Multiparametric Single-Vesicle Flow Cytometry Resolves Extracellular Vesicle Heterogeneity and Reveals Selective Regulation of Biogenesis and Cargo Distribution.

Mammalian cells release a heterogeneous array of extracellular vesicles (EVs) that contribute to intercellular communication by means of the cargo that they carry. To resolve EV heterogeneity and determine if cargo is partitioned into select EV populations, we developed a method named "EV Fingerprinting" that discerns distinct vesicle populations using dimensional reduction of multiparametric data collected by quantitative single-EV flow cytometry. EV populations were found to be discernible by a combination of membrane order and EV size, both of which were obtained through multiparametric analysis of fluorescent features from the lipophilic dye Di-8-ANEPPS incorporated into the lipid bilayer. Molecular perturbation of EV secretion and biogenesis through respective ablation of the small GTPase Rab27a and overexpression of the EV-associated tetraspanin CD63 revealed distinct and selective alterations in EV populations, as well as cargo distribution. While Rab27a disproportionately affects all small EV populations with high membrane order, the overexpression of CD63 selectively increased the production of one small EV population of intermediate membrane order. Multiplexing experiments subsequently revealed that EV cargos have a distinct, nonrandom distribution with CD63 and CD81 selectively partitioning into smaller vs larger EVs, respectively. These studies not only present a method to probe EV biogenesis but also reveal how the selective partitioning of cargo contributes to EV heterogeneity.

Nanoparticle Retinoic Acid-Inducible Gene I Agonist for Cancer Immunotherapy.

Pharmacological activation of the retinoic acid-inducible gene I (RIG-I) pathway holds promise for increasing tumor immunogenicity and improving the response to immune checkpoint inhibitors (ICIs). However, the potency and clinical efficacy of 5'-triphosphate RNA (3pRNA) agonists of RIG-I are hindered by multiple pharmacological barriers, including poor pharmacokinetics, nuclease degradation, and inefficient delivery to the cytosol where RIG-I is localized. Here, we address these challenges through the design and evaluation of ionizable lipid nanoparticles (LNPs) for the delivery of 3p-modified stem-loop RNAs (SLRs). Packaging of SLRs into LNPs (SLR-LNPs) yielded surface charge-neutral nanoparticles with a size of ∼100 nm that activated RIG-I signaling in vitro and in vivo. SLR-LNPs were safely administered to mice via both intratumoral and intravenous routes, resulting in RIG-I activation in the tumor microenvironment (TME) and the inhibition of tumor growth in mouse models of poorly immunogenic melanoma and breast cancer. Significantly, we found that systemic administration of SLR-LNPs reprogrammed the breast TME to enhance the infiltration of CD8+ and CD4+ T cells with antitumor function, resulting in enhanced response to αPD-1 ICI in an orthotopic EO771 model of triple-negative breast cancer. Therapeutic efficacy was further demonstrated in a metastatic B16.F10 melanoma model, with systemically administered SLR-LNPs significantly reducing lung metastatic burden compared to combined αPD-1 + αCTLA-4 ICI. Collectively, these studies have established SLR-LNPs as a translationally promising immunotherapeutic nanomedicine for potent and selective activation of RIG-I with the potential to enhance response to ICIs and other immunotherapeutic modalities.

STING-activating nanoparticles normalize the vascular-immune interface to potentiate cancer immunotherapy.

The tumor-associated vasculature imposes major structural and biochemical barriers to the infiltration of effector T cells and effective tumor control. Correlations between stimulator of interferon genes (STING) pathway activation and spontaneous T cell infiltration in human cancers led us to evaluate the effect of STING-activating nanoparticles (STANs), which are a polymersome-based platform for the delivery of a cyclic dinucleotide STING agonist, on the tumor vasculature and attendant effects on T cell infiltration and antitumor function. In multiple mouse tumor models, intravenous administration of STANs promoted vascular normalization, evidenced by improved vascular integrity, reduced tumor hypoxia, and increased endothelial cell expression of T cell adhesion molecules. STAN-mediated vascular reprogramming enhanced the infiltration, proliferation, and function of antitumor T cells and potentiated the response to immune checkpoint inhibitors and adoptive T cell therapy. We present STANs as a multimodal platform that activates and normalizes the tumor microenvironment to enhance T cell infiltration and function and augments responses to immunotherapy.

