Delivering all in one: Antigen-nanocapsule loaded with dual adjuvant yields superadditive effects by DC-directed T cell stimulation

Therapeutic vaccination is and remains a major challenge, particularly in cancer treatment. In this process, the effective activation of dendritic cells by a combination of distinctly acting adjuvants and an antigen is crucial for success. While most common vaccine formulations lack the efficiency to trigger sufficient T cell responses in a therapeutic tumor treatment, nanovaccines offer unique properties to tackle that challenge. Here, we report the stepwise development of a nanocapsule for vaccination approaches, comprising a shell consisting of antigen and loaded with a superadditive adjuvant combination. In a first initial step, we identified the combination of resiquimod (R848) and mur…

Surface Modification of Polysaccharide-Based Nanoparticles with PEG and Dextran and the Effects on Immune Cell Binding and Stimulatory Characteristics.

Surface modifications of nanoparticles can alter their physical and biological properties significantly. They effect particle aggregation, circulation times, and cellular uptake. This is particularly critical for the interaction with primary immune cells due to their important role in particle processing. We can show that the introduction of a hydrophilic PEG layer on the surface of the polysaccharide-based nanoparticles prevents unwanted aggregation under physiological conditions and decreases unspecific cell uptake in different primary immune cell types. The opposite effect can be observed with a parallel-performed introduction of a layer of low molecular weight dextran (3.5 and 5 kDa) on…

Xylochemical Synthesis of Cytotoxic 2-Aminophenoxazinone-Type Natural Products Through Oxidative Cross Coupling

Today, the total synthesis of natural products mostly relies on the use of petroleum-based starting materials. The commercial availability of building blocks of this type often deflects attention away from lengthy synthetic routes required for the reintroduction of heteroatom substitution patterns lost in the geological process of kerogenesis. Herein, the use of wood-based starting materials, the so-called xylochemicals, for the synthesis of 2-aminophenoxazinone natural products is presented. The inherent heteroatom substitution patterns as well as chemical functionalities were employed and permitted a short and straightforward synthetic strategy. Moreover, a novel and “green” method for co…

Quantum Chemical-Based Protocol for the Rational Design of Covalent Inhibitors.

We propose a structure-based protocol for the development of customized covalent inhibitors. Starting from a known inhibitor, in the first and second steps appropriate substituents of the warhead are selected on the basis of quantum mechanical (QM) computations and hybrid approaches combining QM with molecular mechanics (QM/MM). In the third step the recognition unit is optimized using docking approaches for the noncovalent complex. These predictions are finally verified by QM/MM or molecular dynamic simulations. The applicability of our approach is successfully demonstrated by the design of reversible covalent vinylsulfone-based inhibitors for rhodesain. The examples show that our approach…

Protein-Based Nanoparticles for the Delivery of Enzymes with Antibacterial Activity.

Proteins represent a versatile biopolymer material for the preparation of nanoparticles due to their biocompatibility, biodegradability, and low immunogenicity. This study presents a protein-based nanoparticle system consisting of high surface PEGylated lysozyme polyethylene glycol-modified lysozyme (LYZmPEG ). This protein modification leads to a solubility switch, which allows a nanoparticle preparation using a mild double emulsion method without the need of surfactants. The method allows the encapsulation of large hydrophilic payloads inside of the protein-based nanoparticle system. Native lysozyme (LYZ) was chosen as payload because of its innate activity as natural antibiotic. The mild…

Double stimuli-responsive polysaccharide block copolymers as green macrosurfactants for near-infrared photodynamic therapy

The NIR absorbing photosensitizer phthalocyanine zinc (PC(Zn)) was stabilized in aqueous media as water-dispersible nanoparticles with a reduction- and pH-responsive full polysaccharide block copolymer. A cellular uptake and also photo switchable intracellular activity of the cargo upon irradiation at wavelengths in the near infrared region were shown. The block copolymer was synthesized by applying a copper-free click strategy based on a thiol exchange reaction, creating an amphiphilic double-stimuli-responsive mixed disulfide. The dual-sensitive polysaccharide micelles represent a non-toxic and biodegradable green macrosurfactant for the delivery of phthalocyanine zinc. By encapsulation i…

Methods of protein surface PEGylation under structure preservation for the emulsion-based formation of stable nanoparticles

Proteins show remarkable versatility as multifunctional materials for therapeutic applications. They can be easily modified with the toolkit of bioorganic chemistry and are particularly attractive because of their degradability and biocompatibility. Herein, we evaluate different methods for the attachment of multiple PEG chains on the surface of the enzyme lysozyme. For this, we activated standard 2 kDa mPEG chains with four different electrophilic groups and tested their ability to react with different amino acids on the surface of our model protein. The aim was to find an effective and at the same time mild modification method that preserves the native structure and activity of the enzyme…

A new approach to inhibit human β-tryptase by protein surface binding of four-armed peptide ligands with two different sets of arms

