Chapter 25

Preparation of siRNA-Encapsulated PLGA Nanoparticles

for Sustained Release of siRNA and Evaluation
of Encapsulation Efficiency
Panayotis Pantazis, Konstantinos Dimas, James H. Wyche,
Shrikant Anant, Courtney W. Houchen, Jayanth Panyam,
and Rama P. Ramanujam

Abstract
Nanoparticles (NPs) formulated using poly (D,L-lactide-co-glycolide) (PLGA), a biodegradable, biocompati-
ble, and clinically approved polymer, have been widely used for targeted drug delivery. Here we provide
methods for preparing PLGA NPs that encapsulate small interfering RNA (siRNA). The siRNA NPs are
formulated using a double-emulsion solvent evaporation technique with the addition of a small amount of
the cationic polymer, polyethyleneimine, which significantly increases siRNA encapsulation.

Key words: Poly(lactic-co-glycolic) acid, Cancer therapy, Cancer treatment, Drug delivery, siRNA,
RNAi, Nanoparticles, Cancer therapy

1. Introduction

RNA interference (RNAi) is a cellular pathway that decreases the

expression of specific genes. RNAi are small interfering RNA mol-
ecules (siRNA), also known as short interfering RNAs and silenc-
ing RNAs. siRNAs are 20–25 nucleotide-long double-stranded
RNAs. Since their discovery in 2001 by Baulcombe’s group (1),
siRNAs have gained widespread interest in the biomedical research
and drug development fields because of their ability to interfere
with or “knock down” a particular gene of interest. However, a
major limitation for the use siRNA, both in vitro and in vivo, is the
inability of naked and nuclease-sensitive siRNA to passively diffuse
through cellular membranes due to the strong anionic charge of
the phosphate backbone and consequent electrostatic repulsion

Mikhail Soloviev (ed.), Nanoparticles in Biology and Medicine: Methods and Protocols, Methods in Molecular Biology, vol. 906,
DOI 10.1007/978-1-61779-953-2_25, © Springer Science+Business Media, LLC 2012

311
312 P. Pantazis et al.

from the anionic cell membrane surface. Therefore, the primary

success of siRNA applications depends on the preparation of suit-
able vectors to deliver therapeutic genes to targets of interest.
In recent years, many techniques reliant on therapeutic lipids
and polymers have been developed for specific gene delivery appli-
cations, but their general use is hindered by several drawbacks.
These include the difficulty associated with preparing the needed
copolymers, the limited density of functional groups, poor encap-
sulation efficiency, and targeting effects that decrease with time
due to desorption or degradation of the adsorbed group as the
particle or scaffold erodes (2, 3).
Poly (lactide-co-glycolide) or PLGA is one of the most studied
synthetic polymer because of its biocompatibility, biodegradability,
and approval by the United States FDA for human use. PLGA NPs
have been widely used as a vehicle for pharmaceutical delivery of
nucleotides, hormones, or drugs to target tissues. PLGA polymers
in nanoscale have also been used for the fabrication of bioscaffolds
for tissue engineering. Our studies have shown that addition of a
small amount of the cationic polymer, polyethyleneimine (PEI),
during NP formulation significantly increased siRNA encapsula-
tion in NPs, an improved siRNA release profile, and improved
effectiveness of gene silencing in cultured cancer cell and animal
models of the human tumor/mouse xenograft system (unpub-
lished data). We use polyvinyl alcohol (PVA) to stabilize the emul-
sion during nanoparticle formation, because PVA forms particles
of relatively small sizes and uniform size distribution.
Patil and Panyam (3) reported a variation of the formulation
protocol and used the same to formulate dual action NPs with
both a siRNA targeted to P-glycoprotein and an anti-cancer drug
(4). Unlike the above, the protocol described in this chapter uti-
lizes pre-complexing of siRNA with PEI, followed by encapsula-
tion of the complex in PLGA nanoparticles. A small amount of PEI
is added to the PLGA polymer phase (30:0.1 PLGA-PEI weight
ratio) to improve the encapsulation and release of siRNA.
In our opinion, the likelihood of successful therapy of cancer
genes should be enhanced by the availability of drugs that could be
delivered systemically to have specific antitumor targeting capabil-
ity along with the ability to induce death in primary and metastatic
tumor cells. In addition, we believe that the selective and robust
effect of siRNA on gene expression makes it a valuable research
tool, both in cell culture and in living organisms. However, admin-
istering siRNAs to living animals, and to humans, poses many
challenges. In laboratory conditions, siRNAs show different effec-
tiveness in different cell types in a manner yet poorly understood:
some cells respond well to siRNAs and show a robust knockdown,
whereas others show no such knockdown. Nevertheless, siRNA-
encapsulated in PLGA NPs as described below can be efficiently
used to systematically knockdown cancer signaling markers in
tumor cells.
 25 siRNA-Encapsulated PLGA Nanoparticles for Cancer Therapy 313

