Biopolymeric nanoparticles optimized as carriers of P60 antitumor peptide

Researchers have taken a significant step toward improving cancer immunotherapy by optimizing the preparation of biopolymeric nanoparticles designed to carry P60, a synthetic peptide that inhibits the activity of forkhead box P3 (FOXP3), a transcription factor crucial to the function of regulatory T cells. The study, published in Pharmacia, evaluated two types of nanoparticles—chitosan-alginate and chitosan-albumin—as potential carriers of the peptide, which is characterized by low in vivo stability, poor selectivity, and rapid systemic clearance. These limitations have significantly hindered the clinical application of P60, despite its demonstrated potential in cancer immunotherapies.

FOXP3 is expressed in a subset of CD4+ T lymphocytes known as regulatory T cells (Tregs), where it plays an important role in their development and suppressive immune function. By regulating genes such as CD25, CTLA4, and GITR, and interacting with transcription factors like NFAT and NF-κB, FOXP3 modulates the suppressive function of Tregs and contributes to preventing autoimmunity. However, this same function is unfavorable for tumor progression because Tregs inhibit antitumor immunity. Increased infiltration of FOXP3+ Tregs within the tumor microenvironment is usually associated with tumor progression and poor clinical prognosis. Therefore, selective inhibition of FOXP3 activity in tumor-infiltrating Tregs is considered a promising strategy to enhance antitumor immunity.

P60 binds to FOXP3 and inhibits its activity, thereby offering a route to implement this strategy in cancer immunotherapies. Previous studies have shown that treatment with P60 improved the efficacy of dendritic cell vaccines in breast tumor-bearing mice and inhibited tumor growth in both immunocompetent and immunocompromised mice. Another group developed a proteolysis-targeting chimeric drug by linking P60 to pomalidomide, which decreased tumor growth in mice by enhancing antitumor immunity. However, like many peptides, P60 suffers from low in vivo stability, poor selectivity, and rapid systemic clearance, which significantly hinders its application.

Nanoparticle encapsulation is considered a suitable strategy for facilitating peptide application by increasing stability and prolonging blood circulation. Polymeric nanoparticles, in particular, are regarded as appropriate carriers of peptides and proteins because they show high stability in biological fluids, strong protection of loaded peptides against enzymatic degradation, improved bioavailability, and high functionalization capacity. The use of biopolymers for such nanoparticles provides biocompatibility and biodegradability. Previous studies have successfully loaded oxytocin, gonadotropin-releasing hormone, and liraglutide into polymeric nanoparticles, demonstrating the versatility of these systems.

The current research aimed to optimize the technological procedure for the preparation of biopolymeric nanoparticles as possible carriers of the P60 peptide. Four procedures were tested for the preparation of chitosan-alginate nanoparticles, and P60 was also loaded into chitosan-albumin nanoparticles. The systems were characterized by mean diameter, polydispersity, zeta potential, and release profiles. To the knowledge of the researchers, P60 has not previously been encapsulated in polymeric nanoparticles based on these biopolymers.

Chitosan is a linear polysaccharide derived from chitin, consisting of D-glucosamine and N-acetyl-D-glucosamine units linked via β-(1→4)-glycosidic bonds. The presence of amino groups in its structure allows interactions with oppositely charged polymers or drugs. Its cationic properties in pH-neutral physiological fluids allow electrostatic interactions with the negatively charged membranes of epithelial cells, a prerequisite for enhanced cellular transport. Alginate, a polysaccharide consisting of L-guluronic and D-mannuronic acids, possesses anionic properties, making it very appropriate for interactions with chitosan and the production of nanoparticles under mild conditions. Both polysaccharides are biocompatible, biodegradable, and nontoxic.

P60-loaded chitosan-alginate nanoparticles were developed via the ionic gelation method, with the ratio between P60 and alginate varied at 1:20 and 1:10 wt/wt. The loading degrees of P60 were 124.22 µg/mL and 226.5 µg/mL, and the encapsulation efficiencies were 99.4% and 90.6%, respectively. The nanoparticles with the higher loading degree possessed a mean size of 356 nm, a polydispersity index of 0.268, and a zeta potential of −41.57 mV, allowing high colloidal stability. The nanoparticles with the lower loading degree were slightly smaller at approximately 332 nm, with a slight increase in the polydispersity index (0.318) and a negative zeta potential (−32 mV) with a sufficiently high absolute value.

