Extracellular barriers
Effective siRNA delivery is initially disrupted by the hostile
extracellular environment including all chemical, biological, and
physical barriers, like the immune system reactions, scavengers,
nucleases and proteases together with extreme pH (Hill, Chen, Chen,
Pfeifer, & Jones, 2016). Kidney glomerulus is a critical physically
filtrating challenge in siRNA delivery in which molecules with small
size and water are rapidly cleared into urine whereas molecules with
higher molecular weight are retained in the bloodstream (Choi et al.,
2007). The pore diameter of the glomerular basement membrane (GBM), a
thin (250–400 nm) non-cellular layer of the glomerular filtration
barrier, is demonstrated to be around 6–10 nm. Hence, siRNAs without
any delivery systems due to their small size (i.e. about 7.5 nm in
length and 2 nm in diameter) can be easily filtered within 10 min via
GBM (Abedini, Ebrahimi, & Hosseinkhani, 2018). Therefore, it is
necessary to set a lower size limitation of about 10 nm for the delivery
systems design (Lu & Qiao, 2018). By the way, the defective “leaky”
vascular architecture of several solid tumors, which is related to
immature lymphatic ducts, allow size-dependent accumulation of
nanoparticles (10-100nm) in tumor. This preferential size-dependent
accumulation of drug-loaded nanocarriers in the cancer cells was
initially called the enhanced permeability and retention (EPR) effect in
1986 (Davoodi et al., 2018; Kalyane et al., 2019). To date, the EPR
phenomenon has been broadly established in different mice models of
pancreatic tumors using various types of delivery vehicles (Aghamiri,
Jafarpour, & Shoja, 2019). It is noteworthy that this phenomenon is
strongly affected by pancreatic tumor physiology. While highly permeable
LS174T-transplanted SCID model mice are reported to permit substantial
accumulation of even 400 nm nanoparticles (Yuan et al., 1995), mice
bearing BxPC3 tumors demonstrated hypervascularity and thick fibrotic
stroma impeding tumor accumulation of >50 nm-sized
nanoparticles; unlike 30 nm-sized ones (H. Cabral et al., 2011).
Finally, high antitumor activity offers intriguing glimpses into the
potential of 30 nm-sized nanoparticles in pancreatic cancer therapy
(Horacio Cabral et al., 2013). As a consequence, many nanoparticle-based
delivery systems with a size of less than 50 nm have been designed for
increased accumulation in pancreatic tumor tissues (Maeda, 2015).
Because of high cytidine deaminase (CDA) expression as well as physical
blockage, stroma can contribute to pancreatic cancer chemoresistance and
unfavorable pharmacokinetics and pharmacodynamic profile in vivo ,
which can decrease the systemic circulation time of GEM to
<0.3 hours (Erkan et al., 2012; Meng & Nel, 2018).
The pharmacological reduction of stromal cells is one of the main
strategies for overcoming pancreatic tumor which is shown by
Abraxane®, albumin-bound paclitaxel approved by the
Food and Drug Administration (FDA). Clinical studies have revealed that
the combination of this drug with GEM improves the survival rate. The
underlying mechanism of Abraxane® for the stromal
reduction and reduction of the CDA expression is the reactive oxygen
species generation (Lancet et al., 2014).
Particle dynamics play an important role in overcoming the stromal
barrier (Figure 2) and transportation of nanoparticles from blood
vessels to pancreatic tumor cells as a result. Because of the
hydrodynamic pressure gradient, an opening temporarily generates through
the walls of the blood vessels of pancreatic tumors. Subsequently, fluid
enters the pancreatic tumor interstitial space (termed ‘eruptions’).
