Tag Archives: Proceedings of the National Academy of Sciences

A view to controversies about nanoparticle drug delivery, sticky-flares, and a PNAS surprise

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Despite all the excitement and claims for nanoparticles as vehicles for drug delivery to ‘sick’ cells there is at least one substantive problem, the drug-laden nanoparticles don’t actually enter the interior of the cell. They are held in a kind of cellular ‘waiting room’.

Leonid Schneider in a Nov. 20, 2015 posting on his For Better Science blog describes the process in more detail,

A large body of scientific nanotechnology literature is dedicated to the biomedical aspect of nanoparticle delivery into cells and tissues. The functionalization of the nanoparticle surface is designed to insure their specificity at targeting only a certain type of cells, such as cancers cells. Other technological approaches aim at the cargo design, in order to ensure the targeted release of various biologically active agents: small pharmacological substances, peptides or entire enzymes, or nucleotides such as regulatory small RNAs or even genes. There is however a main limitation to this approach: though cells do readily take up nanoparticles through specific membrane-bound receptor interaction (endocytosis) or randomly (pinocytosis), these nanoparticles hardly ever truly reach the inside of the cell, namely its nucleocytoplasmic space. Solid nanoparticles are namely continuously surrounded by the very same membrane barrier they first interacted with when entering the cell. These outer-cell membrane compartments mature into endosomal and then lysosomal vesicles, where their cargo is subjected to low pH and enzymatic digestion. The nanoparticles, though seemingly inside the cell, remain actually outside. …

What follows is a stellar piece featuring counterclaims about and including Schneider’s own journalistic research into scientific claims that the problem of gaining entry to a cell’s true interior has been addressed by technologies developed in two different labs.

Having featured one of the technologies here in a July 24, 2015 posting titled: Sticky-flares nanotechnology to track and observe RNA (ribonucleic acid) regulation and having been contacted a couple of times by one of the scientists, Raphaël Lévy from the University of Liverpool (UK), challenging the claims made (Lévy’s responses can be found in the comments section of the July 2015 posting), I thought a followup of sorts was in order.

Scientific debates (then and now)

Scientific debates and controversies are part and parcel of the scientific process and what most outsiders, such as myself, don’t realize is how fraught it is. For a good example from the past, there’s Leviathan and the Air-Pump: Hobbes, Boyle, and the Experimental Life (from its Wikipedia entry), Note: Links have been removed),

Leviathan and the Air-Pump: Hobbes, Boyle, and the Experimental Life (published 1985) is a book by Steven Shapin and Simon Schaffer. It examines the debate between Robert Boyle and Thomas Hobbes over Boyle’s air-pump experiments in the 1660s.

The style seems more genteel than what a contemporary Canadian or US audience is accustomed to but Hobbes and Boyle (and proponents of both sides) engaged in bruising communication.

There was a lot at stake then and now. It’s not just the power, prestige, and money, as powerfully motivating as they are, it’s the research itself. Scientists work for years to achieve breakthroughs or to add more to our common store of knowledge. It’s painstaking and if you work at something for a long time, you tend to be invested in it. Saying you’ve wasted ten years of your life looking at the problem the wrong way or have misunderstood your data is not easy.

As for the current debate, Schneider’s description gives no indication that there is rancour between any of the parties but it does provide a fascinating view of two scientists challenging one of the US’s nanomedicine rockstars, Chad Mirkin. The following excerpt follows the latest technical breakthroughs to the interior portion of the cell through three phases of the naming conventions (Nano-Flares, also known by its trade name, SmartFlares, which is a precursor technology to Sticky-Flares), Note: Links have been removed,

The next family of allegedly nucleocytoplasmic nanoparticles which Lévy turned his attention to, was that of the so called “spherical nucleic acids”, developed in the lab of Chad Mirkin, multiple professor and director of the International Institute for Nanotechnology at the Northwestern University, USA. These so called “Nano-Flares” are gold nanoparticles, functionalized with fluorophore-coupled oligonucleotides matching the messenger RNA (mRNA) of interest (Prigodich et al., ACS Nano 3:2147-2152, 2009; Seferos et al., J Am. Chem.Soc. 129:15477-15479, 2007). The mRNA detection method is such that the fluorescence is initially quenched by the gold nanoparticle proximity. Yet when the oligonucleotide is displaced by the specific binding of the mRNA molecules present inside the cell, the fluorescence becomes detectable and serves thus as quantitative read-out for the intracellular mRNA abundance. Exactly this is where concerns arise. To find and bind mRNA, spherical nucleic acids must leave the endosomal compartments. Is there any evidence that Nano-Flares ever achieve this and reach intact the nucleocytoplasmatic space, where their target mRNA is?

