- Top of page
- Materials and methods
- Supporting Information
RNA interference (RNAi) allows the knockdown of any particular gene of interest, offering great potential as a novel therapeutic strategy. It is mediated through approximately 21–23 nt, double-stranded ‘small interfering RNAs’ (siRNAs), which trigger the sequence-specific cleavage of mRNA molecules leading to their subsequent degradation 1. These siRNAs are generated intracellularly through the cleavage of longer double-stranded RNAs 2, 3 or can be directly introduced into the cell as chemically synthesized siRNA molecules 1.
Vascular endothelial growth factor (VEGF) is one of the principal regulators of tumor growth, tumor angiogenesis and metastasis, and is present at significant levels in tumor cells from various origin. Examples include neoplastic, but not benign hyperplastic or normal prostate cells 4, 5, as well as pancreatic tumor cells 6. Inhibition of VEGF or its receptor (VEGFR) inhibits tumor growth and metastasis in animal tumor models 7, 8. Consequently, several approaches have been used to implement the blockade of VEGF or VEGF receptor signaling in cancer treatment, including humanized antibodies to VEGF 9, as well as gene targeting approaches through RNA interference (RNAi). Besides the blockage of tumor angiogenesis upon VEGF inhibition, autocrine/paracrine VEGF/VEGFR signaling, which results in the stimulation of tumor cell proliferation, has also been shown 10–13. Bevacizumab (Avastin®, Roche, Basel, Switzerland), a humanized monoclonal anti-VEGF antibody, is the first VEGF inhibitor approved by the FDA for systemic use in cancer 14 and shows activity in various cancers, including pancreas and prostate carcinoma 15.
Upon RNAi-mediated VEGF down-regulation through in vitro transfection, anti-proliferative and/or anti-tumoral effects in various tumor cell lines including prostate and pancreatic carcinoma cells were oberved 16–18. In vivo, however, synthetic siRNAs display poor stability and poor penetration into the cells and, thus, the main issue is the delivery of therapeutically active siRNAs into the target tissue/target cells 19. To circumvent these problems, the approach of direct intratumoral injection has been employed for (formulated) siRNA molecules in subcutaneous (s.c.) tumor models 20–24 with the local administration, however, probably being of only limited therapeutic relevance in cancer therapy. Alternatively, the viral or nonviral delivery of DNA-based siRNA constructs for RNAi-mediated VEGF down-regulation showed anti-tumorigenic effects in various xenograft models 25–28. However, limited loading capacities, problems in large-scale production and, most importantly, safety risks as a result of their inflammatory and immunogenic effects and their oncogenic potential pose severe limitations to the applicability of viruses. Issues regarding safety and efficacy are also critical with regard to the use of plasmid-based siRNA constructs in vivo. Thus, the direct, systemic, nonviral administration of siRNA molecules allowing their therapeutic use is most desirable.
Previously, we and others have introduced polyethylenimine (PEI) complexation of siRNAs as an efficient tool for in vivo siRNA delivery 19. PEIs are synthetic linear or branched polymers available in a wide range of molecular weights 29, 30 and, because of their high cationic charge density at physiological pH, they are able to form noncovalent complexes with siRNAs 31, 32. This complexation leads to siRNA protection against degradation, efficient cellular uptake through endocytosis and subsequent intracellular release from endosomes based on the so-called ‘proton-sponge effect’ 33, 34. Using the commercially available, linear jetPEI, anti-tumorigenic effects were demonstrated upon targeting of the HER-2 receptor in s.c. ovarian carcinoma xenografts 31 or the growth factor pleiotrophin in s.c. or orthotopic glioblastoma xenografts 32. Modified PEI coupled to polyethylenglycol in combination with a ligand for tissue-specific targeting (RGD peptide for the recognition of tumor vasculature) was used for VEGFR knockdown 35, and polyelectrolyte complex micelles have been employed for VEGF targeting in a s.c. tumor model 36.
