Branching in poly(amine-co-ester) polyplexes impacts mRNA transfection.

Branching is a key structural parameter of polymers, which can have profound impacts on physicochemical properties. It has been demonstrated that branching is a modulating factor for mRNA delivery and transfection using delivery vehicles built from cationic polymers, but the influence of polymer branching on mRNA delivery remains relatively underexplored compared to other polymer features such as monomer composition, hydrophobicity, pKa, or the type of terminal group. In this study, we examined the impact of branching on the physicochemical properties of poly(amine-co-esters) (PACE) and their efficiency in mRNA transfection in vivo and in vitro under various conditions. PACE polymers were synthesized with various degrees of branching ranging from 0 to 0.66, and their transfection efficiency was systemically evaluated. We observed that branching improves the stability of polyplexes but reduces the pH buffering capacity. Therefore, the degree of branching (DB) must be optimized in a delivery route specific manner due to differences in challenges faced by polyplexes in different physiological compartments. Through a systematic analysis of physicochemical properties and mRNA transfection in vivo and in vitro, this study highlights the influence of polymer branching on nucleic acid delivery.

Exploiting Metabolic Defects in Glioma with Nanoparticle Encapsulated NAMPT Inhibitors.

The treatment of primary central nervous system (CNS) tumors is challenging due to the blood-brain barrier and complex mutational profiles, which is associated with low survival rates. However, recent studies have identified common mutations in gliomas (IDH-WT and mutant, WHO grades II-IV; with grade IV tumors referred to as glioblastomas; GBMs). These mutations drive epigenetic changes, leading to promoter methylation at the NAPRT gene locus, which encodes an enzyme involved in generating NAD+. Importantly, NAPRT-silencing introduces a therapeutic vulnerability to inhibitors targeting another NAD+ biogenesis enzyme, NAMPT, rationalizing a treatment for these malignancies. Multiple systemically-administered NAMPTis have been developed and tested in clinical trials, but dose-limiting toxicities-including bone marrow suppression and retinal toxicity-have limited their efficacy. Here, we report a novel approach for the treatment of NAPRT-silenced GBMs using nanoparticle-encapsulated (NP) NAMPT inhibitors (NAMPTis) administered by convection-enhanced delivery (CED). We demonstrate that GMX1778 (a NAMPTi) can be formulated in degradable polymer NPs with retention of potency for NAMPT inhibition and anticancer activity in vitro, plus sustained drug release in vitro and in vivo. Direct injection of these drugs via CED into the brain is associated with reduced retinal toxicity compared with systemic administration. Finally, we show that CED of NP-encapsulated GMX1778 to NAPRT-silenced intracranial GBM xenografts in mice exhibit significant tumor growth delay and extends survival. These data support an approach to treat gliomas harboring defects in NAD+ metabolism using CED of NP-encapsulated NAMPTis to greatly improve the therapeutic index and treatment efficacy for this class of drugs.

Nanoscale Surface Topography of Polyethylene Glycol-Coated Nanoparticles Composed of Bottlebrush Block Copolymers Prolongs Systemic Circulation and Enhances Tumor Uptake.

Improving the performance of nanocarriers remains a major challenge in the clinical translation of nanomedicine. Efforts to optimize nanoparticle formulations typically rely on tuning the surface density and thickness of stealthy polymer coatings, such as poly(ethylene glycol) (PEG). Here, we show that modulating the surface topography of PEGylated nanoparticles using bottlebrush block copolymers (BBCPs) significantly enhances circulation and tumor accumulation, providing an alternative strategy to improve nanoparticle coatings. Specifically, nanoparticles with rough surface topography achieve high tumor cell uptake in vivo due to superior tumor extravasation and distribution compared to conventional smooth-surfaced nanoparticles based on linear block copolymers. Furthermore, surface topography profoundly impacts the interaction with serum proteins, resulting in the adsorption of fundamentally different proteins onto the surface of rough-surfaced nanoparticles formed from BBCPs. We envision that controlling the nanoparticle surface topography of PEGylated nanoparticles will enable the design of improved nanocarriers in various biomedical applications.

Intrathecal delivery of nanoparticle PARP inhibitor to the cerebrospinal fluid for the treatment of metastatic medulloblastoma.