Lévy’s lab has focused its research on the commercially available analogue of the Nano-Flares, based on the patent to Mirkin and Northwestern University and sold by Merck Millipore under the trade name of SmartFlares. These were described by Mirkin as “a powerful and prolific tool in biology and medical diagnostics, with ∼ 1,600 unique forms commercially available today”. The work, led by Lévy’s postdoctoral scientist David Mason, now available in post-publication process at ScienceOpen and on Figshare, found no experimental evidence for SmartFlares to be ever found outside the endosomal membrane vesicles. On the contrary, the analysis by several complementary approaches, i.e., electron, fluorescence and photothermal microscopy, revealed that the probes are retained exclusively within the endosomal compartments.

In fact, even Merck Millipore was apparently well aware of this problem when the product was developed for the market. As I learned, Merck performed a number of assays to address the specificity issue. Multiple hundred-fold induction of mRNA by biological cell stimulation (confirmed by quantitative RT-PCR) led to no significant changes in the corresponding SmartFlare signal. Similarly, biological gene downregulation or experimental siRNA knock-down had no effect on the corresponding SmartFlare fluorescence. Cell lines confirmed as negative for a certain biomarker proved highly positive in a SmartFlare assay.  Live cell imaging showed the SmartFlare signal to be almost entirely mitochondrial, inconsistent with reported patterns of the respective mRNA distributions.  Elsewhere however, cyanine dye-labelled oligonucleotides were found to unspecifically localise to mitochondria   (Orio et al., J. RNAi Gene Silencing 9:479-485, 2013), which might account to the often observed punctate Smart Flare signal.

…

More recently, Mirkin lab has developed a novel version of spherical nucleic acids, named Sticky-Flares (Briley et al., PNAS 112:9591-9595, 2015), which has also been patented for commercial use. The claim is that “the Sticky-flare is capable of entering live cells without the need for transfection agents and recognizing target RNA transcripts in a sequence-specific manner”. To confirm this, Lévy used the same approach as for the striped nanoparticles [not excerpted here]: he approached Mirkin by email and in person, requesting the original microscopy data from this publication. As Mirkin appeared reluctant, Lévy invoked the rules for data sharing by the journal PNAS, the funder NSF as well as the Northwestern University. After finally receiving Mirkin’s thin-optical microscopy data by air mail, Lévy and Mason re-analyzed it and determined the absence of any evidence for endosomal escape, while all Sticky-Flare particles appeared to be localized exclusively inside vesicular membrane compartments, i.e., endosomes (Mason & Levy, bioRxiv 2015).

I encourage you to read Schneider’s Nov. 20, 2015 posting in its entirety as these excerpts can’t do justice to it.

The PNAS surprise

PNAS (Proceedings of the National Academy of Science) published one of Mirkin’s papers on ‘Sticky-flares’ and is where scientists, Raphaël Lévy and David Mason, submitted a letter outlining their concerns with the ‘Sticky-flares’ research. Here’s the response as reproduced in Lévy’s Nov. 16, 2015 posting on his Rapha-Z-Lab blog

Dear Dr. Levy,

I regret to inform you that the PNAS Editorial Board has declined to publish your Letter to the Editor. After careful consideration, the Board has decided that your letter does not contribute significantly to the discussion of this paper.

Thank you for submitting your comments to PNAS.

Sincerely yours,
Inder Verma
Editor-in-Chief

Judge for yourself, Lévy’s and Mason’s letter can be found here (pdf) and here.

Conclusions

My primary interest in this story is in the view it provides of the scientific process and the importance of and difficulty associated with the debates.

I can’t venture an opinion about the research or the counterarguments other than to say that Lévy’s and Mason’s thoughtful challenge bears more examination than PNAS is inclined to accord. If their conclusions or Chad Mirkin’s are wrong, let that be determined in an open process.

I’ll leave the very last comment to Schneider who is both writer and cartoonist, from his Nov. 20, 2015 posting,

LeonidSchneiderImagination

Looking at glass on the molecular scale

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Glass isn’t transparent (at the molecular scale) as it’s cooling and scientists have been curious about this transition from liquid to glass state. According to an Oct. 15, 2012 posting by Carol Clark for Emory University’s eScienceCommons, a team from Emory University (and New York University)  has cracked this mystery. First, here’s more about the mystery (from Clark’s article)

Scientists fully understand the process of water turning to ice. As the temperature cools, the movement of the water molecules slows. At 32 F, the molecules lock into crystal lattices, solidifying into ice. In contrast, the molecules of glasses do not crystallize.The movement of the glass molecules slows as the temperature cools, but they never lock into crystal patterns. Instead, they jumble up and gradually become glassier, or more viscous. No one understands exactly why.

The phenomenon leaves physicists to ponder the molecular question of whether glass is a solid, or merely an extremely slow-moving liquid.

This purely technical physics question has stoked a popular misconception: That the glass in the windowpanes of some centuries-old buildings is thicker at the bottom because the glass flowed downward over time.

