Tumour-associated macrophages act as a slow-release reservoir of nano-therapeutic Pt(IV) pro-drug

Therapeutic nanoparticles (TNPs) aim to deliver drugs more safely and effectively to cancers, yet clinical results have been unpredictable owing to limited in vivo understanding. Here we use single-cell imaging of intratumoral TNP pharmacokinetics and pharmacodynamics to better comprehend their heterogeneous behaviour. Model TNPs comprising a fluorescent platinum(IV) pro-drug and a clinically tested polymer platform (PLGA-b-PEG) promote long drug circulation and alter accumulation by directing cellular uptake toward tumour-associated macrophages (TAMs). Simultaneous imaging of TNP vehicle, its drug payload and single-cell DNA damage response reveals that TAMs serve as a local drug depot that accumulates significant vehicle from which DNA-damaging Pt payload gradually releases to neighbouring tumour cells. Correspondingly, TAM depletion reduces intratumoral TNP accumulation and efficacy. Thus, nanotherapeutics co-opt TAMs for drug delivery, which has implications for TNP design and for selecting patients into trials.National Cancer Institute (U.S.) (Grant RO1-CA034992

Interplay between Lipid Interaction and Homo-coiling of Membrane-Tethered Coiled-Coil Peptides

The designed coiled-coil-forming peptides E [(EIAALEK)₃] and K [(KIAALKE)₃] are known to trigger efficient membrane fusion when they are tethered to lipid vesicles in the form of lipopeptides. Knowledge of their secondary structure is a key element in understanding their role in membrane fusion. Special conditions can be found at the interface of the membrane, where the peptides are confined in close proximity to other peptide molecules as well as to the lipid interface. Consequently, different structural states were proposed for the peptides when tethered to this interface. Due to the multitude of possible states, determining the structure solely on the basis of circular dichroism (CD) spectra at a single temperature can be misleading. In addition, it has not yet been possible to unambiguously distinguish between the membrane-bound and the coiled-coil states of these peptides by means of infrared (IR) spectroscopy due to their very similar amide I′ bands. Here, the molecular basis of this similarity is investigated by means of site-specific ¹³C-labeled FTIR spectroscopy. Structural similarities between the membrane-interacting helix of K and the homo-coiled-coil-forming helix of E are shown to cause the similar spectroscopic properties. Furthermore, the peptide structure is investigated using temperature-dependent CD and IR spectroscopy, and it is shown that the different states can be distinguished on the basis of their thermal behavior. It is shown that the two peptides behave fundamentaly differently when tethered to the lipid membrane, which implies that their role during membrane fusion is different and the mechanism of this process is asymmetric

Membrane Interactions of Fusogenic Coiled-Coil Peptides: Implications for Lipopeptide Mediated Vesicle Fusion

Fusion of lipid membranes is an important natural process for the intra- and intercellular exchange of molecules. However, little is known about the actual fusion mechanism at the molecular level. In this study we examine a system that models the key features of this process. For the molecular recognition between opposing membranes two membrane anchored heterodimer coiled-coil forming peptides called ‘E’ (EIAALEK)₃ and ‘K’ (KIAALKE)₃ were used. Lipid monolayers and IR reflection absorption spectroscopy (IRRAS) revealed the interactions of the peptides ‘E’, ‘K’, and their parallel coiled-coil complex ‘E/K’ with the phospholipid membranes and thereby mimicked the pre- and postfusion states, respectively. The peptides adopted α-helical structures and were incorporated into the monolayers with parallel orientation. The strength of binding to the monolayer differed for the peptides and tethering them to the membrane increased the interactions even further. Remarkably, these interactions played a role even in the postfusion state. These findings shed light on important mechanistic details of the membrane fusion process in this model system. Furthermore, their implications will help to improve the rational design of new artificial membrane fusion systems, which have a wide range of potential applications in supramolecular chemistry and biomedicine

Membrane-Fusogen Distance Is Critical for Efficient Coiled-Coil-Peptide-Mediated Liposome Fusion

We have developed a model system for membrane fusion that utilizes lipidated derivatives of a heterodimeric coiled-coil pair dubbed E₃ (EIAALEK)₃ and K₃ (KIAALKE)₃. In this system, peptides are conjugated to a lipid anchor via a poly­(ethylene glycol) (PEG) spacer, and this contribution studies the influence of the PEG spacer length, coupled with the type of lipid anchor, on liposome–liposome fusion. The effects of these modifications on peptide secondary structure, their interactions with liposomes, and their ability to mediate fusion were studied using a variety of different content mixing experiments and CD spectroscopy. Our results demonstrate the asymmetric role of the peptides in the fusion process because alterations to the PEG spacer length affect E₃ and K₃ differently. We conclude that negatively charged E₃ acts as a “handle” for positively charged K₃ and facilitates liposome docking, the first stage of the fusion process, through coiled-coil formation. The efficacy of this E₃ handle is enhanced by longer spacer lengths. K₃ directs the fusion process via peptide–membrane interactions, but the length of the PEG spacer plays two competing roles: a PEG₄/PEG₈ spacer length is optimal for membrane destabilization; however, a PEG₁₂ spacer increases the fusion efficiency over time by improving the peptide accessibility for successive fusion events. Both the anchor type and spacer length affect the peptide structure; a cholesterol anchor appears to enhance K₃–membrane interactions and thus mediates fusion more efficiently

In Situ Modification of Plain Liposomes with Lipidated Coiled Coil Forming Peptides Induces Membrane Fusion