The morbidity associated with pediatric medulloblastoma, in particular in patients who develop leptomeningeal metastases, remains high in the absence of effective therapies. Administration of substances directly into the cerebrospinal fluid (CSF) is one approach to circumvent the blood-brain barrier and focus delivery of drugs to the site of tumor. However, high rates of CSF turnover prevent adequate drug accumulation and lead to rapid systemic clearance and toxicity. Here, we show that PLA-HPG nanoparticles, made with a single-emulsion, solvent evaporation process, can encapsulate talazoparib, a PARP inhibitor (BMN-673). These degradable polymer nanoparticles improve the therapeutic index when delivered intrathecally and lead to sustained drug retention in the tumor as measured with PET imaging and fluorescence microscopy. We demonstrate that administration of these particles into the CSF, alone or in combination with systemically administered temozolomide, is a highly effective therapy for tumor regression and prevention of leptomeningeal spread in xenograft mouse models of medulloblastoma. These results provide a rationale for harnessing nanoparticles for the delivery of drugs limited by brain penetration and therapeutic index and demonstrate important advantages in tolerability and efficacy for encapsulated drugs delivered locoregionally.

Polymer nanoparticles deliver mRNA to the lung for mucosal vaccination.

An inhalable platform for messenger RNA (mRNA) therapeutics would enable minimally invasive and lung-targeted delivery for a host of pulmonary diseases. Development of lung-targeted mRNA therapeutics has been limited by poor transfection efficiency and risk of vehicle-induced pathology. Here, we report an inhalable polymer-based vehicle for delivery of therapeutic mRNAs to the lung. We optimized biodegradable poly(amine-co-ester) (PACE) polyplexes for mRNA delivery using end-group modifications and polyethylene glycol. These polyplexes achieved high transfection of mRNA throughout the lung, particularly in epithelial and antigen-presenting cells. We applied this technology to develop a mucosal vaccine for severe acute respiratory syndrome coronavirus 2 and found that intranasal vaccination with spike protein-encoding mRNA polyplexes induced potent cellular and humoral adaptive immunity and protected susceptible mice from lethal viral challenge. Together, these results demonstrate the translational potential of PACE polyplexes for therapeutic delivery of mRNA to the lungs.

CMPK2 restricts Zika virus replication by inhibiting viral translation.

Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a pressing need to identify host and viral factors that can be targeted for effective therapeutic intervention. Type I interferon (IFN-I) production in response to microbial products is one of the host's first line of defense against invading pathogens. Cytidine/uridine monophosphate kinase 2 (CMPK2) is a type I interferon-stimulated gene (ISG) that exerts antiviral effects. However, the molecular mechanism by which CMPK2 inhibits viral replication is unclear. Here, we report that CMPK2 expression restricts Zika virus (ZIKV) replication by specifically inhibiting viral translation and that IFN-I- induced CMPK2 contributes significantly to the overall antiviral response against ZIKV. We demonstrate that expression of CMPK2 results in a significant decrease in the replication of other pathogenic flaviviruses including dengue virus (DENV-2), Kunjin virus (KUNV) and yellow fever virus (YFV). Importantly, we determine that the N-terminal domain (NTD) of CMPK2, which lacks kinase activity, is sufficient to restrict viral translation. Thus, its kinase function is not required for CMPK2's antiviral activity. Furthermore, we identify seven conserved cysteine residues within the NTD as critical for CMPK2 antiviral activity. Thus, these residues may form an unknown functional site in the NTD of CMPK2 contributing to its antiviral function. Finally, we show that mitochondrial localization of CMPK2 is required for its antiviral effects. Given its broad antiviral activity against flaviviruses, CMPK2 is a promising potential pan-flavivirus inhibitor.

Anti-seed PNAs targeting multiple oncomiRs for brain tumor therapy.

Glioblastoma (GBM) is one of the most lethal malignancies with poor survival and high recurrence rates. Here, we aimed to simultaneously target oncomiRs 10b and 21, reported to drive GBM progression and invasiveness. We designed short (8-mer) γ-modified peptide nucleic acids (sγPNAs), targeting the seed region of oncomiRs 10b and 21. We entrapped these anti-miR sγPNAs in nanoparticles (NPs) formed from a block copolymer of poly(lactic acid) and hyperbranched polyglycerol (PLA-HPG). The surface of the NPs was functionalized with aldehydes to produce bioadhesive NPs (BNPs) with superior transfection efficiency and tropism for tumor cells. When combined with temozolomide, sγPNA BNPs administered via convection-enhanced delivery (CED) markedly increased the survival (>120 days) of two orthotopic (intracranial) mouse models of GBM. Hence, we established that BNPs loaded with anti-seed sγPNAs targeting multiple oncomiRs are a promising approach to improve the treatment of GBM, with a potential to personalize treatment based on tumor-specific oncomiRs.

Polyglycerol and Poly(ethylene glycol) exhibit different effects on pharmacokinetics and antibody generation when grafted to nanoparticle surfaces.