Nanoparticle STING Agonist Reprograms the Bone Marrow to an Antitumor Phenotype and Protects Against Bone Destruction.

When breast cancer metastasizes to bone, treatment options are limited. Failure to treat bone metastases is thought to be due to therapy-resistant features of the bone marrow microenvironment. Using a murine model of bone metastatic mammary carcinoma, we demonstrate that systemic delivery of polymer nanoparticles loaded with cyclic dinucleotide (CDN) agonists of stimulator of interferon genes (STING) inhibited tumor growth and bone destruction after 7 days of treatment. Each dose of STING-activating nanoparticles trafficked to the bone marrow compartment and was retained within the tumor microenvironment for over 24 hours, enhancing antitumor immunity through proinflammatory cytokine production and early T-cell activation. While acquired resistance mechanisms, including increased levels of immunosuppressive cytokines and the infiltration of regulatory T cells, ultimately limited antitumor efficacy after 2 weeks of treatment, bone protective effects remained. Overall, these studies demonstrate that STING pathway activation, here enabled using a nanomedicine approach to enhance CDN delivery to bone metastatic sites, can reprogram the immune contexture of the bone marrow to an antitumor phenotype that inhibits bone colonization of metastatic breast cancer cells and protects from tumor-mediated bone destruction. Significance: Bone metastases are difficult to treat due to the inaccessibility of the bone marrow compartment and the immunosuppressive microenvironment that protects resident stem cells. Packaging a STING agonist into a nanoparticle that enables systemic administration and drug accumulation at tumor sites overcomes both barriers to stymie metastatic breast cancer growth.

Amphiphilic Polyelectrolyte Graft Copolymers Enhance the Activity of Cyclic Dinucleotide STING Agonists.

Cyclic dinucleotide (CDN) agonists of stimulator of interferon genes (STING) hold great therapeutic potential, but their activity is hindered by poor drug-like properties that restrict cytosolic bioavailability. Here, this challenge is addressed through the synthesis and evaluation of a novel series of PEGMA-co-DEAEMA-co-BMA copolymers with pH-responsive, membrane-destabilizing activity to enhance intracellular delivery of the CDN, cGAMP. Copolymers are synthesized with PEGMA of two different molecular weights (300 and 950 Da) and over a range of PEG mass fraction and polymer molecular weight, and relationships between copolymer structure, self-assembly, endosomal escape, and cGAMP activity are elucidated. A subset of polymers that self-assembled into 50-800 nm nanoparticles is identified, which can be loaded with cGAMP via a simple mixing strategy, resulting in significantly enhanced immunostimulatory activity. Increased cGAMP activity is found to be highly correlated with the capacity of carriers to enhance intracellular CDN uptake and to promote endosomal destabilization, findings that establish efficient cytosolic delivery as a criterion for CDN carriers. Additionally, it is demonstrated that a lead CDN carrier formulation can enhance STING activation in vivo in a model of intratumoral immunotherapy. Collectively, these investigations demonstrate the utility of PEGMA-co-DEAEMA-co-BMA copolymers as carriers for CDNs and potentially other cytosolically-acting drug cargo.

Nanoparticle delivery improves the pharmacokinetic properties of cyclic dinucleotide STING agonists to open a therapeutic window for intravenous administration.

The stimulator of interferon genes (STING) pathway plays an important role in the immune surveillance of cancer and, accordingly, agonists of STING signaling have recently emerged as promising therapeutics for remodeling of the immunosuppressive tumor microenvironment (TME) and enhancing response rates to immune checkpoint inhibitors. 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP) is the endogenous ligand for STING, but is rapidly metabolized and poorly membrane permeable, restricting its use to intratumoral administration. Nanoencapsulation has been shown to allow for systemic administration of cGAMP and other cyclic dinucleotides (CDN), but little is known about how nanocarriers affect important pharmacological properties that impact the efficacy and safety of CDNs. Using STING-activating nanoparticles (STING-NPs) - a polymersome platform designed to enhance cGAMP delivery - we investigate the pharmacokinetic (PK)-pharmacodynamic (PD) relationships that underlie the ability of intravenously (i.v.) administered STING-NPs to induce STING activation and inhibit tumor growth. First, we demonstrate that nanoencapsulation improves the half-life of encapsulated cGAMP by 40-fold, allowing for sufficient accumulation of cGAMP in tumors and activation of the STING pathway in the TME as assessed by western blot analysis and gene expression profiling. Nanoparticle delivery also changes the biodistribution profile, resulting in increased cGAMP accumulation and STING activation in the liver and spleen, which we identify as dose limiting organs. As a consequence of STING activation in tumors, i.v. administered STING-NPs reprogram the TME towards a more immunogenic antitumor milieu, characterized by an influx of >20-fold more CD4+ and CD8+ T-cells. Consequently, STING-NPs increased response rates to αPD-L1 antibodies, resulting in significant improvements in median survival time in a B16-F10 melanoma model. Additionally, we confirmed STING-NP monotherapy in an additional melanoma (YUMM1.7) and breast adenocarcinoma (E0771) models leading to >50% and 80% reduction in tumor burden, respectively, and significant increases in median survival time. Collectively, this work provides an examination of the PK-PD relationship governing STING activation upon systemic delivery using STING-NPs, providing insight for future optimization for nanoparticle-based STING agonists and other immunomodulating nanomedicines.

