Nature article: Orally delivered thioketal nanoparticles loaded with TNF-a-siRNA target inflammation and inhibit gene expression in the intestines
Problems: troubleshooting things
Hypotheses: actual experiments to test effects
· Problem 1:
o How to localize delivery of siRNA to diseased intestinal tissue?
· Solution 1
o Made a chemical that can target high levels of ROS produced in inflamed intestine
§ TKNs made from PPADT, which are stable in acid and base solutions, but break down in high oxygen concentration areas
· PPADT stable in gut, attracted to ROS
o Encapsulated siRNA in TKN, so it is released only in areas with high ROS (inflammation)
· Problem 2
o How specific is PPDAT for ROS?
· Solution 2
o Incubated PPADT in acid, base, or oxide solutions
o Results: figure 2a
§ Chromotograhy separation based on side of particles
§ Fewer PPADT broke down in acid/base solutions, so they showed up at a different molecular weight than the PPADT in KO2 as seen by the 2 different peaks
· Problem 3
o This is all good but, what about in physiological ROS concentrations, where ROS is produced by actual macrophages?
· Solution 3
o Induced a toll-like receptor immune response using LPS and dye-filled capsules
o Compared amount of dye released during activated macrophage response (physiological ROS concentration) with no activation (1st two bars in figure 2b)
o Did a positive control: added TEMPOL so that they can verify that the macrophages producing ROS was actually what caused a release of the dye, not something else. TEMPOL chelates ROS, so a decrease in dye release means that it must be ROS that is making the dye be released
o Results: Figure 2b
§ This is what happened in the 2 bottom bars
· Problem 4
o What is TNF-alpha role in gut inflammation? But first how do you get the TNF-alpha-siRNA ready for delivery?
· Solution 4
o Complex siRNA with DOTAP, which increases siRNA stability, cell internalization, mucosal transport, and endosomal escape
· Hypothesis 1 (in vitro)
o Can TNF-a-siRNA TKNs deliver the active form of siRNA safely to decrease TNF-a expression in inflamed cells?
· Test 1
o Treated LPS-activated macropages with TNF-a-TKNs and controls
o Results
§ Figure 2c
§ TNF-a protein concentration significantly decreased with treatment groups: scrambled siRNA, TNF-a siRNA
· Hypothesis 2 (in vivo)
o Can orally-administered TKNs deliver siRNA to the right tissue?
· Test 2
o Induced ulcerative colitis by DSS in mice, and treated with TKNs loaded with siRNA labeled with a fluorescent probe
o Results
§ Figure 3a
§ TKNS can deliver siRNA to correct sites: proximal and distal colon
§ After 7 days, see fluorescent dye only in colon; would want to see trend over all 7 days? Did it stay in the stomach for a few days?
· Hypothesis 3
o Can orally-administered TKNs deliver siRNA, and silence TNF-a mRNA expression in the same was as it silenced TNF-a protein levels in macrophages?
· Test 3
o DSS mice treated with TNF-a siRNA or scrambled siRNA by different means as seen in fig 3b
o Results
§ Figure 3b
§ TNF-a mRNA expression significantly decreased with treatment group: TNF-a siRNA-TKN by 10 fold
§ Free TNF-a-DOTAP increased expression!
· Hypothesis 4
o Did not get the PLGA stuff
· Hypothesis 5
o What is the smallest dose possible that elicits an adequate decrease in TNF-a mRNA expression
· Test 5
o Lowered treatment dose 1-fold for DSS mice
o Results
§ Figure 3c
§ Still see significant inhibition of mRNA
· Hypothesis 6
o Did not understand why they treated with beta glucan particles
o Seemed like beta-gp cannot target disease tissue, but siRNA can still can get to colon by TNKs, but they the siRNa cannot lower mRNA levels? Why not?
· Hypothesis 7
o What are the clinical/macroscopic and physiological manifestations of DDS, and can TNF-a-TKNs treat this too?
· Test 7
o Do a histological examination, neutrophil accumulation assessment (MPO-lysozyme produced by neutophils), and weight measurement
o Results
§ Figure 4 a-f
§ D looks great: TNF-a-TKN
§ Even with lowered dose, TNF-a-TKNs reduced MPO activity, and DSS animals maintained weight with this treatment
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ReplyDeleteI had one last thought on this article after discussion this afternoon. Does anyone know the significance (if there was any) of weight-gain experienced by the mice treated with TNF-alpha-siRNA? I was wondering if it was a result of improved gut function (benefical), or if it could be an accumulation of fluid in the tissues(an adverse side effect that was not discussed perhaps.) Regardless of the cause, it should be taken into consideration that people may be leary about a treatment that results in weight gain.
ReplyDeleteFirst thank you for posting this it is a nice overview of the whole paper, and allows me to go back over some points that I missed the first time reading it. After I read this paper I was thinking that if this technique were perfected and transferred to human models this could help a lot of people and maybe even be used in other areas of research. One of the problems that I have however has to do with the siRNA getting to the right tissue (hypothesis 2 in vivo). Yes they did show that when the TKNs were administered orally they did reach the target tissue (proximal and distal colon). They also reached places like the small intestine, liver and stomach. It just makes me wonder what the effects of having the siRNA being absorbed into these tissues would have in the long run. I would be nice if they looked more in depth at these results in future research.
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