On the Use of Triethyloxonium Tetrafluoroborate for Chloride Removal during Sarcosine N-Carboxyanhydride Synthesis: Improving NCA purity for controlled ring opening polymerization.

The clinical translation of polysarcosine (pSar) as polyethylene glycol (PEG) replacement in the development of novel nanomedicines creates a broad demand of polymeric material in high-quality making high-purity sarcosine N-carboxyanhydride (Sar-NCA) as monomer for its production inevitable. Within this report, we present the use of triethyloxonium tetrafluoroborate in Sar-NCA synthesis with focus on amino acid and chloride impurities to avoid the sublimation of Sar-NCAs. With a view towards upscaling into kilogram or ton scale, a new methodology of monomer purification is introduced by utilizing the Meerwein's Salt triethyloxonium tetrafluoroborate to remove chloride impurities by covalent binding and converting chloride ions into volatile products within a single step. The novel straightforward technique enables access to monomers with significantly reduced chloride content (<100 ppm) compared to Sar-NCA derived by synthesis or sublimation. The derived monomers enable the controlled-living polymerization in DMF and provide access to pSar polymers with Poisson-like molecular weight distribution within a high range of chain lengths (Xn 25-200). In conclusion, the reported method can be easily applied to Sar-NCA synthesis or purification of commercially available pSar-NCAs and eases access to well-defined hetero-telechelic pSar polymers.

Bottom-Up Preparation of Phase-Separated Polymersomes.

A bottom-up approach to fabricating monodisperse, two-component polymersomes that possess phase-separated ("patchy") chemical topology is presented. This approach is compared with already-existing top-down preparation methods for patchy polymer vesicles, such as film rehydration. These findings demonstrate a bottom-up, solvent-switch self-assembly approach that produces a high yield of nanoparticles of the target size, morphology, and surface topology for drug delivery applications, in this case patchy polymersomes of a diameter of ≈50 nm. In addition, an image processing algorithm to automatically calculate polymersome size distributions from transmission electron microscope images based on a series of pre-processing steps, image segmentation, and round object identification is presented.

A Multiscale Study of Phosphorylcholine Driven Cellular Phenotypic Targeting.

Phenotypic targeting requires the ability of the drug delivery system to discriminate over cell populations expressing a particular receptor combination. Such selectivity control can be achieved using multiplexed-multivalent carriers often decorated with multiple ligands. Here, we demonstrate that the promiscuity of a single ligand can be leveraged to create multiplexed-multivalent carriers achieving phenotypic targeting. We show how the cellular uptake of poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2-(diisopropylamino)ethyl methacry-late) (PMPC-PDPA) polymersomes varies depending on the receptor expression among different cells. We investigate the PMPC-PDPA polymersome insertion at the single chain/receptor level using all-atom molecular modeling. We propose a theoretical statistical mechanics-based model for polymersome-cell association that explicitly considers the interaction of the polymersome with the cell glycocalyx shedding light on its effect on the polymersome binding. We validate our model experimentally and show that the binding energy is a nonlinear function, allowing us to tune the interaction by varying the radius and degree of polymerization. Finally, we show that PMPC-PDPA polymersomes can be used to target monocytes in vivo due to their promiscuous interaction with SRB1, CD36, and CD81.

One-Pot Synthesis of Oxidation-Sensitive Supramolecular Gels and Vesicles.

Polypeptide-based nanoparticles offer unique advantages from a nanomedicine perspective such as biocompatibility, biodegradability, and stimuli-responsive properties to (patho)physiological conditions. Conventionally, self-assembled polypeptide nanostructures are prepared by first synthesizing their constituent amphiphilic polypeptides followed by postpolymerization self-assembly. Herein, we describe the one-pot synthesis of oxidation-sensitive supramolecular micelles and vesicles. This was achieved by polymerization-induced self-assembly (PISA) of the N-carboxyanhydride (NCA) precursor of methionine using poly(ethylene oxide) as a stabilizing and hydrophilic block in dimethyl sulfoxide (DMSO). By adjusting the hydrophobic block length and concentration, we obtained a range of morphologies from spherical to wormlike micelles, to vesicles. Remarkably, the secondary structure of polypeptides greatly influenced the final morphology of the assemblies. Surprisingly, wormlike micellar morphologies were obtained for a wide range of methionine block lengths and solid contents, with spherical micelles restricted to very short hydrophobic lengths. Wormlike micelles further assembled into oxidation-sensitive, self-standing gels in the reaction pot. Both vesicles and wormlike micelles obtained using this method demonstrated to degrade under controlled oxidant conditions, which would expand their biomedical applications such as in sustained drug release or as cellular scaffolds in tissue engineering.

