Transmucosal delivery of protein and peptide drugs by using cell penetrating peptides

Protein drugs are primarily administered through parenteral routes, such as subcutaneous (s.c.) injection, due to their poor membrane permeability and low stability in the gastrointestinal tract. However, needle-based delivery methods pose challenges to treatment adherence due to inconvenience, discomfort, and needle phobia—especially for patients with chronic conditions requiring regular lifelong administrations. Here, we have developed a needle-free delivery platform based on the clinically used cell-penetrating peptide (CPP), protamine, for therapeutic proteins, aiming to enhance treatment adherence and therapeutic outcomes. The first part of this dissertation delves into the transcellular and transmucosal delivery of proteins using protamine via a non-covalent approach (physical mixing). Protamine demonstrated enhanced intracellular protein delivery by mediating actin rearrangement and promoting tubulation. Additionally, protamine aided insulin in overcoming mucus and epithelium barriers, promoting systemic absorption and achieving comparable blood glucose (BG) lowering effects in diabetic mice as its subcutaneous preparation. In the second part, two protamine derivatives, stearic acid-protamine (C18-P) and protamine dimer (P2), were synthesized to further enhance protein absorption when administered via the sublingual route through physical mixing. C18-P and P2 exhibited superior penetration abilities when delivering immunoglobulin G (IgG) compared to protamine in 2D models, 3D models, human sublingual substitutes, and in vivo. When mixed with insulin or semaglutide, C18-P and P2 showed enhanced BG control compared to protamine when delivered sublingually in mice. Pharmacokinetic (PK) studies revealed that C18-P and P2 enable the absorption of proteins up to 150 kDa via the sublingual route in mice, with relative bioavailabilities exceeding 50%. In the third part, Nano-P (allyl glycidyl ether-conjugated protamine) and Dendri-P (polyamidoamine-linked protamine) were synthesized to enhance intranasal delivery of anti-CD124 monoclonal antibody (αCD124) through physical mixing, aiming at chronic rhinosinusitis with nasal polyps (CRSwNP) treatment. The efficacy was found to be improved compared to protamine by evaluating the number of nasal polypoid lesions, epithelium disruptions, and induction of immunoglobulin E (IgE) and type 2 inflammation biomarkers. Both nano-formulations exhibited a superior anti-CRSwNP effect compared to a 12.5-fold higher dose of subcutaneous αCD124.