Category: Formulation and Quality
Purpose: Although the immunomodulatory potentials of nanoparticles have been established1, these are often regarded as toxicological adversities rather than therapeutic benefits. Copolymers of ethyl acrylate, methyl methacrylate, and a low content of methacrylic acid ester with quaternary ammonium groups, ammonio methacrylate copolymers (AMCs) are well-known excipients in pharmaceutical industry2. Herein, we sought to evaluate the immunostimulatory properties of cargo-free AMC nanoparticles (AMCNP) in cell culture, and the resultant immunotherapeutic potentials against a subcutaneous cancer model in-vivo. Furthermore, the effect of particles’ physicochemical properties such as size and surface charge was investigated3.
Nanoparticles were prepared using type A and B AMC (Eudragit® RL (RLNP) and Eudragit® RS (RSNP), with 10 and 5% quaternary ammonium content, respectively2) in two different sizes (100 and 700nm) through a spontaneous emulsification/solvent diffusion technique. The polymer was dissolved in a mixture of ethyl acetate and acetone, the aqueous phase was added under homogenization, and the organic solvents were removed under reduced pressure. Particle size was adjusted through the optimization of the homogenization force, ethyl acetate:acetone ratio, and volume of the aqueous phase. The particles were then characterized in terms of their physicochemical properties.
Within the context of cell culture experiments, the ability of the particles to induce the secretion of pro-inflammatory cytokines from RAW276.4 macrophages and JAWSII DCs was investigated. To this end, the cells were incubated with AMCNP overnight, following which concentration of different proinflammatory cytokines was measured using ELISA. Furthermore, RAW Blue cells with active and blocked TLR4 signaling were used to investigate the potential role of NF-κB in this scenario, and the TLR4-dependency thereof.
To assess the immunotherapeutic potentials of the particles in-vivo, subcutaneous tumors were induced through the injection of 3x105 Colon26 cells in the right flank of male BALB/C mice. Treatment was initiated once the tumor approached a volume of 50 mm3, in form of biweekly peritumoral injections of PBS (control) or different AMCNPs (10mg/ml). The mice were monitored in terms of the tumor volume and the overall wellbeing. Experiments were terminated once the tumor surpassed a volume of 1000mm3.
Results: AMCNP were prepared in different sizes and with different cationic charge densities (RLNP-100nm; 96±17nm and +66±9mV, RSNP-100nm; 88±14nm and +55±2mV, RLNP-700nm; 679±37nm and +42±3mV, and RSNP-100nm; 734±43nm and +34±3mV). All particles induced the secretion of an array of pro-inflammatory cytokines and chemokines from the cells of innate immunity, some of which have been presented in Figure 1. The cytokine induction profile was dependent either on nanoparticle size (TNF-α and IL-6) or cationic ammonium content and surface charge (IL-12, IFN-γ and IL-1β). The immunostimulatory properties of the particles were NF-κB-mediated and TLR4-dependent (Figure 2).
In-vivo, all particles resulted in a significant retardation of the syngeneic tumor, and even a complete retardation thereof. This led to a significant improvement of the animals’ survival in case of the AMCNP-treated groups compared to the PBS-treated control (Figure 3).
Both the size and charge of the particles affected the in vivo immunotherapeutic performance. Generally, while smaller particles were more effective than their larger counterparts, a higher cationic charge improved the efficiency at a given size. Accordingly, the best performance, including a 40% remission rate, was observed in the group treated with RLNP-100nm. Recovered animals were immune to further challenges with Colon26 cells. Additionally, when cultured with Colon26 cells, splenocytes isolated from the nanoparticle-treated animals resulted in a significantly higher level of apoptosis induction therein than those isolated from the control animals, which further indicated the presence of the immunological memory (data not shown).
Conclusion: Nanoparticles possess unique immunological properties, which are often regarded from a nanotoxicological, rather than immunotherapeutic perspective1. Here, we demonstrated that despite being well-recognized excipients in pharmaceutical industry, nanoscaled AMCs possess remarkable immunostimulatory properties exploitable for the treatment of cancer. The particles result in a significant induction of pro-inflammatory cytokines from the cells of innate immunity in an NF-κB-mediated and TLR4-dependent manner. Furthermore, a purposeful manipulation of the nanoparticle size and surface charge allows for the optimization of the eventual immunostimulatory potentials. So promising has been the immunotherapeutic potentials of the particles that preitumoral treatment of the animals therewith has resulted in significant tumor growth retardation, or in cases remission. Additionally, AMCs are associated with a more acceptable safety profile than many of conventional TLR4 ligands such as LPS, and also possess acceptable regulatory status. Further research in this arena can uncover a generation of safe, cost-effective and apparently inert biomaterials capable of exerting pharmacological and therapeutic effects for the treatment of different types of disorders and in particular cancer.