Co-delivery of Peptide Neoantigens and Stimulator of Interferon Genes Agonists Enhances Response to Cancer Vaccines.

Cancer vaccines targeting patient-specific neoantigens have emerged as a promising strategy for improving responses to immune checkpoint blockade. However, neoantigenic peptides are poorly immunogenic and inept at stimulating CD8+ T cell responses, motivating a need for new vaccine technologies that enhance their immunogenicity. The stimulator of interferon genes (STING) pathway is an endogenous mechanism by which the innate immune system generates an immunological context for priming and mobilizing neoantigen-specific T cells. Owing to this critical role in tumor immune surveillance, a synthetic cancer nanovaccine platform (nanoSTING-vax) was developed that mimics immunogenic cancer cells in its capacity to efficiently promote co-delivery of peptide antigens and the STING agonist, cGAMP. The co-loading of cGAMP and peptides into pH-responsive, endosomolytic polymersomes promoted the coordinated delivery of both cGAMP and peptide antigens to the cytosol, thereby eliciting inflammatory cytokine production, co-stimulatory marker expression, and antigen cross-presentation. Consequently, nanoSTING-vax significantly enhanced CD8+ T cell responses to a range of peptide antigens. Therapeutic immunization with nanoSTING-vax, in combination with immune checkpoint blockade, inhibited tumor growth in multiple murine tumor models, even leading to complete tumor rejection and generation of durable antitumor immune memory. Collectively, this work establishes nanoSTING-vax as a versatile platform for enhancing immune responses to neoantigen-targeted cancer vaccines.

Concomitant Chemoradiation Therapy with Gold Nanoparticles and Platinum Drugs Co-Encapsulated in Liposomes.

A liposomal formulation of gold nanoparticles (GNPs) and carboplatin, named LipoGold, was produced with the staggered herringbone microfluidic method. The radiosensitizing potential of LipoGold and similar concentrations of non-liposomal GNPs, carboplatin and oxaliplatin was evaluated in vitro with the human colorectal cancer cell line HCT116 in a clonogenic assay. Progression of HCT116 tumor implanted subcutaneously in NU/NU mice was monitored after an irradiation of 10 Gy combined with either LipoGold, GNPs or carboplatin injected directly into the tumor by convection-enhanced delivery. Radiosensitization by GNPs alone or carboplatin alone was observed only at high concentrations of these compounds. Furthermore, low doses of carboplatin alone or a combination of carboplatin and GNPs did not engender radiosensitization. However, the same low doses of carboplatin and GNPs administered simultaneously by encapsulation in liposomal nanocarriers (LipoGold) led to radiosensitization and efficient control of cell proliferation. Our study shows that the radiosensitizing effect of a combination of carboplatin and GNPs is remarkably more efficient when both compounds are simultaneously delivered to the tumor cells using a liposomal carrier.