“The real reason the bottom is thicker is because they hadn’t yet learned how to make perfectly flat panes of glass,” Weeks says [Emory physicist Eric Weeks]. “For practical purposes, glass is a solid and it will not flow, even over centuries. But there is a kernel of truth in this urban legend: Glasses are different than other solid materials.”

Speaking more technically about the transition,

“Cooling a glass from a liquid into a highly viscous state fundamentally changes the nature of particle diffusion,” says Emory physicist Eric Weeks, whose lab conducted the research. “We have provided the first direct observation of how the particles move and tumble through space during this transition, a key piece to a major puzzle in condensed matter physics.”

Weeks specializes in “soft condensed materials,” substances that cannot be pinned down on the molecular level as a solid or liquid, including everyday substances such as toothpaste, peanut butter, shaving cream, plastic and glass.

The scientists have prepared a video animation of what they believing is occurring as glass cools (no sound),

Here’s what the movie depicts (from the Clark article),

The movie and data from the experiment provide the first clear picture of the particle dynamics for glass formation. As the liquid grows slightly more viscous, both rotational and directional particle motion slows. The amount of rotation and the directional movements of the particles remain correlated.

“Normally, these two types of motion are highly coupled,” Weeks says. “This remains true until the system reaches a viscosity on the verge of being glass. Then the rotation and directional movements become decoupled: The rotation starts slowing down more.”

He uses a gridlocked parking lot as an analogy for how the particles are behaving. “You can’t turn your car around, because it’s not a sphere shape and you would bump into your neighbors. You have to wait until a car in front of you moves, and then you can drive a bit in that direction. This is directional movement, and if you can make a bunch of these, you may eventually be able to turn your car. But turning in a crowded parking lot is still much harder than moving in a straight line.”

There’s more about the work and team in Clark’s article. H/T to the Oct. 16, 2012 news item on Nanowerk for alerting me to this work. You can find the article the researchers have written at the Proceedings of the National Academy of Sciences (PNAS),

Decoupling of rotational and translational diffusion in supercooled colloidal fluids by Kazem V. Edmond, Mark T. Elsesser, Gary L. Hunter, David J. Pine, and Eric R. Weeks. Published online before print October 15, 2012, doi: 10.1073/pnas.1203328109 PNAS October 15, 2012

The article is behind a paywall.

Uncomfortable truths; favouring males a gender bias practiced by male and female scientists

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Nancy Owano’s Sept. 21, 2012 phy.org article on a study about gender bias (early publication Sept. 17, 2012 in the Proceedings of the National Academy of Sciences) describes a situation that can be summed up with this saying ‘we women eat our own’.

The Yale University researchers developed applications for a supposed position in a science faculty and had faculty members assess the applicants’ paper submissions.  From Owano’s article,

Applications were all identical except for the male names and female names. Even though the male and female name applications were identical in competencies, the female student was less likely to be hired, being viewed as less competent and desirable as a new-hire.

Results further showed the faculty members chose higher starting salaries and more career mentoring for applicants with male names.

Interestingly, it made no difference on hiring decisions as to whether the faculty member was male or female. Bias was just as likely to occur at the hands of a female as well as male faculty member.

I tracked down the paper (which is open access), Science faculty’s subtle gender biases favor male students by Corinne A. Moss-Racusin, John F. Dovidio, Victoria L. Bescroll, Mark J. Graham, and Jo Handelsman and found some figures in a table which I can’t reproduce here but suggest the saying ‘we women eat their own’ isn’t far off the mark. In it, you’ll see that while women faculty members will offer less to both genders, they offer significantly less to female applicants.

For a male applicant, here’s the salary offer,

Male Faculty               Female Faculty

30,520.82                    29, 333.33

 

For a female applicant, here’s the salary offer,

Male Faculty               Female Faculty

27,111.11                    25,000.00

To sum this up, the men offered approximately $3000 (9.25%) less to female applicants while the women offered approximately $4000 (14.6%) less. It’s uncomfortable to admit that women may be just as much or even more at fault as men where gender bias is concerned. However, it is necessary if the situation is ever going to change.

The Sept. 24, 2012 news release from Yale University features a quote from the lead author (Note: I have removed a link),

Yale University researchers asked 127 scientists to review a job application of identically qualified male and female students and found that the faculty members – both men and women – consistently scored a male candidate higher on a number of criteria such as competency and were more likely to hire the male. The result came as no surprise to Jo Handelsman, professor of molecular, cellular, and developmental biology (MCDB), a leading microbiologist, and national expert on science education. She is the lead author of the study scheduled to be published the week of Sept. 24 in the Proceedings of the National Academy of Sciences.

“Whenever I give a talk that mentions past findings of implicit gender bias in hiring, inevitably a scientist will say that can’t happen in our labs because we are trained to be objective. I had hoped that they were right,” said Handelsman, who is also a Howard Hughes Medical Institute Professor.

