profiles of therapeutic agents in the future.

In addition to demonstrating the use of a new platform technology, we introduced multiple new characterization methods, which will be of utmost use to researchers developing nanoscale devices for precision medicine. In particular, while QCM methods have been used previously to study the deposition of nanomaterials on solid surfaces or the interfacial interactions governing monolayer self-assembly (3234), the use of QCM to study swelling and biodegradation of nanogel materials is novel. Similarly, while nanomaterial internalization by cells has been an active area of research, using methods such as confocal microscopy and flow cytometry (35, 36), we developed a high-throughput microplate assay for nanogel uptake. As a direct result of having this new analysis tool, we were able to screen the dose-dependent and kinetic uptake of our modified nanogels by three relevant cell lines.

In conclusion, we engineered a new nanogel platform, which is modularly tunable for precision medicine applications. We quantified the extent to which nanogel composition altered drug-material interactions for the loading and release of cargo, transduction of external signals, targeting of proteins, and uptake by cells. Our new methods, described herein, will also provide new tools to the drug delivery field to rapidly screen or precisely quantify biological interactions with engineered nanomaterials in the future.

Nanogels were synthesized by inverse emulsion polymerization, as previously optimized by Zhong et al. (16). Acrylamide [75 mole percent (mol %)], methacrylic acid (22.5 mol %), and methylene bisacrylamide (2.5 mol %) were dissolved in water at 42 wt %. This aqueous phase (2.762 ml) and N,N,N,N-tetramethylethylenediamine (50 l; catalyst) were added slowly to a stirring solution of Brij 30 (151.4 mM) and AOT (dioctyl sulfosuccinate sodium salt; 30.3 mM) in hexanes (50 ml) to form a water-in-oil emulsion. This prepolymer emulsion was purged with nitrogen for 20 min to remove dissolved oxygen, and polymerization was initiated by injecting 10 mg of nitrogen-purged ammonium persulfate [100 l of a freshly prepared stock (100 mg/ml) in ultrapure water]. After 2hours, the reaction was stopped by opening the round-bottom flask to air, and the nanogels were purified by precipitation in ethanol (three times) followed by either ionomer collapse or dialysis against a water:ethanol gradient.

In ionomer collapse, the nanogels were suspended in 0.5 N sodium hydroxide and precipitated with the addition of a threefold volume excess of acetone. Precipitated nanogels were collected by centrifugation (3200g for 5 min), and the collapse procedure was repeated an additional four times. In gradient dialysis, nanogels were suspended in a 50:50 water:ethanol mixture and dialyzed against a decreasing water:ethanol gradient for >5 days with twice-daily dialysate change. Nanogels purified by both ionomer collapse and gradient dialysis were then exchanged into ultrapure water by dialysis. All purified nanogels were lyophilized and stored at room temperature.

For studies involving degradable nanogels, synthesis was conducted in the manner described above, with N,N-bis(acryloyl)cystamine substituted for methylene bisacrylamide. N,N-bis(acryloyl)cystamine is a biodegradable cross-linker that is labile via reduction of its disulfide bond. Cross-linker comprised 2.5 mol % of the monomer feed, and the masses of acrylamide and methacrylic acid were adjusted such that total monomer concentration remained 42wt % in water.

Nanogels were suspended at 10 mg/ml in 1 PBS and adjusted to pH 7.4. Then, 0.5 ml of nanogels and 0.5 ml of DTT or glutathione (20 mM in 1 PBS, pH 7.4) were mixed in a polystyrene cuvette, immediately after which light scattering measurements were recorded. Measurements were recorded using a Zetasizer Nano ZS (Malvern) with a manual attenuation (Attn, 7), measurement position (4.65 mm), and measurement time (10s). Measurements were taken repeatedly for 50 min. In each interval, a hydrodynamic diameter and count rate were recorded. Because count rate trends with the number of particles in solution (37), the count rate at a given time, normalized to the initial count rate, provided a measure of the degree of degradation.

QCM studies were conducted using QSense E4 QCM-D (Biolin Scientific). Uncoated gold sensors were washed in a 5:1:1 volume ratio of ultrapure water, ammonia hydroxide (25 volume %), and hydrogen peroxide (30 volume %) at 75C for 5 min. The sensors were then washed with an excess of water and an excess of ethanol and were dried under nitrogen. Immediately prior to experimentation, clean sensors were treated with ultraviolet/ozone for 10 min.

All experiments were conducted in their entirety at 37C and a flow rate of 0.200 ml/min. A stable baseline for the sensors was achieved by flowing 1 PBS for at least 10 min. Then, the sensors were coated with an amine-terminated monolayer through the addition of cysteamine HCl (10 mg/ml in 1 PBS). Nanogels activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (twofold molar excess relative to MAA) were flowed over the modified sensor at 2 mg/ml. After a stable coating was obtained (as evidenced by no further fluctuation in the frequency or dissipation), 1 PBS was flowed over the sensor to wash away unreacted polymer and remaining catalyst. The change in resonance frequency, as a result of nanogel coupling, was recorded as a measure of the bound nanogel mass.

Nanogels were swelled in a series of buffers differing in ionic strength (PBS buffers at pH 7.4 diluted to 5, 2, 1, 0.5, 0.1, and 0.01 with ultrapure water) as well as 1 PBS buffer adjusted to different pH values (3, 5, 7, 9, and 11). The purpose of these swelling steps was twofold: to quantify the responsiveness of nanogels to environmental conditions and to verify that the nanogels are behaving as expected despite their immobilization. The frequency and dissipation values were monitored to quantify the nanogels water uptake or expulsion in each buffer condition, as well as to determine the viscoelastic properties of the nanogel layer.