Potent STING activation stimulates immunogenic cell death to enhance antitumor immunity in neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is a childhood cancer for which new treatment options are needed. The success of immune checkpoint blockade in the treatment of adult solid tumors has prompted the exploration of immunotherapy in NB; however, clinical evidence indicates that the vast majority of NB patients do not respond to single-agent checkpoint inhibitors. This motivates a need for therapeutic strategies to increase NB tumor immunogenicity. The goal of this study was to evaluate a new immunotherapeutic strategy for NB based on potent activation of the stimulator of interferon genes (STING) pathway. METHODS: To promote STING activation in NB cells and tumors, we utilized STING-activating nanoparticles (STING-NPs) that are designed to mediate efficient cytosolic delivery of the endogenous STING ligand, 2'3'-cGAMP. We investigated tumor-intrinsic responses to STING activation in both MYCN-amplified and non-amplified NB cell lines, evaluating effects on STING signaling, apoptosis, and the induction of immunogenic cell death. The effects of intratumoral administration of STING-NPs on CD8+ T cell infiltration, tumor growth, and response to response to PD-L1 checkpoint blockade were evaluated in syngeneic models of MYCN-amplified and non-amplified NB. RESULTS: The efficient cytosolic delivery of 2'3'-cGAMP enabled by STING-NPs triggered tumor-intrinsic STING signaling effects in both MYCN-amplified and non-amplified NB cell lines, resulting in increased expression of interferon-stimulated genes and pro-inflammatory cytokines as well as NB cell death at concentrations 2000-fold to 10000-fold lower than free 2'3'-cGAMP. STING-mediated cell death in NB was associated with release or expression of several danger associated molecular patterns that are hallmarks of immunogenic cell death, which was further validated via cell-based vaccination and tumor challenge studies. Intratumoral administration of STING-NPs enhanced STING activation relative to free 2'3'-cGAMP in NB tumor models, converting poorly immunogenic tumors into tumoricidal and T cell-inflamed microenvironments and resulting in inhibition of tumor growth, increased survival, and induction of immunological memory that protected against tumor re-challenge. In a model of MYCN-amplified NB, STING-NPs generated an abscopal response that inhibited distal tumor growth and improved response to PD-L1 immune checkpoint blockade. CONCLUSIONS: We have demonstrated that activation of the STING pathway, here enabled by a nanomedicine approach, stimulates immunogenic cell death and remodels the tumor immune microenvironment to inhibit NB tumor growth and improve responses to immune checkpoint blockade, providing a multifaceted immunotherapeutic approach with potential to enhance immunotherapy outcomes in NB.

At the bench: Engineering the next generation of cancer vaccines.

Cancer vaccines hold promise as an immunotherapeutic modality based on their potential to generate tumor antigen-specific T cell responses and long-lived antitumor responses capable of combating metastatic disease and recurrence. However, cancer vaccines have historically failed to deliver significant therapeutic benefit in the clinic, which we maintain is due in part to drug delivery challenges that have limited vaccine immunogenicity and efficacy. In this review, we examine some of the known and putative failure mechanisms of common first-generation clinical cancer vaccines, and describe how the rational design of materials engineered for vaccine delivery and immunomodulation can address these shortcomings. First, we outline vaccine design principles for augmenting cellular immunity to tumor antigens and describe how well-engineered materials can improve vaccine efficacy, highlighting recent innovations in vaccine delivery technology that are primed for integration into neoantigen vaccine development pipelines. We also discuss the importance of sequencing, timing, and kinetics in mounting effective immune responses to cancer vaccines, and highlight examples of materials that potentiate antitumor immunity through spatiotemporal control of immunomodulation. Furthermore, we describe several engineering strategies for improving outcomes of in situ cancer vaccines, which leverage local, intratumoral delivery to stimulate systemic immunity. Finally, we highlight recent innovations leveraging nanotechnology for increasing the immunogenicity of the tumor microenvironment (TME), which is critical to enhancing tumor infiltration and function of T cells elicited in response to cancer vaccines. These immunoengineering strategies and tools complement ongoing advances in cancer vaccines as they reemerge as an important component of the immunotherapeutic armamentarium.

Characterization of a liposomal copper(II)-quercetin formulation suitable for parenteral use.

