Molecular bottlebrush prodrugs as mono- and triplex combination therapies for multiple myeloma.

Cancer therapies often have narrow therapeutic indexes and involve potentially suboptimal combinations due to the dissimilar physical properties of drug molecules. Nanomedicine platforms could address these challenges, but it remains unclear whether synergistic free-drug ratios translate to nanocarriers and whether nanocarriers with multiple drugs outperform mixtures of single-drug nanocarriers at the same dose. Here we report a bottlebrush prodrug (BPD) platform designed to answer these questions in the context of multiple myeloma therapy. We show that proteasome inhibitor (bortezomib)-based BPD monotherapy slows tumour progression in vivo and that mixtures of bortezomib, pomalidomide and dexamethasone BPDs exhibit in vitro synergistic, additive or antagonistic patterns distinct from their corresponding free-drug counterparts. BPDs carrying a statistical mixture of three drugs in a synergistic ratio outperform the free-drug combination at the same ratio as well as a mixture of single-drug BPDs in the same ratio. Our results address unanswered questions in the field of nanomedicine, offering design principles for combination nanomedicines and strategies for improving current front-line monotherapies and combination therapies for multiple myeloma.

Author Correction: Nanoparticle conjugates of a highly potent toxin enhance safety and circumvent platinum resistance in ovarian cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Antibody-targeting of ultra-small nanoparticles enhances imaging sensitivity and enables longitudinal tracking of multiple myeloma.

Monitoring malignant progression and disease recurrence post-therapy are central challenges to improving the outcomes of patients with multiple myeloma (MM). Whereas current detection methods that rely upon bone marrow examination allow for precise monitoring of minimal residual disease and can help to elucidate clonal evolution, they do not take into account the spatial heterogeneity of the tumor microenvironment. As such, they are uninformative as to the localization of malignant plasma cells and may lead to false negative results. With respect to the latter challenge, clinically-available imaging agents are neither sufficiently sensitive nor specific enough to detect minute plasma cell populations. Here, we sought to explore methods by which to improve detection of MM cells within their natural bone marrow environment, using whole-animal magnetic resonance imaging to longitudinally monitor early-stage disease as well as to enhance tumor detection after systemic therapy. We conducted a proof-of-concept study to demonstrate that ultra-small (<5 nm) gadolinium-containing nanoparticles bound to full-length antibodies against the B-cell maturation antigen (BCMA) exhibit rapid tumor uptake followed by renal clearance, improving the signal-to-noise ratio for MM detection beyond levels that are currently afforded by other FDA-approved clinical imaging modalities. We anticipate that when combined with bone marrow or blood biopsy, such imaging constructs could help to augment the effective management of patients with MM.

Triply Loaded Nitroxide Brush-Arm Star Polymers Enable Metal-Free Millimetric Tumor Detection by Magnetic Resonance Imaging.

Nitroxides occupy a privileged position among plausible metal-free magnetic resonance imaging (MRI) contrast agents (CAs) due to their inherently low-toxicity profiles; nevertheless, their translational development has been hindered by a lack of appropriate contrast sensitivity. Nanostructured materials with high nitroxide densities, where each individual nitroxide within a macromolecular construct contributes to the image contrast, could address this limitation, but the synthesis of such materials remains challenging. Here, we report a modular and scalable synthetic approach to nitroxide-based brush-arm star polymer (BASP) organic radical CAs (ORCAs) with high nitroxide loadings. The optimized ∼30 nm diameter "BASP-ORCA3" displays outstanding T2 sensitivity with a very high molecular transverse relaxivity ( r2 > 1000 mM-1 s-1). BASP-ORCA3 further exhibits excellent stability in vivo, no acute toxicity, and highly desirable pharmacokinetic and biodistribution profiles for longitudinal detection of tumors by MRI. When injected intravenously into mice bearing subcutaneous plasmacytomas, BASP-ORCA3 affords distinct in vivo visualization of tumors on translationally relevant time scales. Leveraging its high sensitivity, BASP-ORCA3 enables efficient mapping of tumor necrosis, which is an important biomarker to predict therapeutic outcomes. Moreover, BASP-ORCA3 allows for detection of millimetric tumor implants in a disseminated murine model of advanced-stage human ovarian cancer that possess genetic, histological, and vascular characteristics that are similar to those seen in patients. This work establishes BASP-ORCA3 as a promising metal-free spin contrast agent for MRI.

Targeting the human MUC1-C oncoprotein with an antibody-drug conjugate.

Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed on the surface of diverse human carcinomas and is an attractive target for the development of mAb-based therapeutics. However, attempts at targeting the shed MUC1 N-terminal subunit have been unsuccessful. We report here the generation of mAb 3D1 against the nonshed oncogenic MUC1 C-terminal (MUC1-C) subunit. We show that mAb 3D1 binds with low nM affinity to the MUC1-C extracellular domain at the restricted α3 helix. mAb 3D1 reactivity is selective for MUC1-C-expressing human cancer cell lines and primary cancer cells. Internalization of mAb 3D1 into cancer cells further supported the conjugation of mAb 3D1 to monomethyl auristatin E (MMAE). The mAb 3D1-MMAE antibody-drug conjugate (ADC) (a) kills MUC1-C-positive cells in vitro, (b) is nontoxic in MUC1-transgenic (MUC1.Tg) mice, and (c) is active against human HCC827 lung tumor xenografts. Humanized mAb (humAb) 3D1 conjugated to MMAE also exhibited antitumor activity in (a) MUC1.Tg mice harboring syngeneic MC-38/MUC1 tumors, (b) nude mice bearing human ZR-75-1 breast tumors, and (c) NCG mice engrafted with a patient-derived triple-negative breast cancer. These findings and the absence of associated toxicities support clinical development of humAb 3D1-MMAE ADCs as a therapeutic for the many cancers with MUC1-C overexpression.

Advancements in Nanomedicine for Multiple Myeloma.

In the past decades, considerable progress has been made in our understanding and treatment of multiple myeloma. Several challenges remain including our abilities to longitudinally image tumor responses to treatment, to combine various therapeutic agents with different mechanisms of action but with overlapping toxicities, and to efficiently harness the power of the immune system to augment remission and/or to induce permanent cures. Nanomedicine may help to address many of these outstanding issues, affording novel diagnostic capabilities and offering disruptive technologies that promise to revolutionize treatment. Here, we review recent developments and the future of nanomedicine for multiple myeloma, highlighting new considerations in nanoparticle designs that may help to augment active targeting, to facilitate longitudinal imaging, and to improve drug delivery.

Differences in Nanoparticle Uptake in Transplanted and Autochthonous Models of Pancreatic Cancer.

Human pancreatic ductal adenocarcinoma (PDAC) contains a distinctively dense stroma that limits the accessibility of anticancer drugs, contributing to its poor overall prognosis. Nanoparticles can enhance drug delivery and retention in pancreatic tumors and have been utilized clinically for their treatment. In preclinical studies, various mouse models differentially recapitulate the microenvironmental features of human PDAC. Here, we demonstrate that through utilization of different organic cosolvents and by doping of a homopolymer of poly(ε-caprolactone), a diblock copolymer composition of poly(ethylene oxide)- block-poly(ε-caprolactone) may be utilized to generate biodegradable and nanoscale micelles with different physical properties. Noninvasive optical imaging was employed to examine the pharmacology and biodistribution of these various nanoparticle formulations in both allografted and autochthonous mouse models of PDAC. In contrast to the results reported with transplanted tumors, spherical micelles as large as 300 nm in diameter were found to extravasate in the autochthonous model, reaching a distance of approximately 20 µm from the nearest tumor cell clusters. A lipophilic platinum(IV) prodrug of oxaliplatin was further able to achieve a ∼7-fold higher peak accumulation and a ∼50-fold increase in its retention half-life in pancreatic tumors when delivered with 100 nm long worm-like micelles as when compared to the free drug formulation of oxaliplatin. Through further engineering of nanoparticle properties, as well as by widespread adoption of the autochthonous tumor model for preclinical testing, future therapeutic formulations may further enhance the targeting and penetration of anticancer agents to improve survival outcomes in PDAC.

Publisher Correction: Nanoparticle conjugates of a highly potent toxin enhance safety and circumvent platinum resistance in ovarian cancer.

The original version of this Article contained an error in the spelling of the author Yingjie Yu, which was incorrectly given as Yu Yingjie. Furthermore, in Figure 3a, the labels 'MD | p < 0.05' incorrectly read 'MD | p > 0.05'. These errors have now been corrected in both the PDF and HTML versions of the Article.

Nanoparticle conjugates of a highly potent toxin enhance safety and circumvent platinum resistance in ovarian cancer.