Nanogels were degraded by flowing a 10 mM DTT solution over the nanogel-modified sensors. The frequency and dissipation values were monitored to determine the mass loss during degradation as well as probe changes in viscoelastic properties that indicate the degradation mechanism (i.e., bulk degradation or surface erosion). In each case, the mass immobilized or adsorbed was quantified by the Sauerbrey equationm=Cfnwhere m is the mass adsorbed or immobilized, C is a constant that depends on the intrinsic properties of quartz [for a 5-MHz crystal, C = 17.7 ng/(cm2 Hz)], and n is the overtone number (i.e., 3 and 5). The relative mass was computed by normalizing the change in mass, due to swelling or degradation, to the mass of nanoparticles immobilized (32, 38). It is also equal to the ratio of the frequency changesmdegradationmimmobilized=fdegradationfimmobilized

Purified, dried nanogels were suspended in 10 mM MES buffer and adjusted to pH 4.50.05. Tyramine or N,N-dimethylethylenediamine was dissolved in water at 25 mg/ml. EDC hydrochloride was dissolved immediately prior to use in MES buffer at 56 mg/ml. Each reaction was composed of 5 ml of nanogels (50 mg), 1 ml of EDC solution (56 mg of EDC, a 2:1 molar ratio EDC:MAA by original synthesis feed, and 0.8:1 molar ratio EDC:acid subunit when confirmed by titration analysis), and a variable volume of tyramine or N,N-dimethylethylenediamine (1.6 ml for the highest degree of modification and cut by half for each subsequent reaction). The highest modification ratio (by moles) tested was 2:1 tyramine:MAA and a 3.1:1 N,N-dimethylethylenediamine:MAA. In each highest modification case, the ligand concentration was 80% that of the nanogels by mass. As a control, nanogels were subjected to the reaction conditions (MES buffer, pH 4.5, 56 mg of EDC) in the absence of ligand. Modified nanogels were purified by dialysis against ultrapure water (>72 hours, frequent water changes). Reactions were completed in duplicate.

The physicochemical properties of nanogels were quantified by attenuated total reflectanceFTIR spectroscopy (Nicolet iS10 FTIR Spectrometer; Thermo Fisher Scientific), DLS, and zeta potential measurement (Zetasizer Nano ZS; Malvern). Dried nanogels were pressed in contact with a germanium crystal, and the IR absorption spectrum was recorded from 4000 to 675 cm1. All presented spectra are the average of 64 measurements. All spectra were normalized such that the magnitude of their largest peak was 1U, and their baseline was set at zero. All DLS measurements, unless otherwise stated, were obtained at a nanogel concentration of 2 mg/ml in 1 PBS, adjusted to pH 7.4. All zeta potential measurements, unless otherwise stated, were taken at a nanogel concentration of 2 mg/ml in 5 mM sodium phosphate buffer, adjusted to pH 7.4.

The extent of modification with tyramine, N,N-dimethylethylenediamine, or 5-(aminoacetamido)fluorescein was quantified by potentiometric titration. Ten milligrams of modified or unmodified nanogels was suspended in 60 ml of 5 mM potassium chloride buffer. The suspension was adjusted to pH 10 with 1 N sodium hydroxide to completely deprotonate pendant methacrylic acid groups. The solution was titrated through the equivalence point with 0.01 N hydrochloric acid (HCl) using an autotitrator (Hanna HI901C). We titrated the nanogel suspensions from a basic-to-acidic environment to ensure that the nanogels were swollen during the entirety of adjustment to equivalence. The equivalence point for methacrylic acidcontaining nanogels was consistently at pH 4.8.

We assume that at equivalence (pH 4.8), exactly half of the acid moieties are protonated. We also assume that at a pH three points above equivalence (pH 7.8), 0.1% of the acid groups are protonated. The volume of 0.01 N HCl needed to adjust pure 5 mM KCl buffer from pH 7.8 to pH 4.8, as well as the volume needed to adjust each nanogel suspension the same increment, was recorded. Using these measurements and the stated assumptions, we calculated the mass fraction of methacrylic acid groups usingmMAAmnanogels=(10.499)(VsuspensionVbuffer)NtitrantMWMAA1mnanogelswhere mnanogels was 0.010 g, Ntitrant was 0.01 M, the molecular weight of methacrylic acid (MWMAA) is 86.06 g/mol, and both volumes were measured in liters.

The extent of nanogel functionalization with peptides or proteins was quantified using a Micro BCA colorimetric assay (Thermo Fisher Scientific), as described previously (39). Modified or unmodified nanogels, suspended at 2 mg/ml in 1 PBS (pH 7.40.05), were combined at an equal volume ratio with Micro BCA working reagent and mixed for 2 hours at 37C (constant mixing). The absorbance of the reduced supernatant (=562 nm) was used to quantify the suspensions peptide concentration, relative to standard curves generated for each pure peptide or protein. The background absorbance of unmodified nanogels under the same testing conditions was subtracted from each measurement.

L929 murine fibroblasts, RAW 264.7 murine macrophages, and SW-48 human colorectal epithelial carcinoma cells were chosen as model cells to properly assess nanomaterial interactions with model connective, immune, and epithelial tissues that would interact in vivo. All cells were cultured in T-75 tissue culturetreated flasks and were used at passages ranging from 6 to 20.

Cells were incubated in a sterile 37C, 5% CO2 environment. Culture medium for all three cell lines was phenol redcontaining high-glucose Dulbeccos modified Eagles medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 1% penicillin-streptomycin. Cells were passaged once they reached 80 to 90% confluency. Experiment medium for all three cell lines was phenol redfree, high-glucose DMEM with 2% FBS, 2 mM l-glutamine, and 1% penicillin-streptomycin. Experiments were conducted when cells reached 50 to 70% confluency.

For all cell assays, L929 and RAW 264.7 cells were seeded in tissue culturetreated 96-well plates at a density of 10,000 cells per well. SW-48 cells were seeded in similar plates at 25,000 cells per well. Cells were given a minimum of 24 hours to attach and reach 50 to 70% confluency before cytotoxicity, or nanogel uptake assays were performed.

Methylene blue was selected as a model hydrophilic, cationic therapeutic. Methylene blue is a photosensitizer and was selected because of its similarity in hydrophilicity and ionization to hydrophilic chemotherapeutics (i.e., 5-fluorouracil), as well as its compatibility with our hydrophilic, anionic nanogels. Methylene blue was loaded into modified and/or unmodified nanogels by equilibrium partitioning in ultrapure water. For loading experiments, methylene blue (2 mg/ml) and purified nanogels (2 mg/ml) were mixed for 15 min in distilled water. Drug loading was quantified by removing a sample (500 l) and separating the unbound drug by ultrafiltration (Sartorius Vivaspin 500; 300,000 MWCO). The unbound methylene blue was quantified by absorbance (=590 nm) relative to a standard curve. Loaded or partitioned methylene blue was quantified usingQ=(C0Ce)Vmwhere Q is the mass ratio of loaded methylene blue to nanogels, C0 is the methylene blue concentration in the loading solution (1 mg/ml), Ce is the unbound concentration of methylene blue (that passed through the filter), V is the volume of the loading solution, and m is the mass of nanogels in the loading solution.

