Mitigating The Deleterious Effects

of siRNA-Nanoparticle Therapy
Koushul Ramjattun1, Clyde Overby2, Danielle S. W.Benoit 3-5
1. University of Rochester, Department of Chemical Engineering, Rochester, NY, USA
2. University of Rochester, Department of Biomedical Engineering, Rochester, NY, USA
3. University of Rochester, Department of Biomedical Engineering, Rochester, NY, USA
4. University of Rochester, Department of Chemical Engineering, Rochester, NY, USA
5. University of Rochester Medical Center, Department of Orthopaedics, Center for Musculoskeletal Research, Rochester, NY, USA

Introduction BX795 & Cu-CPT8m Increase Cell Metabolism

polyIC a 24h Post-Treatment b 48h Post-Treatment
ssRNA + Triphosphate
RNA interference (RNAi) is a naturally occurring endogenous 5' PPP - OH3' TLR 3 Human mesenchymal stem cells (hMSCs) were treated with different
or blunt siRNA
regulatory process in which a short single-stranded RNA causes FC99 dsDNA
dosages of drugs B, C and F. AlamarBlue® assay was used to evaluate
sequence-specific post-transcriptional gene silencing. Small C
Hydrochloride siRNA
polyIC
cell metabolic activity at Day 0, Day 1 (24h post-treatment) and Day 2
interfering RNA (siRNA), a specific subset of RNAi, is a potent
MDA-5 Unmethylated mRNA
(48h post-treatment). Comparisons were made against the Day 0
therapeutic approach for several pathological conditions such as viral metabolic rate using multiple paired t-tests, with alpha = 0.05, and using
Apoptosis
infections, autoimmune conditions, genetic diseases and cancer. C C
IPS-1

TLR 7/8
Šidák correction for multiple comparisons. No significant increase in
C

However, siRNA is unstable in the bloodstream, cannot efficiently RIG-1

TLR 3 dsRNA metabolic activity on Day 1 compared to Day 0 was observed.
siRNA
cross cell membranes, and can also trigger an immune response. PKR
P
Endosome Surprisingly, 48h post-treatment, a significant increase in metabolic rate
was observed for high dosages of C and B.
Myd88

The most significant challenges in realizing the broad potential of

PKR
P TRAM p13K

siRNA-based therapeutics for disease therapy is the need for a safe P TRIF
Cu-CPT8m
This suggests that either inhibiting these immune pathways boost cell
IRF7

and effective delivery system[1]. Polymeric nanoparticles have been P

metabolic activity, or that C and B potentially have side effects that

TRAF3

shown to be effective for delivering siRNAs [2] [4] across cell IRF5

Interferon P
IKK ε
stimulate metabolism. No drug treatment resulted in a decrease in cellular
membranes. However, siRNA-NP therapy still has negative side
STAT1
Stimulated TBK1
Genes STAT1
P
BX795 metabolism, indicating that the 3 drugs used by themselves did not have
effects on cell metabolism. We hypothesize that these deleterious
P
IRF3
Figure 3, Relative increase in metabolic activity of hMSCs as evaluated using AlamarBlue® assay.
IRF3
any detectable acute toxic effects after 48h and are well tolerated.
effects are due to cells’ innate immune responses. c-Jun
P
P
IRF3 A: No significant change in metabolic activity was observed on Day 1 relative to Day 0. B: CuCPT8m and BX795
IFN α ATF-2
treated hMSCs showed a significant increase in metabolic rate on Day 2 relative to Day 0. *p < 0.05
P