Complementary coiled coil forming lipidated peptides embedded in liposomal membranes are able to induce rapid, controlled, and targeted membrane fusion. Traditionally, such fusogenic liposomes are prepared by mixing lipids and lipidated peptides in organic solvent (e.g., chloroform). Here we prepared fusogenic liposomes in situ, i.e., by addition of a lipidated peptide solution to plain liposomes. As the lipid anchor is vital for the correct insertion of lipidated peptides into liposomal membranes, a small library of lipidated coiled coil forming peptides was designed in which the lipid structure was varied. The fusogenicity was screened using lipid and content mixing assays showing that cholesterol modified coiled coil peptides induced the most efficient fusion of membranes. Importantly, both lipid and content mixing experiments demonstrated that the in situ modification of plain liposomes with the cholesterol modified peptides yielded highly fusogenic liposomes. This work shows that existing membranes can be activated with lipidated coiled coil forming peptides, which might lead to highly potent applications such as the fusion of liposomes with cells

Library of Random Copolypeptides by Solid Phase Synthesis

Random copolypeptides are promising and versatile bioinspired macromolecules of minimal complexity for studying their interactions with both living and synthetic matter. They provide the opportunity to investigate the role of, for example, total net charge and hydrophobicity through simply changing the monomer composition, without considering the effect of specific sequences or secondary structure. However, synthesizing large libraries of these polymers so far was prohibited by the time-consuming preparation methods available (ring-opening polymerization (ROP) of amino acid <i>N</i>-carboxyanhydrides and enzymatic polymerization of amino acids). Here we report the automated solid phase synthesis (SPS) of a complete library of polypeptides containing Glu, Lys, and Ala monomers with excellent control over the degree of polymerization and composition and with polydispersity indices (PDIs) between 1.01 and 1.001, which is impossible to achieve by other methods. This method provides access to a library of polymers with a precisely defined total charge that can range from approximately −15 to +15 per chain and with a disordered conformation almost completely devoid of any secondary structure. In solution the polymers are largely present as unimers, with only the most hydrophobic polypeptides showing slight signs of aggregation. Our new approach provides convenient access to libraries of this versatile class of polymers with tunable composition, which can be used in a wide variety of physicochemical studies as a tool that allows systematic variation of charge and hydrophobicity, without the interference of secondary structure or aggregation on their performance

Tumor associated macrophages act as a slow-release reservoir of nano-therapeutic Pt(IV) pro-drug

Therapeutic nanoparticles (TNPs) aim to deliver drugs more safely and effectively to cancers, yet clinical results have been unpredictable owing to limited in vivo understanding. Here we use single-cell imaging of intratumoral TNP pharmacokinetics and pharmacodynamics to better comprehend their heterogeneous behavior. Model TNPs comprised of a fluorescent platinum(IV) pro-drug and a clinically-tested polymer platform (PLGA-b-PEG) promote long drug circulation and alter accumulation by directing cellular uptake toward tumor associated macrophages (TAMs). Simultaneous imaging of TNP vehicle, its drug payload, and single-cell DNA damage response reveals that TAMs serve as a local drug depot that accumulates significant vehicle from which DNA damaging Pt payload gradually releases to neighboring tumor cells. Correspondingly, TAM depletion reduces intratumoral TNP accumulation and efficacy. Thus, nanotherapeutics co-opt TAMs for drug delivery, which has implications for TNP design and for selecting patients into trials

Restoration of tumour-growth suppression in vivo via systemic nanoparticle-mediated delivery of PTEN mRNA

© 2018, The Author(s). Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a well-characterized tumour-suppressor gene that is lost or mutated in about half of metastatic castration-resistant prostate cancers and in many other human cancers. The restoration of functional PTEN as a treatment for prostate cancer has, however, proven difficult. Here, we show that PTEN messenger RNA (mRNA) can be reintroduced into PTEN-null prostate cancer cells in vitro and in vivo via its encapsulation in polymer–lipid hybrid nanoparticles coated with a polyethylene glycol shell. The nanoparticles are stable in serum, elicit low toxicity and enable high PTEN mRNA transfection in prostate cancer cells. Moreover, significant inhibition of tumour growth is achieved when delivered systemically in multiple mouse models of prostate cancer. We also show that the restoration of PTEN function in PTEN-null prostate cancer cells inhibits the phosphatidylinositol 3-kinase (PI3K)–AKT pathway and enhances apoptosis. Our findings provide proof-of-principle evidence of the restoration of mRNA-based tumour suppression in vivo

Multifunctional Envelope-Type siRNA Delivery Nanoparticle Platform for Prostate Cancer Therapy

With the capability of specific silencing of target gene expression, RNA interference (RNAi) technology is emerging as a promising therapeutic modality for the treatment of cancer and other diseases. One key challenge for the clinical applications of RNAi is the safe and effective delivery of RNAi agents such as small interfering RNA (siRNA) to a particular nonliver diseased tissue (<i>e</i>.<i>g</i>., tumor) and cell type with sufficient cytosolic transport. In this work, we proposed a multifunctional envelope-type nanoparticle (NP) platform for prostate cancer (PCa)-specific <i>in vivo</i> siRNA delivery. A library of oligoarginine-functionalized and sharp pH-responsive polymers was synthesized and used for self-assembly with siRNA into NPs with the features of long blood circulation and pH-triggered oligoarginine-mediated endosomal membrane penetration. By further modification with ACUPA, a small molecular ligand specifically recognizing prostate-specific membrane antigen (PSMA) receptor, this envelope-type nanoplatform with multifunctional properties can efficiently target PSMA-expressing PCa cells and silence target gene expression. Systemic delivery of the siRNA NPs can efficiently silence the expression of prohibitin 1 (PHB1), which is upregulated in PCa and other cancers, and significantly inhibit PCa tumor growth. These results suggest that this multifunctional envelope-type nanoplatform could become an effective tool for PCa-specific therapy