Prior to drug release experiments, unloaded drug was removed by dialysis against ultrapure water (24 hours, 12,000 to 14,000 MWCO). Methylene blueloaded nanogels [10 ml, nanogels (1 mg/ml), methylene blue (1 mg/ml) in the loading solution, variable methylene blue loaded], still within dialysis tubing, were transferred to 1 PBS solution (400 ml) at pH 4.5 or 7.4 under constant stirring at T=37C. At regular time intervals (15 min, 30 min, 1 hour, 1.5 hours, 2 hours, 4 hours, 8 hours, 24 hours, and 28 hours), samples were taken both from within and outside the dialysis tubing. For samples drawn from within the dialysis tubing, loaded and released methylene blue were separated by ultrafiltration. The dialysate was exchanged for fresh buffer at the 2-hour time point and each time point thereafter to simulate drug metabolism. This dialysate exchange ensured that a concentration gradient (between the nanogel and solution phases) was maintained to facilitate complete methylene blue elution. The total released drug was quantified for the first time point asmreleased=Vwithin tubingCMB,within tubing+VdialysateCMB,dialysatewhere the volume parameters describe the total volume within and outside of the dialysis tubing, respectively, and the concentration parameters capture the released methylene blue present in each solution location.

Note that the volume within the dialysis tubing changes with each time point (as sample is depleted) and that released methylene blue within the dialysis tubing eventually dissipates into the dialysate. It is important to correct for these mathematically. For example, at the 30-min time point, the methylene blue released in the 15-min increment from 15 to 30 min was calculated usingmreleased=(Vwithin tubingCMB,within tubing)t=30(Vwithin tubingCMB,within tubing)t=15+(VdialysateCMB,dialysate)t=30(VdialysateCMB,dialysate)t=15

Please note that this equation holds for all future time points as well, changing the respective time indicators, with the one exception that the final term is omitted if the dialysate buffer was exchanged prior to the interval.

Culture medium was removed by plate inversion and replaced with experiment medium containing hydrogel microparticles (0.0005 to 2 mg/ml). In the case of degraded nanogels, the culture medium containing degradable nanogels was spiked with 10 mM glutathione and incubated at 37C for 24 hours prior to the experiment.

Plate layouts were pseudorandomized. To pseudorandomize, we distributed the samples and controls throughout each microplate to ensure that each sample/control was positioned equitably along the plate exterior or within the interior. This controlled for variation in cell proliferation explained by well location within the plate. Following 24-hour incubation, cytotoxicity was quantified via metabolic activity (MTS) and cell membrane integrity (LDH assay).

For MTS assays, the nanogel-containing experiment medium was removed by plate inversion, and cells were washed with 37C Dulbeccos PBS (DPBS) twice to remove adsorbed nanogels and cell debris. Then, 100 l of MTS assay buffer (MTS diluted 1:6 in experiment medium) was added to each well, and relative metabolic activity of each cell sample was quantified by measuring the MTS metabolism (90 min at 37C) within each well, relative to control, as specified by the manufacturer (Promega).

For LDH assays, LDH assay buffer (100 l) was added directly to the cell media containing nanogels and any cell debris. The relative membrane integrity was calculated by measuring the LDH activity (excitation, 560 nm; emission, 590 nm) according to the following relationRelative membrane integrity=100100sampleblankmax releaseblankwhere the sample measurement is the fluorescence of the treatment media with LDH assay buffer, the blank is the cell culture medium and assay buffer without cells, and the max release is the LDH buffer and treatment media after a 20 min incubation of cells with media and 2 l of lysis buffer (Promega).

A high-throughput fluorescence imaging assay was developed for rapidly screening cells uptake of modified and unmodified nanogels. Fluorescently tagged nanogels, with the addition or lack of tyramine or N,N-dimethylethylenediamine, were suspended in phenol redfree DMEM at concentrations ranging from 1000 to 6.25 g/ml. Cells were dosed with either a gradient of nanogel concentration (24-hour incubation) or a set concentration for a range of time (concentration of 400 g/ml).

For concentration-dependent nanogel uptake assays, culture medium was removed from each well by plate inversion and replaced by treatment medium containing suspended nanogels. Cells were allowed to incubate for 24 hours in the presence of nanogels (100 l per well). Following incubation, the nanogels were removed by aspiration, followed by three washes with cold DPBS. Cells were fixed with cold paraformaldehyde solution for 10 min (50 l per well).

For time-dependent nanogel uptake assays, culture medium was removed from each well and replaced with treatment media containing nanogels (400 g/ml) by aspiration in an inverse time manner (i.e., 24-hour time point first, 15-min time point last) (100 l per well). This was scheduled in such a way that all wells reached their end point simultaneously. Nanogel suspensions were removed from the cells by aspiration, and the cells were washed three times with cold DPBS (100 l per well). These cells were also fixed with cold paraformaldehyde solution (50 l per well).

Cells were stained directly in the microplates for fluorescence imaging. Following fixation, each well was washed three times with cold Hanks balanced salt solution (HBSS) (100 l per well). Then, the cell membranes were stained with a WGA Alexa Fluor 594 conjugate solution (3 g/ml) in cold HBSS (15 min) (50 l per well). After three more washes with cold HBSS (100 l per well), the cells were stained with a DAPI solution (1 g/ml) in cold HBSS for 10 min (50 l per well). Each well was washed three times with cold HBSS prior to imaging (100 l per well). Imaging was conducted with 100 l of fresh HBSS in each well.

