Ferroptosis is a form of cell death that results from the catastrophic accumulation of lipid reactive oxygen species (ROS). Oncogenic signaling elevates lipid ROS production in many tumor types and ...is counteracted by metabolites that are derived from the amino acid cysteine. In this work, we show that the import of oxidized cysteine (cystine) via system x
is a critical dependency of pancreatic ductal adenocarcinoma (PDAC), which is a leading cause of cancer mortality. PDAC cells used cysteine to synthesize glutathione and coenzyme A, which, together, down-regulated ferroptosis. Studying genetically engineered mice, we found that the deletion of a system x
subunit,
, induced tumor-selective ferroptosis and inhibited PDAC growth. This was replicated through the administration of cyst(e)inase, a drug that depletes cysteine and cystine, demonstrating a translatable means to induce ferroptosis in PDAC.
When nanoparticles interact with their environment, the nature of that interaction is governed largely by the properties of its outermost surface layer. Here, we exploit the exceptional properties of ...a common disaccharide, trehalose, which is well-known for its unique biological stabilization effects. To this end, we have developed a synthetic procedure that readily affords a polymer of this disaccharide, poly(methacrylamidotrehalose) or “poly(trehalose)” and diblock copolycations containing this polymer with 51 repeat units chain extended with aminoethylmethacrylamide (AEMA) at three degrees of polymerization (n = 34, 65, and 84). Two series of experiments were conducted to study these diblock copolymers in detail and to compare their properties to two control polymers PEG-P(AEMA) and P(AEMA). First, we demonstrate that the poly(trehalose) coating ensures colloidal stability of polyplexes containing siRNA in the presence of high salt concentrations and serum proteins. Poly(trehalose) retains the ability of trehalose to lower the phase transition energy associated with water freezing and can protect siRNA polyplexes during freeze-drying, allowing complete nanoparticle resuspension without loss of biological function. Second, we show that siRNA polyplexes coated with poly(trehalose) have exceptional cellular internalization into glioblastoma cells that proceeds with zero-order kinetics. Moreover, the amount of siRNA delivered by poly(trehalose) block copolycations can be controlled by the siRNA concentration in cell culture media. Using confocal microscopy we show that trehalose-coated polyplexes undergo active trafficking in cytoplasm upon internalization and significant siRNA-induced target gene down-regulation was achieved with an IC50 of 19 nM. These findings coupled with a negligible cytotoxicity suggests that poly(trehalose) has the potential to serve as an important component of therapeutic nanoparticle formulations of nucleic acids and has great promise to be extended as a new coating for other nanobased technologies and macromolecules, in particular, those related to nanomedicine applications.
Chemoselective oxime formation between aminooxy end-functional polymers and levulinyl-modified proteins is an attractive method to prepare well-defined bioconjugates. We demonstrate the synthesis of ...Boc-protected aminooxy initiators for atom transfer radical polymerization (ATRP) of acrylamide and methacrylate monomers. Copper-mediated ATRP of N-isopropylacrylamide (NIPAAm), 2-hydroxyethyl methacrylate (HEMA), and poly(ethylene glycol) methacrylate (PEGMA) resulted in polymers with polydispersity indices (PDIs) as low as 1.11, 1.18, and 1.24, respectively. Kinetic analysis indicated that ATRP of HEMA was well-controlled. The polymer end groups of polyNIPAAm were deprotected with trifluoroacetic acid, exposing α-aminooxy moieties. Complete removal of the Boc groups was confirmed by 1H NMR, and the ability to form oxime bonds was verified by conjugation to aldehyde fluorescent nanospheres. Chemospecific reaction with N ε-levulinyl lysine-modified bovine serum albumin (BSA) to form “smart” polymer conjugates was demonstrated.
