Biomedical Engineering Reference
In-Depth Information
degradation. Under reductive conditions similar to the cytosolic environment,
an intact form of siRNA was released from the siRNA-PEG conjugate by
cleavage of the disulfide linkage. The VEGF siRNA-PEG/PEI PEC micelles
effectively silenced VEGF gene expression in prostate carcinoma cells (PC-3)
by up to 96.5% under an optimized formulation condition.
107
Intravenous as
well as intratumoral administration of the PEC micelles significantly inhibited
VEGF expression at tumor tissues, leading to tumor growth suppression and
an anti-angiogenesis effect in an animal tumor model without showing any
detectable inflammatory responses in mice. Upon examination of the PEC
micelle distribution and in vivo optical imaging following intravenous injection,
enhanced accumulation of the PEC micelles was observed in the tumor region
by the EPR effect.
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The PEC micelle formulation also showed full penetration
through multiple layers of cancer cells with a unique punctuated distribution
pattern and significant suppression of mRNA and protein expression in a
dose-dependent manner.
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Furthermore, the authors continued this work and generated targeted
siRNA PEC micelles. A cell-specific targeting property was added to the PEC
micellar delivery system by employing luteinizing hormone releasing hormone
(LHRH) as a cancer targeting moiety. For the LHRH receptor overexpressing
ovarian cancer cells (A2780), the PEC micelles with LHRH exhibited enhanced
cellular uptake via receptor-mediated endocytosis, compared to those without
LHRH. As a result, LHRH-modified PEC micelles containing 50 nM siRNA
reduced VEGF expression by 63%, significantly better than the unmodified
counterpart (siRNA-PEG/PEI) that reduced VEGF expression by 50%. In
contrast, in SK-OV3 cells that are LHRH receptor negative, the same level of
VEGF expression was observed after treatment with LHRH-modified and
unmodified PEC micelles containing VEGF siRNA.
108
In a separate study, PEC micelles were prepared by conjugating siRNA to
PEG via a disulfide linkage and further condensing it with cationic fusogenic
KALA peptide (WEAKLAKALAKALAKHLAKALAKALKACEA) instead
of PEI. KALA is an amphipathic peptide that undergoes a conformational
change from pH 5.0 to 7.5, resulting in effective disruption of the endosomal
membrane.
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siRNA-PEG/KALA PEC micelles inhibited gene expression
as effectively as siRNA-PEG/PEI PEC micelles at a much reduced N/P ratio. As
PEI's cytotoxicity might limit clinical applications, KALA could be utilized as a
non-cytotoxic core condensing agent for siRNA-PEG conjugates to form PEC
micelles.
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Multifunctional PEC micelles based on this delivery system are
continually being developed. siRNA was conjugated to a six-arm PEG
derivative and the conjugate was also functionalized with Hph1, a cell
penetrating peptide, to enhance its cellular uptake property (6PEG-siRNA-
Hph1). The 6PEG-siRNA-Hph1 conjugate was electrostatically complexed with
cationic self-crosslinked fusogenic KALA peptide (cl-KALA). The 6PEG-
siRNA-Hph1/cl-KALA complexes showed superior physical stability, resistance
to enzymatic degradation and enhanced gene-silencing efficiency in MDA-MB-
435 cells under serum-containing conditions without eliciting cytotoxicity.
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