Integrated imaging probe and bispecific antibody development enables in vivo targeting of glypican-3-expressing hepatocellular carcinoma.

Glypican-3 (GPC3) is a proteoglycan with high sensitivity and specificity for hepatocellular carcinoma (HCC). We describe the integrated development and validation of a GPC3-targeting optical imaging probe and T-cell redirecting antibody (TRAB) as a theranostic strategy for the detection and treatment of HCC. A novel TRAB targeting GPC3 on HCC tumor cells and the CD3 T-cell receptor as well as a distinct GPC3-specific optical imaging probe were developed from a short peptide. The efficacy of GPC3/CD3 TRAB was evaluated in vitro using interferon-γ release and calcein-AM assays. Patient-derived xenografts (PDX) were used to assess the in vivo efficacy of GPC3/CD3 TRAB and the GPC3 imaging probe for the detection of GPC3+ HCC. GPC3/CD3 TRAB caused a dose-dependent escalation in interferon-γ release from inactive peripheral blood T-cells (P = 0.001) and higher tumor-cell lysis (P = 0.01) compared to controls in vitro. Intratumorally injected GPC3/CD3 TRAB resulted in significant prolongation of tumor doubling time in the GPC3+ PDX mice, with an associated reduction of tumor fluorescent signal from the HiLyte 488- conjugated GPC3 specific peptide on optical imaging. HCC cell targeting using a GPC3/CD3 TRAB derived from a small peptide resulted in effective T-cell activation and induction of a cytotoxic response toward GPC3+ HCC tumor cells both in vitro and in vivo. GPC3-specific optical imaging enabled the detection of the GPC3+ HCC cells and noninvasive monitoring of tumor response to adoptive immunotherapy. The integrated development of a targeted therapeutic and molecular imaging probe provides a novel paradigm for developing cancer theranostics.

Lipid-mediated intracellular delivery of recombinant bioPROTACs for the rapid degradation of undruggable proteins.

Recently, targeted degradation has emerged as a powerful therapeutic modality. Relying on "event-driven" pharmacology, proteolysis targeting chimeras (PROTACs) can degrade targets and are superior to conventional inhibitors against undruggable proteins. Unfortunately, PROTAC discovery is limited by warhead scarcity and laborious optimization campaigns. To address these shortcomings, analogous protein-based heterobifunctional degraders, known as bioPROTACs, have been developed. Compared to small-molecule PROTACs, bioPROTACs have higher success rates and are subject to fewer design constraints. However, the membrane impermeability of proteins severely restricts bioPROTAC deployment as a generalized therapeutic modality. Here, we present an engineered bioPROTAC template able to complex with cationic and ionizable lipids via electrostatic interactions for cytosolic delivery. When delivered by biocompatible lipid nanoparticles, these modified bioPROTACs can rapidly degrade intracellular proteins, exhibiting near-complete elimination (up to 95% clearance) of targets within hours of treatment. Our bioPROTAC format can degrade proteins localized to various subcellular compartments including the mitochondria, nucleus, cytosol, and membrane. Moreover, substrate specificity can be easily reprogrammed, allowing modular design and targeting of clinically-relevant proteins such as Ras, Jnk, and Erk. In summary, this work introduces an inexpensive, flexible, and scalable platform for efficient intracellular degradation of proteins that may elude chemical inhibition.

Lipid Nanoparticle-Associated Inflammation is Triggered by Sensing of Endosomal Damage: Engineering Endosomal Escape Without Side Effects.

