Relationship between the size of nanoparticles and their adjuvant activity: Data from a study with an improved experimental design

Abstract

There is a growing interest in identifying the relationship between the size of nanoparticles and their adjuvant activity, but the results from recent studies remain controversial. To address the controversy, it was thought that one should pay attention to the nanoparticle formulations to make sure that the antigen-loaded nanoparticles to be compared are not only different in particle size, but more importantly, as identical to each other as possible in all other formulation properties. In the present study, using ovalbumin (OVA) as a model antigen conjugated onto nanoparticles engineered from lecithin/glyceryl monostearate-in-water emulsions, we prepared OVAnanoparticles of 230 nm and 708 nm. Before evaluating the immune responses induced by them in a mouse model, we made sure that: i) the sizes of the two OVA-nanoparticles did not extensively overlap, ii) the nanoparticles have similar zeta potentials and comparable antigen-loading, and iii) the nanoparticles did not aggregate when suspended in simulated biological media. We then showed that when subcutaneously injected into mice, the 230 nm OVA-conjugated nanoparticles induced stronger OVA-specific antibody and cellular immune responses than the 708 nm OVAnanoparticles. Future studies attempting to correlate the size of nanoparticles and their adjuvant activities need to consider formulation parameters to ensure that the particles are different only in size and are stable before and after injection.

Multifunctional nanoparticles for cancer immunotherapy

“…8 For uptake into cells, particles of 500 nm or smaller were optimal for uptake by DCs and macrophages. 33 However, the influence of size on the induction of immunity is not clear and may depend on the route of administration. For example, particles administered either orally or intranasally, the increased size of the particles (>500 nm) may facilitate their trapping in gut-associated lymphoid tissue or nasal-associated lymphoid tissue, thus inducing efficient mucosal responses.…”

Permeation of antigen protein-conjugated nanoparticles and live bacteria through microneedle-treated mouse skin

“…27,29 To label the nanoparticles fluorescently, DOPE-fluorescein (5%, m/m of total lipids) was included in the lecithin and glyceryl monostearate mixture during nanoparticle preparation. 27,29 conjugation of ovalbumin onto the nanoparticles…”

“…The endotoxin level in the nanoparticle preparation was estimated to be 0.18-0.57 EU/mL using a ToxinSensor TM chromogenic limulus amebocyte lysate endotoxin assay kit from GenScript (Piscataway, NJ). 29 The size and zeta potential of the nanoparticles were determined using a Malvern Zetasizer ® Nano ZS (Westborough, MA).…”

“…[27][28][29] Prior to conjugation, ovalbumin was thiolated using Traut's reagent. Ovalbumin was diluted in carbonate buffer (0.1 M, pH 9.6), followed by addition of Traut's reagent (20× molar excess) and a 60-minute incubation at room temperature.…”

“…27,28 It was shown that subcutaneous injection of protein antigens conjugated onto the nanoparticles induced strong functional antibody and cellular immune responses. 27,29 In the present study, the antibody responses induced by ovalbumin nanoparticles or ovalbumin alone when applied onto a mouse skin area pretreated with microneedle rollers were evaluated and compared, using ovalbumin as a model antigen chemically conjugated onto nanoparticles (mean diameter 230 nm) and three different microneedle rollers with different-sized microneedles. Prior to an in vivo animal immunization study, permeation of the ovalbumin nanoparticles through mouse skin treated with microneedle rollers was evaluated in vitro.…”