2. Materials

All RNA and especially single-stranded RNA oligonucleotides are

susceptible to degradation by RNase during handling. Wear gloves
and change them frequently. Use RNase-free pipette tips,
plasticware, water, buffers, and treated glassware.

2.1. Preparation 1. siRNA: Custom-made RNA oligonucleotides. These need to

of siRNA NPs be designed by the user depending on the mRNA target. The
oligonucleotides used in our work are listed in Table 1 as
examples only. In general, siRNAs are shipped by vendors as
freeze-dried single-stranded or pre-annealed double-stranded
RNAs. Centrifuge the tube(s) briefly upon receipt to ensure
that the siRNA is collected at the bottom of the well. Store
at −20 °C in a frost-free freezer. If necessary, siRNAs as dry
pellets (unopened) can be stored at 4 °C for several weeks.
2. 5× Annealing buffer: 50 mM Tris, pH 8.0, 100 mM NaCl,
prepared in RNase-free water. This is needed only if single-
stranded oligonucleotides are provided. Buffer can be stored at
−20 °C and freeze-thawed many times. Dilute with RNase-free
water to 1× concentration before use.
3. Control siRNA: We used a random (scrambled) double-
stranded RNA, see Table 1.
4. Chloroform (CHCl3).
5. PEI: Average molecular weight 750,000, make 5 % solution
(w/v) in Chloroform.
6. PLGA: 50:50 (by weight) monomer ratio with molecular
weight of 106 kDa and viscosity of 1.05 dL/g.
7. PVA: 2 % in RNase-free water.
8. Sterile RNase-free water: Sterile. Do not use DEPC-treated water.
9. Ficoll/trehalose solution: 5 % Ficoll 400, 5 % trehalose in
RNase-free water.
10. Analytical balance.

Table 1
siRNAs used for making PLGA nanoparticles

siRNA Sequence

RNA-binding motif protein 3 GGGTATGGATATGGATATG

(RBM3)
Β-catenin CGGGAUGUUCACAACCGAATT
Scrambled RNA UUCUCCGAACGUGUCACGUT
314 P. Pantazis et al.

11. Fume hood.

12. Magnetic stir plate.
13. Probe sonicator: using continuous mode and 100 %
amplitude.
14. Repeat pipettor.
15. Refrigerated fixed-angle centrifuge capable of 20,000 × g.
16. Freeze-drying equipment.

2.2. Characterization 1. Release buffer (TE buffer): 10 mM Tris–HCl, pH 7.4, 1 mM

of siRNA NPs EDTA, prepared in RNase-free water.
2. ZetaPlus particle size and zeta potential analyzer equipped
with a 35 mW solid state laser (658 nm) (Brookhaven
Instruments).
3. Scanning electron microscope (SEM) Hitachi S-900 Cold
Field Emission Gun SEM (Hitachi) or a similar instrument to
characterize the surface morphology of nanoparticles.
4. 300-Mesh copper grid for the above.
5. Ultra sensitive fluorescent nucleic acid stain Quant-iT picogreen
dsDNA reagent (Molecular Probes).
6. FLx-800 fluorescent microplate reader (BIO-TEK Instruments)
or equivalent Spectrophotometer for measuring fluorescence
of Quant-iT picogreen reagent.
7. Orbital shaker at 37 °C.