Quercetin (3,3',4',5,7-pentahydroxyflavone) is a naturally derived flavonoid that is commonly found in fruits and vegetables. There is mounting evidence to suggest that quercetin has potential anticancer effects and appears to interact synergistically when used in combination with approved chemotherapeutic agents such as irinotecan and cisplatin. Unfortunately, quercetin has shown limited clinical utility, partly due to low bioavailability related to its poor aqueous solutions (< 10 µg/mL). In this study, liposomal formulations of quercetin were developed by exploiting quercetin's ability to bind copper. Quercetin powder was added directly to pre-formed copper-containing liposomes (2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CHOL) (55:45 M ratio)). As a function of time and temperature, the formation of copper-quercetin was measured. Using this methodology, a final quercetin-to-lipid (mol:mol) ratio of 0.2 was achievable and solutions containing quercetin at concentrations of > 5 mg/mL were attained, representing at least a > 100-fold increase in apparent solubility. The resulting formulation was suitable for intravenous dosing with no overt toxicities when administered at doses of 50 mg/kg in mice. Pharmacokinetic studies demonstrated that the copper-quercetin formulations had an AUC0-24H of 8382.1 µg h/mL when administered to mice at 50 mg/kg. These studies suggested that quercetin (not copper-quercetin) dissociates from the liposomes after administration. The resulting formulation is suitable for further development and also serves as a proof-of-concept for formulating other flavonoids and flavonoid-like compounds. Given that quercetin exhibits an IC50 of >10 µM when tested against cancer cell lines, we believe that the utility of this novel quercetin formulation for cancer indications will ultimately be as a component of a combination product.

Correction: The efficiency of cytosolic drug delivery using pH-responsive endosomolytic polymers does not correlate with activation of the NLRP3 inflammasome.

Correction for 'The efficiency of cytosolic drug delivery using pH-responsive endosomolytic polymers does not correlate with activation of the NLRP3 inflammasome' by Jessalyn J. Baljon et al., Biomater. Sci., 2019, DOI: 10.1039/c8bm01643g.

The efficiency of cytosolic drug delivery using pH-responsive endosomolytic polymers does not correlate with activation of the NLRP3 inflammasome.

Inefficient cytosolic delivery has limited the development of many promising biomacromolecular drugs, a long-standing challenge that has prompted extensive development of drug carriers that facilitate endosomal escape. Although many such carriers have shown considerable promise for cytosolic delivery of a diversity of therapeutics, the rupture or destabilization of endo/lysosomal membranes has also been associated with activation of the inflammasome with attendant risk of inflammation and toxicity. In this study, we investigated relationships between pH-dependent membrane destabilization, cytosolic drug delivery, and inflammasome activation using a series of well-defined poly[(ethylene glycol)-block-[(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate)] copolymers of variable second block composition and pH-responsive properties. We found that polymers that demonstrated the most potent membrane-destabilizing activity at early endosomal pH values in an erythrocyte hemolysis assay were most efficient at delivery of siRNA, yet tended to be associated with the least amount of NOD-like related protein 3 (NLRP3) inflammasome activation. By contrast, polymers that displayed minimal hemolysis activity and poor siRNA knockdown, and instead mediated lysosomal rupture likely due to a proton sponge mechanism, strongly induced NLPR3 inflammasome activation in a caspase- and cathepsin-dependent manner. Collectively, these findings reinforce the importance of early endosomal escape in minimizing inflammasome activation and also demonstrate the ability to tune the degree inflammasome activation via control of polymer structure with potential implications for design of vaccine adjuvants and immunotherapeutics.

Liposomal formulations of carboplatin injected by convection-enhanced delivery increases the median survival time of F98 glioma bearing rats.

BACKGROUND: Effectiveness of chemotherapy for treating glioblastoma (GBM) brain tumors is hampered by the blood-brain barrier which limits the entry into the brain of most drugs from the blood. To bypass this barrier, convection-enhanced delivery (CED) was proposed to directly inject drugs in tumor. However, the benefit of CED may be hampered when drugs diffuse outside the tumor to then induce neurotoxicity. Encapsulation of drugs into liposome aims at increasing tumor cells specificity and reduces neurotoxicity. However, the most appropriate liposomal formulation to inject drugs into brain tumor by CED still remains to be determined. In this study, four liposomal carboplatin formulations were prepared and tested in vitro on F98 glioma cells and in Fischer rats carrying F98 tumor implanted in the brain. Impact of pegylation on liposomal surface and relevance of positive or negative charge were assessed. RESULTS: The cationic non-pegylated (L1) and pegylated (L2) liposomes greatly improved the toxicity of carboplatin in vitro compared to free carboplatin, whereas only a modest improvement and even a reduction of efficiency were measured with the anionic non-pegylated (L3) and the pegylated (L4) liposomes. Conversely, only the L4 liposome significantly increased the median survival time of Fisher rats implanted with the F98 tumor, compared to free carboplatin. Neurotoxicity assays performed with the empty L4' liposome showed that the lipid components of L4 were not toxic. These results suggest that the positive charge on liposomes L1 and L2, which is known to promote binding to cell membrane, facilitates carboplatin accumulation in cancer cells explaining their higher efficacy in vitro. Conversely, negatively charged and pegylated liposome (L4) seems to diffuse over a larger distance in the tumor, and consequently significantly increased the median survival time of the animals. CONCLUSIONS: Selection of the best liposomal formulation based on in vitro studies or animal model can result in contradictory conclusions. The negatively charged and pegylated liposome (L4) which was the less efficient formulation in vitro showed the best therapeutic effect in animal model of GBM. These results support that relevant animal model of GBM must be considered to determine the optimal physicochemical properties of liposomal formulations.