Lipid nanoparticles (LNPs) have emerged as the dominant platform for RNA delivery, based on their success in the COVID-19 vaccines and late-stage clinical studies in other indications. However, we and others have shown that LNPs induce severe inflammation, and massively aggravate pre-existing inflammation. Here, using structure-function screening of lipids and analyses of signaling pathways, we elucidate the mechanisms of LNP-associated inflammation and demonstrate solutions. We show that LNPs' hallmark feature, endosomal escape, which is necessary for RNA expression, also directly triggers inflammation by causing endosomal membrane damage. Large, irreparable, endosomal holes are recognized by cytosolic proteins called galectins, which bind to sugars on the inner endosomal membrane and then regulate downstream inflammation. We find that inhibition of galectins abrogates LNP-associated inflammation, both in vitro and in vivo . We show that rapidly biodegradable ionizable lipids can preferentially create endosomal holes that are smaller in size and reparable by the endosomal sorting complex required for transport (ESCRT) pathway. Ionizable lipids producing such ESCRT-recruiting endosomal holes can produce high expression from cargo mRNA with minimal inflammation. Finally, we show that both routes to non-inflammatory LNPs, either galectin inhibition or ESCRT-recruiting ionizable lipids, are compatible with therapeutic mRNAs that ameliorate inflammation in disease models. LNPs without galectin inhibition or biodegradable ionizable lipids lead to severe exacerbation of inflammation in these models. In summary, endosomal escape induces endosomal membrane damage that can lead to inflammation. However, the inflammation can be controlled by inhibiting galectins (large hole detectors) or by using biodegradable lipids, which create smaller holes that are reparable by the ESCRT pathway. These strategies should lead to generally safer LNPs that can be used to treat inflammatory diseases.

High-Throughput Single-Cell, Single-Mitochondrial DNA Assay Using Hydrogel Droplet Microfluidics.

There is growing interest in understanding the biological implications of single cell heterogeneity and heteroplasmy of mitochondrial DNA (mtDNA), but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi-step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95 % mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single-mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.

High-throughput single-cell, single-mitochondrial DNA assay using hydrogel droplet microfluidics.

There is growing interest in understanding the biological implications of single cell heterogeneity and intracellular heteroplasmy of mtDNA, but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi-step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95% mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.

Intracellular Protein Delivery: Approaches, Challenges, and Clinical Applications.

Protein biologics are powerful therapeutic agents with diverse inhibitory and enzymatic functions. However, their clinical use has been limited to extracellular applications due to their inability to cross plasma membranes. Overcoming this physiological barrier would unlock the potential of protein drugs for the treatment of many intractable diseases. In this review, we highlight progress made toward achieving cytosolic delivery of recombinant proteins. We start by first considering intracellular protein delivery as a drug modality compared to existing Food and Drug Administration-approved drug modalities. Then, we summarize strategies that have been reported to achieve protein internalization. These techniques can be broadly classified into 3 categories: physical methods, direct protein engineering, and nanocarrier-mediated delivery. Finally, we highlight existing challenges for cytosolic protein delivery and offer an outlook for future advances.

Exploring the Sensitivity of Antibody-Drug Conjugate Efficacy to the Selection of Payload, Antibody, and Cell line.

Antibody-drug conjugates (ADCs) make up a growing class of targeted therapeutics with important applications in cancer treatment. ADCs are highly modular in nature and thus can be engineered to target any cancer type, but their efficacy is strongly influenced by the specific choice of payload, antibody, and target cell. Considering the number of possible antibody-payload combinations, ADC development would benefit from an efficient method to narrow the number of ADC compositions to those with the highest and most universal potency prior to assessing pharmacokinetics and pharmacodynamics in animal models. To facilitate the identification of optimal ADC compositions, we describe the use of photoreactive antibody-binding domain-drug conjugates (known commercially as oYo-Link) to enable the site-specific labeling of off-the-shelf antibodies. This approach allows for the rapid generation of ADCs with a drug-to-antibody ratio of ∼2 with no subsequent purification required. As a demonstration of this approach, ADCs were generated with different combinations of tubulin-inhibitor drugs (DM1, DM4, VcMMAE, and VcMMAF) and anti-EGFR antibodies (cetuximab, panitumumab, anti-EGFR clone 425, and anti-EGFR clone 528) and were delivered to three EGFR-expressing cell lines (A431, A549, and MDA-MB-231). Real-time cytolysis assays indicated that the most effective antibody varied based on the choice of cell line: cetuximab was most potent against A431 cells, while 425 and 528 led to the greatest cytotoxicity against A549 and MDA-MB-231 cells. These results did not correlate with differences in measured anti-EGFR binding affinity as cetuximab had the highest affinity across all three cell lines, while 425 and 528 had the lowest affinities for all three cell lines. Panitumumab, which had the second-highest anti-EGFR affinity, exhibited the least effective cytolysis across A431, A549, and MDA-MB-231 cells. By demonstrating that ADC potency toward a given target is dependent on both the antibody and drug chosen, these findings can guide the selection of ADCs for further in vivo analysis.

