Targeting and monitoring ovarian cancer invasion with an RNAi and peptide delivery system.

RNA interference (RNAi) therapeutics are an emerging class of medicines that selectively target mRNA transcripts to silence protein production and combat disease. Despite the recent progress, a generalizable approach for monitoring the efficacy of RNAi therapeutics without invasive biopsy remains a challenge. Here, we describe the development of a self-reporting, theranostic nanoparticle that delivers siRNA to silence a protein that drives cancer progression while also monitoring the functional activity of its downstream targets. Our therapeutic target is the transcription factor SMARCE1, which was previously identified as a key driver of invasion in early-stage breast cancer. Using a doxycycline-inducible shRNA knockdown in OVCAR8 ovarian cancer cells both in vitro and in vivo, we demonstrate that SMARCE1 is a master regulator of genes encoding proinvasive proteases in a model of human ovarian cancer. We additionally map the peptide cleavage profiles of SMARCE1-regulated proteases so as to design a readout for downstream enzymatic activity. To demonstrate the therapeutic and diagnostic potential of our approach, we engineered self-assembled layer-by-layer nanoparticles that can encapsulate nucleic acid cargo and be decorated with peptide substrates that release a urinary reporter upon exposure to SMARCE1-related proteases. In an orthotopic ovarian cancer xenograft model, theranostic nanoparticles were able to knockdown SMARCE1 which was in turn reported through a reduction in protease-activated urinary reporters. These LBL nanoparticles both silence gene products by delivering siRNA and noninvasively report on downstream target activity by delivering synthetic biomarkers to sites of disease, enabling dose-finding studies as well as longitudinal assessments of efficacy.

Plasticity of ether lipids promotes ferroptosis susceptibility and evasion.

Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers1. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions2-5. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.

Consequences of poly(ethylene oxide) and poloxamer P188 on transcription in healthy and stressed myoblasts.

Poly(ethylene oxide) (PEO) and poloxamers, a class of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers, have many personal and medical care applications, including the stabilization of stressed cellular membranes. Despite the widespread use, the cellular transcriptional response to these molecules is relatively unknown. C2C12 myoblasts, a model muscle cell, were subjected to short-term Poloxamer 188 (P188) and PEO181 (8,000 g/mol) treatment in culture. RNA was extracted and sequenced to quantify transcriptomic impact. The addition of moderate concentrations (14 µM) of either polymer to unstressed cells caused substantial differential gene expression, including at least twofold modulation of 357 and 588 genes, respectively. In addition, evaluation of the transcriptome response to osmotic stress without polymer treatment revealed dramatic change in RNA expression. Interestingly, the addition of polymer to stressed cells-at concentrations that provide physiological protection-did not yield a significant difference in expression of any gene relative to stress alone. Genome-scale expression analysis was corroborated by single-gene quantitative real-time PCR. Changes in protein expression were measured via western blot, which revealed partial alignment with the RNA results. Collectively, the significant changes to expression of multiple genes and resultant protein translation demonstrates an unexpectedly broad biochemical response to these polymers in healthy myoblasts in vitro. Meanwhile, the lack of substantial transcriptional response to polymer treatment in stressed cells highlights the physical nature of that protective mechanism.

A predictive microfluidic model of human glioblastoma to assess trafficking of blood-brain barrier-penetrant nanoparticles.

The blood­brain barrier represents a significant challenge for the treatment of high-grade gliomas, and our understanding of drug transport across this critical biointerface remains limited. To advance preclinical therapeutic development for gliomas, there is an urgent need for predictive in vitro models with realistic blood­brain-barrier vasculature. Here, we report a vascularized human glioblastoma multiforme (GBM) model in a microfluidic device that accurately recapitulates brain tumor vasculature with self-assembled endothelial cells, astrocytes, and pericytes to investigate the transport of targeted nanotherapeutics across the blood­brain barrier and into GBM cells. Using modular layer-by-layer assembly, we functionalized the surface of nanoparticles with GBM-targeting motifs to improve trafficking to tumors. We directly compared nanoparticle transport in our in vitro platform with transport across mouse brain capillaries using intravital imaging, validating the ability of the platform to model in vivo blood­brain-barrier transport. We investigated the therapeutic potential of functionalized nanoparticles by encapsulating cisplatin and showed improved efficacy of these GBM-targeted nanoparticles both in vitro and in an in vivo orthotopic xenograft model. Our vascularized GBM model represents a significant biomaterials advance, enabling in-depth investigation of brain tumor vasculature and accelerating the development of targeted nanotherapeutics.

