Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and adenosine triphosphate citrate lyase (ACL) in the cytosol1-3. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle (TCA) and fatty acid synthesis4. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis5, the regulation and utilization of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells employ reductive metabolism of alpha-ketoglutarate (αKG) to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase 1 (IDH1) dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived αKG for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau (VHL) tumor suppressor protein preferentially utilize reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon utilization in order to produce AcCoA and support lipid synthesis in mammalian cells.

Nanoscale objects are typically internalized by cells into membrane-bounded endosomes and fail to access the cytosolic cell machinery. Whereas some biomacromolecules may penetrate or fuse with cell membranes without overt membrane disruption, no synthetic material of comparable size has shown this property yet. Cationic nano-objects pass through cell membranes by generating transient holes, a process associated with cytotoxicity. Studies aimed at generating cell-penetrating nanomaterials have focused on the effect of size, shape and composition. Here, we compare membrane penetration by two nanoparticle 'isomers' with similar composition (same hydrophobic content), one coated with subnanometre striations of alternating anionic and hydrophobic groups, and the other coated with the same moieties but in a random distribution. We show that the former particles penetrate the plasma membrane without bilayer disruption, whereas the latter are mostly trapped in endosomes. Our results offer a paradigm for analysing the fundamental problem of cell-membrane-penetrating bio-and macro-molecules. Nanomaterials are of great interest for use in biomedicine as imaging tools 1-3 , phototherapy agents 4,5 and gene delivery carriers 6,7 . Their interactions with cell membranes are of central importance for all such applications. For example, many drugdelivery systems are based on the transport of therapeutic agents to the cytosol or nucleus of cells by nanoparticles; efficient delivery must be achieved while avoiding cytotoxicity during passage through cell membranes to reach intracellular target compartments 8,9 . Indeed, membrane penetration by synthetic 10 as well as by biologically derived 11 molecules/particles is currently under intense investigation. Some biomacromolecules, such as cell-penetrating peptides (CPPs), may be capable of penetrating membranes without overt lipid bilayer disruption/poration 12-15 . Likewise, synthetic nanomaterials with very small dimensions (molecules, metal nanoclusters 16 , small dendrimers 10 and carbon nanotubes 17 ) can also pass through cell membranes. However, to the best of our knowledge, no synthetic material larger than a few nanometres in size can pass through membranes without disrupting the integrity of these biological barriers. For example, charged particles (such as cationic quantum dots or dendrimers, mostly assisted by some degree of hydrophobicity) induce transient poration of cell membranes to enter cells, a process associated with cytotoxicity 18 . Alternatively, nanoparticles have been designed to explicitly disrupt endolysosomal membranes to enter the cell by force 19 or enter the cell aided by exogenous agents such as CPP chaperones 20 . In contrast, most nanoparticles are trapped in endosomes 21 and hence do not reach the cytosol.The surface properties of nanomaterials play a critical role in determining the outcome of their interactions with cells 22 . Recently, we found that when gold nanoparticles are coated with binary mixtures of hydrophobic and hydrophilic organic mo...

In cancer patients, visual identification of sentinel lymph nodes (LNs) is achieved by the injection of dyes that bind avidly to endogenous albumin, targeting these compounds to LNs where they are efficiently filtered by resident phagocytes1,2. Here we translate this “albumin hitchhiking” approach to molecular vaccines, via the synthesis of amphiphiles (amph-vaccines) comprised of an antigen or adjuvant cargo linked to a lipophilic albumin-binding tail by a solubility-promoting polar polymer chain. Structurally-optimized CpG-DNA/peptide amph-vaccines exhibited dramatic increases in LN accumulation and decreased systemic dissemination relative to their parent compounds, leading to 30-fold increases in T-cell priming and enhanced anti-tumor efficacy while greatly reducing systemic toxicity. Amph-vaccines provide a simple, broadly-applicable strategy to simultaneously increase the potency and safety of subunit vaccines.

The activation of T cells through interaction of their T-cell receptors with antigenic peptide bound to major histocompatibility complex (MHC) on the surface of antigen presenting cells (APCs) is a crucial step in adaptive immunity. Here we use three-dimensional fluorescence microscopy to visualize individual peptide-I-E(k) class II MHC complexes labelled with the phycobiliprotein phycoerythrin in an effort to characterize T-cell sensitivity and the requirements for forming an immunological synapse in single cells. We show that T cells expressing the CD4 antigen respond with transient calcium signalling to even a single agonist peptide-MHC ligand, and that the organization of molecules in the contact zone of the T cell and APC takes on the characteristics of an immunological synapse when only about ten agonists are present. This sensitivity is highly dependent on CD4, because blocking this molecule with antibodies renders T cells unable to detect less than about 30 ligands.

Synthetic carriers such as polymer and lipid particles often struggle to meet clinical expectations. Natural particulates - that range from pathogens to mammalian cells - are therefore worth examining in more depth, as they are highly optimized for their specific functions in vivo and possess features that are often desired in drug delivery carriers. With a better understanding of these biological systems, in conjunction with the availability of advanced biotechnology tools that are useful for re-engineering the various natural systems, researchers have started to exploit natural particulates for multiple applications in the delivery of proteins, small interfering RNA and other therapeutic agents. Here, we review the natural drug delivery carriers that have provided the basis and inspiration for new drug delivery systems.

A major limitation of cell therapies is the rapid decline in viability and function of transplanted cells. Here we describe a strategy to enhance cell therapy via the conjugation of adjuvant drug-loaded nanoparticles to the surfaces of therapeutic cells. Using this method to provide sustained pseudo-autocrine stimulation to donor cells, we elicited dramatic enhancements in tumor elimination in a model of adoptive T-cell therapy for cancer and increased the in vivo repopulation rate of hematopoietic stem cell grafts, using very low doses of adjuvant drugs that were ineffective when given systemically. This approach is a facile and generalizable strategy to augment cytoreagents while minimizing systemic side effects of adjuvant drugs. In addition, these results suggest therapeutic cells are promising vectors for actively targeted drug delivery.

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Vaccines based on recombinant proteins avoid toxicity and anti-vector immunity associated with live vaccine (e.g., viral) vectors, but their immunogenicity is poor, particularly for CD8+ T-cell (CD8T) responses. Synthetic particles carrying antigens and adjuvant molecules have been developed to enhance subunit vaccines, but in general these materials have failed to elicit CD8T responses comparable to live vectors in preclinical animal models. Here, we describe interbilayer-crosslinked multilamellar vesicles (ICMVs) formed by crosslinking headgroups of adjacent lipid bilayers within multilamellar vesicles. ICMVs stably entrapped protein antigens in the vesicle core and lipid-based immunostimulatory molecules in the vesicle walls under extracellular conditions, but exhibited rapid release in the presence of endolysosomal lipases. We found that these antigen/adjuvant-carrying ICMVs form an extremely potent whole-protein vaccine, eliciting endogenous T-cell and antibody responses comparable to the strongest vaccine vectors. These materials should enable a range of subunit vaccines and provide new possibilities for protein therapeutic delivery.