Theranostic Phthalocyanine and Naphthalocyanine Nanoparticles for Photoacoustic Imaging and Photothermal Therapy of Tumors.

Background: Phthalocyanine (PC) and naphthalocyanine (NC) dyes have long garnered interest as theranostic agents for optical imaging and phototherapy due to their near-infrared absorbance, photostability, imaging contrast, and proven safety in clinical trials. Yet, only a small fraction of these dyes has been evaluated as photothermal therapy (PTT) agents for cancer treatment. Methods: Nearly 40 distinct NC and PC dyes were encapsulated within polymeric PEG-PCL micelles via oil-in-water emulsions. The optimal NC/PC-loaded micelle formulations for PTT and photoacoustic (PA) imaging were identified through in vivo and in vitro studies. Results: The most promising candidate, CuNC(Octa)-loaded micelles, demonstrated a strong PA signal with a peak absorbance at ~870 nm, high photothermal efficiency, and photostability. The CuNC(Octa)-loaded micelles exhibited heat generation as good or better than gold nanorods/nanoshells and >10-fold higher photoacoustic signals. Micelle preparation was reproducible/scalable, and the CuNC(Octa)-loaded micelles are highly stable under physiological conditions. The CuNC(Octa)-loaded micelles localize within tumors via enhanced permeability and retention and are readily detectable by PA imaging. In a syngeneic murine tumor model of triple-negative breast cancer, CuNC(Octa)-loaded micelles demonstrate efficient heat generation with PTT, leading to the complete eradication of tumors. Conclusions: CuNC(Octa)-loaded micelles represent a promising theranostic agent for PA imaging and PTT. The ability to utilize conventional ultrasound in combination with PA imaging enables the simultaneous acquisition of information about tumor morphology and micelle accumulation. PTT with CuNC(Octa)-loaded micelles can lead to the complete eradication of highly invasive tumors.

Phthalocyanine-Blue Nanoparticles for the Direct Visualization of Tumors with White Light Illumination.

The current standard of care for colon cancer surveillance relies heavily on white light endoscopy (WLE). However, dysplastic lesions that are not visible to the naked eye are often missed when conventional WLE equipment is used. Although dye-based chromoendoscopy shows promise, current dyes cannot delineate tumor tissues from surrounding healthy tissues accurately. The goal of the present study was to screen various phthalocyanine (PC) dye-loaded micelles for their ability to improve the direct visualization of tumor tissues under white light following intravenous administration. Zinc PC (tetra-tert-butyl)-loaded micelles were identified as the optimal formulation. Their accumulation within syngeneic breast tumors led the tumors to turn dark blue in color, making them clearly visible to the naked eye. These micelles were similarly able to turn spontaneous colorectal adenomas in Apc+/Min mice a dark blue color for easy identification and could enable clinicians to more effectively detect and remove colonic polyps.

Lipid Nanoparticle Delivery of Small Proteins for Potent In Vivo RAS Inhibition.

Mutated RAS proteins are potent oncogenic drivers and have long been considered "undruggable". While RAS-targeting therapies have recently shown promise, there remains a clinical need for RAS inhibitors with more diverse targets. Small proteins represent a potential new therapeutic option, including K27, a designed ankyrin repeat protein (DARPin) engineered to inhibit RAS. However, K27 functions intracellularly and is incapable of entering the cytosol on its own, currently limiting its utility. To overcome this barrier, we have engineered a lipid nanoparticle (LNP) platform for potent delivery of functional K27-D30─a charge-modified version of the protein─intracellularly in vitro and in vivo. This system efficiently encapsulates charge-modified proteins, facilitates delivery in up to 90% of cells in vitro, and maintains potency after at least 45 days of storage. In vivo, these LNPs deliver K27-D30 to the cytosol of cancerous cells in the liver, inhibiting RAS-driven growth and ultimately reducing tumor load in an HTVI-induced mouse model of hepatocellular carcinoma. This work shows that K27 holds promise as a new cancer therapeutic when delivered using this LNP platform. Furthermore, this technology has the potential to broaden the use of LNPs to include new cargo types─beyond RNA─for diverse therapeutic applications.

Labeling of Phospho-Specific Antibodies with oYo-Link® Epitope Tags for Multiplex Immunostaining.