The 1:10 wt/wt ratio between P60 and alginate was considered more appropriate regarding the loading of the peptide. Since the nanoparticles’ size was slightly larger, the next step was to perform sonication for different durations, an important factor affecting the size of colloidal systems. Increasing the duration of sonication led to a significant decrease in size to 203 nm, which is important for cellular uptake and especially for the enhanced permeability and retention (EPR) effect in tumor therapies. The polydispersity index of these particles was low (0.269), and the zeta potential was −38.8 mV. An additional sonication step after stirring was also examined, but the properties of these nanoparticles were almost similar, while the loading degree and encapsulation efficiency were lower than those of the first sample, particularly 174.72 µg/mL and 69.9% and 162.62 µg/mL and 65.1%, respectively. Thus, the conditions applied for the first preparation regime were considered optimal.

The second part of the study focused on loading P60 into chitosan-albumin nanoparticles. Albumin is a globular protein with high aqueous solubility, biocompatibility, and biodegradability. The carboxylic and amino groups in its structure can participate in electrostatic interactions with oppositely charged polymers such as chitosan at a specific pH. At high temperatures, albumin can partially denature, resulting in the formation of a three-dimensional gel structure. Albumin could allow prolonged blood circulation of the loaded drugs by reducing macrophage phagocytosis, which is particularly important for P60 peptide efficacy. Moreover, albumin can undergo gp60- and SPARC-mediated receptor endocytosis, allowing for inherent targeting. The loading degree and encapsulation efficiency of P60 were higher in the chitosan-albumin nanoparticles (423 µg/mL and 98.7%) than in the chitosan-alginate nanoparticles, probably due to the greater number of functional groups provided by albumin. The P60-chitosan-albumin nanoparticles were characterized by a mean diameter of 34 nm, a narrow size distribution (0.234), and a positive zeta potential (29.28 mV). The smaller size is important for the cellular transport of the peptide.

Finally, the release profiles of P60 from both types of nanoparticles were evaluated in media at pH 7.4, typical for healthy cells, and pH 5.0, typical for the tumor microenvironment. There was an initial burst release, which was more pronounced for the chitosan-alginate particles, probably due to the hydrophilic properties of the polymers and peptides. Sustained release was observed for both types of polymeric systems. After 8 hours, the release from the albumin-based nanoparticles was 38.6% in the medium at pH 7.4 and 51.4% in the medium at pH 5.0, whereas the release from the alginate-based system was 59.5% in the slightly alkaline medium and 65.5% in the slightly acidic medium. The slightly slower release from the albumin-based nanoparticles supported the suggestion of a greater number of bonds between P60 and albumin. More importantly, the release from both systems was faster in the slightly acidic medium, probably due to the pH-dependent solubility of chitosan, which is more soluble in acidic media. This result is particularly important and advantageous for the selective delivery of the loaded peptide to tumor tissue. Both types of the developed biopolymeric nanoparticles possessed optimal physicochemical properties and can be further evaluated as possible carriers of the P60 peptide.

Subject of Research: Optimization of the procedure for preparation of biopolymeric nanoparticles as carriers of P60 antitumor peptide

Article Title: Optimization of the procedure for preparation of biopolymeric nanoparticles as carriers of P60 antitumor peptide

Article References: Radeva, L., Serrano, A., Garrido, M. J., & Yoncheva, K. (2026). Optimization of the procedure for preparation of biopolymeric nanoparticles as carriers of P60 antitumor peptide. Pharmacia, 73, Article e206826. https://doi.org/10.3897/pharmacia.73.e206826

Image Credits: AI Generated

DOI: 10.3897/pharmacia.73.e206826

Keywords: Optimization, procedure, preparation, biopolymeric, nanoparticles, carriers, antitumor, peptide, scientific research