Poly(ethylene glycol) (PEG) is widely employed for passivating nanoparticle (NP) surfaces to prolong blood circulation and enhance localization of NPs to target tissue. However, the immune response of PEGylated NPs-including anti-PEG antibody generation, accelerated blood clearance (ABC), and loss of delivery efficacy-is of some concern, especially for treatments that require repeat administrations. Although polyglycerol (PG), which has the same ethylene oxide backbone as PEG, has received attention as an alternative to PEG for NP coatings, the pharmacokinetic and immunogenic impact of PG has not been studied systematically. Here, linear PG, hyperbranched PG (hPG), and PEG-coated polylactide (PLA) NPs with varying surface densities were studied in parallel to determine the pharmacokinetics and immunogenicity of PG and hPG grafting, in comparison with PEG. We found that linear PG imparted the NPs a stealth property comparable to PEG, while hPG-grafted NPs needed a higher surface density to achieve the same pharmacokinetic impact. While linear PG-grafted NPs induced anti-PEG antibody production in mice, they exhibited minimal accelerated blood clearance (ABC) effects due to the poor interaction with anti-PEG immunoglobulin M (IgM). Further, we observed no anti-polymer IgM responses or ABC effects for hPG-grafted NPs.

Inhalable polymer nanoparticles for versatile mRNA delivery and mucosal vaccination.

An inhalable platform for mRNA therapeutics would enable minimally invasive and lung targeted delivery for a host of pulmonary diseases. Development of lung targeted mRNA therapeutics has been limited by poor transfection efficiency and risk of vehicle-induced pathology. Here we report an inhalable polymer-based vehicle for delivery of therapeutic mRNAs to the lung. We optimized biodegradable poly(amine-co-ester) polyplexes for mRNA delivery using end group modifications and polyethylene glycol. Our polyplexes achieved high transfection of mRNA throughout the lung, particularly in epithelial and antigen-presenting cells. We applied this technology to develop a mucosal vaccine for SARS-CoV-2. Intranasal vaccination with spike protein mRNA polyplexes induced potent cellular and humoral adaptive immunity and protected K18-hACE2 mice from lethal viral challenge. One-sentence summary: Inhaled polymer nanoparticles (NPs) achieve high mRNA expression in the lung and induce protective immunity against SARS-CoV-2.

Direct targeting of amplified gene loci for proapoptotic anticancer therapy.

Gene amplification drives oncogenesis in a broad spectrum of cancers. A number of drugs have been developed to inhibit the protein products of amplified driver genes, but their clinical efficacy is often hampered by drug resistance. Here, we introduce a therapeutic strategy for targeting cancer-associated gene amplifications by activating the DNA damage response with triplex-forming oligonucleotides (TFOs), which drive the induction of apoptosis in tumors, whereas cells without amplifications process lower levels of DNA damage. Focusing on cancers driven by HER2 amplification, we find that TFOs targeting HER2 induce copy number-dependent DNA double-strand breaks (DSBs) and activate p53-independent apoptosis in HER2-positive cancer cells and human tumor xenografts via a mechanism that is independent of HER2 cellular function. This strategy has demonstrated in vivo efficacy comparable to that of current precision medicines and provided a feasible alternative to combat drug resistance in HER2-positive breast and ovarian cancer models. These findings offer a general strategy for targeting tumors with amplified genomic loci.

Nonsurgical treatment of skin cancer with local delivery of bioadhesive nanoparticles.

Keratinocyte-derived carcinomas, including squamous cell carcinoma (SCC), comprise the most common malignancies. Surgical excision is the therapeutic standard but is not always clinically feasible, and currently available alternatives are limited to superficial tumors. To address the need for a nonsurgical treatment for nodular skin cancers like SCC, we developed a bioadhesive nanoparticle (BNP) drug delivery system composed of biodegradable polymer, poly(lactic acid)-hyperbranched polyglycerol (PLA-HPG), encapsulating camptothecin (CPT). Nanoparticles (NPs) of PLA-HPG are nonadhesive NPs (NNPs), which are stealthy in their native state, but we have previously shown that conversion of the vicinal diols of HPG to aldehydes conferred NPs the ability to form strong covalent bonds with amine-rich surfaces. Herein, we show that these BNPs have significantly enhanced binding to SCC tumor cell surfaces and matrix proteins, thereby significantly enhancing the therapeutic efficacy of intratumoral drug delivery. Tumor injection of BNP-CPT resulted in tumor retention of CPT at ∼50% at 10 d postinjection, while CPT was undetectable in NNP-CPT or free (intralipid) CPT-injected tumors at that time. BNP-CPT also significantly reduced tumor burden, with a portion (∼20%) of BNP-CPT-treated established tumors showing histologic cure. Larger, more fully established PDV SCC tumors treated with a combination of BNP-CPT and immunostimulating CpG oligodeoxynucleotides exhibited enhanced survival relative to controls, revealing the potential for BNP delivery to be used along with local tumor immunotherapy. Taken together, these results indicate that percutaneous delivery of a chemotherapeutic agent via BNPs, with or without adjuvant immunostimulation, represents a viable, nonsurgical alternative for treating cutaneous malignancy.

