In the past decade, significant advances have been made in cancer immunotherapy, demonstrating that the immune system, and in particular T cells, can effectively attack cancerous tissue. The immune system is tightly controlled by co-stimulatory and co-inhibitory receptors, which determine the signal transduction of T cell receptor (TCR) and are important for T cell activation. Co-inhibitory receptors are also known as immune checkpoint molecules [1]. Blocking the immune checkpoint by antibodies can restore the function of T lymphocytes and eventually eradicate the tumor in some patients [2,3]. However, the response rate of immune checkpoint blockade remains to be improved in some cancers, such as microsatellite stable colorectal cancer etc. Compared with antibodies, other immune checkpoint inhibitors (ICIs), such as small molecules and peptides, have many advantages, especially better tumor penetration and easier manufacturing [4]. Especially, peptides exhibit higher specificity and less toxicity than small molecules. However, there are two major challenges to the development of peptide drugs, overcoming enzymatic degradation and efficient oral delivery.

Oral delivery of peptides is convenient, non-invasive, and safe with high patient compliance [5]. In the last decade, many attempts have been made to improve oral bioavailability of peptide drugs. The significant breakthrough in oral peptide delivery is semaglutide, which was approved by the U.S. Food and Drug Administration, but it still has low oral bioavailability [6]. To promote peptide drug absorption, strategies have been developed to instantaneously disrupt the intestinal epithelium, such as opening the tight junction [[7], [8], [9]]. This may result in nonspecific absorption of pathogens and toxins, which lead to adverse effects in long-term treatment [10]. Another commonly used strategy is to incorporate nanocarriers to enhance the active transportation of peptides. However, it often uses various synthetic materials or toxic organic solvents during preparation, which may also lead to side effects [11]. In the previous study, we developed a 12-mer peptide (OPBP-1) targeting PD-1/PD-L1 using mirror-imaged phage display technology, which significantly inhibited tumor growth and increased infiltration of CD8+ T cells and IFN-γ secretion in mouse models of colorectal cancer and melanoma when administered intraperitoneally at a relatively low dose of 0.5 mg/kg. This peptide is entirely made up of D-amino acids, which may effectively prevent gastrointestinal enzyme degradation and retain long-term integrity in the gastrointestinal tract (GIT). It also has the qualities of facile synthesis, good specificity and low toxicity. And it can be efficiently delivered orally by N-trimethyl-chitosan (TMC) hydrogel capsulation [[12], [13], [14]]. However, TMC can also open the intestinal tight junction and cause unwanted adverse effects. Thus, there is an urgent need for a green and biocompatible delivery system to overcome the physical and chemical barriers, and maximize the oral bioavailability of peptide drugs.

Microemulsions are nano-sized, thermodynamically stable isotropic systems in which two immiscible liquids (water and oil) are mixed to form a single phase by means of a surfactant and a co-surfactant [15,16]. Microemulsions are easily fabricated and demonstrate good stability, which are ideal delivery systems for improving oral absorption of peptide drugs [17]. Fish oil, a dietary source of long-chain fatty acids, is well known as a nutritional supplement to lower plasma cholesterol [18]. Omega-3 and omega-6 polyunsaturated fatty acids are the major components of fish oil and are essential fats that have numerous health benefits. Additionally, the most important types of fatty acid in omega-3 are docosahexaenoic (DHA) and eicosapentaenoic (EPA), which can repair disrupted tight junctions in a peroxisome proliferators-activated receptors γ (PPAPγ)-dependent manner [19], as well as to enhance intestinal cellular penetration of drugs without damaging the intestinal barriers [20], and have been utilized as drug delivery vehicles [21,22].

Besides the potential application as the component of oral peptide delivery microemulsion, Cockbain et al have reported that polyunsaturated fatty acid (PUFA) enhanced reactive oxygen species (ROS) within the colonocytes, which led to apoptosis of the cancer cells [23]. Recently, it has been reported that the peroxidation of polyunsaturated fatty acid (PUFA) can lead to the ferroptosis of cancer cells [24]. These results indicated that the fish oil microemulsion might be used as a functional delivery system, which might not only deliver peptide drugs orally but also induce cancer cell death. Interestingly, it has been reported that the anti-PD-1 immunotherapy-activated CD8+ T cells could enhance ferroptosis-specific lipid peroxidation in cancer cells [25]. Therefore, it is worth investigating whether fish oil-based microemulsion can act as a peptide ICI oral delivery system and lead to synergistic killing effects on cancer cells through ferroptosis.

In the present study, an optimal fish oil-based microemulsion was developed for oral delivery of PD-1/PD-L1 blocking model peptide, OPBP-1. The microemulsion was characterized, and intestinal cell uptake and transportation with associated mechanisms were investigated. Additionally, the in vivo pharmacokinetic and pharmacodynamic studies were performed. Lastly, the effect of fish oil on ROS-related ferroptosis of tumor cells was also studied. This study is the first time to develop a fish oil-based delivery system which has great potential for oral delivery of peptide drugs. Meanwhile, this functional nutraceutical delivery system also demonstrated a promising antitumor effect, since the fish oil could induce ferroptosis in tumor cells, and exerted a synergistic effect with peptide ICIs for cancer immunotherapy (Scheme 1).