1. Graduate
Category: Health Sciences
Degree Level: MS
Abstract ID#140
Kamaljeet Singh Sandhu, Arun K. Iyer, Qiong L. Zhou, and Mansoor Amiji
Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University,
Boston, MA 02115 (Email: sandhu.k@husky.neu.edu)
Abstract
Purpose: The pro-inflammatory cytokine TNF-α gene silencing was
evaluated using a non-viral hyaluronic acid-poly(ethylene imine (HA-PEI)
conjugates in macrophages for potential therapeutic approach in the
treatment of type 1 diabetes-associated inflammation.
Methods: HA-PEI conjugates were synthesized and characterized by
1H-NMR. Small interfering RNA (siRNA) duplexes for silencing TNF-α
gene were complexed with HA-PEI and the nanoparticles formed were
characterized for size, surface charge, transmission electron microscopy
(TEM), and siRNA loading and stability. Intracellular delivery and TNF-a
gene silencing efficacy was evaluated in J774.A1 adherent murine
macrophages that were stimulated with lipopolysaccharide (LPS). RT-
PCR was used for qualitative and quantitative determinations of TNF-α
levels post-administration of siRNA.
Results: HA-PEI conjugates efficiently encapsulated negatively-charged
siRNA duplexes against TNF-α to form self-assembled core-shell
nanoparticles of approximately 90 nm in diameter. The average surface
charge was -36.0 mV due to the HA core and PEI-siRNA shell structure
of the assembled nanoparticles. TEM imaging confirmed the core-shell
structure of the nanoparticles with the nucleic acid confined to the center.
The HA-PEI nanoparticles were found to efficiently deliver siRNA in
J774.A1 adherent murine macrophages. RT-PCR results showed that
TNF-α in LPS-stimulated cells was significantly lower following
transfection with siRNA-encapsulated in HA-PEI relatively to all other
controls including Lipofectin®-complexed siRNA.
Conclusions: The preliminary results obtained here are highly
encouraging for development of HA-PEI self-assembled nanoparticle
system for encapsulation and delivery of TNF-α silencing siRNA in
macrophages to limit inflammation. This approach has significant utility,
including in the treatment of type 1 diabetes-related inflammation and
insulin resistance.
Introduction
Methods
 Diabetes is a global problem and one with the highest healthcare
expenditure. Over 230,000 deaths were recorded in 2007 alone due to
this disease.
 Diabetes mellitus (or type 1 diabetes) is a group of metabolic diseases
in which the body loses its ability to either produce or use insulin,
resulting in higher blood glucose levels which can gradually lead to life
threatening long term complications such as myocardial infarction,
cerebrovascular stroke, retinopathy, neuropathy, nephropathy, end
stage renal diseases, periodontal pathologies, etc.
 Current treatment strategies include administration of insulin pills, oral
hypoglycaemics and intensive insulin therapy. Moreover
transplantation of pancreas or just islet cells of langerhans have been
tested for treatment of type-1 diabetes, but safety concerns have
limited the success of these strategies.
 Increased levels of TNF-α, a pro-inflammatory cytokine, has been
implicated in patients with diabetes. Thus, strategies targeting down-
regulation of TNF- α can be beneficial in the treatment of diabetes.
 RNA interference therapy has emerged as a powerful strategy to
down-regulate TNF-α. However, intracellular delivery of small
interfering RNA is a major challenge as the nucleic acid payload must
navigate through the circulatory system of body and avoid filtration by
kidney, aggregation with serum proteins, uptake by phagocytes and
degradation by endogenous nucleases enzymes. These physiological
parameters must be overcome by a delivery system for efficient use of
siRNA as a clinically viable therapeutic strategy for diabetic patients
RNA Interference Results
Conclusions
References
Figure 1. Mechanism of RNA intereference1.
 TNF-α silencing HA-PEI siRNA nanoparticles: 3mg/ml of HA-PEI was
taken in PBS and added to 0.5mg/ml of TNF-silencing siRNA. Then
the mixture was vortexed for1 minute. Mixture was then kept for about
20 minutes at RT and then refrigerated at 4⁰C until further used.
 The formulations were characterized for size, surface charge with zeta
sizer and transmission electron microscopy (TEM). RNA loading
efficiency was examined with RiboGreen® RNA assay.
 Gel electrophoresis was used to check the stability of RNA under
treatment of 2% poly(acrylic acid).
 Intracellular delivery and TNF-a gene silencing efficacy was evaluated
in J774.A1 adherent murine macrophages that were stimulated with
lipopolysaccharide (LPS). RT-PCR was used for qualitative and
quantitative determinations of TNF-α levels post-administration of
siRNA
Kim, D.H. and J.J. Rossi, Strategies for silencing human disease using RNA interference. Nat Rev Genetics, 2007. 8(3): p. 173-84.