A targeted polypeptide-based nanoconjugate as a nanotherapeutic for alcohol-induced neuroinflammation.

Alcohol abuse induces the expression of inflammatory mediators by activating the immune receptors to trigger neuroinflammation and brain damage; however, therapies that reduce neuroimmune system activation may protect against alcohol's damaging effects. Curcuminoids possess anti-inflammatory properties but suffer from low bioavailability; therefore, we designed a new receptor-targeted biodegradable star-shaped crosslinked polypeptide polymer that bears propargylamine moieties and bisdemethoxycurcumin (StClPr-BDMC-ANG) as an enhanced anti-inflammatory therapeutic that penetrates the blood-brain-barrier and ameliorates alcohol-induced neuroinflammation. StClPr-BDMC-ANG administration maintains the viability of primary glia and inhibits the ethanol-induced upregulation of crucial inflammatory mediators in the prefrontal and medial cortex in a mouse model of chronic ethanol consumption. StClPr-BDMC-ANG treatment also suppresses the ethanol-mediated downregulation of microRNAs known to negatively modulate neuroinflammation in the brain cortex (miRs 146a-5p and let-7b-5p). In summary, our results demonstrate the attenuation of alcohol-induced neuroinflammation by an optimized and targeted polypeptide-based nanoconjugate of a curcuminoid.

Polyglutamic acid-based crosslinked doxorubicin nanogels as an anti-metastatic treatment for triple negative breast cancer.

Treatment of triple negative breast cancer (TNBC)-associated metastasis represents an unmet clinical need, and we lack effective therapeutics for a disease that exhibits high relapse rates and associates with poor patient outcomes. Advanced nanosized drug delivery systems may enhance the efficacy of first-line chemotherapeutics by altering drug pharmacokinetics and enhancing tumor/metastasis targeting to significantly improve efficacy and safety. Herein, we propose the application of injectable poly-amino acid-based nanogels (NGs) as a versatile hydrophilic drug delivery platform for the treatment of TNBC lung metastasis. We prepared biocompatible and biodegradable cross-linked NGs from polyglutamic acid (PGA) loaded with the chemotherapeutic agent doxorubicin (DOX). Our optimized synthetic procedures generated NGs of ~100 nm in size and 25 wt% drug loading content that became rapidly internalized in TNBC cell lines and displayed IC50 values comparable to the free form of DOX. Importantly, PGA-DOX NGs significantly inhibited lung metastases and almost completely suppressed lymph node metastases in a spontaneously metastatic orthotopic mouse TNBC model. Overall, our newly developed PGA-DOX NGs represent a potentially effective therapeutic strategy for the treatment of TNBC metastases.

Combinatorial Intracellular Delivery Screening of Anticancer Drugs.

Conventional drug solubilization strategies limit the understanding of the full potential of poorly water-soluble drugs during drug screening. Here, we propose a screening approach in which poorly water-soluble drugs are entrapped in poly(2-(methacryloyloxyethyl phosphorylcholine)-poly(2-(diisopropylaminoethyl methacryate) (PMPC-PDPA) polymersomes (POs) to enhance drug solubility and facilitate intracellular delivery. By using a human pediatric glioma cell model, we demonstrated that PMPC-PDPA POs mediated intracellular delivery of cytotoxic and epigenetic drugs by receptor-mediated endocytosis. Additionally, when delivered in combination, drug-loaded PMPC-PDPA POs triggered both an enhanced drug efficacy and synergy compared to that of a conventional combinatorial screening. Hence, our comprehensive synergy analysis illustrates that our screening methodology, in which PMPC-PDPA POs are used for intracellular codelivery of drugs, allows us to identify potent synergistic profiles of anticancer drugs.