Cell imaging was conducted at high throughput using a Cytation 3 plate reader (BioTek) with Gen5 software (version 3.04) equipped with DAPI, GFP, and Texas Red filters (DAPI: excitation, 377 nm; emission, 447 nm; Texas Red: excitation, 586 nm; emission, 647 nm; and GFP: excitation, 469 nm; emission, 525 nm) and an Olympus 20 objective. Imaging parameters were optimized to the most fluorescent samples to prevent saturation and were held constant to enable both qualitative and quantitative image analyses between cell lines and treatments [DAPI: light-emitting diode (LED) intensity, 5; integration time, 63; and gain, 0; Texas Red: LED intensity, 10; integration time, 100; and gain, 13.8; and GFP: LED intensity, 10; integration time, 158; and gain, 15]. Four images were taken for each well, and images were preprocessed with a background subtraction step prior to qualitative analysis.

For quantitative analysis, the fluorescence of the whole well was taken for each relevant channel (DAPI, Texas Red, and GFP with gain values of 60, 100, and 120, respectively). To normalize the nanoparticle signal intensity to the cell count, we normalized the fluorescence intensity of GFP to the DAPI channel. The relative nanogel uptake for each cell linecondition pair is given as this ratio.

To precipitate gold nanoparticles within the hydrogel nanogels, chloroauric acid (0.05 wt %) and nanogels (1 mg/ml) were suspended in ultrapure water and mixed (Eppendorf ThermoMixer) at 1000 rpm and 60C for 1 hour. Nanogels with precipitated nanoparticles were used in further experiments without purification. These composite nanogels were characterized by their visible absorption spectra (300 to 1000 nm in 1-nm intervals using a Cytation 3 microplate reader) as well as by TEM (FEI Tecnai Transmission Electron Microscope, operating at 80 kV, cast on carbon-coated grid, and stained with uranyl acetate).

Photothermal experiments were conducted as previously described (40, 41). For photothermal therapy experiments, a 532-nm laser diode (PN156-10.07-0447) was used. This laser wavelength was within the maximum absorbance peak of the gold nanoparticlecontaining nanogels. The nanogels were suspended in ultrapure water at 1 mg/ml, and 1 ml of each nanogel suspension was added to a 24-well microplate. The laser, operated at a power of 200 mW, was focused on a circular area with a 6-mm diameter using a convex lens (Thorlabs Inc.), which was mounted at a 30 angle. Dynamic fluctuation in temperature, within the circular area, was measured using an indium antimonide IR camera (FLIR Systems Inc.).

For peptide modification through a thiol-maleimide click reaction, the nanogels were first modified with N-(2-aminoethyl)maleimide. Purified, dried nanogels were suspended in 10 mM MES at 10 mg/ml and adjusted to pH 4.5. Carboxylic acids were first activated by the addition of a twofold molar excess EDC (relative to MAA content), after which the N-(2-aminoethyl)maleimide trifluoroacetate salt was added. The amount of this linker molecule added was calculated such that if 100% peptide coupling were achieved, then the final peptide concentration would be 2 wt % of the dry nanogel. During this modification reaction, the pH was carefully maintained at 4.5. After 30 min, the pH of the solution was raised to 7.0 with 1 N sodium hydroxide (to terminate the carboxylic acidamine reaction), and the thiol-containing hexamer peptides (FAHWWC, HAHWEC, CDNWQY, ADCFLQ, and CDHFAI) were dissolved in 0.1 PBS at 10 mg/ml, adjusted to pH 7, and added (final peptide concentration of 2 wt % relative to the nanogels). This thiol-maleimide reaction was allowed to proceed overnight at room temperature under constant mixing. The nanogels were purified by dialysis against ultrapure water (12,000 to 14,000 MWCO, >72 hours, frequent water changes).

For peptide modification through a carboxylic acidamine reaction, the nanogels were suspended in 10 mM MES, and pH was adjusted to 5.5. Carboxylic acids were activated with a twofold molar excess (relative to MAA) of EDC. Peptides were dissolved in 10 mM MES at 10 mg/ml and adjusted to pH 5.5. The proper volume of the peptide solution was added to each modification reaction to achieve the desired extent of peptide decoration (i.e., 0 to 10 wt %, relative to the dried nanogels). Nanogels were purified by dialysis against ultrapure water.

Nanogel modification with bioactive proteins was conducted in the same manner as the peptide carboxylic acidamine coupling, except for that WGA Alexa Fluor 594 (Thermo Fisher Scientific) or HRP (Worthington) was dissolved at 1 mg/ml in 10 mM MES and added to the modification reaction at a final protein concentration of 2 wt % (relative to the dried nanogels).

HRP bioactivity within modified nanogels was quantified by its ability to convert TMB substrate, relative to free HRP. Nanogels were dissolved at 2 mg/ml in 1 PBS (pH 7.4 0.05) and diluted 1:8000 for a final concentration of 0.25 g/ml. Lyophilized HRP (Worthington) was also dissolved at 2 mg/ml in 1 PBS and diluted 1:1,000,000 for a final concentration of 0.002 g/ml. A calibration curve for HRP activity was generated via serial dilution with a maximum concentration of 0.002 g/ml. In a 96-well microplate, 100 l of HRP solution or nanogel suspension was mixed with 100 l of TMB substrate solution (Pierce). After 10 min of incubation at ambient conditions, the reaction was stopped by adding 50 l of 1 N sulfuric acid. The reaction product was quantified by visible absorbance at =450 nm.

WGA bioactivity was quantified by its ability to recognize fibroblast cell membrane (via interaction with sialic acid and N-acetylglucosaminyl residues in the membrane). Fibroblasts were seeded in 96-well microplates at 10,000 cells per well and allowed to attach overnight. Cells were incubated in phenol red-free DMEM, supplemented with 2% FBS, containing WGA-conjugated nanogels at 1 mg/ml for 2 hours. As control samples, separate wells were incubated in media alone or media with unmodified nanogels (1 mg/ml) (2 hours). All wells were washed three times with cold DPBS and fixed with cold paraformaldehyde (IC Fixation Buffer; Invitrogen) for 10 min (50 l per well).

After fixation, the nuclei of all cells were stained with DAPI (1 g/ml in cold HBSS, 10 min). The plasma membranes of positive control cells were stained with WGAAlexa Fluor 594 (3 g/ml in cold HBSS, 15 min). After each staining step, all wells were washed three times with cold HBSS (100 l per well).

