(M1130-12-72) Inulin-Based Dual-Function Polymer for Enhanced Delivery of Nucleic Acids to the Kidney

Purpose: Acute kidney injury (AKI) is a global public health concern affecting over 13 million people each year. AKI is a major unmet medical need due to the lack of effective therapeutic interventions and the significant healthcare burden of the disease. Unfortunately, pharmacological treatments of AKI are often associated with unfavorable off-target effects. In addition, pathological conditions associated with AKI may affect the distribution of drugs to the kidney. Therefore, renal-targeted drug delivery is of great significance in improving the therapeutic outcome in renal diseases. The use of polymers with natural tropism to specific organs is a strategy long used in the development of targeted delivery systems. Inulin is a biologically inert plant polysaccharide that is freely filtered through the glomerulus and is almost exclusively cleared by the kidneys. This indicates that inulin has excellent access to the urinary space and particularly to the renal tubules, which are the primary site of injury in AKI. Thus, inulin could be used as a renal targeted polymeric drug carrier. Recent studies have shown that the chemokine receptor CXCR4 is overexpressed on renal tubules after AKI and plays a role in mediating AKI-associated inflammation [1, 2]. CXCR4 inhibition has been demonstrated to improve the therapeutic efficacy in AKI by decreasing inflammatory cell infiltration and expression of pro-inflammatory cytokines. In addition, enhanced nanoparticle endocytosis through CXCR4 binding has been reported in several CXCR4 targeted systems, further underscoring its potential as a target in AKI [3, 4]. With the aim of leveraging the natural renal access of inulin and with the importance of CXCR4 for the AKI pathology in mind, the purpose of this project was to synthesize a novel inulin based polymeric CXCR4 inhibitor and evaluate its ability to form renal targeted nanoparticles for efficient delivery of therapeutic siRNA to the kidneys.

Methods: Inulin-Cyclam (IC), a novel polymeric CXCR4 inhibitor, was synthesized by conjugating a pharmacophore for CXCR4 inhibition (α-cyclam-p-toluic acid) to inulin. Stable nanoparticles were formulated based on electrostatic interactions between the cationic polymer and the anionic siRNA by mixing the polymer with the siRNA at an optimum polymer to siRNA ratio. In vitro uptake in healthy cells and cells injured with a nephrotoxic drug - cisplatin (mimicking AKI) was determined by confocal microscopy and flow cytometry. Given that CPTA conjugation could introduce the polymer with CXCR4 antagonism, we performed a CXCR4 redistribution assay to assess the CXCR4 inhibitory potential of the polymer. The in vivo renal targeting ability was tested in a murine ischemia-reperfusion (I/R) AKI model [5]. Fluorescently labeled nanoparticles were injected intravenously 24 h after the I/R surgery. Mice were sacrificed at specific time points after administration, and major organs were isolated for ex-vivo fluorescence imaging.

Results: The polymer (IC) could efficiently condense siRNA, forming stable, relatively non-toxic nanoparticles. The nanoparticles were successfully taken up by the cells, with a higher uptake seen in injured cells. In the CXCR4 redistribution assay, IC inhibited the CXCR4 receptor and blocked its internalization following the addition of an agonist (SDF-1), confirming its CXCR4 antagonist properties. The nanoparticles displayed selective accumulation in the injured kidneys 24 hours after intravenous administration. Since liver is the major off-target organ for many cationic formulations, we calculated the kidney-to-liver ratio to determine the renal targeting ability of the polymer. We found that IC had about 5-times higher accumulation in the injured kidneys than the liver at 24 h.

Conclusion: A novel polymeric CXCR4 inhibitor that can selectively deliver siRNA to the injured kidneys was developed. The selective uptake and retention in the injured kidneys could be due to the increased renal CXCR4 expression following AKI. Based on this, we hypothesize that the innovative design of our polymer will establish a widely applicable approach that leverages the natural renal access of inulin combined with overexpression of the CXCR4 receptor for the specific delivery of therapeutics to the injured kidney.

References: 1. Tögel, F., et al., Renal SDF-1 signals mobilization and homing of CXCR4-positive cells to the kidney after ischemic injury. Kidney international, 2005. 67(5): p. 1772-1784.
2. Zuk, A., et al., CXCR4 antagonism as a therapeutic approach to prevent acute kidney injury. American Journal of Physiology-Renal Physiology, 2014. 307(7): p. F783-F797.
3. Misra, A.C., et al., CXCR4-targeted nanocarriers for triple-negative breast cancers. Biomacromolecules, 2015. 16(8): p. 2412-2417.
4. Gao, D.-Y., et al., CXCR4-targeted lipid-coated PLGA nanoparticles deliver sorafenib and overcome acquired drug resistance in liver cancer. Biomaterials, 2015. 67: p. 194-203.
5. Wei, Q. and Z. Dong, Mouse model of ischemic acute kidney injury: technical notes and tricks. American Journal of Physiology-Renal Physiology, 2012. 303(11): p. F1487-F1494.

Figure 1 – Nanoparticle biodistribution in an Ischemia-Reperfusion induced AKI model.



Figure 2 - CXCR4 redistribution assay



Figure 3 – In vitro uptake of the nanoparticles by A) Confocal microscopy B) Flow Cytometry