3. Methods

3.1. Preparation of We recommend making a separate PREPARATION OF PLGA

siRNA-Encapsulated NPs with scrambled siRNA for use as a negative control for in vitro
PLGA NPs and in vivo experiments. In our studies, we use siRNA with a ran-
dom (scrambled) sequence. Steps 3–7 should be performed in a
fume hood. Synthesis of siRNA encapsulated poly PLGA nanopar-
ticles using a double-emulsion solvent evaporation (w1/o/w2)
technique is summarized in Fig. 1.
1. Resuspend the RNAs in RNase-free water. Anneal single
stranded oligonucleotides if not supplied as double-stranded
oligonucleotides (see Note 1). Briefly centrifuge the tube to
bring down all droplets from the wall and lid of the tube
(see Note 2).
2. Add 200 μL of siRNA to 400 μL of 5 % PEI. Incubate at room
temperature for 30 min.
3. Dispense 1 mL of CHCl3 into a glass vial containing a magnetic
stir bar. Add 30 mg of PLGA. Stir to dissolve (see Note 3).
 25 siRNA-Encapsulated PLGA Nanoparticles for Cancer Therapy 315

Fig. 1. Schematic representation of synthesis of siRNA encapsulated poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles
using a double-emulsion solvent evaporation (w1/o/w2) technique.
316 P. Pantazis et al.

4. Sonicate on ice for 30 s.

5. Add 6 mL of 2 % PVA to a glass vial with a magnetic stir bar. Add
the emulsion from step 4 to the PVA solution (see Note 4).
6. Sonicate on ice for 1 min.
7. Allow the organic solvent (CHCl3) to evaporate overnight
(18 h) under continuous stirring (see Note 5).
8. Recover the PLGA NPs by centrifugation at 20,000 × g for
20 min at 4 °C. Remove the supernatant and save it for later
evaluation (see Note 6).
9. Wash the pellet three times with 10 mL of RNase-free water to
remove any residual PVA and free (i.e., non-encapsulated)
siRNA. Save the supernatant from each wash separately for
later evaluation of un-encapsulated RNA remaining in the wash
solutions (see Note 7).
10. Resuspend the NPs in 5 mL of 5 % Ficoll/5 % trehalose solu-
tion to stabilize the siRNA inside the NPs. Sonicate the NPs
for 30 s over an ice bath. (see Note 8).
11. Freeze-dry the PLGA NPs. The lyophilized NPs are stable up
to 6 months at −20 °C.

3.2. Characterization 1. Use diffraction light scattering (DLS) to determine the NP

of the siRNA NPs size, the polydispersity index (PI) and zeta potential (see Notes
9 and 10) (Fig. 2).
2. Use a UV spectrophotometer to obtain absorbance measure-
ments at 260 nm for all four supernatants from the NP formu-
lation performed in Subheading 3.1.
3. Determine the siRNA release kinetics using Quant-iT picogreen
reagent (see Note 11). Prepare several tubes containing 1 mg

Fig. 2. SEM image of dual agent nanoparticles without surface functionalization (a) and with biotin functionalization (b).
Nanoparticles were platinum-coated for visualization. Bar is 670 nm. Reproduced from (4) with permission from Elsevier.
 25 siRNA-Encapsulated PLGA Nanoparticles for Cancer Therapy 317

of siRNA nanoparticles in 1 mL of release buffer for each tube.

Incubate all the tubes on an orbital shaker at 100 rpm and
37 °C. At pre-defined time points (e.g., “zero incubation
time,” 1, 3, 5, 7, 14, 21 and 30 days), take out sets of three
tubes and centrifuge at 20,000 × g for 20 min at 4 °C. To deter-
mine the amount of oligonucleotide or siRNA in the supernatant
measure the fluorescence generated by the binding of picogreen
to double-stranded siRNA in triplicate for each time point.
The siRNA encapsulation in nanoparticles can be determined
by subtracting the amount of siRNA recovered in the wash
solutions from the initial amount of siRNA added. We typically
obtain >75 % encapsulation efficiencies.