Only certain PEIs are capable of successfully delivering siRNAs and, more recently, Werth et al.37 described the preparation of PEI F25-LMW, a low molecular weight 4–10 kDa branched PEI. The in vitro data demonstrate low toxicity, high cellular uptake efficacy and efficient protection/intracellular release of siRNA molecules 37, as well as favorable features regarding the preparation, handling and long-term storage of the complexes. In particular, without the addition of any lyoprotectant, PEI F25-LMW/siRNA complexes can be stored frozen for several months with full retention of their in vitro bioactivity 38.
In the present study, we explored the in vivo efficacy and safety of PEI F25-LMW-mediated gene targeting of VEGF in mouse tumor xenograft models and present optimized administration strategies by performing biodistribution studies. Using novel, chemically modified siRNAs, we analysed the anti-tumorigenic effects of PEI/VEGF siRNA or Bevacizumab treatment alone, as well as the synergistic effects of a combination of both, and establish PEI F25-LMW/VEGF siRNA-mediated VEGF targeting as a novel strategy in tumor therapy.
- Top of page
- Materials and methods
- Supporting Information
The poor prognosis of advanced pancreatic and prostate carcinoma, as well as the limited treatment options, emphasize the need for novel therapeutic strategies. In addition to cytostatic drugs, inhibitory antibodies or small molecule inhibitors, the discovery of RNAi has extended the portfolio towards highly specific gene-targeting approaches. However, this requires the efficient systemic delivery of RNAi-inducing agents such as siRNAs, based on the generation and in vivo testing of appropriate delivery vehicles and their optimal modes of administration.
PEIs were introduced previously as a delivery platform for siRNAs in vitro and in vivo19. Although several, if not all, PEIs are able to complex and protect siRNAs, efficient PEI/siRNA-mediated gene targeting in vivo relies on the stability of the complexes including that in the presence of serum (i.e. little aggregation or extracellular complex disruption), their penetration into tissues, the efficient cellular uptake of the complexes, as well as the subsequent intracellular release of the siRNA. Additional issues are the biocompatibility of the complexes, as well as of the single components, also upon repeated treatments. In the present study, we demonstrate the in vivo efficacy and safety of branched 4–10 kDa polyethylenimine PEI F25-LMW 37 and, for PEI-mediated delivery, we employed for the first time chemically modified siRNA. The analysis of in vitro targeting efficacies and the in vivo delivery (as reported in the present study as well as unpublished data) reveals that the chemical modification neither impairs nor enhances PEI-mediated siRNA delivery and targeting efficacy, which rather relies on the PEI as a delivery vehicle than the introduction of chemical siRNA modifications.
In both tumor models, the comparison between PEI F25-LMW/siRNA-mediated VEGF targeting and the treatment with Bevacizumab shows similar anti-tumorigenic efficacies. This is despite their completely different mechanisms of action at transcriptional and post-translational levels, respectively. Thus, it can be assumed that a combination may enhance the anti-tumorigenic efficacies exerted through VEGF blockade based on different mechanisms of action, as observed in the present study with regard to the synergistic effects in prostate carcinoma xenografts. This may be relevant with regard to avoiding excess siRNAs, which can compete for the intracellular RNAi machinery as suggested previously 46, and may allow the enhancement of individual drug effects and/or the reduction of therapeutically relevant doses. The latter aspect is particularly important when aiming to avoid nonspecific siRNA side-effects exerted, for example, through the activation of toll-like receptors of the innate immune system, which have been shown to be dependent on siRNA concentrations and to increase upon siRNA delivery in liposomal formulations 47, 48. In this context, it should also be noted that we always employed relatively low siRNA amounts compared to other studies that relied on large siRNA quantities.