On the shuttling across the blood-brain barrier via tubule formation: Mechanism and cargo avidity bias.

The blood-brain barrier is made of polarized brain endothelial cells (BECs) phenotypically conditioned by the central nervous system (CNS). Although transport across BECs is of paramount importance for nutrient uptake as well as ridding the brain of waste products, the intracellular sorting mechanisms that regulate successful receptor-mediated transcytosis in BECs remain to be elucidated. Here, we used a synthetic multivalent system with tunable avidity to the low-density lipoprotein receptor-related protein 1 (LRP1) to investigate the mechanisms of transport across BECs. We used a combination of conventional and super-resolution microscopy, both in vivo and in vitro, accompanied with biophysical modeling of transport kinetics and membrane-bound interactions to elucidate the role of membrane-sculpting protein syndapin-2 on fast transport via tubule formation. We show that high-avidity cargo biases the LRP1 toward internalization associated with fast degradation, while mid-avidity augments the formation of syndapin-2 tubular carriers promoting a fast shuttling across.

Designing peptide nanoparticles for efficient brain delivery.

The targeted delivery of therapeutic compounds to the brain is arguably the most significant open problem in drug delivery today. Nanoparticles (NPs) based on peptides and designed using the emerging principles of molecular engineering show enormous promise in overcoming many of the barriers to brain delivery faced by NPs made of more traditional materials. However, shortcomings in our understanding of peptide self-assembly and blood-brain barrier (BBB) transport mechanisms pose significant obstacles to progress in this area. In this review, we discuss recent work in engineering peptide nanocarriers for the delivery of therapeutic compounds to the brain: from synthesis, to self-assembly, to in vivo studies, as well as discussing in detail the biological hurdles that a nanoparticle must overcome to reach the brain.

Effective Nephroprotection Against Acute Kidney Injury with a Star-Shaped Polyglutamate-Curcuminoid Conjugate.

The lack of effective pharmacological treatments for acute kidney injury (AKI) remains a significant public health problem. Given the involvement of apoptosis and regulated necrosis in the initiation and progression of AKI, the inhibition of cell death may contribute to AKI prevention/recovery. Curcuminoids are a family of plant polyphenols that exhibit attractive biological properties that make them potentially suitable for AKI treatment. Now, in cultured tubular cells, we demonstrated that a crosslinked self-assembled star-shaped polyglutamate (PGA) conjugate of bisdemethoxycurcumin (St-PGA-CL-BDMC) inhibits apoptosis and necroptosis induced by Tweak/TNFα/IFNγ alone or concomitant to caspase inhibition. St-PGA-CL-BDMC also reduced NF-κB activation and subsequent gene transcription. In vivo, St-PGA-CL-BDMC prevented renal cell loss and preserved renal function in mice with folic acid-induced AKI. Mechanistically, St-PGA-CL-BDMC inhibited AKI-induced apoptosis and expression of ferroptosis markers and also decreased the kidney expression of genes involved in tubular damage and inflammation, while preserving the kidney expression of the protective factor, Klotho. Thus, due to renal accumulation and attractive pharmacological properties, the application of PGA-based therapeutics may improve nephroprotective properties of current AKI treatments.

l-Asparaginase Encapsulation into Asymmetric Permeable Polymersomes.

This work reports, for the encapsulation of l-asparaginase, an anticancer enzyme into hybrid PMPC25-PDPA70/PEO16-PBO22 asymmetric polymersomes previously developed by our group, with loading capacities with over 800 molecules per vesicle. Enzyme-loaded polymersomes show permeability and capacity to hydrolyze l-asparagine, which is essential to cancer cells. The nanoreactors proposed in this work can be potentially used in further studies to develop novel therapeutic alternatives based on l-asparaginase.

Envisioning the future of polymer therapeutics for brain disorders.

