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Diblock copolymer hydrophobicity facilitates efficient gene silencing and cytocompatible nanoparticlemediated siRNA delivery to musculoskeletal cell types Dominic W. Malcolm, Margaret A.T. Freeberg, Yuchen Wang, Kenneth R. Sims Jr., Hani A. Awad, and Danielle S. W. Benoit Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01349 • Publication Date (Web): 29 Sep 2017 Downloaded from http://pubs.acs.org on September 29, 2017
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Diblock copolymer hydrophobicity facilitates efficient gene silencing and cytocompatible
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nanoparticle-mediated siRNA delivery to musculoskeletal cell types.
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Dominic W. Malcolm1,2, Margaret A.T. Freeberg1,2, Yuchen Wang1,2, Kenneth R. Sims Jr.3, Hani
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A. Awad1,2,4, and Danielle S. W. Benoit1,2,5 *
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1. Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA
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2. Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, NY,
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USA 3. Translational Biomedical Science, University of Rochester School of Medicine & Dentistry, Rochester, NY, USA
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4. Department of Orthopedics, University of Rochester Medical Center, Rochester, NY, USA
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5. Department of Chemical Engineering, University of Rochester, Rochester, NY, USA
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*Corresponding Author Contact Information:
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Danielle S. W. Benoit, Ph.D.
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207 Robert B. Goergen Hall,
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Department of Biomedical Engineering,
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University of Rochester,
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Rochester, NY 14627, USA.
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[email protected] 22 23
Conflict of Interest Statement: The authors have no conflicts of interest related to this work.
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Abstract
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pH-responsive diblock copolymers provide tailorable nanoparticle (NP) architecture and
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chemistry critical for siRNA delivery. Here, diblock polymers varying in first (corona) and second
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(core) block molecular weight (Mn), Corona:Core ratio and core hydrophobicity (%BMA) were
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synthesized to determine their effect on siRNA delivery in murine tenocytes (mTenocyte) and
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murine and human mesenchymal stem cells (mMSC and hMSCs, respectively). NP-mediated
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siRNA uptake, gene silencing, and cytocompatibility were quantified. Uptake is positively
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correlated with first block Mn in mTenocytes and hMSCs (p≤0.0005). All NP resulted in
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significant gene silencing that was positively correlated with %BMA (p 90% knockdown
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in GAPDH expression in mTenocytes, except for NP H, which resulted in ~85% gene
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knockdown. These effects were greater than the knockdown achieved using Lipofectamine2000.
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Control NP X resulted in ~70% gene silencing. All NP except NP X also showed significantly
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reduced GAPDH expression in mMSCs compared to untreated cells (Figure 3B). However,
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knockdown using NP C, F, G, H, and I were significantly smaller than that achieved with
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Lipofecamine2000, indicating differential gene silencing abilities in mMSCs across the NP
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library. Similarly, all NP achieved significant reductions in GAPDH expression in hMSCs (Figure
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3C). All NP except for H and X resulted in > 90% reductions compared to untreated controls,
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and performed no differently than Lipofectamine2000. Two-way ANOVA using Tukey’s post-hoc
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test shows that overall gene silencing in mMSCs was significantly reduced compared to
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mTenocytes (p < 0.0001) and hMSCs (p < 0.0001).
Figure 3: NP-mediated delivery of siRNA results in robust gene silencing across multiple cell types and species. Murine tenocytes (A), murine MSCs (mMSC) (B), and human MSCs (hMSC) (C) were treated with 30 nM siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) complexed to NP at a charge ratio of 4:1. Gene expression was detected using RTPCR and normalized to negative controls in each cell type. GAPDH expression was normalized to B-Actin expression for murine cell types and peptidylprolyl isomerase B (PPIB) expression for hMSCs. *p < 0.0001 compared to negative control, #p < 0.001 compared to positive controls (+) control. Negative controls (-) are comprised of untreated cells and positive controls (+) are cells treated with 30 nM anti-GAPDH siRNA using Lipofectamine2000.
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To determine the extent to which polymer parameters control NP-mediated gene silencing, MPR
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analysis was performed on NP-mediated gene expression. NP gene silencing capability is
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significantly dependent on NP properties in all three cell types (mTenocyte p = 0.0011, mMSC p
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< 0.0001, hMSC p < 0.0001) (Figure 4A). Furthermore, relative GAPDH expression is
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significantly dependent on and negatively correlated with %BMA content in all three cell types
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(mTenocyte E = -1.7, p < 0.0001; mMSC E = -0.55, p = 0.038; hMSC E = -4.2, p < 0.0001)
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(Figure 4B), suggesting %BMA may be an important parameter governing gene silencing.
Figure 4: Relative GAPDH Expression data was fit using a multi-parameter linear regression to determine if gene-silencing capability is dependent on NP properties. A) p-values less than 0.05 (bold) indicates that gene silencing is dependent on NP parameters, and the R2 value describes what percentage of the behavior can be adequately described by the predictive model. B) Effect sizes (E) from significant regression models show the extent that a single property correlates
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with MFI. p < 0.05 indicates the polymer property significantly influences GAPDH expression. Significant effects are bolded.
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pH-dependent NP membrane disruption behavior.
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A hemolysis assay was performed to determine the impact of polymer properties on NP pH-
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dependent membrane disruption ability, as this behavior is required for endosomal escape and
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subsequent gene knockdown. In this assay, human erythrocytes are isolated and incubated with
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NP complexed to negative control non-targeting siRNA under normal treatment conditions, and
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incubated at various pH that mimic the various stages of the endosomal environment. NP-
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mediated membrane disruption is assayed by measuring absorbance of hemoglobin released
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by lysed erythrocytes. Triton-X is used as a positive control to set 100% lysis for normalization.
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Figure 5 shows that all NPs display pH-dependent membrane lysis ability, except the control NP
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X, which contains no pH responsive core components. A two-way ANOVA using Tukey’s test
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shows that mean membrane lysis significantly increases as %BMA increases (p < 0.001). No
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significant differences were observed for the other polymer properties.
Figure 5: pH-dependent hemolysis ability of NPs. *p