Cells evolved Pattern Recognition Receptors (PRRs) as part of their P

IFN β

siRNA-NP Antagonizes BX795 Metabolic Effects

IRF5
RISC Complex
innate immune system, acting as sensors against pathogens. For
IRF7
P
P Type 1
IRF5 Interferons
example, toll-like receptors (TLRs) and RIG-I like recognize highly IRF5
P siRNA - mRNA

conserved bits and pieces of invading pathogens such as viral RNA. Inflammatory ucl
eus Complex
48h Post-Treatment 48h Post-Treatment
hMSCs were treated with 30nM siRNA-NP solution, loaded in a 1:4
N
When PRRs identify a piece of viral RNA, an immune response is
Cytokines siRNA:NP charge ratio, simultaneously with two different dosages of each
triggered in order to fight the invader. Unfortunately, siRNA-NP have drug. Comparisons were made against the Day 0 metabolic rate using
also been shown to activate the innate immune system, resulting in
Mitochondria is the
power house of multiple paired t-tests, with alpha = 0.05, and using Šidák correction for
the cell Sliced mRNA
alterations in cell metabolism and proliferation which limits NP multiple comparisons. We found a significant difference between drug C
delivery safety and efficacy[3]. with siRNA-NP treatment versus the control using siRNA-NP only.
No Protein
However, when comparing siRNA-NP + drug C to drug C treatment alone
The goal of this project is to demonstrate that small molecules
Translation Figure 1. Cellular at the highest concentration (Figure 4), there was no significant difference.
innate immune Surprisingly, 80nM B treatment had the opposite effect of what we
inhibitors that suppress specific pathways in the innate immune
Disassembled siRNA-NP
at low pH inside endosome response pathways
response of cells can be used simultaneously with nanoparticles involved in sensing expected to observe. 48h post-treatment, hMSCs treated with B and
loaded with siRNA in order to mitigate the deleterious effects of Cytoplasm
foreign genetic siRNA-NP showed a significant decrease in metabolic activity versus only
material. RNA
siRNA-NP treatment on metabolism. interference pathway. drug B, while cells treated with only drug B showed an increase in
metabolic activity instead. (Figure 3b and Figure 4) This suggests that the
siRNA-NP antagonizes the metabolic increase cause by drug B.
Small Molecule Inhibitors Suppress Innate Immune Responses
It should also be noted that there was no significant difference between
Since toll-like receptors play an important role in sensing invading pathogens and triggering an immune response, we identified and purchased 2 inhibitor Figure 4. Comparing the relative increase in metabolic Figure 5. Relative increase in metabolic activity of the siRNA-NP treated cells and the untreated cells. This could indicate
drugs targeting TLR3 and TLR8. We also purchased BX795, a known inhibitor of TANK-binding kinase 1 (TBK1) which is involved in inflammatory responses activity of hMSCs 48h post-treatment for NP+Drug hMSCs 48h post-treatment. S-NP = siRNA-NP. No
treatment versus drug only. *p < 0.05 significant difference between siRNA-NP treated cells that a 30nM siRNA-NP concentration was too low to trigger an immune
and mediates the transcription of IRF3. The IC50 of a particular drug is the inhibitor concentration at which 50% of the inhibitory action occurs. The drug versus Untreated cells. *p < 0.05 response to begin with.
dosages in this experiment were chosen accordingly.
FC 99 hydrochloride inhibits TLR3 Conclusions and Future Directions
I O

Cu-CPT8m inhibits N

BX795 inhibits H
NH2

expression and suppresses

N

TLR8 through HN N
H
N NH

phosphorylation of TBK1.
inflammatory responses induced by
N

stabilization of its resting

N
BX795

IC50 = 11-30nM The goal of this project was to use small molecules to selectively inhibit cells’ ability to trigger an innate immune response when exposed to siRNA-NP. We
O FC 99

a synthetic dsRNA. IC50 = 67nM

S

state. IC50 = 50µM.

N

did not see any significant benefit of the inhibitor drugs in modifying the response of cells to siRNA-NP compared to the drug alone. We believe higher
HN