Genome editing therapies hold great promise for the cure of monogenic and other diseases; however, the application of nonviral gene delivery methods is limited by both a lack of fundamental knowledge ...of interactions of the gene-carrier in complex animals and biocompatibility. Herein, we characterize nonviral gene delivery vehicle formulations that are based on diblock polycations containing a hydrophilic and neutral glucose block chain extended with cationic secondary amines of three lengths, poly(methacrylamido glucopyranose-block-2-methylaminoethyl methacrylate) P(MAG-b-MAEMt)-1, -2, -3. These polymers were formulated with plasmid DNA to prepare polyelectrolyte complexes (polyplexes). In addition, two controls, P(EG-b-MAEMt) and P(MAEMt), were synthesized, formulated into polyplexes and the ex vivo hemocompatibility, or blood compatibility, and in vivo biodistribution of the formulations were compared to the glycopolymers. While both polymer structure and N/P (amine to phosphate) ratio were important factors affecting hemocompatibility, N/P ratio played a stronger role in determining polyplex biodistribution. P(EG-b-MAEMt) and P(MAEMt) lysed red blood cells at both high and low N/P formulations while P(MAG-b-MAEMt) did not significantly lyse cells at either formulation at short and medium polymer lengths. Conversely, P(MAG-b-MAEMt) did not affect coagulation at N/P = 5, but significantly delayed coagulation at N/P = 15. P(EG-b-MAEMt) and P(MAEMt) did not affect coagulation at either formulation. After polymer and pDNA cargo distribution was observed in vivo, P(EG-b-MAEMt) N/P = 5 and P(MAG-b-MAEMt) N/P = 5 both dissociated and deposited polymer in the liver, while pDNA cargo from P(MAG-b-MAEMt) N/P = 15 was found in the liver, lungs, and spleen. The contrast between P(MAG-b-MAEMt) at N/P = 5 and 15 demonstrates that polyplex stability in the blood can be improved with N/P ratio and potentially aid polyplex biodistribution through simply varying the formulation ratios.
Glioblastoma (GBM) is highly resistant to chemotherapies, immune-based therapies, and targeted inhibitors. To identify novel drug targets, we screened orthotopically implanted, patient-derived ...glioblastoma sphere-forming cells using an RNAi library to probe essential tumor cell metabolic programs. This identified high dependence on mitochondrial fatty acid metabolism. We focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids (MCFA), due to its consistently high score and high expression among models and upregulation in GBM compared with normal brain. Beyond the expected energetics impairment, MCAD depletion in primary GBM models induced an irreversible cascade of detrimental metabolic effects characterized by accumulation of unmetabolized MCFAs, which induced lipid peroxidation and oxidative stress, irreversible mitochondrial damage, and apoptosis. Our data uncover a novel protective role for MCAD to clear lipid molecules that may cause lethal cell damage, suggesting that therapeutic targeting of MCFA catabolism may exploit a key metabolic feature of GBM. SIGNIFICANCE: MCAD exerts a protective role to prevent accumulation of toxic metabolic by-products in glioma cells, actively catabolizing lipid species that would otherwise affect mitochondrial integrity and induce cell death. This work represents a first demonstration of a nonenergetic role for dependence on fatty acid metabolism in cancer.
.
A pyridyl disulfide end-functionalized polymer with N-acetyl-d-glucosamine pendant side-chains was synthesized by atom transfer radical polymerization (ATRP). The glycopolymer was prepared from a ...pyridyl disulfide initiator catalyzed by a Cu(I)/Cu(II)/2,2′-bipyridine system in a mixture of methanol and water at 30 °C. The final polymer had a number-average molecular weight (M n) of 13.0 kDa determined by 1H NMR spectroscopy and a narrow polydispersity index (1.12) determined by gel permeation chromatography (GPC). The pyridyl disulfide end-group was then utilized to conjugate the glycopolymer to a double-stranded short interfering RNA (siRNA). Characterization of the glycopolymer-siRNA by polyacrylamide gel electrophoresis (PAGE) showed 97% conjugation. The activated disulfide polymer was also patterned on gold via microcontact printing. The pyridyl disulfide allowed for ready immobilization of the glycopolymer into 200 μm sized features on the surface.
Polymeric excipients are crucial ingredients in modern pills, increasing the therapeutic bioavailability, safety, stability, and accessibility of lifesaving products to combat diseases in developed ...and developing countries worldwide. Because many early-pipeline drugs are clinically intractable due to hydrophobicity and crystallinity, new solubilizing excipients can reposition successful and even failed compounds to more effective and inexpensive oral formulations. With assistance from high-throughput controlled polymerization and screening tools, we employed a strategic, molecular evolution approach to systematically modulate designer excipients based on the cyclic imide chemical groups of an important (yet relatively insoluble) drug phenytoin. In these acrylamide- and methacrylate-containing polymers, a synthon approach was employed: one monomer served as a precipitation inhibitor for phenytoin recrystallization, while the comonomer provided hydrophilicity. Systems that maintained drug supersaturation in amorphous solid dispersions were identified with molecular-level understanding of noncovalent interactions using NOESY and DOSY NMR spectroscopy. Poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (poly(NIPAm-co-DMA)) at 70 mol % NIPAm exhibited the highest drug solubilization, in which phenytoin associated with inhibiting NIPAm units only with lowered diffusivity in solution. In vitro dissolution tests of select spray-dried dispersions corroborated the screening trends between polymer chemical composition and solubilization performance, where the best NIPAm/DMA polymer elevated the mean area-under-the-dissolution-curve by 21 times its crystalline state at 10 wt % drug loading. When administered to rats for pharmacokinetic evaluation, the same leading poly(NIPAm-co-DMA) formulation tripled the oral bioavailability compared to a leading commercial excipient, HPMCAS, and translated to a remarkable 23-fold improvement over crystalline phenytoin.