Elbashir, S.M., et al., Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001. 411(6836): p. 494-8.
Whitehead, K.A., R. Langer, and D.G. Anderson, Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discovery, 2009 8(2):p.129-38.
 The preliminary results show that siRNA can be encapsulated in self-assembled HA-PEI nanoparticles and these can be efficiently delivered to
silence TNF-α gene with potential to treat diabetes-related inflammation.
 Studies showed that biological activity of siRNA was preserved while formulating the nanoparticles. TEM imaging confirmed the core-shell structure
of the nanoparticles with the nucleic acid confined to the center.
 These nanoparticles are efficiently taken up by J774.A1 adherent murine macrophage cells.
 Also these HA-PEI nanoparticles are capable of efficiently silencing TNF-α expression in these cells as confirmed by RT-PCR.
Particle Size and Zeta Potential:TNF-α containing HA-PEI
nanoparticles were characterized by dynamic light scattering (DLS),
using the Malvern Zetasizer. Nanoparticles showed unimodal
distribution with a mean particle diameter of ~90nm and zeta potential
of -36mV and were stable at that size when stored at 4ºC. These
results are summarized in Table 1.
Table 1: Characterization of the nanoparticle formulations.
Formulations Size
(nm)
Polydispersity
Index
Zeta Potential
(mV)
TNFα siRNA encapsulated in HA-
PEI Nanoparticles in PBS
92.0 ± 3.0 0.21 -35.9 ± 4.2
TNFα siRNA encapsulated in HA-
PEI Nanoparticles in DI Water
164.4 ± 6.7 0.34 -32.7 ± 9.5
Scramble siRNA encapsulated in
HA-PEI Nanoparticles in PBS
105.9 ± 6.0 0.22 -35.1 ± 3.3
Figure 3. Transmission electron microscopy images of siRNA
encapsulated in HA-PEI self assembled nanoparticles.
Figure 4. DIC and fluorescence microscopy images of siRNA-
encapsulated HA-PEI nanoparticles in J774.A1 cells
DIC Cy3-Labeled siRNA FITC-Labeled HA-PEI
Figure 4 Cont’d. The epifluorescence images confirm internalization of
Cy3 labeled TNF-α siRNA by the cells. For this study nanoparticles were
formulated using Cy3 labeled TNF-α siRNA and FITC labeled HA-PEI to
perform epifluorescence microscopic analysis of cell uptake. siRNA
internalization was best seen after12 hours of incubation.
Lane 1: Ladder
Lane 2: No Treatment
Lane 3: Scramble Nanoparticles
Lane 4: 50 nM HA-PEI Nanoparticles
Lane 5: 75 nM HA-PEI Nanoparticles
Lane 6: 100 nm HA-PEI Nanoparticles
Lane 7: Lipofectin-50 nm siRNA
Lane 8: Lipofectin-75 nm siRNA
Lane 9: Lipofectin-100 nm siRNA
Lane 10: Ladder
Figure 5. RT-PCR analysis data of TNF-α gene silencing after
treatment with siRNA duplexes complexed with Lipofectin®, or in HA-
PEI nanoparticles (a) and semi-quantitative analysis of expression
profile (b). The adherent murine macrophages J774.A1 cells were
stimulated with lipopolysaccharide (LPS) for 6 hours prior to dosing
with native and scrambled (as control) siRNA sequences. The siRNA
dose ranged from 50 to 100 nM. HA-PEI nanoparticles were effective
in TNF-α gene silencing similar to the levels obtained with
Lipofectin®-complexed siRNA . HA-PEI, on the other hand, was
shown significantly less toxic that Lipofectin® in intracellular siRNA
delivery.
No Scramble 50nM HA-PEI 75 nM HA-PEI 100 nM HA-PEI Lipofectin Lipofectin Lipofectin
Treatment Nanoparticles Nanoparticles Nanoparticles Nanoparticles 50nM siRNA 75 nM siRNA 100nM siRNA
b
Figure 2. Self assembled HA-PEI/siRNA nanoparticles. Figure illustrating
core-shell structure of Cy3-labeled siRNA conjugated to PEI modified HA
(with FITC) (a) and 1H-NMR of HA-PEI-FITC Conjugate (b).
In Vitro Evaluation of TNF-a Gene Silencing in Macrophages using Hyaluronic Acid-Based Self-Assembled
Nanoparticles for Anti-Inflammatory Therapy
a
B
Free NH2
(Peak from
PEI) (Peaks
from
PEI)
Peak from
FITC
Peaks
from HA
Solvent
peak
(D2O)
b
PEI modified HA (with FITC label)
HA-PEI/siRNA (nanoparticle)
Size of nanoparticles:
~ 80 to 100 nm (by Dynamic
Light Scattering analysis)
a