Cu-CPT8m NH2 Referred to as B

O

Referred to as C
O N
Referred to as F. dosages of siRNA-NP are required since the 30nM showed to significant decrease in cell metabolic activity. More work is needed to model the siRNA-NP
treatment to screen for interactions with drugs inhibiting the innate immune system.
Synthesis of pH-Responsive siRNA Loaded Polymeric Nanoparticles via RAFT Polymerization All 3 inhibitor drugs were not only very well tolerated by hMSCs, cells treated with drugs C and B even showed significant increase in metabolic activity 48h
The hydrophilic block 1 chain was synthesized by mixing distilled DMAEMA, AIBN and a trithiocarbonyl RAFT agent 4-cyano-4-[(ethylsulfanyl- after the treatment. The significant and large increase in cell metabolism resulting from 160nM C treatment is very intriguing and suggests that inhibiting the
thiocarbonyl)sulfanyl]pentanoic acid, referred to as ECT, in dimethylformamide (DMF) at 25 wt for 6 h at 60 °C. The hydrophobic pH-Responsive hydrophobic TLR3 has positive metabolic side effects or drug C itself is affecting other metabolic pathways.
block 2 chain was synthesized by combining the purified block 1 with 25% DMAEMA, 25% propylacrylic acid (PAA) and 50% butyl methacrylate (BMA), at 60 Previous work in our lab [3] has shown that multiple pathways are upregulated in the presence of
°C for 24 h. The resulting polymer was dialyzed and lyophilized for storage. These polymers were synthesized to match previous polymers used in siRNA
siRNA-NP in cells. Toll-Like receptors were only one of these. Since we did not see significant
delivery by Malcolm et al.[3]
+
effect when inhibiting TLR3 and TLR8, future work will involve probing and inhibiting different
+
innate immune response pathways, such as apoptotic ones and PKR signaling to investigate if
M.W. = 23 kDa
+
they contribute more to the deleterious effects of siRNA-NP treatment.
+
+
Composition: DMAEMA = 157.2 Da
+
BMA = 142.2 Da
pD

Block 1 & 2 +
+ + Inhibitor
Future work will also potentially involve loading the drugs directly into the core of the
M

+ -
+ PAA = 114.1 Da
AE

+ + + Drug
pD

- +
nanoparticles for treatment, or even use multiple siRNAs on the same nanoparticle to knock
A-
M

+ + + +
AE

pB

+
- siRNA:NP charge ratio = 1:4
M

+
M
A

+
down receptor proteins involved in sensing foreign RNAs, effectively masking the nanoparticle
A-

-
pP
AA

-
Hydrophilic Block 1
Hydrophobic Block 2
d = 31.77 +/- 0.05 nm
-
- -
Figure 6. Loading siRNA-NP with hydrophobic drugs from the cell’s sensors.
Assembled Nanoparticle siRNA Figure 2. Nanoparticles were synthesized via reversible addition-fragmentation
Hydrophilic Positively Charged Corona Negatively chain transfer (RAFT) polymerizations. Polymer diblocks form micelles due to
Charged
Hydrophobic Neutral Core solution thermodynamics when mixed with PBS. siRNA was loaded onto the
corona via electrostatic attraction. GPC, NMR, DLS and PRODAN assays were
used to characterize the nanoparticles.

1. Whitehead, K. A., Langer, R., & Anderson, D. G. (2010). Erratum: Knocking down barriers: Advances in siRNA delivery. Nature Reviews Drug Discovery,9(5), 412-412.
doi:10.1038/nrd3182 Kearns
2. Tatiparti, K., Sau, S., Kashaw, S. K., & Iyer, A. K. (2017). siRNA Delivery Strategies: A Comprehensive Review of Recent Developments. Nanomaterials, 7(4), 77.
http://doi.org/10.3390/nano7040077 This research was funded by the generous gift from the Xerox Research
3. Malcolm, D. W., Sorrells, J. E., Van Twisk, D., Thakar, J., & Benoit, D. S. W. (2016). Evaluating side effects of nanoparticle-mediated siRNA delivery to mesenchymal stem cells using
next generation sequencing and enrichment analysis. Bioengineering & Translational Medicine, 1(2), 193–206. http://doi.org/10.1002/btm2.10035313-333. Corporation and the Office of The President Experience
4. Cavallaro, G., Sardo, C., Craparo, E. F., Porsio, B., & Giammona, G. (2017). Polymeric nanoparticles for siRNA delivery: Production and applications. International journal of
pharmaceutics, 525(2),