Fibroblasts were imaged using the fluorescence imaging capabilities of the Cytation 3 microplate reader, equipped with a 20 Olympus objective. So that images could be compared qualitatively, common imaging parameters were used for all images [DAPI (nucleus): LED intensity, 5; integration time, 50; and gain, 0; Texas Red (WGA-nanogels and membrane stain): LED intensity, 10; integration time, 130; gain, 13.6]. Images were processed using Gen5 software (version 3.04), where the background fluorescence was subtracted from each image.

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Synthetic networks with tunable responsiveness, biodegradation, and molecular recognition for precision medicine applications - Science Advances

Self-Assembly Of Retinoid Nanoparticles For Melanoma Therapy | IJN – Dove Medical Press

Han Liao,1,2 Shan Zhao,1,2 Huihui Wang,1,2 Yang Liu,1 Ying Zhang,1 Guangwei Sun1

1Scientific Research Center for Translational Medicine, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, Peoples Republic of China; 2University of Chinese Academy of Sciences, Beijing 100049, Peoples Republic of China

Correspondence: Guangwei SunScientific Research Center for Translational Medicine, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, Peoples Republic of ChinaTel/Fax +86-411-82463027Email sungw@dicp.ac.cn

Background: Amphiphilic fusion drugs are covalent conjugates of a lipophilic drug and a hydrophilic drug or their active fragments. These carrier-free self-assembly nanomaterials are helpful to co-deliver two synergic drugs to the same site regardless of pharmacokinetic properties of individual drugs. Retinoic hydroxamic acid (RHA) is a fusion drug of all-trans retinoic acid (ATRA) and vorinostat, a histone deacetylase (HDAC) inhibitor showing synergic effect with ATRA on cancer therapy. Although RHA was synthesized in 2005, its nanoscale self-assembly property, anticancer activity, and possible related mechanism are still unclear.Methods: RHA nanoparticles were observed under transmission electron microscope (TEM). Both in vitro cell viability, colony formation assay, and in vivo xenograft mouse tumor model were employed here to study anticancer activity of RHA nanoparticles. The putative synergic anticancer mechanism of activating retinoic acid receptor (RAR) and inhibiting HDAC were investigated via receptor inhibitor rescue assay and in vitro enzyme activity assay, respectively.Results: RHA could form nanoparticle formation by self-assembly and abrogates growth of several solid tumor cell lines even after RHA nanoparticles washout. However, opposite to our initial hypothesis, pre-treating the melanoma cells with RAR antagonists showed no impact on inhibitory effect of RHA nanoparticles, which suggested that the target of the molecule on melanoma cells is not RAR and retinoid X receptor (RXR). Importantly, RHA nanoparticles inhibited the growth of xenograft tumors without obvious impact on haematological indexes and hepatorenal function of these tumor-bearing mice.Conclusion: Our findings demonstrate the promise of RHA nanoparticles in treating malignant melanoma tumors with high efficacy and low toxicity.

Keywords: nano-drugs, self-assembly, retinoid, cancer therapy, melanoma

This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License.By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.

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Self-Assembly Of Retinoid Nanoparticles For Melanoma Therapy | IJN - Dove Medical Press

Nanomanipulator Market Upcoming Trends, Revenue, Key Manufactures and Competitive Analysis till 2025 – One Digi Click

Los Angeles, United State, Oct 01, 2019 QY Research provides an authentic report about the global Nanomanipulator Market. It includes market figures, both historical as well as estimates. The research report details the milestones that the global market for Nanomanipulator has achieved and discusses the potential opportunities for the players operating in the market. The publication titled Nanomanipulator includes Porters five forces analysis and SWOT analysis to give its readers a holistic outlook.

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In this section of the report, the global Nanomanipulator market focuses on the major players that are operating in the market and their competitive landscape present in the market. The report includes a list of initiatives taken by the companies in the past years along with the ones, which are likely to happen in the coming years. Analysts have also made a note of their expansion plans for the near future, financial analysis of these companies, and their research and development activities. This research report includes a complete dashboard view of the global Nanomanipulator market, which helps the readers to view an in-depth knowledge about the report.

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Global Nanomanipulator Market: Research Methodology

QY Research has gathered the data from various sources such as secondary and primary research to provide an authenticate result of the global Nanomanipulator market. To validate the data, the team of analysts has gone through the discussion with the panel members by taking their interviews and conducting the market size to analyze the global structure of Nanomanipulator market and getting the feedback from the product manufacturers.

The report also considers the various factors based on secondary sources, market size, key data parameters such as the market positioning of key players in terms of the regional revenue, segmental revenue. Geographic penetration also shows the market potential, market risk, industry trends, and opportunities. Secondary sources mainly include journals, companies annual reports, website, public and paid databases, and press releases.

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QYResearch always pursuits high product quality with the belief that quality is the soul of business. Through years of effort and supports from the huge number of customer supports, QYResearch consulting group has accumulated creative design methods on many high-quality markets investigation and research team with rich experience. Today, QYResearch has become a brand of quality assurance in the consulting industry.

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Nanomanipulator Market Upcoming Trends, Revenue, Key Manufactures and Competitive Analysis till 2025 - One Digi Click

First-of-its-kind trial in ALS spurs hope for brutal disease – BioPharma Dive

Until the day she dies, Sandy Morris is hunting for a comma and a clause.

In May 2018, the 53-year-old mother of three learned she has ALS, a diagnosis made even more devastating by a somber directive to get her affairs in order period. The finality speaks to the absence of an effective treatment, yet alone a cure, for the neurodegenerative disease also known as Lou Gehrig's disease.

Since the diagnosis, Morris has sought to give future ALS patients some measure of optimism, amending the 'get your affairs in order' instruction to add 'but, there's something worth looking into.' While medical advances in areas like gene therapy show potential to change the course of other potentially fatal diseases, a disease-modifying treatment in ALS has proved elusive.

"In cancer, you have a shot," Morris said in an interview. "Not always a great shot, but you have one. In this land, everybody is just like, 'We don't have anything.'"

But now, Morris said she has reason for hope, stemming from a first-of-its-kind study in ALS that will test five experimental drugs simultaneously in a platform trial. Five drugmakers selected for the trial were announced last week.

After years of work, the broader promise of these adaptive trials, which allow more flexibility to make changes throughout, is beginning to be put into action. This trial aims to accelerate the pace of ALS research and may prove to be a template for other rare diseases as well, experts said.