4. Notes

1. Gene silencing is triggered by using siRNA molecules, that are

about 20–25 base pairs (bp) long. Upon introduction into
cells, siRNAs assemble into ribonuclease-containing complexes
known as RNA-induced silencing complexes (RISCs). The
siRNA strands guide the RISCs to complementary RNA
molecules, where they cleave and destroy the target RNA.
Therefore, double-stranded RNAs are used for this procedure.
To anneal single stranded oligonucleotides, dissolve them in
1× annealing buffer to a final concentration of 50 μM each.
Gently pipette up and down three times to mix well, aliquot
into sterile tubes, and store at −20 °C if not used immediately.
For annealed double-stranded RNAs, resuspend to a final
concentration of 20 μM (for each of the two oligonucleotides
in the tube (5)).
2. Aliquot the annealed siRNA into RNase-free tubes and store
at −20 °C. Do not freeze-thaw more than five times.
3. For water-in-oil (W/O) emulsion preparations, utilizing glass
vials with magnetic stirring provides consistent results than
harsh vortexing.
4. PVA acts as a surfactant.
5. Alternatively, the sample can be stirred overnight with the cap
on, after which the solvent can be evaporated under vacuum.
6. Use a pipette to remove the supernatant. Do not invert the
tube, as this may dislodge the aggregated NPs. These should
be visible, whitish in color, at the bottom of the tube.
7. Use a pipette to resuspend the NPs, then centrifuge to sedi-
ment them after each wash. To quantify the remaining RNA in
the wash solutions use Picogreen dsDNA quantitation reagent
(Invitrogen), which is suitable for determining RNA ranging
from 100 ng to 5 μg of siRNA/mL.
318 P. Pantazis et al.

8. We use a final 5 % Ficoll/5 % trehalose concentration to achieve

a Tg (glass transition temperature; (6)) of greater than 60 °C.
This increases the shelf-life of the freeze-dried NPs and pro-
vides good solubility when the NPs are resuspended in water.
9. We calculate mean hydrodynamic diameters for size distribu-
tion by weight, assuming a lognormal distribution. We use
Smoluchowski’s equation to calculate Zeta potential values
from measured velocities. In each case, distilled water may be
used as solvent for the nanoparticles and the results should be
expressed as means SEM of five runs. To study the surface mor-
phology of the NPs, use SEM. Nanoparticle suspension can be
in water (2 mg/mL), and a single drop should be air-dried on
a 300-mesh copper grid for SEM. Figure 2 shows a typical
SEM image of dual agent nanoparticles without surface func-
tionalization (A) and with biotin functionalization (B).
10. In our experiments, DLS measurements yielded the average
particle size (hydrodynamic diameter) of about ~100–250 nm.
With these NPs, initially, a burst release was seen for both
siRNA (15–20 % in the first 24 h); and a more sustained and
continuous release was observed over the next 10–20 days.
11. siRNA can be quantified using UV absorbance methods or
Quant-iT picogreen reagent (PicoGreen) which is ~10,000-
fold more sensitive than UV absorbance.
For hydrophilic drugs encapsulated inside polymeric nano-
particles, an initial burst followed by a relatively constant release
over a number of days (e.g., 5–28 days) is very characteristic.
This burst release could be explained by the fact that the hydro-
philic drug has readily escaped or diffused into the aqueous
medium under a concentration gradient. The relatively con-
stant release that follows is primarily due to the hydrolysis of
the ester bonds between the individual monomer, which causes
the degradation of the nanoparticles and hence the sustained
release and bioavailability of encapsulated siRNA (7–9).

Acknowledgement

This was partly funded by NIH grant 3R44AT004118-03S1

(Rama P. Ramanujam) and NIH grant 7R21CA116641 (Jayanth
Panyam). We thank Fadee G. Mondalek and Sivapriya Ponnurangam
for technical assistance.
 25 siRNA-Encapsulated PLGA Nanoparticles for Cancer Therapy
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