Copper-CX-5461: A novel liposomal formulation for a small molecule rRNA synthesis inhibitor.

CX-5461 is currently in Phase I/II clinical trials for advanced hematologic malignancies and triple negative or BRCA-deficient breast cancer. The compound is currently administered to patients intravenously (i.v.) at low pH (3.5) due to solubility challenges. Reliance of low pH to enhance solubility of CX-5461 can adversely impact pharmacokinetics, biodistribution and therapeutic potential. We have addressed this solubility issue through a formulation method that relies on the interactions between CX-5461 and copper. Copper binds CX-5461 through the nitrogens of the pyrazine ring. Here, we describe synthesizing this copper-complexed CX-5461 (Cu(CX-5461)) within liposomes. CX-5461 was added to copper-containing liposomes and incubated at 60 °C for 30 min. The pharmacokinetics of CX-5461 was assessed in mice following a single i.v. injection at 30 mg/kg. Efficacy studies were completed in multiple subcutaneous mouse xenografts as well as in a bone marrow engraftment model of acute myeloid leukemia (AML). The novel Cu(CX-5461) formulation was stable at pH 7.4 and exhibited increased plasma circulation longevity, increasing the total exposure to CX5461 by an order of magnitude. Cu(CX-5461) was more active than CX-5461 in AML models in vivo. In HCT116-B46 and Capan-1 solid tumour models that are BRCA-deficient, the Cu(CX-5461) formulation engendered activity that was comparable to that of the low pH CX-5461 formulation. We have generated the first Cu(CX-5461) formulation suitable for i.v. administration that is more efficacious than the existing low-pH formulation in pre-clinical models of AML. The Cu(CX-5461) formulation may serve as an alternative formulation for CX-5461 in BRCA-deficient cancers.

A Perspective - can copper complexes be developed as a novel class of therapeutics?

Although copper-ligand complexes appear to be promising as a new class of therapeutics, other than the family of copper(ii) coordination compounds referred to as casiopeínas these compounds have yet to reach the clinic for human use. The pharmaceutical challenges associated with developing copper-based therapeutics will be presented in this article along with a discussion of the potential for high-throughput chemistry, computer-aided drug design, and nanotechnology to address the development of this important class of drug candidates.

Copper (II) complexes of bidentate ligands exhibit potent anti-cancer activity regardless of platinum sensitivity status.

Insensitivity to platinum, either through inherent or acquired resistance, is a major clinical problem in the treatment of many solid tumors. Here, we explored the therapeutic potential of diethyldithiocarbamate (DDC), pyrithione (Pyr), plumbagin (Plum), 8-hydroxyquinoline (8-HQ), clioquinol (CQ) copper complexes in a panel of cancer cell lines that differ in their sensitivity to platins (cisplatin/carboplatin) using a high-content imaging system. Our data suggest that the copper complexes were effective against both platinum sensitive (IC50 ~ 1 µM platinum) and insensitive (IC50 > 5 µM platinum) cell lines. Furthermore, copper complexes of DDC, Pyr and 8-HQ had greater therapeutic activity compared to the copper-free ligands in all cell lines; whereas the copper-dependent activities of Plum and CQ were cell-line specific. Four of the copper complexes (Cu(DDC)2, Cu(Pyr)2, Cu(Plum)2 and Cu(8-HQ)2) showed IC50 values less than that of cisplatin in all tested cell lines. The complex copper DDC (Cu(DDC)2) was selected for in vivo evaluation due to its low nano-molar range activity in vitro and the availability of an injectable liposomal formulation. Liposomal (Cu(DDC)2) was tested in a fast-growing platinum-resistant A2780-CP ovarian xenograft model and was found to achieve a statistically significant reduction (50%; p < 0.05) in tumour size. This work supports the potential use of copper-based therapeutics to treat cancers that are insensitive to platinum drugs.

