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).