Aminooxy α- and α,ω-end-functionalized polystyrene were synthesized via atom transfer radical polymerization (ATRP) and atom transfer radical (ATR) coupling. A 1-bromoethylphenyl initiator possessing ...an N-hydroxyphthalimide group was used for copper-mediated ATRP of styrene. The polymerization kinetics indicated good control with high initiator efficiency, and the resulting polymers had polydispersity indices (PDIs) as low as 1.12. N-Hydroxyphthalimide−polystyrene was then dimerized using Cu(0)-mediated ATR coupling, and GPC results indicated high coupling efficiency. Hydrazine deprotection of both the α and α,ω-end-functionalized polystyrene to the aminooxy groups was confirmed by 1H NMR spectroscopy. End-group reactivity was verified by reaction with 4-bromobenzaldehyde to form the oxime linkages.
A Boc-protected aminooxy end-functionalized poly(N-isopropylacrylamide) (pNIPAAm) was synthesized by reversible addition−fragmentation chain transfer (RAFT) polymerization. The monomer was ...polymerized in the presence of a Boc-protected aminooxy trithiocarbonate chain transfer agent (CTA) utilizing 2,2′-azobis(2-isobutyronitrile) (AIBN) as the initiator in dimethylformamide (DMF) at 70 °C. The final polymer had a number-average molecular weight (M n) of 4200 Da as determined by 1H NMR spectroscopy and a narrow polydispersity index (1.14) by gel permeation chromatography (GPC). The Boc group was removed, and the polymer was then incubated with N ε-levulinyllysine-modified bovine serum albumin (BSA). Gel electrophoresis confirmed that the conjugation was successful. The aminooxy end-functionalized pNIPAAm was also immobilized on a gold surface after reduction of the trithiocarbonate end-group. The pNIPAAm surface was then incubated with an aldehyde-modified heparin to yield the polysaccharide-functionalized surface. All surface modifications were monitored by FT-IR spectroscopy.
The development and thorough characterization of nonviral delivery agents for nucleic acid and genome editing therapies are of high interest to the field of nanomedicine. Indeed, this vehicle class ...offers the ability to tune chemical architecture/biological activity and readily package nucleic acids of various sizes and morphologies for a variety of applications. Herein, we present the synthesis and characterization of a class of trehalose-based block copolycations designed to stabilize polyplex formulations for lyophilization and in vivo administration. A 6-methacrylamido-6-deoxy trehalose (MAT) monomer was synthesized from trehalose and polymerized via reversible addition–fragmentation chain transfer (RAFT) polymerization to yield pMAT43. The pMAT43 macro-chain transfer agent was then chain-extended with aminoethylmethacrylamide (AEMA) to yield three different pMAT-b-AEMA cationic-block copolymers, pMAT-b-AEMA-1 (21 AEMA repeats), -2 (44 AEMA repeats), and -3 (57 AEMA repeats). These polymers along with a series of controls were used to form polyplexes with plasmids encoding firefly luciferase behind a strong ubiquitous promoter. The trehalose-coated polyplexes were characterized in detail and found to be resistant to colloidal aggregation in culture media containing salt and serum. The trehalose-polyplexes also retained colloidal stability and promoted high gene expression following lyophilization and reconstitution. Cytotoxicity, cellular uptake, and transfection ability were assessed in vitro using both human glioblastoma (U87) and human liver carcinoma (HepG2) cell lines wherein pMAT-b-AEMA-2 was found to have the optimal combination of high gene expression and low toxicity. pMAT-b-AEMA-2 polyplexes were evaluated in mice via slow tail vein infusion. The vehicle displayed minimal toxicity and discouraged nonspecific internalization in the liver, kidney, spleen, and lungs as determined by quantitative polymerase chain reaction (qPCR) and fluorescence imaging experiments. Hydrodynamic infusion of the polyplexes, however, led to very specific localization of the polyplexes to the mouse liver and promoted excellent gene expression in vivo.
PDF