So Handelsman and Corinne A. Moss-Racusin, a postdoctoral associate in MCDB and psychology, as well as colleagues in social psychology decided to test whether this bias among researchers might help explain why fewer women than men have careers in science. They provided about 200 academic researchers with an application from a senior undergraduate student ostensibly applying for a job as lab manager. The faculty participants all received the same application, which was randomly assigned a male or female name. The faculty were asked to judge the applicants’ competency, how much they should be paid, and whether or not they would be willing to mentor the student.

In the end, scientists responded no differently than other groups tested for bias. Both men and women science faculty were more likely to hire the male, ranked him higher in competency, and were willing to pay him $4000 more than the woman. [emphasis mine] They were also more willing to provide mentoring to the male than to the female candidate.

I highlighted the sentence in the excerpt since the portion about the salary difference somewhat contradicts my own reading of the information in the study. If you are female, you will still be offered less money by male faculty but the percentage (9% less) is an improvement over the 14% differential offered by female faculty.  I do appreciate that these numbers have been crunched together and there will be individual differences, as well as, outliers but this finding certainly confirms ‘folk wisdom’ and points to the difficulty of facing uncomfortable truths for even the researchers and their sponsoring institutions.

ETA Sept. 25, 2012: There have been some comments about the research and the methodology on Uta Frith’s Science&shopping website:

Research on gender bias

Comments by David Attwell on Moss-Racusin et al. ‘Science faculty’s subtle gender biases’

Comments on comments by Virginia Valian

Comments on comments by Dorothy Bishop

H/T to Jenny Rohn for the information about Uta Frith’s coverage of the issue which I found in Rohn’s Sept. 25, 2012 posting about women, science, and bias (she mentions this recent research from Yale but in the context of other research and broader issues of gender bias in the sciences) for the Guardian science blogs.

ETA Sept. 26, 2012: The Canadian Broadcasting Corporation’s As It Happens radio show features an interview with Corinne A. Moss-Racusin about the paper in their Sept. 25, 2012 broadcast. Click here and scroll down to the Sept. 25, 2012 entry and keep scrolling until you see the speaker icon and Listen, click on Listen and the popup menu will appear. Scroll down to part 3 and click again (it’s the second interview). There’s also a Sept. 25, 2012 podcast in the left column of today’s front page screen of As It Happens, which I did not test.

Squeezing blood from rice

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They are squeezing the equivalent of human blood protein (blood-derived human serum albumin [HSA]) from transgenic rice according to the research paper (open access) published in the Proceedings of the National Academy of Sciences (PNAS). From the abstract in PNAS,

Human serum albumin (HSA) is widely used in clinical and cell culture applications. Conventional production of HSA from human blood is limited by the availability of blood donation and the high risk of viral transmission from donors. Here, we report the production of Oryza sativa recombinant HSA (OsrHSA) from transgenic rice seeds. … Physical and biochemical characterization of OsrHSA revealed it to be equivalent to plasma-derived HSA (pHSA). The efficiency of OsrHSA in promoting cell growth and treating liver cirrhosis in rats was similar to that of pHSA. Furthermore, OsrHSA displays similar in vitro and in vivo immunogenicity as pHSA. Our results suggest that a rice seed bioreactor produces cost-effective recombinant HSA that is safe and can help to satisfy an increasing worldwide demand for human serum albumin.

The Oct. 31, 2011 news item about this research on physorg.com notes this about the demand for HSA,

“Our results suggest that a rice seed bioreactor produces cost-effective recombinant HSA that is safe and can help to satisfy an increasing worldwide demand for human serum albumin,” said the study.

The protein is often used in the manufacture of vaccines and drugs and is given to patients with serious burn injuries, hemorrhagic shock and liver disease, the researchers said.

In 2007, a shortage in China led to price spikes and a brief rise in the number of fraudulent albumin medicines on the market.

Concerns have also been raised about the potential for the transmission of hepatitis and HIV, since the protein comes from human blood.

The lead author, Yang He is from Wuhan University, China. Other listed authors are:  Tingting Ning, Tingting Xie, Qingchuan Qiu, Liping Zhang, Yunfang Sun, Daiming Jiang, Kai Fu, Fei Yin, Wenjing Zhang, Lang Shen, Hui Wang, Jianjun Li, Qishan Lin, Yunxia Sun, Hongzhen Li, Yingguo Zhu, and Daichang Yang. This list gets more interesting if you have time to check out their affiliations (at the PNAS website) which include the National Research Council of Canada’s Institutes for Biological Sciences, University of Albany, New York State and Joinn Laboratory, Beijing and you get a sense of how much cooperation it takes to do this research. Finally, the paper is titled, Large-scale production of functional human serum albumin from transgenic rice seeds.