The growing incidence of brain-related pathologies and the problems that undermine the development of efficient and effective treatments have prompted both researchers and the pharmaceutical industry to search for novel therapeutic alternatives. Polymer therapeutics (PT) display properties well suited to the treatment of neuro-related disorders, which help to overcome the many hidden obstacles on the journey to the central nervous system (CNS). The inherent features of PT, derived from drug(s) conjugation, in parallel with the progress in synthesis and analytical methods, the increasing knowledge in molecular basis of diseases, and collected clinical data through the last four decades, have driven the translation from "bench to bedside" for various biomedical applications. However, since the approval of Gliadel® wafers, little progress has been made in the CNS field, even though brain targeting represents an ever-growing challenge. A thorough assessment of the steps required for successful brain delivery via different administration routes and the consideration of the disease-specific hallmarks are essential to progress in the field. Within this review, we hope to summarize the latest developments, successes, and failures and discuss considerations on designs and strategies for PT in the treatment of CNS disorders. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Capturing "Extraordinary" Soft-Assembled Charge-Like Polypeptides as a Strategy for Nanocarrier Design.

The rational design of nanomedicines is a challenging task given the complex architectures required for the construction of nanosized carriers with embedded therapeutic properties and the complex interface of these materials with the biological environment. Herein, an unexpected charge-like attraction mechanism of self-assembly for star-shaped polyglutamates in nonsalty aqueous solutions is identified, which matches the ubiquitous "ordinary-extraordinary" phenomenon previously described by physicists. For the first time, a bottom-up methodology for the stabilization of these nanosized soft-assembled star-shaped polyglutamates is also described, enabling the translation of theoretical research into nanomaterials with applicability within the drug-delivery field. Covalent capture of these labile assemblies provides access to unprecedented architectures to be used as nanocarriers. The enhanced in vitro and in vivo properties of these novel nanoconstructs as drug-delivery systems highlight the potential of this approach for tumor-localized as well as lymphotropic delivery.

Modulating angiogenesis with integrin-targeted nanomedicines.

Targeting angiogenesis-related pathologies, which include tumorigenesis and metastatic processes, has become an attractive strategy for the development of efficient guided nanomedicines. In this respect, integrins are cell-adhesion molecules involved in angiogenesis signaling pathways and are overexpressed in many angiogenic processes. Therefore, they represent specific biomarkers not only to monitor disease progression but also to rationally design targeted nanomedicines. Arginine-glycine-aspartic (RGD) containing peptides that bind to specific integrins have been widely utilized to provide ligand-mediated targeting capabilities to small molecules, peptides, proteins, and antibodies, as well as to drug/imaging agent-containing nanomedicines, with the final aim of maximizing their therapeutic index. Within this review, we aim to cover recent and relevant examples of different integrin-assisted nanosystems including polymeric nanoconstructs, liposomes, and inorganic nanoparticles applied in drug/gene therapy as well as imaging and theranostics. We will also critically address the overall benefits of integrin-targeting.

Well-Defined Star-Shaped Polyglutamates with Improved Pharmacokinetic Profiles As Excellent Candidates for Biomedical Applications.

There is a need to develop new and innovative polymer carriers to be used as drug delivery systems and/or imaging agents owing to the fact that there is no universal polymeric system that can be used in the treatment of all diseases. Additionally, limitations with existing systems, such as a lack of biodegradability and biocompatibility, inevitably lead to side effects and poor patient compliance. New polymer therapeutics based on amino acids are excellent candidates for drug delivery, as they do not suffer from these limitations. This article reports on a simple yet powerful methodology for the synthesis of 3-arm star-shaped polyglutamic acid with well-defined structures, precise molecular weights (MW), and low polydispersity (D = <1.3). These were synthesized by ring-opening polymerization (ROP) of N-carboxyanhydrides (NCA) in a divergent method from novel multifunctional initiators. Herein, their exhaustive physicochemical characterization is presented. Furthermore, preliminary in vitro evaluation in selected cell models, and exhaustive in vivo biodistribution and pharmacokinetics, highlighted the advantages of these branched systems when compared with their linear counterparts in terms of cell uptake enhancement and prolonged plasma half-life.