Surface Presentation of Hyaluronic Acid Modulates Nanoparticle-Cell Association.

Nanoparticle (NP) drug carriers have revolutionized medicine and increased patient quality of life. Clinically approved formulations typically succeed because of reduced off-target toxicity of the cargo. However, increasing carrier accumulation at disease sites through precise targeting remains one of the biggest challenges in the field. Novel multivalent ligand presentations and self-assembled constructs can enhance cell association, but an inability to draw direct comparisons across formulations has hindered progress. Furthermore, how nanoparticle structure influences function often is unclear. In this report, we leverage the well-characterized hyaluronic acid (HA)-CD44 binding pair to investigate how the surface architecture of modified NPs impacts their association with ovarian cancer cells that overexpress CD44. We functionalized anionic liposomes with 5 kDa HA by either covalent conjugation via surface coupling or electrostatic self-assembly using the layer-by-layer (LbL) adsorption method. Comparing these two methods, we observed a consistent enhancement of NP-cell association with the self-assembly LbL technique, particularly with higher molecular weight (≥10 kDa) HA. To further optimize association, we increased the surface-available HA. We synthesized a bottlebrush glycopolymer composed of a polynorbornene backbone and pendant 5 kDa HA and layered this macromolecule onto NPs. Flow cytometry revealed that the LbL HA bottlebrush NP outperformed the LbL linear display of HA. Cellular visualization by deconvolution optical microscopy corroborated results from all three constructs. Using exogenous HA to block NP-CD44 interactions, we found the LbL HA bottlebrush NP had a 4-fold higher binding avidity than the best-performing LbL linear HA NP. We further observed that decreasing the density of HA bottlebrush side chains to 75% had minimal impact on LbL NP stability or cell association, though we did see a reduction in binding avidity with this side-chain-modified NP. Our studies indicate that LbL surfaces are highly effective for multivalent displays, and the mode in which they present a targeting ligand can be optimized for NP cell targeting.

Electrostatic Conjugation of Nanoparticle Surfaces with Functional Peptide Motifs.

We report the surface functionalization of anionic layer by layer nanoparticles (LbL NPs) with cationic tumor-penetrating peptides (TPPs) via electrostatic adsorption while retaining particle stability and charge characteristics. This strategy eliminates the need for structural modifications of the peptide and enables facile functionalization of surface chemistries difficult to modify or inaccessible via covalent conjugation strategies. We show that both carboxylated and sulfated LbL NPs are able to accommodate linear and cyclic TPPs and used fluorescence-based detection assays to quantify peptide loading per NP. We also demonstrate that TPP activity is retained upon adsorption, implying sufficient numbers of peptides take on the appropriate surface orientation, enabling efficient uptake of functionalized NPs in vitro, as characterized via flow cytometry and deconvolution microscopy. Overall, we believe that this strategy will serve as a broadly applicable approach to impart electrostatically assembled NPs with bioactive peptide motifs.

Theranostic Layer-by-Layer Nanoparticles for Simultaneous Tumor Detection and Gene Silencing.

Layer-by-layer nanoparticles (NPs) are modular drug delivery vehicles that incorporate multiple functional materials through sequential deposition of polyelectrolytes onto charged nanoparticle cores. Herein, we combined the multicomponent features and tumor targeting capabilities of layer-by-layer assembly with functional biosensing peptides to create a new class of nanotheranostics. These NPs encapsulate a high weight percentage of siRNA while also carrying a synthetic biosensing peptide on the surface that is cleaved into a urinary reporter upon exposure to specific proteases overexpressed in the tumor microenvironment. Importantly, this biosensor reports back on a molecular signature characteristic to metastatic tumors and associated with poor prognosis, MMP9 protease overexpression. This nanotheranostic mediates noninvasive urinary-based diagnostics in mouse models of three different cancers with simultaneous gene silencing in flank and metastatic mouse models of ovarian cancer.

Design of modular dual enzyme-responsive peptides.

Dual enzyme-responsive peptides were synthesized by masking the ɛ-amine of lysine with various enzyme substrates. Enzymatic cleavage of these sequences unmasked the ɛ-amine, allowing for further digestion by a second enzyme, which was monitored colorimetrically. This modular peptide design should provide substrates for a large combination of clinically relevant enzymes.

Imine Hydrogels with Tunable Degradability for Tissue Engineering.

A shortage of available organ donors has created a need for engineered tissues. In this context, polymer-based hydrogels that break down inside the body are often used as constructs for growth factors and cells. Herein, we report imine cross-linked gels where degradation is controllable by the introduction of mixed imine cross-links. Specifically, hydrazide-functionalized poly(ethylene glycol) (PEG) reacts with aldehyde-functionalized PEG (PEG-CHO) to form hydrazone linked hydrogels that degrade quickly in media. The time to degradation can be controlled by changing the structure of the hydrazide group or by introducing hydroxylamines to form nonreversible oxime linkages. Hydrogels containing adipohydrazide-functionalized PEG (PEG-ADH) and PEG-CHO were found to degrade more rapidly than gels formed from carbodihydrazide-functionalized PEG (PEG-CDH). Incorporating oxime linkages via aminooxy-functionalized PEG (PEG-AO) into the hydrazone cross-linked gels further stabilized the hydrogels. This imine cross-linking approach should be useful for modulating the degradation characteristics of 3D cell culture supports for controlled cell release.

Protein-polymer conjugation via ligand affinity and photoactivation of glutathione S-transferase.

A photoactivated, site-selective conjugation of poly(ethylene glycol) (PEG) to the glutathione (GSH) binding pocket of glutathione S-transferase (GST) is described. To achieve this, a GSH analogue (GSH-BP) was designed and chemically synthesized with three functionalities: (1) the binding affinity of GSH to GST, (2) a free thiol for polymer functionalization, and (3) a photoreactive benzophenone (BP) component. Different molecular weights (2 kDa, 5 kDa, and 20 kDa) of GSH-BP modified PEGs (GSBP-PEGs) were synthesized and showed conjugation efficiencies between 52% and 76% to GST. Diazirine (DA) PEG were also prepared but gave conjugation yields lower than for GSBP-PEGs. PEGs with different end-groups were also synthesized to validate the importance of each component in the end-group design. End-groups included glutathione (GS-PEG) and benzophenone (BP-PEG). Results showed that both GSH and BP were crucial for successful conjugation to GST. In addition, conjugations of 5 kDa GSBP-PEG to different proteins were investigated, including bovine serum albumin (BSA), lysozyme (Lyz), ubiquitin (Ubq), and GST-fused ubiquitin (GST-Ubq) to ensure specific binding to GST. By combining noncovalent and covalent interactions, we have developed a new phototriggered protein-polymer conjugation method that is generally applicable to GST-fusion proteins.

Ether lipids influence cancer cell fate by modulating iron uptake.

Cancer cell fate has been widely ascribed to mutational changes within protein-coding genes associated with tumor suppressors and oncogenes. In contrast, the mechanisms through which the biophysical properties of membrane lipids influence cancer cell survival, dedifferentiation and metastasis have received little scrutiny. Here, we report that cancer cells endowed with a high metastatic ability and cancer stem cell-like traits employ ether lipids to maintain low membrane tension and high membrane fluidity. Using genetic approaches and lipid reconstitution assays, we show that these ether lipid-regulated biophysical properties permit non-clathrin-mediated iron endocytosis via CD44, leading directly to significant increases in intracellular redox-active iron and enhanced ferroptosis susceptibility. Using a combination of in vitro three-dimensional microvascular network systems and in vivo animal models, we show that loss of ether lipids also strongly attenuates extravasation, metastatic burden and cancer stemness. These findings illuminate a mechanism whereby ether lipids in carcinoma cells serve as key regulators of malignant progression while conferring a unique vulnerability that can be exploited for therapeutic intervention.

Synergistic combination therapy delivered via layer-by-layer nanoparticles induces solid tumor regression of ovarian cancer.

