Hydrogel microspheres for spatiotemporally controlled delivery of RNA and silencing gene expression within scaffold-free tissue engineered constructs.

Delivery systems for controlled release of RNA interference (RNAi) molecules, including small interfering (siRNA) and microRNA (miRNA), have the potential to direct stem cell differentiation for regenerative musculoskeletal applications. To date, localized RNA delivery platforms in this area have focused predominantly on bulk scaffold-based approaches, which can interfere with cell-cell interactions important for recapitulating some native musculoskeletal developmental and healing processes in tissue regeneration strategies. In contrast, scaffold-free, high density human mesenchymal stem cell (hMSC) aggregates may provide an avenue for creating a more biomimetic microenvironment. Here, photocrosslinkable dextran microspheres (MS) encapsulating siRNA-micelles were prepared via an aqueous emulsion method and incorporated within hMSC aggregates for localized and sustained delivery of bioactive siRNA. siRNA-micelles released from MS in a sustained fashion over the course of 28 days, and the released siRNA retained its ability to transfect cells for gene silencing. Incorporation of fluorescently labeled siRNA (siGLO)-laden MS within hMSC aggregates exhibited tunable siGLO delivery and uptake by stem cells. Incorporation of MS loaded with siRNA targeting green fluorescent protein (siGFP) within GFP-hMSC aggregates provided sustained presentation of siGFP within the constructs and prolonged GFP silencing for up to 15 days. This platform system enables sustained gene silencing within stem cell aggregates and thus shows great potential in tissue regeneration applications. STATEMENT OF SIGNIFICANCE: This work presents a new strategy to deliver RNA-nanocomplexes from photocrosslinked dextran microspheres for tunable presentation of bioactive RNA. These microspheres were embedded within scaffold-free, human mesenchymal stem cell (hMSC) aggregates for sustained gene silencing within three-dimensional cell constructs while maintaining cell viability. Unlike exogenous delivery of RNA within culture medium that suffers from diffusion limitations and potential need for repeated transfections, this strategy provides local and sustained RNA presentation from the microspheres to cells in the constructs. This system has the potential to inhibit translation of hMSC differentiation antagonists and drive hMSC differentiation toward desired specific lineages, and is an important step in the engineering of high-density stem cell systems with incorporated instructive genetic cues for application in tissue regeneration.

Selective mTORC2 Inhibitor Therapeutically Blocks Breast Cancer Cell Growth and Survival.

Small-molecule inhibitors of the mTORC2 kinase (torkinibs) have shown efficacy in early clinical trials. However, the torkinibs under study also inhibit the other mTOR-containing complex mTORC1. While mTORC1/mTORC2 combined inhibition may be beneficial in cancer cells, recent reports describe compensatory cell survival upon mTORC1 inhibition due to loss of negative feedback on PI3K, increased autophagy, and increased macropinocytosis. Genetic models suggest that selective mTORC2 inhibition would be effective in breast cancers, but the lack of selective small-molecule inhibitors of mTORC2 have precluded testing of this hypothesis to date. Here we report the engineering of a nanoparticle-based RNAi therapeutic that can effectively silence the mTORC2 obligate cofactor Rictor. Nanoparticle-based Rictor ablation in HER2-amplified breast tumors was achieved following intratumoral and intravenous delivery, decreasing Akt phosphorylation and increasing tumor cell killing. Selective mTORC2 inhibition in vivo, combined with the HER2 inhibitor lapatinib, decreased the growth of HER2-amplified breast cancers to a greater extent than either agent alone, suggesting that mTORC2 promotes lapatinib resistance, but is overcome by mTORC2 inhibition. Importantly, selective mTORC2 inhibition was effective in a triple-negative breast cancer (TNBC) model, decreasing Akt phosphorylation and tumor growth, consistent with our findings that RICTOR mRNA correlates with worse outcome in patients with basal-like TNBC. Together, our results offer preclinical validation of a novel RNAi delivery platform for therapeutic gene ablation in breast cancer, and they show that mTORC2-selective targeting is feasible and efficacious in this disease setting.Significance: This study describes a nanomedicine to effectively inhibit the growth regulatory kinase mTORC2 in a preclinical model of breast cancer, targeting an important pathogenic enzyme in that setting that has been undruggable to date. Cancer Res; 78(7); 1845-58. ©2018 AACR.

Porcine Ischemic Wound-Healing Model for Preclinical Testing of Degradable Biomaterials.