Hence, not only 30 nm diameter but also 70 nm diameter nanocarriers can
enter into the interstitial spaces of pancreatic tumors. The
nanocarriers with 30 nm diameter can rapidly extravasate into the PDAC
tumor microenvironment; however, the extravasation of the nanocarriers
with 70 nm diameter can be blocked by an abundant dysplastic stroma
which can interfere with the drug delivery and cause chemoresistance in
pancreatic cancer (Matsumoto et al., 2016). Therefore, concerning tumors
with high content, drug delivery systems penetration into the stroma
tissue is necessary to reach the tumor cells environment. As a result,
the size of delivery systems plays a significant role in the
penetrability of the nanocarriers. Many studies showed that smaller
delivery systems are preferred to distribute across extracellular matrix
with high content of stroma cells (Perrault, Walkey, Jennings, Fischer,
& Chan, 2009). Furthermore, fast growth of pancreatic tumor cells and
further compression of blood and lymphatic vessel leads to an increase
in fluid pressure throughout the cancer interstitial region, impeding
effective penetration of nanocarriers from
intravascular region to the
pancreatic tumor cells (Kurtanich et al., 2018). Administrating
collagenase and transforming growth factor-β inhibitor can respectively
decrease the pericyte coverage of endothelium and fibrosis in the
pancreatic cancer milieu in order to increase the diffusion of
nanocarriers (Samanta et al., 2019). Stromal targeting therapy is
another stromal barrier overcoming strategy. Numerous studies have shown
that cyclical iRGD peptides bind to tumor-specific integrins. αvβ3 and
αvβ5 integrins are then proteolytically processed to reveal a C-terminal
(CendR) motif that binds to NRP-1 which could act to induce the
formation of grape-like cytoplasmic vesicles and vacuoles, termed the
Vesiculo–Vacuolar organelle (VVO) (Ding et al., 2019; Dvorak et al.,
1996; Sugahara et al., 2010). This strategy is believed to mediate the
transcytosis of nanoparticle-based delivery vehicles into pancreatic
tumor cells (Liu et al., 2017).
Figure 2. Schematic
illustration of extracellular barriers in pancreatic cancer siRNA
delivery.
It is of note that the previous mentioned size of nanoparticle-based
delivery systems must be maintained even in the blood circulation
containing various cells and biomacromolecules. Therefore, nanocarriers
should be rigorously developed to minimize fast dissociation and
unwanted aggregation in the biological environment. In particular, the
nanoparticle-based delivery systems with positive charge can
electrostatically interact with proteoglycans and proteins with anionic
charge in the serum, like heparan sulfate and albumin, leading to their
dissociation and/or aggregation (Hui et al., 2019). In an important
study, transmission electron microscopy (TEM) has demonstrated that the
CALAA-01 in blood (with a zeta-potential of 10–30 mV) can be filtered
through GBMs due to the aggregation with a high density of heparan
sulfate. Therefore, the structural integrity of several delivery systems
will be lost. On the other hand, it has been reported that the excretion
of siRNA-conjugated cationic polysaccharide nanocarriers is considerably
lower than siRNA without delivery systems. Although the initial size of
the delivery systems (220-230 nm) was bigger than the GBMs pore size,
some siRNAs were excreted through the urine. This suggests that siRNA
payloads were slowly disengaged from the nanoparticles. Furthermore, it
seems that GBM slightly enhances disassembly of the nanocarriers through
IV route (Naeye et al., 2013; Zuckerman, Choi, Han, & Davis, 2012). It
is noteworthy that large nanocarriers, <300 nm, can be
captured and phagocytosed by Kupffer cells and removed from body
(Gustafson, Holt-Casper, Grainger, & Ghandehari, 2015). Hence, the
opsonization of nanoparticles can make them more accessible to
phagocytes. A robust strategy to limit nanoparticle opsonization by
serum proteins is surface modification with hydrophilic, non-ionic, and
flexible polymer chains such as poly(oxazoline),
poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), poly(Nvinyl
pyrrolidone) (PVP), and polyethylene glycol (PEG), which all improve
colloidal stability of nanoparticle-based delivery systems and hinder
the non-specific interactions with serum protein (Adler & Leong, 2010;
J. Hu et al., 2018). Among these polymers, PEG, an injectable
biocompatible, hydrophilic, and biologically-inert material, is the most
typically utilized polymer for nanoparticle modification and it is
approved by FDA in United States for numerous applications (Adiseshaiah,
Crist, Hook, & McNeil, 2016).