The design offers advantages for each key stakeholder in drug development: patients have a lower probability of receiving placebo due to the study's shared control arm; companies get quicker topline results at lower cost; and researchers talk of the study as an "endpoint engine" that could advance their scientific understanding of the disease.

Formidable challenges remain in the way of broader adoption, particularly among leaders of the drug industry. The ALS study uses experimental treatments from small biotechs instead of industry giants, which can afford to prioritize control and pay for their own studies instead.

Even so, it's an exciting advancement in ALS research. Morris, who is also a leader of the patient group I Am ALS, said the trial can bring hope to future patients, even if it comes too late to help her.

"It's not going to save me. I'm not in time for any of this," Morris said. "But, dammit, we have to make it better for the rest."

Merit Cudkowicz understands speed. After 25 years specializing in ALS research, Cudkowicz is fully aware of how ALS changes a patient's life, shortening their life expectancy from decades to just years. The disease has a roughly three-year median survival.

Merit Cudkowicz

Massachusetts General Hospital

That life-changing diagnosis came in May 2018 for Sean Healey, then the CEO of an asset management firm worth more than $8 billion. After resigning from that post, Healey connected with Cudkowicz as he searched for treatment options and realized there was an opportunity to accelerate the pace of research.

Just six months from his diagnosis, Healey raised $40 million to launch a research center at Massachusetts General Hospital. Now, slightly less than a year after being established, the Sean M. Healey & AMG Center for ALS Research is aiming to start the platform trial in the first few months of 2020.

And earlier this month, the Healey Center announced the first five therapies to be tested in the study, including drugs from Biohaven Pharmaceutical and Ra Pharmaceuticals.

Cudkowicz, who leads the study, as well as the Healey Center and Mass General's neurology department, said in an interview the trial aims to have 160 patients for each of five treatments. The primary outcome will be whether or not the drug boosted a functional rating score for ALS after six months. One placebo group will be shared for all treatment arms, and more drugs can be added as the study continues. (Cudkowicz cautioned the plan is nearing finalization with the Food and Drug Administration, so specifics could still change.)

Moving quickly is the trial's aim. Cudkowicz called the study "an endpoint engine" that can help create better outcome measurements, including future surrogate markets, pushing ALS research forward. She estimated topline results to come 12 to 18 months after the study begins, depending on enrollment speed.

But those goals are steps toward the actual mission: developing a cure, Healey wrote in an email to BioPharma Dive.

"Of course, we all understand that the most meaningful measure of success will be the development of effective treatments and ultimately a cure," Healey wrote. "I am convinced that the Platform Trial, along with other initiatives we are supporting, will substantially accelerate the achievement of this ultimate goal."

For the first five biotechs, the decision to participate was a no-brainer. The center and other groups are footing the majority of the cost, leaving the companies' primary expense to simply provide their drug.

Out of about 30 applicants, the Healey Center selected five drugs for the study, most notably Biohaven's verdiperstat and Ra Pharma's zilucoplan. The trial also will test therapies from Implicit Bioscience, Prilenia Therapeutics and Clene Nanomedicine.

Cudkowicz said the typical applicants were small biotechs that "have great ideas but not deep pockets."

The expense of clinical trials limits the ability for biotechs to run multiple studies simultaneously across a range of indications. Instead, companies typically focus on a lead indication, with others following in succession.

"As a small company, we live and die by being able to run efficiently and test our hypotheses," said Irfan Qureshi, Biohaven's vice president of neurology.

For instance, Biohaven's verdiperstat is in Phase 3 testing for multiple system atrophy, and Ra Pharma's zilucoplan is focused on a different neuromuscular disorder called generalized myasthenia gravis.

"We wanted to do the study anyway, but to be honest, we probably wouldn't have gotten to it for years if this had not come along," Ra Pharma CEO Doug Treco said in an interview.

While smaller biotechs have bought into the platform trial and its efficiencies, missing from the list of initial participants are industry leaders. With a market value of about $2 billion, Biohaven is the largest company involved.

Cudkowicz noted larger drugmakers have shown interest in roundtable discussions that shaped the trial's design, and there's always potential to add additional therapies after the trial starts.

"We're talking to the other ones like Biogen and Sanofi," she said. "They are interested and came to these meetings, but they have the money to do it on their own."

But the main hang-up for these companies is ceding control, said Scott Berry, a senior statistical scientist and co-founder of Berry Consultants, which worked on the ALS platform trial.

"All these companies have people that this is what they do for a living they run trials, and they know how to do it," Berry said. "To hand your drug to somebody else and you don't have control over making sure that happens is uncomfortable for companies, and it's different than what they usually do."

Additionally, with lengthy protocols and many moving parts, the trials are complex and typically require extensive consulting to get off the ground. Running a multi-arm study also brings statistical pitfalls that can make it harder to interpret results.

Beyond ALS, adaptive trials have been started in breast cancer, Alzheimer's disease and glioblastoma. Experts say they anticipate other rare diseases as logical future targets for these studies.

"As these start to get developed and people see them, in most rare diseases there will be people jumping on board," predicted Berry.

Biohaven's Qureshi added that once companies are willing to experiment beyond typical drug development, these trials could be particularly attractive for rare diseases by easing enrollment concerns where there are "not patients growing on trees," he said.

But the ultimate test to get the industry's full attention, Qureshi said, would be such a study yielding an approved drug.

In the meantime, platform trials appear here to stay. Janet Woodcock, the long-time leader of the FDA's drug review center, has been an influential supporter for these study designs. Just this month, the agency finalized guidance on ALS research that advised companies to consider adaptive trial designs.

Earlier this year, Woodcock told BioPharma Dive she believes these types of studies will gain ground as patients become more vocal about how trials are conducted.

Morris said the platform trial has struck her with its compassion. While no patient wants to be put on placebo, it's of particular importance in a disease that progresses as rapidly and severely as ALS, leaving most patients the time to try one study in their life.

She already took her shot, enrolling in a clinical trial that required a three-month observation period before receiving treatment, which then carried a 50% chance of being placebo. "We are humans," she said. "We aren't zebrafish."

