(M1230-07-45) Development of CXCR4 Targeted Polymeric Polyplexes for Delivery of MicroRNA Therapeutics in Alcohol Associated Liver Disease Treatment

Purpose: Alcohol-associated liver disease (AALD) is a major health concern with limited therapeutic modalities. About 50% of liver-related deaths each year in the US are attributed to alcohol consumption, which includes but is not limited to alcoholic fatty liver, liver fibrosis, hepatitis, and cirrhosis¹. The absence of an FDA-approved drug for AALD treatment underscores the urgent need for effective intervention. The overexpressed chemokine receptor CXCR4 on hepatic stellate cells (HSC) and microRNA-155 in Kupffer cells (KC) in liver are the two key players in AALD^2-4. We developed a microRNA delivery platform based on self-assembled polyplexes using CXCR4 antagonizing polymeric Plerixafor. We hypothesized that the CXCR4 antagonizing polymer used in this delivery system can simultaneously deliver the therapeutic miRNA in KC and inhibit CXCR4 on HSC, attenuating AALD.

Methods: We synthesized the polymer PAMD via Michael addition of AMD3100 and HMBA and modified it with cholesterol (17 wt%) by nucleophilic substitution reaction of cholesteryl chloroformate and secondary amines in cyclam groups of PAMD. PEGylation to PAMD-Ch (5 and 10 wt%) is done by Michael addition of secondary amines of PAMD and the acrylamide group of mPEG-acrylamide. Products are validated with GPC and proton NMR spectroscopy. Polymers or copolymers are mixed with anti-miR155 in 0.1 M acetate buffer (pH 5.02) through a pipette mixing approach at different w/w ratios to prepare the polyplexes. Nucleic acid gel retardation and RiboGreen assays are done to determine RNA complexation and encapsulation efficiency of polymer and copolymers. Dynamic light scattering and transmission electron microscopy are used to determine the size, charge, or shape of polyplexes. Colloidal stability of polyplexes is tested to assess aggregation properties in physiological conditions by incubating polyplexes in PBS for 24 h. Time-dependent usability of polyplexes is determined by storing them at 4°C and monitoring size and PDI over one month. Fluorescence resonance energy transfer (FRET) study is performed to simulate polyplex resistance to the protein adsorption-caused dissociation. The polymer (or copolymers) and microRNA were labeled with the FRET couple CY3 and Cy5, and formulated polyplexes were incubated in human blood plasma at 37°C. FRET ratio (FR) is calculated from the emission spectra of donor and acceptor and was used to analyze the comparative decrease in FRET ratios for different polyplexes over 6 h. Heparin release study is performed to mimic the in vivo stability against polyelectrolyte exchange by incubating polyplexes in increasing concentrations of polyanion heparin for 30 min. For in vitro studies, we used macrophage RAW 264.7. Cell titer blue assay is conducted to analyze the cytotoxicity of free polymer (or copolymer) and polyplexes. FACS and confocal microscopy are performed to analyze cellular uptake of polyplexes where the polyplexes are formulated with Cy5.5-labeled miR and Cy3-labeled polymer (or copolymer). Real-time PCR is performed to determine the RNA transfection efficiency of polyplexes in lipopolysaccharide-induced miR155 overexpressed³ and polyplex-treated cells.

Results: GPC and NMR demonstrate successful synthesis of polymer and copolymers (Fig. 1a, 1b, 1c), which completely bind RNA at w/w ratios ≥1.5 with encapsulation efficiency >98% (Fig. 1d, 1e). Compared to the PAMD/miR (324 nm), the PAMD-Ch/miR forms polyplexes of reduced size (~86 nm). PEG-PAMD-Ch/miR forms even smaller polyplexes, ranging from 65-83 nm (Fig. 1f, 1g). All polyplexes are cationic with zeta potential 7 to 14 mV and nearly spherical, facilitating endocytosis (Fig. 1h, 1i). PAMD-Ch/miR and PEG-PAMD-Ch/miR polyplexes maintain improved colloidal stability in PBS. Only 2% increase in hydrodynamic size of PAMD-Ch/miR and 9% increase for either 5- or 10PEG-PAMD-Ch/miR are observed compared to 74% increase in PAMD/miR over 24 h (Fig. 2a). The size of polyplexes remains < 118 nm at 4°C for a month, with PDI ranging from 0.29-0.46 (Fig. 2e). 10PEG-PAMD-Ch/miR polyplexes exhibit the highest resistance to both plasma and heparin, with only 11% release of RNA at a 300 µg concentration of heparin (Fig. 2b) and only 8% decrease in FRET efficiency in plasma over 6 hours (Fig. 2c, 2d). More cells uptake the PAMD-Ch/miR compared to PAMD/miR. However, PEG-PAMD-Ch/miR are taken up by fewer cells due to their neutralized surface charge (Fig. 3a, 3b, 3c). All polyplexes retain >95% cell viability (Fig. 3d) and have microRNA transfection efficiency demonstrated by miR155 gene expression inhibition in the LPS-induced miR155 overexpressed cells in vitro (Fig. 3e).

Conclusion: Overall, we developed potential delivery platform for liver delivery of microRNA therapeutics in AALD treatment. Our formulated cationic polyplexes show significant RNA transfection efficiency. Modification of PAMD with cholesterol reduces the hydrodynamic size of polyplexes and improves their colloidal stability via the capping effect of cholesterol, enhancing their potential to cross the liver fenestrae. Cholesterol modification of PAMD also improved the hydrophobicity of polyplexes, increasing their cellular accumulation. Additionally, PEGylation to PAMD-Ch improves the polyplex resistance in blood plasma and heparin, which makes the novel PEG-PAMD-Ch/miR a promising miRNA delivery platform, conferring improved miRNA delivery and in vivo stability.

