Thomas A. Werfel

Tuning PEGylation of mixed micelles to overcome intracellular and systemic siRNA delivery barriers.

A series of endosomolytic mixed micelles was synthesized from two diblock polymers, poly[ethylene glycol-b-(dimethylaminoethyl methacrylate-co-propylacrylic acid-co-butyl methacrylate)] (PEG-b-pDPB) and poly[dimethylaminoethyl methacrylate-b-(dimethylaminoethyl methacrylate-co-propylacrylic acid-co-butyl methacrylate)] (pD-b-pDPB), and used to determine the impact of both surface PEG density and PEG molecular weight on overcoming both intracellular and systemic siRNA delivery barriers. As expected, the percent PEG composition and PEG molecular weight in the corona had an inverse relationship with mixed micelle zeta potential and rate of cellular internalization. Although mixed micelles were internalized more slowly, they generally produced similar gene silencing bioactivity (∼ 80% or greater) in MDA-MB-231 breast cancer cells as the micelles containing no PEG (100 D/no PEG). The mechanistic explanation for the potent bioactivity of the promising 50 mol% PEG-b-DPB/50 mol% pD-b-pDPB (50 D) mixed micelle formulation, despite its relatively low rate of cellular internalization, was further investigated as a function of PEG molecular weight (5 k, 10 k, or 20 k PEG). Results indicated that, although larger molecular weight PEG decreased cellular internalization, it improved cytoplasmic bioavailability due to increased intracellular unpackaging (quantitatively measured via FRET) and endosomal release. When delivered intravenously in vivo, 50 D mixed micelles with a larger molecular weight PEG in the corona also demonstrated significantly improved blood circulation half-life (17.8 min for 20 k PEG micelles vs. 4.6 min for 5 kDa PEG micelles) and a 4-fold decrease in lung accumulation. These studies provide new mechanistic insights into the functional effects of mixed micelle-based approaches to nanocarrier surface PEGylation. Furthermore, the ideal mixed micelle formulation identified (50 D/20 k PEG) demonstrated desirable intracellular and systemic pharmacokinetics and thus has strong potential for in vivo therapeutic use.

Enhanced performance of plasmid DNA polyplexes stabilized by a combination of core hydrophobicity and surface PEGylation

Nonviral gene therapy has high potential for safely promoting tissue restoration and for treating various genetic diseases. One current limitation is that conventional transfection reagents such as polyethylenimine (PEI) form electrostatically stabilized plasmid DNA (pDNA) polyplexes with poor colloidal stability. In this study, a library of poly(ethylene glycol-b-(dimethylaminoethyl methacrylate-co-butyl methacrylate)) [poly(EG-b-(DMAEMA-co-BMA))] polymers were synthesized and screened for improved colloidal stability and nucleic acid transfection following lyophilization. When added to pDNA in the appropriate pH buffer, the DMAEMA moieties initiate formation of electrostatic polyplexes that are internally stabilized by hydrophobic interactions of the core BMA blocks and sterically stabilized against aggregation by a PEG corona. The BMA content was varied from 0% to 60% in the second polymer block in order to optimally tune the balance of electrostatic and hydrophobic interactions in the polyplex core, and polymers with 40 and 50 mol% BMA achieved the highest transfection efficiency. Diblock copolymers were more stable than PEI in physiologic buffers. Consequently, diblock copolymer polyplexes aggregated more slowly and followed a reaction-limited colloidal aggregation model, while fast aggregation of PEI polyplexes was governed by a diffusion-limited model. Polymers with 40% BMA did not aggregate significantly after lyophilization and produced up to 20-fold higher transfection efficiency than PEI polyplexes both before and after lyophilization. Furthermore, poly(EG-b-(DMAEMA-co-BMA)) polyplexes exhibited pH-dependent membrane disruption in a red blood cell hemolysis assay and endosomal escape as observed by confocal microscopy.Lyophilized polyplexes made with the lead candidate diblock copolymer (40% BMA) also successfully transfected cells in vitro following incorporation into gas-foamed polymeric scaffolds. In summary, the enhanced colloidal stability, endosomal escape, and resultant high transfection efficiency of poly(EG-b-(DMAEMA-co-BMA))-pDNA polyplexes underscores their potential utility both for local delivery from scaffolds as well as systemic, intravenous delivery.

Porous Silicon and Polymer Nanocomposites for Delivery of Peptide Nucleic Acids as Anti-MicroRNA Therapies

Self-assembled polymer/porous silicon nanocomposites overcome intracellular and systemic barriers for in vivo application of peptide nucleic acid (PNA) anti-microRNA therapeutics. Porous silicon (PSi) is leveraged as a biodegradable scaffold with high drug-cargo-loading capacity. Functionalization with a diblock polymer improves PSi nanoparticle colloidal stability, in vivo pharmacokinetics, and intracellular bioavailability through endosomal escape, enabling PNA to inhibit miR-122 in vivo.

Fluorocoxib A loaded nanoparticles enable targeted visualization of cyclooxygenase-2 in inflammation and cancer.