Advanced-stage epithelial ovarian cancers are amongst the most difficult to treat tumors and have proven to be refractory to most cytotoxic, molecularly targeted, or immunotherapeutic approaches. Here, we report that nanoparticle-drug conjugates (NDCs) of monomethyl auristatin E (MMAE) significantly increase loading on a per-vehicle basis as compared to antibody-drug conjugates (ADCs). Their intraperitoneal administration enabled triggered release of the active MMAE toxin to inhibit tumor growth and to extend animal survival to >90 days in a cell-line xenograft model of disseminated ovarian cancer. In a patient-derived xenograft model of advanced-stage and platinum-resistant ovarian cancer, an MMAE-based NDC doubled the duration of tumor growth inhibition as compared to cisplatin. NDCs of highly potent toxins thus introduce a translatable platform that may be exploited to maximize the safety and efficacy of cytotoxic chemotherapies, combining the best features of ADCs with those of nanoparticle-based therapeutics.

NIR-emissive PEG-b-TCL micelles for breast tumor imaging and minimally invasive pharmacokinetic analysis.

Motivated by the goal of developing a fully biodegradable optical contrast agent with translational clinical potential, a nanoparticle delivery vehicle was generated from the self-assembly of poly(ethylene-glycol)-block-poly(trimethylene carbonate-co-caprolactone) (PEG-b-TCL) copolymers. Cryogenic transmission electron microscopy verified that PEG-b-TCL-based micelles were formed at low solution temperatures (∼38 °C). Detailed spectroscopic experiments validated facile loading of large quantities of the high emission dipole strength, tris(porphyrin)-based fluorophore PZn3 within their cores, and the micelles displayed negligible in vitro and in vivo toxicities in model systems. The pharmacokinetics and biodistribution of PZn3-loaded PEG-b-TCL-based micelles injected intravenously were determined via ex vivo near-infrared (NIR) imaging of PZn3 emission in microcapillary tubes containing minute quantities of blood, to establish a novel method for minimally invasive pharmacokinetic monitoring. The in vivo circulatory half-life of the PEG-b-TCL-based micelles was found to be ∼19.6 h. Additionally, longitudinal in vivo imaging of orthotopically transplanted breast tumors enabled determination of micelle biodistribution that correlated to ex vivo imaging results, demonstrating accumulation predominantly within the tumors and livers of mice. The PEG-b-TCL-based micelles quickly extravasated within 4T1 orthotopic mammary carcinomas, exhibiting peak accumulation at ∼48 h following intravenous tail-vein injection. In summary, PEG-b-TCL-based micelles demonstrated favorable characteristics for further development as in vivo optical contrast agents for minimally invasive imaging of breast tumors.

Early tumor detection afforded by in vivo imaging of near-infrared II fluorescence.

Cell-intrinsic reporters such as luciferase (LUC) and red fluorescent protein (RFP) have been commonly utilized in preclinical studies to image tumor growth and to monitor therapeutic responses. While extrinsic reporters that emit near infrared I (NIR-I: 650-950 nm) or near-infrared II (NIR-II: 1000-1700 nm) optical signals have enabled minimization of tissue autofluorescence and light scattering, it has remained unclear as to whether their use has afforded more accurate tumor imaging in small animals. Here, we developed a novel optical imaging construct comprised of rare earth lanthanide nanoparticles coated with biodegradable diblock copolymers and doped with organic fluorophores, generating NIR-I and NIR-II emissive bands upon optical excitation. Simultaneous injection of multiple spectrally-unique nanoparticles into mice bearing tumor implants established via intraperitoneal dissemination of LUC+/RFP+ OVCAR-8 ovarian cancer cells enabled direct comparisons of imaging with extrinsic vs. intrinsic reporters, NIR-II vs. NIR-I signals, as well as targeted vs. untargeted exogenous contrast agents in the same animal and over time. We discovered that in vivo optical imaging at NIR-II wavelengths facilitates more accurate detection of smaller and earlier tumor deposits, offering enhanced sensitivity, improved spatial contrast, and increased depths of tissue penetration as compared to imaging with visible or NIR-I fluorescent agents. Our work further highlights the hitherto underappreciated enhancements in tumor accumulation that may be achieved with intraperitoneal as opposed to intravenous administration of nanoparticles. Lastly, we found discrepancies in the fidelity of tumor uptake that could be obtained by utilizing small molecules for in vivo as opposed to in vitro targeting of nanoparticles to disseminated tumors.

Maximizing Synergistic Activity When Combining RNAi and Platinum-Based Anticancer Agents.

