Nanotechnology and Protein Engineering


Many active pharmaceutical ingredients (APIs) have a poor solubility in water, which limits their potential in clinics, especially for intraveneous (i.v.) administration. Drug discovery has led to an increasing number of new hydrophobic chemicals to the point where 90% of small molecules in discovery are considered poorly water-soluble, which is one of the main reasons why many new drug candidates do not reach the market. Moreover, commercially available formulations of hydrophobic APIs are composed of excipients and/or co-solvents to improve their solubility, but may lead to poor safety and tolerability. A good example is paclitaxel (PTX), one of the most widely used chemotherapeutic agents due to its potency against a variety of solid tumors including breast, ovarian, lung and head and neck cancers. The first PTX formulation approved in 1998 by the Food and Drug Administration (FDA) for parenteral administration, Taxol®, was composed of PTX in a co-solvent system comprising polyoxyethylated castor oil (Cremophor® EL) and absolute ethanol at a 50/50 v/v ratio. Although it allowed the dissolution of PTX at 6 mg.mL, this vehicle was responsible for severe side effects like hypersensitivity reactions. To avoid the use of a co-solvent, new PTX formulations have been developed and are in various stages of clinical trials: an albumin-bound PTX Abraxane®, a liposome Lipusu®, and polymeric nanoparticles (NPs) Cynviloq IG-001/Genexol-PM and NK105.

Such nano-sized drug delivery systems (DDSs) allow the solubilization of the hydrophobic API in water, but also have various advantages such as preventing premature drug degradation, enhancing drug uptake into tumors by passive targeting, controlling the drug’s pharmacokinetic profile and thus improving its bioavailability. As DDSs, polymeric nanoparticles made from amphiphilic block copolymers have attracted increasing interest. Among them, poly(amino acid) (PAA)-based NPs have the advantage of being biocompatible and biodegradable. In the case of PTX, NK105 is an example among others of a polymeric formulation where PTX is loaded in poly(ethylene glycol)-b-poly(amino acid) (PEG-b-PAA) micelles. Even if poly(ethylene glycol) (PEG) is widely used as a hydrophilic block for its water-solubility and its stealthiness, it presents major drawbacks as it is not biodegradable and can trigger immunogenic responses. Polysarcosine (PSar) has recently demonstrated its potential as a PAA alternative to PEG since it has similar properties such as its hydrophilicity, stealthiness and low toxicity but has the advantage of being biodegradable as it is based on the endogeneous amino acid derivative sarcosine, N-methyl glycine. Notably, copolymers based on PSar-b-PAA have been investigated as excipients to develop new DDSs. Poly(γ-benzyl-L-glutamate) (PLGluOBn, pure L block) was found to be a suitable PAA hydrophobic block to load PTX, and is a well studied α-helical rod-like polymer to form DDSs. PSar-b-PLGluOBn copolymers with a PSar block of 200 or 400 repeat units were investigated for their capacity to load a hydrophobic adenylate cyclase inhibitor. For this study, we designed various polysarcosine-b-poly(γ-benzyl glutamate) (PSar-b-PGluOBn) copolymers of varying molar mass, hydrophilic fraction and PGluOBn block configuration (racemic, pure L or pure D), to investigate the impact on the self-assembly and PTX loading. The technical and cost constraints of industrial scale-up were also considered. For this purpose, copolymers with shorter molar masses than those of the prior art were designed to facilitate synthesis and yield less expensive products in the perspective of commercial applications. We report the synthesis and characterization of different PSar-b-PGluOBn copolymers as well as the formulation and characterization of PTX-loaded nanoparticles resulting from their self-assembly.

Lipid nanoparticles (LNPs) are shown to be effective for siRNA delivery in Onpattro and in mRNA-based COVID-19 vaccines Comirnaty and Spikevax, all of which are stabilized with PEG-lipids. A potential application of PSar is to replace the PEG-lipids with a PSar-lipid, which may improve the safety of the LNPs.

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Besides, tumor-targeting ligands such as peptides and antibodies may effectively aid in delivery of certain cytotoxic agents (either biological or synthetic) to the tumor cells, thereby improving therapeutic efficacy while limiting the exposure of normal tissues to the cytotoxic agents. Therapeutic approaches have included the use of unarmed monoclonal antibodies (MAbs), radiolabeled MAbs, MAbs conjugated to immunotoxins, or boronated dendrimers. A single-chain variable fragment (scFv) specific to EGFRvIII was discovered and integrated in a tandem antibody (TandAb) construct. Small peptides (< 5 kDa) that selectively recognize tumor cells have advantages over MAbs (150 kDa), TandAbs (100 kDa) and scFvs (20–30 kDa) since they are easy to synthesize and modify due to their much lower size, have higher cell membrane penetration, and possess less immunogenicity. Even if the binding affinity of peptides is lower compared to antibodies and fragments, avidity can be increased by incorporating multiple copies of peptides on the surface of nanostructures when developing tumor-targeting delivery systems.

We report the selection and characterization of a novel peptide ligand using phage display targeted against the cancer specific epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII). EGFRvIII is present in several human malignancies such as glioblastoma, lung cancer, and breast cancer, but not in normal tissues. Two short peptides intended for EGFR targeting of tumor cells have been described in the prior art. The FALGEA peptide is described as binding to both EGFR wild type (EGFR WT) and EGFRvIII. The YHWYGYTPENVI peptide, discovered from the GE11 peptide, has been proven to bind to EGFR WT, while its potential binding to EGFRvIII is unknown. We screened a 12 mer random peptide library against EGFRvIII. Phage selected peptides were sequenced in high throughput by next generation sequencing (NGS), and their diversity was studied to identify highly abundant clones expected to bind with the highest affinities to EGFRvIII. The enriched peptides were characterized and their binding capacity towards stable cell lines expressing EGFRvIII, EGFR WT, or a low endogenous level of EGFR WT was confirmed by flow cytometry analysis. The best peptide candidate was synthesized, and its binding specificity towards EGFRvIII was validated in vitro. Additionally, computational docking analysis suggested that the identified peptide binds selectively to EGFRvIII. The novel peptide is thus a promising EGFRvIII targeting agent for future applications in cancer diagnosis and therapy.

We are also applying engineered peptides and peptide nucleic acids to the programmable self-assembly of nanostructures and reported the first study on the use of peptide nucleic acids in nanostructure self-assembly.

Selected Publications: