"Target-and-release" nanoparticles for effective immunotherapy of metastatic ovarian cancer.

Immunotherapies such as checkpoint inhibitors (CPI) are effective in treating several advanced cancers, but these treatments have had limited success in metastatic ovarian cancer (OC). Here, we engineered liposomal nanoparticles (NPs) carrying a layer-by-layer (LbL) polymer coating that promotes their binding to the surface of OC cells. Covalent anchoring of the potent immunostimulatory cytokine interleukin-12 (IL-12) to phospholipid headgroups of the liposome core enabled the LbL particles to concentrate IL-12 in disseminated OC tumors following intraperitoneal administration. Shedding of the LbL coating and serum protein-mediated extraction of IL-12-conjugated lipids from the liposomal core over time enabled IL-12 to disseminate in the tumor bed following rapid NP localization in tumor nodules. Optimized IL-12 LbL-NPs promoted robust T cell accumulation in ascites and tumors in mouse models, extending survival compared to free IL-12 and remarkedly sensitizing tumors to CPI, leading to curative treatments and immune memory.

Development of a two-hit lethal liver injury model in swine.

PURPOSE: Noncompressible truncal hemorrhage remains a leading cause of preventable death in the prehospital setting. Standardized and reproducible large animal models are essential to test new therapeutic strategies. However, existing injury models vary significantly in consistency and clinical accuracy. This study aims to develop a lethal porcine model to test hemostatic agents targeting noncompressible abdominal hemorrhages. METHODS: We developed a two-hit injury model in Yorkshire swine, consisting of a grade IV liver injury combined with hemodilution. The hemodilution was induced by controlled exsanguination of 30% of the total blood volume and a 3:1 resuscitation with crystalloids. Subsequently, a grade IV liver injury was performed by sharp transection of both median lobes of the liver, resulting in major bleeding and severe hypotension. The abdominal incision was closed within 60 s from the injury. The endpoints included mortality, survival time, serum lab values, and blood loss within the abdomen. RESULTS: This model was lethal in all animals (5/5), with a mean survival time of 24.4 ± 3.8 min. The standardized liver resection was uniform at 14.4 ± 2.1% of the total liver weight. Following the injury, the MAP dropped by 27 ± 8mmHg within the first 10 min. The use of a mixed injury model (i.e., open injury, closed hemorrhage) was instrumental in creating a standardized injury while allowing for a clinically significant hemorrhage. CONCLUSION: This novel highly lethal, consistent, and clinically relevant translational model can be used to test and develop life-saving interventions for massive noncompressible abdominal hemorrhage.

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.

Poly(ß-aminoester) Physicochemical Properties Govern the Delivery of siRNA from Electrostatically Assembled Coatings.

Localized short interfering RNA (siRNA) therapy has the potential to drive high-specificity molecular-level treatment of a variety of disease states. Unfortunately, effective siRNA therapy suffers from several barriers to its intracellular delivery. Thus, drug delivery systems that package and control the release of therapeutic siRNAs are necessary to overcome these obstacles to clinical translation. Layer-by-layer (LbL) electrostatic assembly of thin film coatings containing siRNA and protonatable, hydrolyzable poly(ß-aminoester) (PBAE) polymers is one such drug delivery strategy. However, the impact of PBAE physicochemical properties on the transfection efficacy of siRNA released from LbL thin film coatings has not been systematically characterized. In this study, we investigate the siRNA transfection efficacy of four structurally similar PBAEs in vitro. We demonstrate that small changes in structure yield large changes in physicochemical properties, such as hydrophobicity, pKa, and amine chemical structure, driving differences in the interactions between PBAEs and siRNA in polyplexes and in LbL thin film coatings for wound dressings. In our polymer set, Poly3 forms the most stable interactions with siRNA (Keff,w/w = 0.298) to slow release kinetics and enhance transfection of reporter cells in both colloidal and thin film coating approaches. This is due to its unique physiochemical properties: high hydrophobicity (clog P = 7.86), effective pKa closest to endosomal pH (pKa = 6.21), and high cooperativity in buffering (nhill = 7.2). These properties bestow Poly3 with enhanced endosomal buffering and escape properties. Taken together, this work elucidates the connections between small changes in polymer structure, emergent properties, and polyelectrolyte theory to better understand PBAE transfection efficacy.

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.

Carboxylated Nanoparticle Surfaces Enhance Association with Mucoid Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa biofilms comprise three main polysaccharides: alginate, psl, and pel, which all imbue tolerance against exogenous antimicrobials. Nanoparticles (NPs) are an exciting new strategy to overcome the biofilm matrix for therapeutic delivery applications; however, zero existing FDA approvals for biofilm-specific NP formulations can be attributed to the complex interplay of physiochemical forces at the biofilm-NP interface. Here, we leverage a set of inducible, polysaccharide-specific, expressing isogenic P. aeruginosa mutants coupled with an assembled layer-by-layer NP (LbL NP) panel to characterize biofilm-NP interactions. When investigating these interactions using confocal microscopy, alginate-layered NPs associated more than dextran-sulfate-layered NPs with biofilms that had increased alginate production, including biofilms produced by mucoid P. aeruginosa isolates from people with cystic fibrosis. These differences were further confirmed in LbL NPs layered with polysaccharide- or hydrocarbon-based polymers with pendent carboxylate or sulfate functional groups. These data suggest carboxylated NP surfaces have enhanced interactions specifically with mucoid biofilms as compared to sulfated surfaces and lay the foundation for their inclusion as a design element for increasing biofilm-NP interactions and efficacious drug delivery.

Hydrogel dressings with intrinsic antibiofilm and antioxidative dual functionalities accelerate infected diabetic wound healing.

Chronic wounds are often infected with biofilm bacteria and characterized by high oxidative stress. Current dressings that promote chronic wound healing either require additional processes such as photothermal irradiation or leave behind gross amounts of undesirable residues. We report a dual-functionality hydrogel dressing with intrinsic antibiofilm and antioxidative properties that are synergistic and low-leaching. The hydrogel is a crosslinked network with tethered antibacterial cationic polyimidazolium and antioxidative N-acetylcysteine. In a murine diabetic wound model, the hydrogel accelerates the closure of wounds infected with methicillin-resistant Staphylococcus aureus or carbapenem-resistant Pseudomonas aeruginosa biofilm. Furthermore, a three-dimensional ex vivo human skin equivalent model shows that N-acetylcysteine promotes the keratinocyte differentiation and accelerates the re-epithelialization process. Our hydrogel dressing can be made into different formats for the healing of both flat and deep infected chronic wounds without contamination of the wound or needing other modalities such as photothermal irradiation.

Layer-by-Layer Polymer Functionalization Improves Nanoparticle Penetration and Glioblastoma Targeting in the Brain.

Glioblastoma is characterized by diffuse infiltration into surrounding healthy brain tissues, which makes it challenging to treat. Complete surgical resection is often impossible, and systemically delivered drugs cannot achieve adequate tumor exposure to prevent local recurrence. Convection-enhanced delivery (CED) offers a method for administering therapeutics directly into brain tumor tissue, but its impact has been limited by rapid clearance and off-target cellular uptake. Nanoparticle (NP) encapsulation presents a promising strategy for extending the retention time of locally delivered therapies while specifically targeting glioblastoma cells. However, the brain's extracellular structure poses challenges for NP distribution due to its narrow, tortuous pores and a harsh ionic environment. In this study, we investigated the impact of NP surface chemistry using layer-by-layer (LbL) assembly to design drug carriers for broad spatial distribution in brain tissue and specific glioblastoma cell targeting. We found that poly-l-glutamate and hyaluronate were effective surface chemistries for targeting glioblastoma cells in vitro. Coadsorbing either polymer with a small fraction of PEGylated polyelectrolytes improved the colloidal stability without sacrificing cancer cell selectivity. Following CED in vivo, gadolinium-functionalized LbL NPs enabled MRI visualization and exhibited a distribution volume up to three times larger than liposomes and doubled the retention half-time up to 13.5 days. Flow cytometric analysis of CED-treated murine orthotopic brain tumors indicated greater cancer cell uptake and reduced healthy cell uptake for LbL NPs compared to nonfunctionalized liposomes. The distinct cellular outcomes for different colayered LbL NPs provide opportunities to tailor this modular delivery system for various therapeutic applications.

Transforming ovarian cancer care by targeting minimal residual disease.

Frontline treatment and resultant cure rates in patients with advanced ovarian cancer have changed little over the past several decades. Here, we outline a multidisciplinary approach aimed at gaining novel therapeutic insights by focusing on the poorly understood minimal residual disease phase of ovarian cancer that leads to eventual incurable recurrences.

pH-Responsive, Charge-Reversing Layer-by-Layer Nanoparticle Surfaces Enhance Biofilm Penetration and Eradication.

Microbes entrenched within biofilms can withstand 1000-fold higher concentrations of antibiotics, in part due to the viscous extracellular matrix that sequesters and attenuates antimicrobial activity. Nanoparticle (NP)-based therapeutics can aid in delivering higher local concentrations throughout biofilms as compared to free drugs alone, thereby enhancing the efficacy. Canonical design criteria dictate that positively charged nanoparticles can multivalently bind to anionic biofilm components and increase biofilm penetration. However, cationic particles are toxic and are rapidly cleared from circulation in vivo, limiting their use. Therefore, we sought to design pH-responsive NPs that change their surface charge from negative to positive in response to the reduced biofilm pH microenvironment. We synthesized a family of pH-dependent, hydrolyzable polymers and employed the layer-by-layer (LbL) electrostatic assembly method to fabricate biocompatible NPs with these polymers as the outermost surface. The NP charge conversion rate, dictated by polymer hydrophilicity and the side-chain structure, ranged from hours to undetectable within the experimental timeframe. LbL NPs with an increasingly fast charge conversion rate more effectively penetrated through, and accumulated throughout, wildtype (PAO1) and mutant overexpressing biomass (ΔwspF) Pseudomonas aeruginosa biofilms. Finally, tobramycin, an antibiotic known to be trapped by anionic biofilm components, was loaded into the final layer of the LbL NP. There was a 3.2-fold reduction in ΔwspF colony forming units for the fastest charge-converting NP as compared to both the slowest charge converter and free tobramycin. These studies provide a framework for the design of biofilm-penetrating NPs that respond to matrix interactions, ultimately increasing the efficacious delivery of antimicrobials.

STING Protein-Based In Situ Vaccine Synergizes CD4+ T, CD8+ T, and NK Cells for Tumor Eradication.

Stimulator of interferon genes (STING) signaling is a promising target in cancer immunotherapy, with many ongoing clinical studies in combination with immune checkpoint blockade (ICB). Existing STING-based therapies largely focus on activating CD8+ T cell or NK cell-mediated cytotoxicity, while the role of CD4+ T cells in STING signaling has yet to be extensively studied in vivo. Here, a distinct CD4-mediated, protein-based combination therapy of STING and ICB as an in situ vaccine, is reported. The treatment eliminates subcutaneous MC38 and YUMM1.7 tumors in 70-100% of mice and protected all cured mice against rechallenge. Mechanistic studies reveal a robust TH 1 polarization and suppression of Treg of CD4+ T cells, followed by an effective collaboration of CD4+ T, CD8+ T, and NK cells to eliminate tumors. Finally, the potential to overcome host STING deficiency by significantly decreasing MC38 tumor burden in STING KO mice is demonstrated, addressing the translational challenge for the 19% of human population with loss-of-function STING variants.

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.

Layer-by-layer interleukin-12 nanoparticles drive a safe and effective response in ovarian tumors.

Ovarian cancer is especially deadly, challenging to treat, and has proven refractory to known immunotherapies. Cytokine therapy is an attractive strategy to drive a proinflammatory immune response in immunologically cold tumors such as many high grade ovarian cancers; however, this strategy has been limited in the past due to severe toxicity. We previously demonstrated the use of a layer-by-layer (LbL) nanoparticle (NP) delivery vehicle in subcutaneous flank tumors to reduce the toxicity of interleukin-12 (IL-12) therapy upon intratumoral injection. However, ovarian cancer cannot be treated by local injection as it presents as dispersed metastases. Herein, we demonstrate the use of systemically delivered LbL NPs using a cancer cell membrane-binding outer layer to effectively target and engage the adaptive immune system as a treatment in multiple orthotopic ovarian tumor models, including immunologically cold tumors. IL-12 therapy from systemically delivered LbL NPs shows reduced severe toxicity and maintained anti-tumor efficacy compared to carrier-free IL-12 or layer-free liposomal NPs leading to a 30% complete survival rate.

Designer co-beta-peptide copolymer selectively targets resistant and biofilm Gram-negative bacteria.

New antimicrobials are urgently needed to combat Gram-negative bacteria, particularly multi-drug resistant (MDR) and phenotypically resistant biofilm species. At present, only sequence-defined alpha-peptides (e.g. polymyxin B) can selectively target Gram-negative bacterial lipopolysaccharides. We show that a copolymer, without a defined sequence, shows good potency against MDR Gram-negative bacteria including its biofilm form. The tapered blocky co-beta-peptide with controlled N-terminal hydrophobicity (#4) has strong interaction with the Gram-negative bacterial lipopolysaccharides via its backbone through electrostatic and hydrogen bonding interactions but not the Gram-positive bacterial and mammalian cell membranes so that this copolymer is non-toxic to these two latter cell types. The new #4 co-beta-peptide selectively kills Gram-negative bacteria with low cytotoxicity both in vitro and in a mouse biofilm wound infection model. This strategy provides a new concept for the design of Gram-negative selective antimicrobial peptidomimetics against MDR and biofilm species.

A new label-free optical imaging method for the lymphatic system enhanced by deep learning.

Our understanding of the lymphatic vascular system lags far behind that of the blood vascular system, limited by available imaging technologies. We present a label-free optical imaging method that visualizes the lymphatic system with high contrast. We developed an orthogonal polarization imaging (OPI) in the shortwave infrared range (SWIR) and imaged both lymph nodes and lymphatic vessels of mice and rats in vivo through intact skin, as well as human mesenteric lymph nodes in colectomy specimens. By integrating SWIR-OPI with U-Net, a deep learning image segmentation algorithm, we automated the lymph node size measurement process. Changes in lymph nodes in response to cancer progression were monitored in two separate mouse cancer models, through which we obtained insights into pre-metastatic niches and correlation between lymph node masses and many important biomarkers. In a human pilot study, we demonstrated the effectiveness of SWIR-OPI to detect human lymph nodes in real time with clinical colectomy specimens. One Sentence Summary: We develop a real-time high contrast optical technique for imaging the lymphatic system, and apply it to anatomical pathology gross examination in a clinical setting, as well as real-time monitoring of tumor microenvironment in animal studies.

Predicting transport of intra-articularly injected growth factor fusion proteins into human knee joint cartilage.

There are no drugs or treatment methods known to prevent the development of post-traumatic osteoarthritis (PTOA), a type of osteoarthritis (OA) that is triggered by traumatic joint injuries and accounts for ∼12% of the nearly 600 million OA cases worldwide. Lack of effective drug delivery techniques remains a major challenge in developing clinically effective treatments, but cationic delivery carriers can help overcome this challenge. Scaling up treatments that are effective in in vitro models to achieve success in preclinical in vivo models and clinical trials is also a challenging problem in the field. Here we use a cationic green fluorescent protein (GFP) as a carrier to deliver Insulin-Like Growth Factor 1 (IGF-1), a drug considered as a potential therapeutic for PTOA. GFP-IGF-1 conjugates were first synthesized as fusion proteins with different polypeptide linkers, and their transport properties were characterized in human cartilage explants. In vitro experimental data were used to develop a predictive mathematical transport model that was validated using an independent in vitro experimental data set. The model was used to predict the transport of these fusion proteins upon intra-articular injection into human knee joints. The predictions included results for the rate and extent of fusion protein penetration into cartilage, and the maximum levels of fusion proteins that would escape into systemic circulation through the joint capsule. Together, our transport measurements and model set the stage for translation of such explant culture studies to in vivo preclinical studies and potentially clinical application. STATEMENT OF SIGNIFICANCE: The lack of blood supply in cartilage and rapid clearance of drugs injected into human knees presents a major challenge in developing clinically effective treatments for osteoarthritis. Cationic delivery carriers can target negatively charged cartilage and help overcome this problem. Scaling up treatments that are effective in vitro to achieve success in vivo is also challenging. Here, we use a cationic green fluorescent protein (GFP) to deliver Insulin-Like Growth Factor-1 (IGF-1) into cartilage. Experiments measuring transport of GFP-IGF-1 fusion proteins in human cartilage explants were used to develop and validate a mathematical model to predict fusion protein transport upon injection into human knee joints. This work translates such explant culture studies to in vivo preclinical studies and potentially clinical application.

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.

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.

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.