Spatial proteomics has recently garnered significant interest, as it offers to provide unprecedented insight into biological processes in both health and disease, by connecting protein expression patterns from the subcellular level to the tissue or even organism level. These high-content approaches generally rely on a high degree of multiplexing, whereby multiple proteins can be detected simultaneously. The most versatile multiplexing approaches utilize antibodies to confer specificity for various intracellular proteins of interest. Therefore, these methods must be able to differentiate many antibodies at once. In this chapter, we describe a simple and rapid approach to labeling antibodies with distinct epitope tags in a site-specific manner. This allows multiple antibodies, even from the same host species, to be uniquely identified and detected and offers a simple approach for spatial proteomic applications.

Activating EGFR Signaling Attenuates Osteoarthritis Development Following Loading Injury in Mice.

Posttraumatic osteoarthritis (PTOA) results in joint pain, loss of joint function, and impaired quality of daily life in patients with limited treatment options. We previously demonstrated that epidermal growth factor receptor (EGFR) signaling is essential for maintaining chondroprogenitors during articular cartilage development and homeostasis. Here, we used a nonsurgical, loading-induced PTOA mouse model to investigate the protective action of EGFR signaling. A single bout of cyclic tibial loading at a peak force of 6 N injured cartilage at the posterior aspect of lateral femoral condyle. Similar loading at a peak force of 9 N ruptured the anterior cruciate ligament, causing additional cartilage damage at the medial compartment and ectopic cartilage formation in meniscus and synovium. Constitutively overexpression of an EGFR ligand, heparin binding EGF-like growth factor (HBEGF), in chondrocytes significantly reduced cartilage injury length, synovitis, and pain after 6 N loading and mitigated medial side cartilage damage and ectopic cartilage formation after 9 N loading. Mechanistically, overactivation of EGFR signaling protected chondrocytes from loading-induced apoptosis and loss of proliferative ability and lubricant synthesis. Overexpressing HBEGF in adult cartilage starting right before 6 N loading had similar beneficial effects. In contrast, inactivating EGFR in adult cartilage led to accelerated PTOA progression with elevated cartilage Mankin score and synovitis score and increased ectopic cartilage formation. As a therapeutic approach, we constructed a nanoparticle conjugated with the EGFR ligand TGFα. Intra-articular injections of this nanoconstruct once every 3 weeks for 12 weeks partially mitigated PTOA symptoms in cartilage and synovium after 6 N loading. Our findings demonstrate the anabolic actions of EGFR signaling in maintaining articular cartilage during PTOA development and shed light on developing a novel nanomedicine for PTOA. © 2022 American Society for Bone and Mineral Research (ASBMR).

Rapid, site-specific labeling of "off-the-shelf" and native serum autoantibodies with T cell-redirecting domains.

Extensive antibody engineering and cloning is typically required to generate new bispecific antibodies. Made-to-order genes, advanced expression systems, and high-efficiency cloning can simplify and accelerate this process, but it still can take months before a functional product is realized. We developed a simple method to site-specifically and covalently attach a T cell-redirecting domain to any off-the-shelf, human immunoglobulin G (IgG) or native IgG isolated from serum. No antibody engineering, cloning, or knowledge of the antibody sequence is required. Bispecific antibodies are generated in just hours. By labeling antibodies isolated from tumor-bearing mice, including two syngeneic models, we generated T cell-redirecting autoantibodies (TRAAbs) that act as an effective therapeutic. TRAAbs preferentially bind tumor tissue over healthy tissue, indicating a previously unexplored therapeutic window. The use of autoantibodies to direct the tumor targeting of bispecific antibodies represents a new paradigm in personalized medicine that eliminates the need to identify tumor biomarkers.

Superoxide dismutase-loaded porous polymersomes as highly efficient antioxidant nanoparticles targeting synovium for osteoarthritis therapy.