Development and optimization of an injectable formulation of copper diethyldithiocarbamate, an active anticancer agent.

Copper diethyldithiocarbamate (Cu(DDC)2) is the active anticancer agent generated when disulfiram (DSF) is provided in the presence of copper. To date, research directed toward repurposing DSF as an anticancer drug has focused on administration of DSF and copper in combination, efforts that have proven unsuccessful in clinical trials. This is likely due to the inability to form Cu(DDC)2 at relevant concentrations in regions of tumor growth. Little effort has been directed toward the development of Cu(DDC)2 because of the inherent aqueous insolubility of the complex. Here, we describe an injectable Cu(DDC)2 formulation prepared through a method that involves synthesis of Cu(DDC)2 inside the aqueous core of liposomes. Convection-enhanced delivery of a Cu(DDC)2 formulation prepared using 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol liposomes into a rat model of F98 glioma engendered a 25% increase in median survival time relative to vehicle-treated animals. In a murine subcutaneous MV-4-11 model, treatment resulted in a 45% reduction in tumor burden when compared to controls. Pharmacokinetic studies indicated that the Cu(DDC)2 was rapidly eliminated after intravenous administration while the liposomes remained in circulation. To test whether liposomal lipid composition could increase Cu(DDC)2 circulation lifetime, a number of different formulations were evaluated. Studies demonstrated that liposomes composed of DSPC and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-(carboxy[polyethylene glycol]-2000) (95:5) enhanced Cu(DDC)2 concentrations in the circulation as reflected by a 4.2-fold increase in plasma AUC(0-∞) relative to the DSPC/cholesterol formulation. The anticancer activity of this Cu(DDC)2 formulation was subsequently evaluated in the MV-4-11 model. At its maximum tolerated dose, this formulation exhibited comparable activity to the DSPC/cholesterol formulation. This is the first report demonstrating the therapeutic effects of an injectable Cu(DDC)2 formulation in vivo.

PRCosomes: pretty reactive complexes formed in liposomes.

As a tribute to Pieter R. Cullis, this manuscript identifies a liposomal formulation that bears his initials: the PRCosomes. "Pretty" Reactive Complexes within liposomes were observed, while the senior author of this manuscript completed his Ph.D. thesis under Pieter's supervision. The dye (safranine) was used as a tool to measure the magnitude of the transmembrane gradient generated with liposomes. The dye's redistribution is easily detected by eye and correlates with >98% encapsulation of the dye. This observation became the basis from which remote drug loading methods developed. Remote loading methodology involves the addition of drugs to pre-formed liposomes with a transmembrane gradient, which results in drug redistribution to the liposome interior. Doxorubicin, as an example drug candidate, complexes manganese trapped within the liposome. A color change accompanied drug encapsulation as the solution went from an orange to purple. This manuscript reviews and adds a novel perspective on the use of metal complexation reactions to prepare PRCosomes. The technology described provides a versatile method to form metal-drug complexed within liposomes. The purpose of this work is to differentiation between drug candidate loading that is caused by metal-drug complexation and loading driven by formation of a pH gradient.

Nanoscale Reaction Vessels Designed for Synthesis of Copper-Drug Complexes Suitable for Preclinical Development.

The development of copper-drug complexes (CDCs) is hindered due to their very poor aqueous solubility. Diethyldithiocarbamate (DDC) is the primary metabolite of disulfiram, an approved drug for alcoholism that is being repurposed for cancer. The anticancer activity of DDC is dependent on complexation with copper to form copper bis-diethyldithiocarbamate (Cu(DDC)2), a highly insoluble complex that has not been possible to develop for indications requiring parenteral administration. We have resolved this issue by synthesizing Cu(DDC)2 inside liposomes. DDC crosses the liposomal lipid bilayer, reacting with the entrapped copper; a reaction that can be observed through a colour change as the solution goes from a light blue to dark brown. This method is successfully applied to other CDCs including the anti-parasitic drug clioquinol, the natural product quercetin and the novel targeted agent CX-5461. Our method provides a simple, transformative solution enabling, for the first time, the development of CDCs as viable candidate anticancer drugs; drugs that would represent a brand new class of therapeutics for cancer patients.