Manipulation of Leukocytes Using Therapeutic RNAi Delivered by Targeted and Stabilized Nanoparticles - Nanotechnology for the Delivery of Therapeutic Nucleic Acids - page 187

with the glycosaminoglycan hyaluronan (HA), which functions as a
cryoprotectant [23], stabilizing the particles during lyophilization
and rehydration. Moreover, hyaluronan serves as a scaffold for the
binding of the antibodies, which endow the targeting capability
to the nanoparticles [11]. The encapsulation of siRNAs within the
I-tsNPs occurs when the lyophilized particles are rehydrated with
water containing protamine-condensed siRNAs. Approximately
80% of the siRNAs are encapsulated, with a payload of approxi-
mately 4000 siRNA molecules per particle. I-tsNPs protect
the entrapped siRNAs from degradation and lead to efficient
silencing, even after pre-incubation with RNase A or 50% serum
for 2 h prior to cell transfection. Moreover, when assayed ,

I-tsNP-encapsulated siRNAs do not induce interferon responses or
lymphocyte activation.

in vitro

Hyaluronan Integrin mAb Condensed siRNA

Hyaluronan Integrin mAb Condensed siRNA

NH 2

COOH
COOH

NH 2

COOH

NH 2

MLV

I-tsNP
entrapping siRNAs

MLV I-tsNP

entrapping siRNAs sNP I-tsNP ULV sNP I-tsNP

ULV

COOH

COOH

COOH
COOH
COOH

COOH

COOH

Attachment
of hyaluronan

Extrusion Attachment

of mAb

COOH

Rehydration Extrusion Attachment

of hyaluronan

Attachment
of mAb

Lyophilization

Lyophilization Rehydration

Figure 8.3 The preparation steps of integrin-targeted and stabilized

nanoparticles.

Multilamellar vesicles (MLV) are extruded into nanoscale
unilamellar vesicles (ULV), which are then surface-modified with
high molecular weight (850 kDa) HA to form stabilized nanoparticles
(sNPs). The HA-modified liposomes are coupled to the mAb using
an amine-coupling method, and then the I-tsNPs are purified
and lyophilized. The siRNA entrapment procedure is achieved
by rehydrating the lyophilized I-tsNPs with nucleases-free water
containing protamine-condensed siRNAs.

8.9 In vivo Gene Silencing Using I-tsNP-

Entrapping siRNAs

A single intravenous injection of siRNA (2.5 mg/kg) targeted against
the DNA repair-associated gene

Ku70 was determined to be sufficient