Now, she wants to take the baton from "the voices in the graveyard" and pass it onto the next generation. Platform trials may allow her to cover a bit more ground before that hand-off, eventually reaching a day when people might be able to live with ALS, like HIV.

"ALS's day is coming," Morris said.

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First-of-its-kind trial in ALS spurs hope for brutal disease - BioPharma Dive

FBI Investigation Targets Trump Booster Peter Thiel’s VC Firm

The FBI has reportedly begun investigating Mithril Capital, the venture capital firm co-founded by Silicon Valley tycoon Peter Thiel.

Heightened Scrutiny

The FBI has turned its all-seeing eye toward Mithril Capital, the venture capital firm co-founded by Silicon Valley tycoon Peter Thiel — an industry kingmaker who’s one of the most prominent supporters of the Trump administration in tech.

It’s unclear specifically what prompted the FBI to scrutinize the firm’s business practices, but Recode reports a host of possibilities ranging from questionable tax strategies to mismanagement of investors’ money — a shakeup, in either case, at the explosive intersection of tech and politics.

And My Axe

The FBI isn’t alone in its heightened scrutiny of Mithril, which borrows its name from a fictional metal in “Lord of the Rings.”

Also probing the company’s finances is Cambridge Associates, a prominent investment adviser for the mega-rich that has put millions of dollars into Mithril’s coffers, according to Recode. Cambridge is looking into whether the firm has been overly-selective with investments, choosing to sit on investors’ cash and collect higher management fees as a result.

All-Seeing Eye

While Thiel isn’t involved in day-to-day operations at Mithril, his money and reputation are inexorably linked with its performance.

That’s relevant because the firm is also dealing with low morale, per Recode, related to the dual investigations into its practices. It reportedly once collected staffers’ computers and phones in a bid to determine who’d been talking to the press and investigators — though a spokesperson told Recode that it was merely a “device upgrade.”

Disclosure: The Thiel Foundation previously provided grant funding to Futurism’s parent company, Singularity University.

READ MORE: The FBI is investigating a venture capital fund started by Peter Thiel for financial misconduct [Recode]

More on Mithril Capital: The Next Head of the FDA May be a Man Who is Fighting a War Against Aging

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FBI Investigation Targets Trump Booster Peter Thiel’s VC Firm

YouTube Influencers Are Mentally Collapsing From Stress

YouTube influencers feel constant pressure to churn out content, prompting some — including Christine Sydelko — to give up the gig.

Dark Underbelly

It’s easy to see why so many peopleparticularly kids — long to be YouTube influencers. Make a living just by posting videos of yourself on the internet? Sounds great!

But popular YouTuber Christine Sydelko wants people to know the job isn’t all it’s cracked up to be.

In fact, she’s decided to quit the influencer game altogether, calling it “trivial,” “unfulfilling,” and the “main cause of [her] anxiety and depression”a grim insider’s look at the toll a career in digital content can take on influencers’ mental health.

Abnormal Life

Sydelko started vlogging in 2015, and she now has 1.3 million subscribers on YouTube. But despite her popularity, she tweeted on September 6 that she has decided to “quit the internet stuff.”

That post was preceded by a June tweet in which Sydelko noted the toll being an influencer was taking on her mental health and lamented her decision to forgo a “normal life.”

Breaking Point

Sydelko isn’t the first influencer to struggle under the weight of internet fame. In January, Insider published a story highlighting several YouTube influencers who experienced so much mental anguish, they felt a need to quit the site, too — at least temporarily.

“[YouTube] kind of is a machine and it makes creators believe that we have to pump out content consistently,” Lilly Singh told her 14 million YouTube subscribers in a November video announcing her own break, “even at the cost of our health and our life and our mental happiness.”

READ MORE: Christine Sydelko, a YouTuber with 1.3 million subscribers, decided to quit the internet for good because being an influencer is ‘such a trivial job’ [Business Insider]

More on YouTube: Astronauts Are Out. American Kids Would Rather Be YouTubers.

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YouTube Influencers Are Mentally Collapsing From Stress

Watch SpaceX Test Its Crew Dragon’s Escape System

SpaceX just posted dramatic footage of its commercial space shuttle Crew Dragon undergoing hundreds of rigorous tests of its emergency abort system.

Abort! Abort!

SpaceX just posted dramatic footage of its commercial space taxi Crew Dragon undergoing rigorous tests of its emergency abort system.

The idea is that if something goes wrong with the rocket carrying the Crew Dragon to orbit, the module can engage its own thrusters to quickly escape the danger — and then coast down safely on a parachute.

Ahead of our in-flight abort test for @Commercial_Crew—which will demonstrate Crew Dragon's ability to safely carry astronauts away from the rocket in the unlikely event of an emergency—our team has completed over 700 tests of the spacecraft's SuperDraco engines pic.twitter.com/nswMPCK3F9

— SpaceX (@SpaceX) September 12, 2019

Ejector Button

The spacecraft is outfitted with eight SuperDraco engines, allowing it to cover half a mile in just 7.5 seconds during an emergency, reaching a top speed of 436 mph, according to a follow-up tweet. Parachutes ensure that the craft safely lands back on Earth after the system deploys.

But testing hasn’t always gone according to plan. The same engines were responsible for blowing up the first Crew Dragon capsule during a system test in April.

Last week, SpaceX tested the first stage of its Falcon 9 boosters that will be responsible for launching two NASA astronauts into orbit as part of Crew Dragon’s first-ever crewed test flight. When exactly that test flight will take place is still uncertain.

READ MORE: SpaceX on Twitter

More on Crew Dragon: SpaceX Confirms That Accident Destroyed Crew Dragon Capsule

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Watch SpaceX Test Its Crew Dragon’s Escape System

Residents Are Trying to Flee Russian Town Where Snow Turned Black

People from the Russian town Kiselyovsk want to gain entry to Canada as environmental refugees. Coal mining operations in their town have made it unlivable.


Residents of the Russian town of Kiselyovsk have a simple request for Canada’s Prime Minister Justin Trudeau: let us in.

That’s because the town, infamous for gray-black, coal-polluted snow has proven inhospitable for residents who now seek status as environmental refugees, according to CBC News. But because of complicated legal technicalities — fleeing pollution is seen differently from escaping climate change — experts say they don’t stand much of a chance.


