Lipid-based nanocarriers for oral delivery of peptides

Therapeutic peptides can treat a wide variety of diseases with selective and potent action. Their oral bioavailability is strongly limited by an important proteolytic activity in the intestinal lumen and poor permeation across the intestinal border. We have evaluated the capacity of solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) to overcome both oral bioavailability limiting aspects, using leuprolide (LEU) as model peptide. Lipidization of LEU by formation of a hydrophobic ion pair (HIP) with sodium docusate enables a significant increase of peptide encapsulation efficiency in both SLN and NLC. The nanocarriers, obtained by high-pressure homogenization, measured 120 nm and were platelet shaped. Regarding the protective effect towards proteolytic degradation, only NLC maintained LEU integrity in presence of trypsin. Intestinal transport, evaluated on Caco-2 (enterocyte-like model) and Caco-2/HT29-MTX (mucin-secreting model) monolayers, showed nanocarriers internalization by enterocytes but no improvement of LEU permeability. Indeed, the combination of nanoparticles platelet-shape with the poor stability of the HIP in the transport medium induces a high burst release of the peptide, limiting nanoparticles capacity to transport LEU across the intestinal border. Stability of peptide lipidization needs to be improved to withstand biorelevant medium to benefit from the advantages of encapsulation in solid lipid nanocarriers and consequently improve their oral bioavailability.

Conjugation with gold nanoparticles improves the stability of the KT2 peptide and maintains its anticancer properties

One of the major weaknesses of therapeutic peptides is their sensitivity to degradation by proteolytic enzymes in vivo.

TP-MV: Therapeutic protein prediction by multi-view learning

Background: Therapeutic peptide prediction is critical for drug development and therapy. Researchers have been studying this essential task, developing several computational methods to identify different therapeutic peptide types. Objective: Most predictors are the specific methods for certain peptides. Currently, developing methods to predict the presence of multiple peptides remains a challenging problem. Moreover, it is still challenging to combine different features to make the therapeutic prediction. Method: In this paper, we proposed a new ensemble method TP-MV for general therapeutic peptide recognition. TP-MV is developed using the stacking framework in conjunction with the KNN, SVM, ET, RF, and XGB. Then TP-MV constructs a multi-view learning model as meta-classifiers to extract the discriminative feature for different peptides. Results: In the experiment, the proposed method outperforms the other existing methods on the benchmark datasets, indicating that the proposed method has the ability to predict multiple therapeutic peptides simultaneously. Conclusion: The TP-MV is a useful tool for predicting therapeutic peptides.

Rational domestication of a plant-based recombinant expression system expands its biosynthetic range.

Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead and an orally active analgesic are demonstrated.

Enhanced Skin Delivery of Therapeutic Peptides Using Spicule-Based Topical Delivery Systems

This study reports two therapeutic peptides, insulin (INS, as a hydrophilic model peptide) and cyclosporine A (CysA, as a hydrophobic one), that can be administrated through a transdermal or dermal route by using spicule-based topical delivery systems in vitro and in vivo. We obtained a series of spicules with different shapes and sizes from five kinds of marine sponges and found a good correlation between the skin permeability enhancement induced by these spicules and their aspect ratio L/D. In the case of INS, Sponge Haliclona sp. spicules (SHS) dramatically increased the transdermal flux of INS (457.0 ± 32.3 ng/cm2/h) compared to its passive penetration (5.0 ± 2.2 ng/cm2/h) in vitro. Further, SHS treatment slowly and gradually reduced blood glucose to 13.1 ± 6.3% of the initial level in 8 h, while subcutaneous injection resulted in a rapid blood glucose reduction to 15.9 ± 1.4% of the initial level in 4 h, followed by a rise back to 75.1 ± 24.0% of the initial level in 8 h. In the case of CysA, SHS in combination with ethosomes (SpEt) significantly (p < 0.05) increased the accumulation of CysA in viable epidermis compared to other groups. Further, SpEt reduced the epidermis thickness by 41.5 ± 9.4% in 7 days, which was significantly more effective than all other groups. Spicule-based topical delivery systems offer promising strategies for delivering therapeutic peptides via a transdermal or dermal route.

Harnessing Extracellular Vesicles from Red Blood Cells for Targeted Delivery of Therapeutic Peptides and RNAs for Leukemia Treatment

Extracellular vesicles (EVs) are emerging as a new class of natural drug carriers with intrinsic ability to deliver bioactive cargo at high efficiency and low toxicity. However, clinical applications of EVs are limited by the production scale and the delivery specificity. We have recently established a new platform for purification and surface modification of EVs from red blood cells (RBCs) that are scalable and versatile for targeted delivery of small-molecule drugs and RNA therapeutics (Usman et al, Nat. Com. 2019 and Pham et al, J. Extracell. Vesicles 2021). Here, we describe a new development of the method for conjugation of RBC-EVs to obtain a higher targeting efficiency. We conjugate RBC-EVs, not only with peptides and nanobodies, but also with monoclonal antibodies, by coupling peptide conjugated EVs with streptavidin-bound biotinylated antibodies. Our data demonstrate that the conjugation is stable and does not affect the physicochemical characteristics of EVs. Conjugation of EVs with a cyclic peptide targeting CXCR4 or a monoclonal antibody targeting CD33 promotes specific binding and uptake of the conjugated EVs by leukemia cells expressing the corresponding receptors. We further use CXCR4-targeting RBC-EVs to specifically deliver the pro-apoptotic peptide KLA to CXCR4-expressing leukemia cells. Delivery of KLA using CXCR4-targeting EVs significantly suppresses leukemia burden and increases survival in a leukemia xenografted mouse model. Antibody-conjugated RBC-EVs are used to deliver RNA antisense oligonucleotides to knockdown FLT3 and miR-125b in cell lines and in patient-derived xenograft models of leukemia. Finally, we demonstrate that peptide/antibody conjugated RBC-EVs are biocompatible and nonimmunogenic. Our study provides a new platform for targeted delivery of therapeutic peptides and RNAs that is highly efficient, stable, versatile and biocompatible for potential clinical applications in leukemia treatment. Migara Kavishka Jayasinghe, Marco Pirisinu, Huan Chen, and Yuqi Yang contributed equally to this work as first authors. Disclosures Jayasinghe: Carmine Therapeutics: Patents & Royalties. Shi: Carmine Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Le: Carmine Therapeutics: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.