A series of six new tetravalent ligands (1-6) with two different sets of arms bind to the surface of β-tryptase, a tetrameric enzyme with an A(2)B(2) arrangement of its four monomers and two different binding sites on its protein surface (as suggested by a docking study). Besides proteinogenic amino acids also the guanidiniocarbonyl pyrrole cation (abbreviated as GCP), as an artificial arginine analog, was introduced into the arms of the ligands to investigate its influence on protein surface binding and enzyme inhibition. Furthermore, four ligands (7-10) with four identical arms also containing the GCP group were additionally synthesized to study the influence of the GCP moiety on the inhi…

pH-Responsive protein nanoparticlesviaconjugation of degradable PEG to the surface of cytochromec

Proteins represent a versatile biopolymer material for the preparation of nanoparticles. For drug delivery applications an acid-triggered disassembly and payload release is preferred. Herein, we present a protein nanoparticle system based on cytochrome c, which is surface-modified with acid-degradable polyethylene glycol (PEGylation). pH-Sensitivity was obtained through vinyl ether moieties distributed in the polyether backbone. When PEGylated, cytochrome c shows a different solubility behaviour in organic solvents, which allows for particle preparation using an emulsion-based solvent evaporation method. The resulting particles are stable under physiological conditions but degrade at acidic…

Development of Novel Peptide-Based Michael Acceptors Targeting Rhodesain and Falcipain-2 for the Treatment of Neglected Tropical Diseases (NTDs)

This paper describes the development of a class of peptide-based inhibitors as novel antitrypanosomal and antimalarial agents. The inhibitors are based on a characteristic peptide sequence for the inhibition of the cysteine proteases rhodesain of Trypanosoma brucei rhodesiense and falcipain-2 of Plasmodium falciparum. We exploited the reactivity of novel unsaturated electrophilic functions such as vinyl-sulfones, -ketones, -esters, and -nitriles. The Michael acceptors inhibited both rhodesain and falcipain-2, at nanomolar and micromolar levels, respectively. In particular, the vinyl ketone 3b has emerged as a potent rhodesain inhibitor (k2nd = 67 × 106 M-1 min-1), endowed with a picomolar b…

Asymmetric Disulfanylbenzamides as Irreversible and Selective Inhibitors of Staphylococcus aureus Sortase A

Abstract Staphylococcus aureus is one of the most frequent causes of nosocomial and community‐acquired infections, with drug‐resistant strains being responsible for tens of thousands of deaths per year. S. aureus sortase A inhibitors are designed to interfere with virulence determinants. We have identified disulfanylbenzamides as a new class of potent inhibitors against sortase A that act by covalent modification of the active‐site cysteine. A broad series of derivatives were synthesized to derive structure‐activity relationships (SAR). In vitro and in silico methods allowed the experimentally observed binding affinities and selectivities to be rationalized. The most active compounds were f…

P0312 : Preclinical evaluation of dextran-based therapeutic nanoparticles for hepatic drug delivery

Amphiphilic Polysaccharide Block Copolymers for pH-Responsive Micellar Nanoparticles

A full polysaccharide amphiphilic block copolymer was prepared from end group-functionalized dextrans using copper-mediated azide-alkyne click chemistry. Sufficient modification of the reducing end in both blocks was achieved by microwave-enhanced reductive amination in a borate-buffer/methanol solvent system. The combination of a hydrophilic dextran block with a hydrophobic acetalated dextran block results in an amphiphilic structure that turns water-soluble upon acid treatment. The material has a low critical micelle concentration and self-assembles in water to spherical micellar nanoparticles. The formed nanoparticles have a narrow size distribution below 70 nm in diameter and disassembl…

Dextran-based therapeutic nanoparticles for hepatic drug delivery.

Aim: Evaluation of dextran-based nanoparticles (DNP) as a drug delivery system to target myeloid cells of the liver. Materials & methods: DNP were synthesized and optionally PEGylated. Their toxicity and cellular uptake were studied in vitro. Empty and siRNA-carrying DNP were tested in vivo with regard to biodistribution and cellular uptake. Results: In vitro, DNP were taken up by cells of the myeloid lineage without compromising their viability. In vivo, empty and siRNA-carrying DNP distributed to the liver where a single treatment addressed approximately 70% of macrophages and dendritic cells. Serum parameters indicated no in vivo toxicity. Conclusion: DNP are multifunctional liver-s…

Nanoparticle Assembly of Surface-Modified Proteins

Nature's biomaterials such as peptides and proteins represent a valuable source of highly defined macromolecules. Herein we developed a nanoparticle drug delivery system based on the assembly of surface-modified proteins that can be transferred into organic solvents and represent the structural material of the carrier system. The particles are prepared by an oil-in-water nanoemulsion technique without the need of additional denaturation or cross-linking steps for stabilization. We achieve the necessary lipophilic solubility switch of the protein material by high surface PEGylation under conservation of the native three-dimensional protein structure. This study focuses on lysozyme as model e…