About 80 percent of Kiselyovsk’s population lives near an open-pit coal mine, according to Motherboard. As a result, the town’s water and food are highly contaminated. Cancer and tuberculosis are common, and the life expectancy has plummeted as toxic dust blankets the region.

All in all, it makes Canada look great.

“We chose Canada because the climate there is similar to our region,” an exodus organizer told a local newspaper, per CBC. “So that they [Russian authorities] wouldn’t say that we just wanted to move to warm countries.”


The residents have publicized their plea for asylum through a series of YouTube videos. But Canada typically doesn’t welcome environmental refugees, especially when other parts of Russia remain hospitable — a stance that Queens University law professor Sharry Aiken told Motherboard she finds outdated in the face of an increasingly-inhospitable planet.

“There is no question,” Aiken told Motherboard, “that our laws and policies will need to adapt to this new reality.”

READ MORE: Residents of a Siberian Town With Black Snow Are Pleading for Asylum in Canada [Motherboard]

More on refugees: New Research: Climate Change Causes Migration and Conflict

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Residents Are Trying to Flee Russian Town Where Snow Turned Black

This Startup Will Send DNA From Your Spit to the Moon for $99

LifeShip will send your DNA to the Moon for $99 — a whimsical undertaking that could also ensure humanity lives on even if Earth becomes uninhabitable. 

Sticker Shock

A private trip to the Moon’s orbit could cost you anywhere from $35 million to $150 million. But now, one startup is saying it’ll charge just $99 to put you on the Moon.

Part of you, anyway.

In a newly published IEEE Spectrum story, LifeShip founder Ben Haldeman shares his startup’s plan to send human DNA to the Moon — a whimsical undertaking that could also ensure humanity lives on even if Earth becomes uninhabitable.

Spit Collector

The project will launch this fall with a Kickstarter campaign through which LifeShip will sell $99 kits that backers can use to collect samples of their spit.

LifeShip will then extract DNA from the saliva and preserve it in an “artificial amber” that will reach the Moon as part of a future Arch Mission journey.

“You provide a saliva sample and then we take your DNA, your source code, and preserve it up in space for eternity,” Haldeman told IEEE Spectrum.

Survival Unknown

“Eternity” might be a stretch, though.

While your DNA might land on the Moon through the LifeShip project, even Haldeman later notes that he isn’t sure how long it’ll survive once it reaches its lunar destination.

“We can’t guarantee a million years on the Moon but it should be up there for a while,” he told IEEE Spectrum. “It’s about archiving life and saving for the future but also the wonder of traveling into space and what could happen with this DNA in the future.”

READ MORE: This Startup Wants to Stash Your DNA on the Moon [IEEE Spectrum]

More on the Arch Mission: Israeli Moon Lander Is Carrying a Vast Backup of Human Knowledge

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This Startup Will Send DNA From Your Spit to the Moon for $99

MIT Team “Accidentally” Invents Blackest Material in Existence

On Thursday, a team of MIT engineers reported that they’d created a material that absorbs 99.995 percent of light that reaches it — making it even blacker than Surrey NanoSystems’ mind-bending Vantablack, which absorbs 99.965 percent of light.

But perhaps even more remarkable than the light-absorbing capabilities of the new super black material is how the MIT team discovered it: by accident.

In an attempt to boost certain properties of electrically conductive materials, the MIT team removed the oxide layer from aluminum and began growing carbon nanotubes on it.

“I remember noticing how black it was before growing carbon nanotubes on it, and then after growth, it looked even darker,” researcher Kehang Cui told MIT News. “So I thought I should measure the optical reflectance of the sample.”

That’s when the team discovered that it had created the world’s new blackest material, which it detailed in a study published in the journal ACS-Applied Materials and Interfaces on Thursday.

The MIT researchers almost immediately partnered with artist Diemut Strebe to coat a $2 million diamond in the material as part of an art exhibit that launched at the New York Stock Exchange on Friday.

The MIT team has since offered up the material to any interested artists. But the substance could have many applications beyond the artistic, such as helping astronomers remove unwanted glare from telescopes.

As black as this material is, though, researcher Brian Wardle doesn’t expect it to hold the title of blackest black forever.

“I think the blackest black is a constantly moving target,” he told MIT News. “Someone will find a blacker material, and eventually we’ll understand all the underlying mechanisms, and will be able to properly engineer the ultimate black.”

READ MORE: MIT engineers develop “blackest black” material to date [MIT News]

More on Vantablack: BMW Unveils a Car Coated in Vantablack

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MIT Team “Accidentally” Invents Blackest Material in Existence

A Driver Tricked Uber’s Algorithm, Sexually Assaulted a Passenger

After a horrifying case where an unauthorized Uber driver raped his passenger, the company says its inroducing human oversight to its driver-verifying tech.

Terrifying Flaws

In December 2018, a man driving an authorized Uber vehicle picked up an intoxicated woman leaving a Christmas party — and then brought her to his home and raped her. But the man, who The Age reports was sentenced to five and a half years in prison on Wednesday, was not an authorized Uber driver. Rather, he was able to easily fool Uber’s verification system by holding up a photo of a real driver.

In other words, the AI technology that Uber uses to verify that its drivers are who they claim to be — like Amazon delivery drivers, Uber contractors take a selfie when signing on — wasn’t sophisticated enough to spot a printed headshot. It’s a horrifying story that illustrates the perils of big tech offloading security to dodgy AI systems.


Uber told Business Insider that it deployed a fix in response to the December incident.

The company hired an undisclosed number of humans to review the driver-verifying selfies this year, it said — and also implemented better AI.

Higher Standards

Apparently, it works — The Age reports that drivers posting in online forums are talking less about operating scam rings and more about how difficult it is to get the system to approve a selfie.

But it all raises the question of why the improved security wasn’t in place from the start ­— the unauthorized driver may have been fooling Uber’s security protocols since 2016, according to The Age.

READ MORE: Uber introduces human reviewers to crack down on drivers evading security selfie system with printed photos after Australian rape case [Business Insider]

More on Uber drivers: Uber and Lyft Still Allow Racist Behavior, but Not as Much as Taxi Services

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A Driver Tricked Uber’s Algorithm, Sexually Assaulted a Passenger