Combinations may also include PEI/siRNA-mediated VEGF targeting and established cytostatics, as described previously for Bevacizumab 14, 15. With regard to optimal therapeutic regimens, however, it should also be taken into consideration that anti-angiogenic effects may impair the delivery of other anti-cancer drugs as a result of the reduction of tumor vasculature, or may, in contrast, transiently increase drug delivery as a result of the ‘normalization’ of the abnormal structure and function of tumor vasculature 49. This emphasizes the need for optimized treatment protocols.
The RNase protection assay proved to be a very sensitive and accurate method for quantifying full-length siRNAs, and is therefore superior to other protocols relying on labeled siRNA and the detection of either the label alone (without assessing siRNA integrity or the presence of free label) or the use of radioactively labeled siRNAs. Stronger anti-tumorigenic effects are observed in pancreatic tumor compared to the prostate carcinoma xenografts, despite less siRNA delivery and less VEGF reduction, and rather low initial VEGF expression levels in PANC-1 cells 6. This also indicates that profound biological effects of RNAi may not necessarily rely on particularly high levels of the target gene overexpression or on maximum targeting efficacies. It should also be noted that the VEGF-specific siRNAs used in the present study are also able to target mouse VEGF, which parallels a therapeutic setting in humans where a distinction between tumor and stroma VEGF would neither be made nor wanted. Consequently, the determination of tumor VEGF levels by the ELISA specific for human VEGF may underestimate the total VEGF knockdown. Finally, the differences in VEGF protein levels between the PEI/siRNA and PEI/siRNA + Avastin treatment groups were less prominent than the effects on prostate carcinoma xenograft growth. Although this finding could reflect the already very low VEGF levels in the Avastin treatment group, making it difficult to detect further reduced levels with sufficient accuracy, it should also be noted that RNAi-mediated knockdown of VEGF expression leads to reduced VEGF levels already in the target cells. Because VEGF is able to exert a ‘direct’ proliferative effect on tumor cells, as shown in the present study as well as previously 10–13, the reduced VEGF expression close to its site of action may exert (anti-tumoral) effects that are stronger than expected from the overall decrease of VEGF in the whole tumor mass.
Our qualitative and quantitative assessment of the biodistribution profiles in various organs and the siRNA uptake into the tumors further reveals that the mode of administration is of critical importance. Indeed, i.v. injection leads to poor siRNA levels in the tumors and, concomitantly, to the absence of anti-tumorigenic effects. By contrast, i.p. injection, which is already a relevant administration route in tumor therapy, results in both siRNA uptake and a profound inhibition of tumor growth. This also demonstrates the direct correlation between siRNA levels and VEGF down-regulation, and further excludes nonspecific effects of the PEI or the siRNA with regard to the reduction of tumor growth. A ‘depot effect’ may be beneficial for i.p. administration leading to prolonged release of partially aggregated complexes from the site of injection, thus increasing the time period of siRNA delivery into the tumor. For i.v. application, PEI with modifications to increase the complex circulation half-lives and/or to increase tumor-specific uptake may be required. Nevertheless, the nonmodified PEI F25-LMW introduced in the present study for in vivo use, already demonstrates good results with regard to efficacy and biocompatibility. The toxicity data obtained reveal no side-effects of the complexes or the free carrier at the concentrations used. This is also true for the absence of hepatotoxic or immunostimulatory effects of the PEI/siRNA complexes. In proof-of-principle studies with regard to the relevance of a given gene product, direct injection into the tumor may represent an alternative, reasonable mode of administration. However, because of other therapeutic options, including surgery, this approach would be of little therapeutic value and would be limited to a few tumors not accessible for surgery. Therefore, we did not pursue this further. It should also be noted that PEI F25-LMW/siRNA complexes can be stored in the freezer for several months with full retention of their bioactivity 38. Thus, in contrast to many other delivery platforms, this provides a standardized, ready-to-use formulation that will be particularly important for therapeutic applications. Taken together, PEI F25-LMW/siRNA complexes represent efficient and safe tools for VEGF depletion in vivo and show great promise in comparison to, or in addition to, conventional treatment with Bevacizumab.