Cyclooxygenase-2 (COX-2) is expressed in virtually all solid tumors and its overexpression is a hallmark of inflammation. Thus, it is a potentially powerful biomarker for the early clinical detection of inflammatory disease and human cancers. We report a reactive oxygen species (ROS) responsive micellar nanoparticle, PPS-b-POEGA, that solubilizes the first fluorescent COX-2-selective inhibitor fluorocoxib A (FA) for COX-2 visualization in vivo. Pharmacokinetics and biodistribution of FA-PPS-b-POEGA nanoparticles (FA-NPs) were assessed after a fully-aqueous intravenous (i.v.) administration in wild-type mice and revealed 4-8 h post-injection as an optimal fluorescent imaging window. Carrageenan-induced inflammation in the rat and mouse footpads and 1483 HNSCC tumor xenografts were successfully visualized by FA-NPs with fluorescence up to 10-fold higher than that of normal tissues. The targeted binding of the FA cargo was blocked by pretreatment with the COX-2 inhibitor indomethacin, confirming COX-2-specific binding and local retention of FA at pathological sites. Our collective data indicate that FA-NPs are the first i.v.-ready FA formulation, provide high signal-to-noise in inflamed, premalignant, and malignant tissues, and will uniquely enable clinical translation of the poorly water-soluble FA compound.

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

Significance Small interfering RNA (siRNA) has the capacity to silence traditionally undruggable targets, but in vivo delivery barriers limit clinical translation of siRNA, especially for nonhepatic targets such as solid tumors. Most delivery strategies for RNAi cancer therapies focus on synthetic nanocarriers, but their shortcomings include limited delivery to and variable distribution throughout the target site and low therapeutic indices due to nonspecific, carrier-associated toxicities. A diacyl lipid-modified siRNA can leverage albumin as an endogenous carrier, resulting in comprehensively enhanced pharmacokinetic properties that translate to greater quantity and homogeneity of tumor accumulation relative to nanocarriers. The albumin-binding siRNA conjugate strategy is synthetically simple and safe at high doses, and thus is a translatable and potentially transformative option for RNAi oncology therapies. Clinical translation of therapies based on small interfering RNA (siRNA) is hampered by siRNAs 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.

Combinatorial optimization of PEG architecture and hydrophobic content improves ternary siRNA polyplex stability, pharmacokinetics, and potency in vivo

&NA; A rationally‐designed library of ternary siRNA polyplexes was developed and screened for gene silencing efficacy in vitro and in vivo with the goal of overcoming both cell‐level and systemic delivery barriers. [2‐(dimethylamino)ethyl methacrylate] (DMAEMA) was homopolymerized or copolymerized (50 mol% each) with butyl methacrylate (BMA) from a reversible addition – fragmentation chain transfer (RAFT) chain transfer agent, with and without pre‐conjugation to polyethylene glycol (PEG). Both single block polymers were tested as core‐forming units, and both PEGylated, diblock polymers were screened as corona‐forming units. Ternary siRNA polyplexes were assembled with varied amounts and ratios of core‐forming polymers to PEGylated corona‐forming polymers. The impact of polymer composition/ratio, hydrophobe (BMA) placement, and surface PEGylation density was correlated to important outcomes such as polyplex size, stability, pH‐dependent membrane disruptive activity, biocompatibility, and gene silencing efficiency. The lead formulation, DB4‐PDB12, was optimally PEGylated not only to ensure colloidal stability (no change in size by DLS between 0 and 24 h) and neutral surface charge (0.139 mV) but also to maintain higher cell uptake (> 90% positive cells) than the most densely PEGylated particles. The DB4‐PDB12 polyplexes also incorporated BMA in both the polyplex core‐ and corona‐forming polymers, resulting in robust endosomolysis and in vitro siRNA silencing (˜ 85% protein level knockdown) of the model gene luciferase across multiple cell types. Further, the DB4‐PDB12 polyplexes exhibited greater stability, increased blood circulation time, reduced renal clearance, increased tumor biodistribution, and greater silencing of luciferase compared to our previously‐optimized, binary parent formulation following intravenous (i.v.) delivery. This polyplex library approach enabled concomitant optimization of the composition and ratio of core‐ and corona‐forming polymers (indirectly tuning PEGylation density) and identification of a ternary nanomedicine optimized to overcome important siRNA delivery barriers in vitro and in vivo. Graphical abstract Figure. No caption available.

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.

Particle-based technologies for osteoarthritis detection and therapy

Osteoarthritis (OA) is a disease characterized by degradation of joints with the development of painful osteophytes in the surrounding tissues. Currently, there are a limited number of treatments for this disease, and many of these only provide temporary, palliative relief. In this review, we discuss particle-based drug delivery systems that can provide targeted and sustained delivery of imaging and therapeutic agents to OA-affected sites. We focus on technologies such as polymeric micelles and nano-/microparticles, liposomes, and dendrimers for their potential treatment and/or diagnosis of OA. Several promising studies are highlighted, motivating the continued development of delivery technologies to improve treatments for OA.

Future nanomedicine for the diagnosis and treatment of osteoarthritis

Current treatments for osteoarthritis (OA) are largely palliative until the joints become totally dysfunctional and prosthetic replacement becomes necessary. Effective methods are needed for diagnosing OA and monitoring its progression during its early stages, when the effects of therapeutic drugs or biological agents are most likely to be effective. Theranostic nanosomes and nanoparticles have the potential to noninvasively detect, track and treat the early stages of OA. As articular cartilage does not regenerate once it is degraded, cell-based treatments aided by superparamagnetic iron oxide nanoparticle tracking are attractive future treatment modalities for the later stages of OA progression, when significant cartilage replacement is needed. This article will describe the current and future translational approaches for the detection and noninvasive treatment of degenerative OA.

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.