The majority of patients with high grade serous ovarian cancer (HGSOC) develop recurrent disease and chemotherapy resistance. To identify drug combinations that would be effective in treatment of chemotherapy resistant disease, we examined the efficacy of drug combinations that target the three antiapoptotic proteins most commonly expressed in HGSOC-BCL2, BCL-XL, and MCL1. Co-inhibition of BCL2 and BCL-XL (ABT-263) with inhibition of MCL1 (S63845) induces potent synergistic cytotoxicity in multiple HGSOC models. Since this drug combination is predicted to be toxic to patients due to the known clinical morbidities of each drug, we developed layer-by-layer nanoparticles (LbL NPs) that co-encapsulate these inhibitors in order to target HGSOC tumor cells and reduce systemic toxicities. We show that the LbL NPs can be designed to have high association with specific ovarian tumor cell types targeted in these studies, thus enabling a more selective uptake when delivered via intraperitoneal injection. Treatment with these LbL NPs displayed better potency than free drugs in vitro and resulted in near-complete elimination of solid tumor metastases of ovarian cancer xenografts. Thus, these results support the exploration of LbL NPs as a strategy to deliver potent drug combinations to recurrent HGSOC. While these findings are described for co-encapsulation of a BCL2/XL and a MCL1 inhibitor, the modular nature of LbL assembly provides flexibility in the range of therapies that can be incorporated, making LbL NPs an adaptable vehicle for delivery of additional combinations of pathway inhibitors and other oncology drugs.

Power in Numbers: Harnessing Combinatorial and Integrated Screens to Advance Nanomedicine.

Nanocarriers have significant potential to advance personalized medicine through targeted drug delivery. However, to date, efforts to improve nanoparticle accumulation at target disease sites have largely failed to translate clinically, stemming from an incomplete understanding of nano-bio interactions. While progress has been made to evaluate the effects of specific physical and chemical nanoparticle properties on trafficking and uptake, there is much to be gained from controlling these properties singularly and in combination to determine their interactions with different cell types. We and others have recently begun leveraging library-based nanoparticle screens to study structure-function relationships of lipid- and polymer-based drug delivery systems to guide nanoparticle design. These combinatorial screening efforts are showing promise in leading to the successful identification of critical characteristics that yield improved and specific accumulation at target sites. However, there is a crucial need to equally consider the influence of biological complexity on nanoparticle delivery, particularly in the context of clinical translation. For example, tissue and cellular heterogeneity presents an additional dimension to nanoparticle trafficking, uptake, and accumulation; applying imaging and screening tools as well as bioinformatics may further expand our understanding of how nanoparticles engage with cells and tissues. Given recent advances in the fields of omics and machine learning, there is substantial promise to revolutionize nanocarrier development through the use of integrated screens, harnessing the combinatorial parameter space afforded both by nanoparticle libraries and clinically annotated biological data sets in combination with high throughput in vivo studies.

Massively parallel pooled screening reveals genomic determinants of nanoparticle delivery.

To accelerate the translation of cancer nanomedicine, we used an integrated genomic approach to improve our understanding of the cellular processes that govern nanoparticle trafficking. We developed a massively parallel screen that leverages barcoded, pooled cancer cell lines annotated with multiomic data to investigate cell association patterns across a nanoparticle library spanning a range of formulations with clinical potential. We identified both materials properties and cell-intrinsic features that mediate nanoparticle-cell association. Using machine learning algorithms, we constructed genomic nanoparticle trafficking networks and identified nanoparticle-specific biomarkers. We validated one such biomarker: gene expression of SLC46A3, which inversely predicts lipid-based nanoparticle uptake in vitro and in vivo. Our work establishes the power of integrated screens for nanoparticle delivery and enables the identification and utilization of biomarkers to rationally design nanoformulations.

Lipidome-based Targeting of STAT3-driven Breast Cancer Cells Using Poly-l-glutamic Acid-coated Layer-by-Layer Nanoparticles.