Impaired wound healing that mimics chronic human skin pathologies is difficult to achieve in current animal models, hindering testing and development of new therapeutic biomaterials that promote wound healing. In this article, we describe a refinement and simplification of the porcine ischemic wound model that increases the size and number of experimental sites per animal. By comparing three flap geometries, we adopted a superior configuration (15 × 10 cm) that enabled testing of twenty 1 cm2 wounds in each animal: 8 total ischemic wounds within 4 bipedicle flaps and 12 nonischemic wounds. The ischemic wounds exhibited impaired skin perfusion for ∼1 week. To demonstrate the utility of the model for comparative testing of tissue regenerative biomaterials, we evaluated the healing process in wounds implanted with highly porous poly (thioketal) urethane (PTK-UR) scaffolds that were fabricated through reaction of reactive oxygen species (ROS)-cleavable PTK macrodiols with isocyanates. PTK-lysine triisocyanate (LTI) scaffolds degraded significantly in vitro under both oxidative and hydrolytic conditions whereas PTK-hexamethylene diisocyanate trimer (HDIt) scaffolds were resistant to hydrolytic breakdown and degraded exclusively through an ROS-dependent mechanism. Upon placement into porcine wounds, both types of PTK-UR materials fostered new tissue ingrowth over 10 days in both ischemic and nonischemic tissue. However, wound perfusion, tissue infiltration and the abundance of pro-regenerative, M2-polarized macrophages were markedly lower in ischemic wounds independent of scaffold type. The PTK-LTI implants significantly improved tissue infiltration and perfusion compared with analogous PTK-HDIt scaffolds in ischemic wounds. Both LTI and HDIt-based PTK-UR implants enhanced M2 macrophage activity, and these cells were selectively localized at the scaffold/tissue interface. In sum, this modified porcine wound-healing model decreased animal usage, simplified procedures, and permitted a more robust evaluation of tissue engineering materials in preclinical wound healing research. Deployment of the model for a relevant biomaterial comparison yielded results that support the use of the PTK-LTI over the PTK-HDIt scaffold formulation for future advanced therapeutic studies.

Lipophilic siRNA targets albumin in situ and promotes bioavailability, tumor penetration, and carrier-free gene silencing.

Clinical translation of therapies based on small interfering RNA (siRNA) is hampered by siRNA's comprehensively poor pharmacokinetic properties, which necessitate molecule modifications and complex delivery strategies. We sought an alternative approach to commonly used nanoparticle carriers by leveraging the long-lived endogenous serum protein albumin as an siRNA carrier. We synthesized siRNA conjugated to a diacyl lipid moiety (siRNA-L2), which rapidly binds albumin in situ. siRNA-L2, in comparison with unmodified siRNA, exhibited a 5.7-fold increase in circulation half-life, an 8.6-fold increase in bioavailability, and reduced renal accumulation. Benchmarked against leading commercial siRNA nanocarrier in vivo jetPEI, siRNA-L2 achieved 19-fold greater tumor accumulation and 46-fold increase in per-tumor-cell uptake in a mouse orthotopic model of human triple-negative breast cancer. siRNA-L2 penetrated tumor tissue rapidly and homogeneously; 30 min after i.v. injection, siRNA-L2 achieved uptake in 99% of tumor cells, compared with 60% for jetPEI. Remarkably, siRNA-L2 achieved a tumor:liver accumulation ratio >40:1 vs. <3:1 for jetPEI. The improved pharmacokinetic properties of siRNA-L2 facilitated significant tumor gene silencing for 7 d after two i.v. doses. Proof-of-concept was extended to a patient-derived xenograft model, in which jetPEI tumor accumulation was reduced fourfold relative to the same formulation in the orthotopic model. The siRNA-L2 tumor accumulation diminished only twofold, suggesting that the superior tumor distribution of the conjugate over nanoparticles will be accentuated in clinical situations. These data reveal the immense promise of in situ albumin targeting for development of translational, carrier-free RNAi-based cancer therapies.

Zwitterionic Nanocarrier Surface Chemistry Improves siRNA Tumor Delivery and Silencing Activity Relative to Polyethylene Glycol.

Although siRNA-based nanomedicines hold promise for cancer treatment, conventional siRNA-polymer complex (polyplex) nanocarrier systems have poor pharmacokinetics following intravenous delivery, hindering tumor accumulation. Here, we determined the impact of surface chemistry on the in vivo pharmacokinetics and tumor delivery of siRNA polyplexes. A library of diblock polymers was synthesized, all containing the same pH-responsive, endosomolytic polyplex core-forming block but different corona blocks: 5 kDa (benchmark) and 20 kDa linear polyethylene glycol (PEG), 10 kDa and 20 kDa brush-like poly(oligo ethylene glycol), and 10 kDa and 20 kDa zwitterionic phosphorylcholine-based polymers (PMPC). In vitro, it was found that 20 kDa PEG and 20 kDa PMPC had the highest stability in the presence of salt or heparin and were the most effective at blocking protein adsorption. Following intravenous delivery, 20 kDa PEG and PMPC coronas both extended circulation half-lives 5-fold compared to 5 kDa PEG. However, in mouse orthotopic xenograft tumors, zwitterionic PMPC-based polyplexes showed highest in vivo luciferase silencing (>75% knockdown for 10 days with single IV 1 mg/kg dose) and 3-fold higher average tumor cell uptake than 5 kDa PEG polyplexes (20 kDa PEG polyplexes were only 2-fold higher than 5 kDa PEG). These results show that high molecular weight zwitterionic polyplex coronas significantly enhance siRNA polyplex pharmacokinetics without sacrificing polyplex uptake and bioactivity within tumors when compared to traditional PEG architectures.