RNAi approaches have been widely combined with platinum-based anticancer agents to elucidate cellular responses and to target gene products that mediate acquired resistance. Recent work has demonstrated that platination of siRNA prior to transfection may negatively influence RNAi efficiency based on the position and sequence of its guanosine nucleosides. Here, we used detailed spectroscopic characterization to demonstrate rapid formation of Pt-guanosine adducts within 30 min after coincubation of oxaliplatin [OxaPt(II)] or cisplatin [CisPt(II)] with either guanosine monophosphate or B-cell lymphoma 2 (BCL-2) siRNA. After 3 h of exposure to these platinum(II) agents, >50% of BCL-2 siRNA transcripts were platinated and unable to effectively suppress mRNA levels. Platinum(IV) analogues [OxaPt(IV) or CisPt(IV)] did not form Pt-siRNA adducts but did display decreased in vitro uptake and reduced potency. To overcome these challenges, we utilized biodegradable methoxyl-poly(ethylene glycol)-block-poly(ε-caprolactone)-block-poly(l-lysine) (mPEG-b-PCL-b-PLL) to generate self-assembled micelles that covalently conjugated OxaPt(IV) and/or electrostatically complexed siRNA. We then compared multiple strategies by which to combine BCL-2 siRNA with either OxaPt(II) or OxaPt(IV). Overall, we determined that the concentrations of siRNA (nM) and platinum(II)-based anticancer agents (µM) that are typically used for in vitro experiments led to rapid Pt-siRNA adduct formation and ineffective RNAi. Coincorporation of BCL-2 siRNA and platinum(IV) analogues in a single micelle enabled maximal suppression of BCL-2 mRNA levels (to <10% of baseline), augmented the intracellular levels of platinum (by ∼4×) and the numbers of resultant Pt-DNA adducts (by >5×), increased the cellular fractions that underwent apoptosis (by ∼4×), and enhanced the in vitro antiproliferative activity of the corresponding platinum(II) agent (by 10-100×, depending on the cancer cell line). When combining RNAi and platinum-based anticancer agents, this generalizable strategy may be adopted to maximize synergy during screening or for therapeutic delivery.

Aqueous self-assembly of poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) copolymers: disparate diblock copolymer compositions give rise to nano- and meso-scale bilayered vesicles.

Nanoparticles formed from diblock copolymers of FDA approved PEO and PCL have generated considerable interest as in vivo drug delivery vehicles. Herein, we report the synthesis of the most extensive family PEO-b-PCL copolymers that vary over the largest range of number-average molecular weights (Mn: 3.6-57k), PEO weight fractions (fPEO: 0.08-0.33), and PEO chain lengths (0.75-5.8k) reported to date. These polymers were synthesized in order to establish the full range of aqueous phase behaviours of these diblock copolymers and to specifically identify formulations that were able to generate bilayered vesicles (polymersomes). Cryogenic transmission electron microscopy (cryo-TEM) was utilized in order to visualize the morphology of these structures upon aqueous self-assembly of dry polymer films. Nanoscale polymersomes were formed from PEO-b-PCL copolymers over a wide range of PEO weight fractions (fPEO: 0.14-0.27) and PEO molecular weights (0.75-3.8k) after extrusion of aqueous suspensions. Comparative morphology diagrams, which describe the nature of self-assembled structures as a function of diblock copolymer molecular weight and PEO weight fraction, show that in contrast to micron-scale polymersomes, which form only from a limited range of PEO-b-PCL diblock copolymer compositions, a multiplicity of PEO-b-PCL diblock copolymer compositions are able to give rise to nanoscale vesicles. These data underscore that PEO-b-PCL compositions that spontaneously form micron-sized polymersomes, as well as those that have previously been reported to form polymersomes via a cosolvent fabrication system, provide only limited insights into the distribution of PEO-b-PCL diblocks that give rise to nanoscale vesicles. The broad range of polymersome-forming PEO-b-PCL compositions described herein suggest the ability to construct extensive families of nanoscale vesicles of varied bilayer thickness, providing the ability to tune the timescales of vesicle degradation and encapsulant release based on the intended in vivo application.

Leuko-polymersomes.