Optimizing biodegradable nanoparticle size for tissue-specific delivery.

Nanoparticles (NPs) are promising vehicles for drug delivery because of their potential to target specific tissues [1]. Although it is known that NP size plays a critical role in determining their biological activity, there are few quantitative studies of the role of NP size in determining biodistribution after systemic administration. Here, we engineered fluorescent, biodegradable poly(lactic-co-glycolic acid) (PLGA) NPs in a range of sizes (120-440nm) utilizing a microfluidic platform and used these NPs to determine the effect of diameter on bulk tissue and cellular distribution after systemic administration. We demonstrate that small NPs (∼120nm) exhibit enhanced uptake in bulk lung and bone marrow, while larger NPs are sequestered in the liver and spleen. We also show that small NPs (∼120nm) access specific alveolar cell populations and hematopoietic stem and progenitor cells more readily than larger NPs. Our results suggest that size of PLGA NPs can be used to tune delivery to certain tissues and cell populations in vivo.

Nanoparticle-mediated intratumoral inhibition of miR-21 for improved survival in glioblastoma.

Glioblastoma (GBM) is the most common and deadly form of malignant brain tumor in the United States, and current therapies fail to provide significant improvement in survival. Local delivery of nanoparticles is a promising therapeutic strategy that bypasses the blood-brain barrier, minimizes systemic toxicity, and enhances intracranial drug distribution and retention. Here, we developed nanoparticles loaded with agents that inhibit miR-21, an oncogenic microRNA (miRNA) that is strongly overexpressed in GBM compared to normal brain tissue. We synthesized, engineered, and characterized two different delivery systems. One was designed around an anti-miR-21 composed of RNA and employed a cationic poly(amine-co-ester) (PACE). The other was designed around an anti-miR-21 composed of peptide nucleic acid (PNA) and employed a block copolymer of poly(lactic acid) and hyperbranched polyglycerol (PLA-HPG). We show that both nanoparticle products facilitate efficient intracellular delivery and miR-21 suppression that leads to PTEN upregulation and apoptosis of human GBM cells. Further, when administered by convection-enhanced delivery (CED) to animals with intracranial gliomas, they both induced significant miR-21 knockdown and provided chemosensitization, resulting in improved survival when combined with chemotherapy. The challenges involved in optimizing the two delivery systems differed, and despite offering distinct advantages and limitations, results showed significant therapeutic efficacy with both methods of treatment. This study demonstrates the feasibility and promise of local administration of miR-21 inhibiting nanoparticles as an adjuvant therapy for GBM.

Surface chemistry governs cellular tropism of nanoparticles in the brain.

Nanoparticles are of long-standing interest for the treatment of neurological diseases such as glioblastoma. Most past work focused on methods to introduce nanoparticles into the brain, suggesting that reaching the brain interstitium will be sufficient to ensure therapeutic efficacy. However, optimized nanoparticle design for drug delivery to the central nervous system is limited by our understanding of their cellular deposition in the brain. Here, we investigated the cellular fate of poly(lactic acid) nanoparticles presenting different surface chemistries, after administration by convection-enhanced delivery. We demonstrate that nanoparticles with 'stealth' properties mostly avoid internalization by all cell types, but internalization can be enhanced by functionalization with bio-adhesive end-groups. We also show that association rates measured in cultured cells predict the extent of internalization of nanoparticles in cell populations. Finally, evaluating therapeutic efficacy in an orthotopic model of glioblastoma highlights the need to balance significant uptake without inducing adverse toxicity.

An unusual example of hypervalent silicon: a five-coordinate silyl group bridging two palladium or nickel centers through a nonsymmetrical four-center two-electron bond.

Pd and Ni dimers supported by PSiP ligands in which two hypervalent five-coordinate Si atoms bridge the two metal centers are reported. Crystallographic characterization revealed a rare square-pyramidal geometry at Si and an unusual asymmetric M2 Si2 core (M=Pd or Ni). DFT calculations showed that the unusual structure of the core is also found in a model in which the phosphine and Si centers are not part of a pincer group, thus indicating that the observed geometry is not imposed by the PSiP ligand. NBO analysis showed that an asymmetric four-center two-electron (4c-2e) bond stabilizes the hypervalent Si atoms in the M2 Si2 core.