The oncogenic transcription factor STAT3 is aberrantly activated in 70% of breast cancers, including nearly all triple-negative breast cancers (TNBCs). Because STAT3 is difficult to target directly, we considered whether metabolic changes driven by activated STAT3 could provide a therapeutic opportunity. We found that STAT3 prominently modulated several lipid classes, with most profound effects on N-acyl taurine and arachidonic acid, both of which are involved in plasma membrane remodeling. To exploit these metabolic changes therapeutically, we screened a library of layer-by-layer (LbL) nanoparticles (NPs) differing in the surface layer that modulates interactivity with the cell membrane. We found that poly-l-glutamic acid (PLE)-coated NPs bind to STAT3-transformed breast cancer cells with 50% greater efficiency than to nontransformed cells, and the heightened PLE-NP binding to TNBC cells was attenuated by STAT3 inhibition. This effect was also observed in densely packed three-dimensional breast cancer organoids. As STAT3-transformed cells show greater resistance to cytotoxic agents, we evaluated whether enhanced targeted delivery via PLE-NPs would provide a therapeutic advantage. We found that cisplatin-loaded PLE-NPs induced apoptosis of STAT3-driven cells at lower doses compared with both unencapsulated cisplatin and cisplatin-loaded nontargeted NPs. In addition, because radiation is commonly used in breast cancer treatment, and may alter cellular lipid distribution, we analyzed its effect on PLE-NP-cell binding. Irradiation of cells enhanced the STAT3-targeting properties of PLE-NPs in a dose-dependent manner, suggesting potential synergies between these therapeutic modalities. These findings suggest that cellular lipid changes driven by activated STAT3 may be exploited therapeutically using unique LbL NPs.

Tuning Nanoparticle Interactions with Ovarian Cancer through Layer-by-Layer Modification of Surface Chemistry.

Nanoparticle surface chemistry is a fundamental engineering parameter that governs tumor-targeting activity. Electrostatic assembly generates controlled polyelectrolyte complexes through the process of adsorption and charge overcompensation utilizing synthetic polyions and natural biomacromolecules; it can yield films with distinctive hydration, charge, and presentation of functional groups. Here, we used electrostatic layer-by-layer (LbL) assembly to screen 10 different surface chemistries for their ability to preferentially target human ovarian cancer in vitro. Our screen identified that poly-l-aspartate, poly-l-glutamate, and hyaluronate-coated LbL nanoparticles have striking specificity for ovarian cancer, while sulfated poly(ß-cyclodextrin) nanoparticles target noncancerous stromal cells. We validated top candidates for tumor-homing ability with a murine model of metastatic disease and with patient-derived ovarian cancer spheroids. Nanoparticle surface chemistry also influenced subcellular trafficking, indicating strategies to target the cell membrane, caveolae, and perinuclear vesicles. Our results confirm LbL is a powerful tool to systematically engineer nanoparticles and achieve specific targeting.

Solution Conditions Tune and Optimize Loading of Therapeutic Polyelectrolytes into Layer-by-Layer Functionalized Liposomes.

Layer-by-layer (LbL) nanoparticles offer great potential to the field of drug delivery, where these nanocomposites have been studied for their ability to deliver chemotherapeutic agents, small molecule inhibitors, and nucleic acids. Most exciting is their ability to encapsulate multiple functional elements, which allow nanocarriers to deliver complex combination therapies with staged release. However, relative to planar LbL constructs, colloidal LbL systems have not undergone extensive systematic studies that outline critical synthetic solution conditions needed for robust and efficient assembly. The multistaged process of adsorbing a series of materials onto a nanoscopic template is inherently complex, and facilitating the self-assembly of these materials depends on identifying proper solution conditions for each synthetic step and adsorbed material. Here, we focus on addressing some of the fundamental questions that must be answered in order to obtain a reliable and robust synthesis of nucleic acid-containing LbL liposomes. This includes a study of solution conditions, such as pH, ionic strength, salt composition, and valency, and their impact on the preparation of LbL nanoparticles. Our results provide insight into the selection of solution conditions to control the degree of ionization and the electrostatic screening length to suit the adsorption of nucleic acids and synthetic polypeptides. The optimization of these parameters led to a roughly 8-fold improvement in nucleic acid loading in LbL liposomes, indicating the importance of optimizing solution conditions in the preparation of therapeutic LbL nanoparticles. These results highlight the benefits of defining principles for constructing highly effective nanoparticle systems.