Polymersomes are vesicles whose membranes are comprised of self-assembled amphiphilic block co-polymers. Synthetic control of block co-polymer chemistry provides an advantageous diversity of polymersome functions, ranging from tunable materials strength, superior encaspulation of hydrophobic and hydrophilic drugs and optical dyes, and facile functionalization. We have exploited polymersome tunability to make leuko-polymersomes: polymersomes with the adhesive properties of leukocytes. By functionalizing the terminal groups on the outer shell of the vesicle with biotin, we have used modular avidin-biotin chemistry to attach adhesion ligands that mimic the two critical adhesion pathways that leukocytes utilize to achieve adhesion in the fast fluid flow of blood vessels--selectins and integrins. We demonstrate that adhesion is specific and is supported at hydrodynamic flow rates at which leukocytes adhere. We envision the use of such particles for monitoring or treating inflammation, cancer and cardiovascular disease.

Polymersomes: a new multi-functional tool for cancer diagnosis and therapy.

Nanoparticles are being developed as delivery vehicles for therapeutic pharmaceuticals and contrast imaging agents. Polymersomes (mesoscopic polymer vesicles) possess a number of attractive biomaterial properties that make them ideal for these applications. Synthetic control over block copolymer chemistry enables tunable design of polymersome material properties. The polymersome architecture, with its large hydrophilic reservoir and its thick hydrophobic lamellar membrane, provides significant storage capacity for both water soluble and insoluble substances (such as drugs and imaging probes). Further, the brush-like architecture of the polymersome outer shell can potentially increase biocompatibility and blood circulation times. A further recent advance is the development of multi-functional polymersomes that carry pharmaceuticals and imaging agents simultaneously. The ability to conjugate biologically active ligands to the brush surface provides a further means for targeted therapy and imaging. Hence, polymersomes hold enormous potential as nanostructured biomaterials for future in vivo drug delivery and diagnostic imaging applications.

Near-infrared optical imaging of B16 melanoma cells via low-density lipoprotein-mediated uptake and delivery of high emission dipole strength tris[(porphinato)zinc(II)] fluorophores.

Meso-to-meso ethyne-bridged tris[(porphinato)zinc(II)] (PZn(3)) near-infrared (NIR) fluorophores (lambda(em)(max) approximately 800 nm) can be rendered sufficiently amphiphilic to enable their facile incorporation into the hydrophobic core of the apo form of low-density lipoprotein (apo-LDL). These NIR fluorophores are notable in that they manifest low energy excited states polarized exclusively along the long axis of the supermolecule, broad spectral coverage of the visible and high energy NIR spectral domains, intense S(0)-->S(1) transition moments, and comparably large S(1)-->S(0) emission dipole strengths. The reconstituted LDL(PZn(3)) proteins can be used to deliver rapidly hundreds of copies of PZn(3) to a given murine B16 melanoma cell via LDL receptor-mediated endocytosis. PZn(3)-based NIRFs and their corresponding LDL(PZn(3)) proteins have been shown to display minimal cytotoxicity. Confocal NIR fluorescence microscopy evinces that B16 cells can be imaged at very low doses (approximately nM) of NIRF. The highly attractive photophysical properties of PZn(3) and closely related chromophores, coupled with their lack of toxicity and compatibility with uptake into apo-LDL and subsequent rapid delivery to B16 cells via LDLr-mediated endocytosis, suggest the potential utility of this platform for NIR optical imaging of cancer cells in vivo.

Near-infrared-emissive polymersomes: self-assembled soft matter for in vivo optical imaging.

We demonstrate that synthetic soft materials can extend the utility of natural vesicles, from predominantly hydrophilic reservoirs to functional colloidal carriers that facilitate the biomedical application of large aqueous-insoluble compounds. Near-infrared (NIR)-emissive polymersomes (50-nm- to 50-microm-diameter polymer vesicles) were generated through cooperative self assembly of amphiphilic diblock copolymers and conjugated multi(porphyrin)-based NIR fluorophores (NIRFs). When compared with natural vesicles comprised of phospholipids, polymersomes were uniquely capable of incorporating and uniformly distributing numerous large hydrophobic NIRFs exclusively in their lamellar membranes. Within these sequestered compartments, long polymer chains regulate the mean fluorophore-fluorophore interspatial separation as well as the fluorophore-localized electronic environment. Porphyrin-based NIRFs manifest photophysical properties within the polymersomal matrix akin to those established for these high-emission dipole strength fluorophores in organic solvents, thereby yielding uniquely emissive vesicles. Furthermore, the total fluorescence emanating from the assemblies gives rise to a localized optical signal of sufficient intensity to penetrate through the dense tumor tissue of a live animal. Robust NIR-emissive polymersomes thus define a soft matter platform with exceptional potential to facilitate deep-tissue fluorescence-based imaging for in vivo diagnostic and drug-delivery applications.