Oxidative stress and the reactive oxygen species (ROS) have important roles in osteoarthritis (OA) development and progression. Scavenging ROS by exogenous antioxidant enzymes could be a promising approach for OA treatment. However, the direct use of antioxidant enzymes, such as superoxide dismutase (SOD), is challenging due to a lack of effective drug delivery system to knee joints. This study utilized a highly efficient antioxidative nanoparticle based on SOD-loaded porous polymersome nanoparticles (SOD-NPs) for delivery of SOD to mouse knee joints. The resultant SOD-NPs had prolonged mouse joint retention time with predominant accumulation in synovium but not in articular cartilage. Examining human synovial explants revealed that SOD-NPs minimize oxidative damages induced by OA-like insults. Intra-articular injections of SOD-NPs in mice receiving OA surgery were effective in attenuating OA initiation and preventing its further progression. Mechanistically, SOD-NPs reduced ROS production and the synthesis of catabolic proteases in both articular cartilage and synovium. Hence, our work demonstrates the therapeutic potential of SOD-NPs and indicate that targeting synovium holds a great promise for OA therapy.

Cytosolic Delivery of Small Protein Scaffolds Enables Efficient Inhibition of Ras and Myc.

The ability to deliver small protein scaffolds intracellularly could enable the targeting and inhibition of many therapeutic targets that are not currently amenable to inhibition with small-molecule drugs. Here, we report the engineering of small protein scaffolds with anionic polypeptides (ApPs) to promote electrostatic interactions with positively charged nonviral lipid-based delivery systems. Proteins fused with ApPs are either complexed with off-the-shelf cationic lipids or encapsulated within ionizable lipid nanoparticles for highly efficient cytosolic delivery (up to 90%). The delivery of protein inhibitors is used to inhibit two common proto-oncogenes, Ras and Myc, in two cancer cell lines. This report demonstrates the feasibility of combining minimally engineered small protein scaffolds with tractable nanocarriers to inhibit intracellular proteins that are generally considered "undruggable" with current small molecule drugs and biologics.

Rapid Production of Bispecific Antibodies from Off-the-Shelf IgGs with High Yield and Purity.

Bispecific antibodies (BsAb) refer to a class of biomacromolecules that are capable of binding two antigens or epitopes simultaneously. This can elicit unique biological effects that cannot be achieved with either individual antibody or two unlinked antibodies. Bispecific antibodies have been used for targeting effector cells to tumor cells, preferential targeting of cells expressing two target biomarkers over cells expressing either target biomarker individually, or to couple two molecular targets on the same cell surface to trigger unique intracellular signaling pathways. Here, we present two related methods that enable direct, rapid assembly of bispecific antibodies from any two "off-the-shelf" Immunoglobulin G (IgG) antibodies, in as little as 1 day. Both workflows can be summarized into two steps: (1) attach a small photoreactive antibody binding domain (pAbBD) fused to SpyCatcher or SpyTag (peptide-protein partners derived from the S. pyogenes fibronectin-binding protein FbaB) to each component IgG, respectively; (2) assemble the BsAb through the spontaneous isopeptide bond formation that occurs between SpyTag and SpyCatcher. These approaches enable production of BsAbs from any two IgG molecules without the need to elucidate their amino acid sequences or genetically alter their structure. Binding assays and T cell-mediated cytolysis assays were performed to validate the binding and functional properties of Trastuzumab × Cetuximab BsAb and Cetuximab × OKT3 BsAb, respectively. This approach enables rapid, low-cost production of highly homogeneous tetravalent BsAbs in a modular fashion, presenting an opportunity to quickly evaluate antibody pairs in a BsAb format for unique or synergistic functionalities.

Superoxide Dismutase-Loaded Nanoparticles Attenuate Myocardial Ischemia-Reperfusion Injury and Protect Against Chronic Adverse Ventricular Remodeling.

Early revascularization is critical to reduce morbidity after myocardial infarction, although reperfusion incites additional oxidative injury. Superoxide dismutase (SOD) is an antioxidant that scavenges reactive oxygen species (ROS) but has low endogenous expression and rapid myocardial washout when administered exogenously. This study utilizes a novel nanoparticle carrier to improve exogeneous SOD retention while preserving enzyme function. Its role is assessed in preserving cardiac function after myocardial ischemia-reperfusion (I/R) injury. Here, nanoparticle-encapsulated SOD (NP-SOD) exhibits similar enzyme activity as free SOD, measured by ferricytochrome-c assay. In an in vitro I/R model, free and NP-SOD reduce active ROS, preserve mitochondrial integrity and improve cell viability compared to controls. In a rat in vivo I/R injury model, NP-encapsulation of fluorescent-tagged SOD improves intramyocardial retention after direct injection. Intramyocardial NP-SOD administration in vivo improves left ventricular contractility at 3-hours post-reperfusion by echocardiography and 4-weeks by echocardiography and invasive pressure-volume catheter analysis. These findings suggest that NP-SOD mitigates ROS damage in cardiac I/R injury in vitro and maximizes retention in vivo. NP-SOD further attenuates acute injury and protects against myocyte loss and chronic adverse ventricular remodeling, demonstrating potential for translating NP-SOD as a therapy to mitigate myocardial I/R injury.