References: 1. National Center for Health Statistics (NCHS)-Mortality data (2022) CDC
2. Matsuoka M, Tsukamoto H. Stimulation of hepatic lipocyte collagen production by Kupffer cell-derived transforming growth factor beta: implication for a pathogenetic role in alcoholic liver fibrogenesis. Hepatology. 1990 Apr;11(4):599-605. doi: 10.1002/hep.1840110412. PMID: 2328954.
3. Nieto N. Oxidative-stress and IL-6 mediate the fibrogenic effects of [corrected] Kupffer cells on stellate cells. Hepatology. 2006 Dec;44(6):1487-501. doi: 10.1002/hep.21427. Erratum in: Hepatology. 2007 Feb;45(2):546. PMID: 17133487.
4. Ju C, Tacke F. Hepatic macrophages in homeostasis and liver diseases: from pathogenesis to novel therapeutic strategies. Cell Mol Immunol. 2016 May;13(3):316-27. doi: 10.1038/cmi.2015.104. Epub 2016 Feb 24. PMID: 26908374; PMCID: PMC4856798.
5. Wang Y, Li J, Oupický D. Polymeric Plerixafor: effect of PEGylation on CXCR4 antagonism, cancer cell invasion, and DNA transfection. Pharm Res. 2014 Dec;31(12):3538-48. doi: 10.1007/s11095-014-1440-1. Epub 2014 Jun 19. PMID: 24942536; PMCID: PMC4225219.

Acknowledgements:

Funding: NIH grant R01 AA027695. Nanocore facility (UNMC), Electron Microscopy facility (UNMC), Advanced Microscopy Core Facility (UNMC), Flow Cytometry Research Facility (UNMC)


Figure 1. Characterization of polymer and polyplexes. (a) Molecular weight of polymer and copolymers determined by gel permeation chromatography (b) chemical structure of PEG-PAMD-Ch (c) 1H NMR spectrum of PEG-PAMD-Ch with peak ‘a’ for the aromatic phenylene protons of AMD3100, peak ‘b’ for cholesterol methyl group, and peak ‘c’ for methylene protons of PEG (d) Agarose gel retardation assay showing fully complexed microRNA by PAMD, PAMD-Ch, and PEG-PAMD-Ch at w/w ratios ≥1.5 (e) Quantification of RNA encapsulation efficiency from RiboGreen assay. Experiment was performed with two replicates (f, g) Hydrodynamic size and representative size ditribution curve of polyplexes obtained from dynamic light scattering (DLS). Experiment was performed with three replicates (h) Zeta potential of polyplexes obtained from DLS (i) Morphology of polyplexes observed in Transmission electron microscopy (scale bar 500 nm) Results are expressed as mean ± SD. *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001.


Figure 2. Stability of polyplexes (a) Colloidal stability of polyplexes; compared to the 74% increase in the hydrodynamic size of PAMD/miR, only 2% increase for PAMD-Ch/miR and 9% increase for either 5- or 10PEG-PAMD-Ch/miR are observed over 24 h in PBS. Experiment was performed with three replicates. Results are expressed as mean ± SD (b) In heparin assay, comparatively less RNA is released from PAMD-Ch/miR than PAMD-Ch/miR. 10PEG-PAMD-Ch/miR shows the highest resistance against the same concentration of heparin with a release of only 11% RNA. (c) Representative FRET signal assessed at 670 nm, which is the emission spectrum of the acceptor Cy5. In Fluorescence resonance energy transfer (FRET) study, polymer (or copolymers) and miRNA are labeled with the FRET couple Cy3 and Cy5 respectively (d) The quantitative analysis of FRET ratio (FR) calculated from the emission spectra of donor and acceptor shows the lowest FR decrease for 10PEG-PAMD-Ch/miR polyplexes over 6 h period in blood plasma at 370C (e) Stability of polyplexes at 40C; the hydrodynamic size of PAMD-Ch/miR, 5PEG-PAMD-Ch/miR, and 10PEG-PAMD-Ch/miR increase up to 102-, 118-, and 102 nm respectively by day 30 whereas PAMD/miR reaches its maximum size 1136 nm at day 3 followed by a dissociation due to hydration. Experiment was performed with three replicates. Results are expressed as mean ± SD ****P ≤ 0.0001, ns= non-significant.


Figure 3. In vitro cellular uptake and miR-155 inhibition efficacy study of polyplexes in Macrophage RAW 264.7. (a) Quantification of cellular uptake of polyplexes in Macrophage RAW 264.7 with FACS analysis; polymer or copolymers are labeled with CY3 and miRNA is labeled with Cy 5.5 in polyplexes (b) Mean fluorescence intensity for RNA and polymer or copolymer in cells from FACS analysis. Experiment was performed with three replicates. (c) Representative image for cellular uptake of polyplexes with confocal microscopy (scale bar 20 µm) where polymer or copolymers are labeled with CY3 (green) and miRNA is labeled with Cy 5.5 (red); cholesterol modification converting the hydrophilic PAMD into amphiphilic PAMD-Ch increases cellular uptake of polyplexes whereas, PEGylation results in lower cell uptake of PEG-PAMD-Ch/miR than PAMD-Ch/miR due to charge neutralization of polyplexes. Experiment was performed with two replicates. (d) Cell viability with free polymer/copolymers and with increasing concentrations of anti-miR155 containing polyplexes. Experiment was performed with six replicates. (e) RT-PCR for miR-155 gene expression showing a significant level of miR-155 inhibition in LPS induced miR-155 overexpressed and polyplex treated cells. Experiment was performed with three replicates. Results are expressed as mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.