Local Delivery of PHD2 siRNA from ROS-Degradable Scaffolds to Promote Diabetic Wound Healing.

Small interfering RNA (siRNA) delivered from reactive oxygen species-degradable tissue engineering scaffolds promotes diabetic wound healing in rats. Porous poly(thioketal-urethane) scaffolds implanted in diabetic wounds locally deliver siRNA that inhibits the expression of prolyl hydroxylase domain protein 2, thereby increasing the expression of progrowth genes and increasing vasculature, proliferating cells, and tissue development in diabetic wounds.

Hydrophobic interactions between polymeric carrier and palmitic acid-conjugated siRNA improve PEGylated polyplex stability and enhance in vivo pharmacokinetics and tumor gene silencing.

Formation of stable, long-circulating siRNA polyplexes is a significant challenge in translation of intravenously-delivered, polymeric RNAi cancer therapies. Here, we report that siRNA hydrophobization through conjugation to palmitic acid (siPA) improves stability, in vivo pharmacokinetics, and tumor gene silencing of PEGylated nanopolyplexes (siPA-NPs) with balanced cationic and hydrophobic content in the core relative to the analogous polyplexes formed with unmodified siRNA, si-NPs. Hydrophobized siPA loaded into the NPs at a lower charge ratio (N(+):P(-)) relative to unmodified siRNA, and siPA-NPs had superior resistance to siRNA cargo unpackaging in comparison to si-NPs upon exposure to the competing polyanion heparin and serum. In vitro, siPA-NPs increased uptake in MDA-MB-231 breast cancer cells (100% positive cells vs. 60% positive cells) but exhibited equivalent silencing of the model gene luciferase relative to si-NPs. In vivo in a murine model, the circulation half-life of intravenously-injected siPA-NPs was double that of si-NPs, resulting in a >2-fold increase in siRNA biodistribution to orthotopic MDA-MB-231 mammary tumors. The increased circulation half-life of siPA-NPs was dependent upon the hydrophobic interactions of the siRNA and the NP core component and not just siRNA hydrophobization, as siPA did not contribute to improved circulation time relative to unmodified siRNA when delivered using polyplexes with a fully cationic core. Intravenous delivery of siPA-NPs also achieved significant silencing of the model gene luciferase in vivo (∼40% at 24 h after one treatment and ∼60% at 48 h after two treatments) in the murine MDA-MB-231 tumor model, while si-NPs only produced a significant silencing effect after two treatments. These data suggest that stabilization of PEGylated siRNA polyplexes through a combination of hydrophobic and electrostatic interactions between siRNA cargo and the polymeric carrier improves in vivo pharmacokinetics and tumor gene silencing relative to conventional formulations that are stabilized solely by electrostatic interactions.

Conjugation of palmitic acid improves potency and longevity of siRNA delivered via endosomolytic polymer nanoparticles.

Clinical translation of siRNA therapeutics has been limited by the inability to effectively overcome the rigorous delivery barriers associated with intracellular-acting biologics. Here, to address both potency and longevity of siRNA gene silencing, pH-responsive micellar nanoparticle (NP) carriers loaded with siRNA conjugated to palmitic acid (siRNA-PA) were investigated as a combined approach to improve siRNA endosomal escape and stability. Conjugation to hydrophobic PA improved NP loading efficiency relative to unmodified siRNA, enabling complete packaging of siRNA-PA at a lower polymer:siRNA ratio. PA conjugation also increased intracellular uptake of the nucleic acid cargo by 35-fold and produced a 3.1-fold increase in intracellular half-life. The higher uptake and improved retention of siRNA-PA NPs correlated to a 2- and 11-fold decrease in gene silencing IC50 in comparison to siRNA NPs in fibroblasts and mesenchymal stem cells, respectively, for both the model gene luciferase and the therapeutically relevant gene prolyl hydroxylase domain protein 2 (PHD2) . PA conjugation also significantly increased longevity of silencing activity following a single treatment in fibroblasts. Thus, conjugation of PA to siRNA paired with endosomolytic NPs is a promising approach to enhance the functional efficacy of siRNA in tissue regenerative and other applications.