Efficient Labeling of Native Human IgG by Proximity-Based Sortase-Mediated Isopeptide Ligation.

Antibody-drug conjugates (ADCs) have demonstrated great therapeutic potential due to their ability to target the delivery of potent cytotoxins. However, the heterogeneous nature of conventional drug conjugation strategies can affect the safety, efficacy, and stability of ADCs. Site-specific conjugations can resolve these issues, but often require genetic modification of Immunoglobulin G (IgG), which can impact yield or cost of production, or require undesirable chemical linkages. Here, we describe a near-traceless conjugation method that enables the efficient modification of native IgG, without the need for genetic engineering or glycan modification. This method utilizes engineered variants of sortase A to catalyze noncanonical isopeptide ligation. Sortase A was fused to an antibody-binding domain to improve ligation efficiency. Antibody labeling is limited to five lysine residues on the heavy chain and one on the light chain of human IgG1. The ADCs exhibit conserved antigen and Fc-receptor interactions, as well as potent cytolytic activity.

Phospholipase A2 inhibitor-loaded micellar nanoparticles attenuate inflammation and mitigate osteoarthritis progression.

Treating osteoarthritis (OA) remains a major clinical challenge. Despite recent advances in drug discovery and development, no disease-modifying drug for knee OA has emerged with any notable clinical success, in part, due to the lack of valid and responsive therapeutic targets and poor drug delivery within knee joints. In this work, we show that the amount of secretory phospholipase A2 (sPLA2) enzyme increases in the articular cartilage in human and mouse OA cartilage tissues. We hypothesize that the inhibition of sPLA2 activity may be an effective treatment strategy for OA. To develop an sPLA2-responsive and nanoparticle (NP)-based interventional platform for OA management, we incorporated an sPLA2 inhibitor (sPLA2i) into the phospholipid membrane of micelles. The engineered sPLA2i-loaded micellar NPs (sPLA2i-NPs) were able to penetrate deep into the cartilage matrix, prolong retention in the joint space, and mitigate OA progression. These findings suggest that sPLA2i-NPs can be promising therapeutic agents for OA treatment.

Targeting cartilage EGFR pathway for osteoarthritis treatment.

Osteoarthritis (OA) is a widespread joint disease for which there are no disease-modifying treatments. Previously, we found that mice with cartilage-specific epidermal growth factor receptor (EGFR) deficiency developed accelerated knee OA. To test whether the EGFR pathway can be targeted as a potential OA therapy, we constructed two cartilage-specific EGFR overactivation models in mice by overexpressing heparin binding EGF-like growth factor (HBEGF), an EGFR ligand. Compared to wild type, Col2-Cre HBEGF-overexpressing mice had persistently enlarged articular cartilage from adolescence, due to an expanded pool of chondroprogenitors with elevated proliferation ability, survival rate, and lubricant production. Adult Col2-Cre HBEGF-overexpressing mice and Aggrecan-CreER HBEGF-overexpressing mice were resistant to cartilage degeneration and other signs of OA after surgical destabilization of the medial meniscus (DMM). Treating mice with gefitinib, an EGFR inhibitor, abolished the protective action against OA in HBEGF-overexpressing mice. Polymeric micellar nanoparticles (NPs) conjugated with transforming growth factor-α (TGFα), a potent EGFR ligand, were stable and nontoxic and had long joint retention, high cartilage uptake, and penetration capabilities. Intra-articular delivery of TGFα-NPs effectively attenuated surgery-induced OA cartilage degeneration, subchondral bone plate sclerosis, and joint pain. Genetic or pharmacologic activation of EGFR revealed no obvious side effects in knee joints and major vital organs in mice. Together, our studies demonstrate the feasibility of using nanotechnology to target EGFR signaling for OA treatment.