Effect of nanoparticle size on their distribution and retention in chronic inflammation sites.

Nanomedicines are increasingly researched and used for the treatment of chronic inflammatory diseases. Herein, the effect of the size of nanoparticles on their distribution and retention in chronic inflammatory sites, as compared to healthy tissues, was studied in a mouse model with chronic inflammation in one of the hind footpads. Using PEGylated gold nanoparticles of 2, 10, 100, and 200 nm, we found that although the smaller nanoparticles of 2 and 10 nm showed greater distribution and slower clearance in the inflamed footpad than the relatively larger nanoparticles of 100 and 200 nm, the larger nanoparticles of 100 and 200 nm were more selectively distributed in the inflamed hind footpad than in the healthy hind footpad in the same mouse. Based on these findings, we prepared protein nanoparticles of 100-200 nm with albumin, IgG antibody, or anti-TNF-α monoclonal antibody (mAb). The nanoparticles can release proteins in response to high redox activity and/or low pH, conditions seen in chronic inflammation sites. We then showed that upon intravenous injection, those stimuli-responsive protein nanoparticles distributed more selectively in the inflamed footpad than free proteins and remained longer in the inflamed footpad than similar protein nanoparticles that are not sensitive to high redox activity or low pH. These findings support the feasibility of increasing the selectivity of nanomedicines and protein therapeutics to chronic inflammation sites and prolonging their retention at the sites by innovative nanoparticle engineering.

Effect of the Ratio of Betamethasone to TNF-α siRNA Coencapsulated in Solid Lipid Nanoparticles on the Acute Proinflammatory Activity of the Nanoparticles.

There is evidence that encapsulating glucocorticoids into nucleic acid-containing nanoparticles reduces the inflammatory toxicities of the nanoparticles. Herein, using betamethasone acetate (BA), a glucocorticoid, and a solid lipid nanoparticle formulation of siRNA, we confirmed that coencapsulating BA into the siRNA solid lipid nanoparticles significantly reduced the proinflammatory activity of the siRNA nanoparticles in a mouse model. Using TNF-α siRNA, we then showed that the BA and TNF-α siRNA coencapsulated into the solid lipid nanoparticles acted as a dual anti-inflammatory and synergistically reduced TNF-α release by mouse macrophages in culture following stimulation with lipopolysaccharide, as compared to solid lipid nanoparticles encapsulated with TNF-α siRNA or BA alone. Importantly, upon studying the effect of the ratio of BA and TNF-α siRNA on the proinflammatory activity of the resultant nanoparticles, we identified that BA and TNF-α siRNA coencapsulated solid lipid nanoparticles prepared with a BA to TNF-α siRNA weight ratio of 2:1 induced the lowest proinflammatory cytokine production by macrophages in culture. This result was in comparison to nanoparticles prepared with BA to TNF-α siRNA ratios both higher and lower than 2:1 (i.e., 4:1, 1:1, and 0.5:1) and is likely due to differences in molecular interactions among the various components in the BA and TNF-α-siRNA coencapsulated solid lipid nanoparticles at these ratios. Encapsulating glucocorticoids into siRNA-nanoparticles represents a viable strategy to reduce the proinflammatory activity of the nanoparticles; however, the ratio of the glucocorticoid to siRNA in the nanoparticles requires optimization.

Lipid nanoparticles with minimum burst release of TNF-α siRNA show strong activity against rheumatoid arthritis unresponsive to methotrexate.

TNF-α siRNA has shown promising therapeutic benefits in animal models of rheumatoid arthritis. However, there continues to be a need for siRNA delivery systems that have high siRNA encapsulation efficiency and minimum burst release of TNF-α siRNA, and can target inflamed tissues after intravenous administration. Herein we report a novel acid-sensitive sheddable PEGylated solid-lipid nanoparticle formulation of TNF-α-siRNA, AS-TNF-α-siRNA-SLNs, prepared by incorporating lipophilized TNF-α-siRNA into solid-lipid nanoparticles composed of biocompatible lipids such as lecithin and cholesterol. The nanoparticles are approximately 120 nm in diameter, have a high siRNA encapsulation efficiency (>90%) and a minimum burst release of siRNA (<5%), and increase the deilvery of the siRNA in chronic inflammation sites in mouse models, including in a mouse model with collagen-induced arthritis. Importantly, in a mouse model of collagen antibody-induced arthritis that does not respond to methotrexate therapy, intravenous injection of the AS-TNF-α-siRNA-SLNs significantly reduced paw thickness, bone loss, and histopathological scores. These findings highlight the potential of using this novel siRNA nanoparticle formulation to effectively treat arthritis, potentially in patients who do not respond adequately to methotrexate.

Acid-Sensitive Sheddable PEGylated, Mannose-Modified Nanoparticles Increase the Delivery of Betamethasone to Chronic Inflammation Sites in a Mouse Model.

Inflammation is implicated in a host of chronic illnesses. Within these inflamed tissues, the pH of the microenvironment is decreased and immune cells, particularly macrophages, infiltrate the area. Additionally, the vascular integrity of these sites is altered with increased fenestrations between endothelial cells. These distinctive properties may be exploited to enhance targeted delivery of anti-inflammatory therapies. Using a mouse model of chronic inflammation, we previously showed that acid-sensitive sheddable PEGylation increases the distribution and retention of nanoparticles in chronic inflammation sites. Here we demonstrated that surface modification of the acid-sensitive sheddable PEGylated nanoparticles with mannose, a ligand to mannose receptors present in chronic inflammation sites, significantly increases the targeted delivery of the nanoparticles to these areas. Furthermore, we showed that the acid-sensitive sheddable PEGylated, mannose-modified nanoparticles are able to significantly increase the delivery of betamethasone-21-acetate (BA), a model anti-inflammatory compound, to chronic inflammation sites as compared to free BA. These results highlight the ability to engineer formulations to target chronic inflammation sites by exploiting the microenvironment of these regions.

Acid-Sensitive Sheddable PEGylated PLGA Nanoparticles Increase the Delivery of TNF-α siRNA in Chronic Inflammation Sites.

There has been growing interest in utilizing small interfering RNA (siRNA) specific to pro-inflammatory cytokines, such as tumor necrosis factor-α ( TNF-α), in chronic inflammation therapy. However, delivery systems that can increase the distribution of the siRNA in chronic inflammation sites after intravenous administration are needed. Herein we report that innovative functionalization of the surface of siRNA-incorporated poly (lactic-co-glycolic) acid (PLGA) nanoparticles significantly increases the delivery of the siRNA in the chronic inflammation sites in a mouse model. The TNF-α siRNA incorporated PLGA nanoparticles were prepared by the standard double emulsion method, but using stearoyl-hydrazone-polyethylene glycol 2000, a unique acid-sensitive surface active agent, as the emulsifying agent, which renders (i) the nanoparticles PEGylated and (ii) the PEGylation sheddable in low pH environment such as that in chronic inflammation sites. In a mouse model of lipopolysaccharide-induced chronic inflammation, the acid-sensitive sheddable PEGylated PLGA nanoparticles showed significantly higher accumulation or distribution in chronic inflammation sites than PLGA nanoparticles prepared with an acid-insensitive emulsifying agent (i.e., stearoyl-amide-polyethylene glycol 2000) and significantly increased the distribution of the TNF-α siRNA incorporated into the nanoparticles in inflamed mouse foot.

The effect of the acid-sensitivity of 4-(N)-stearoyl gemcitabine-loaded micelles on drug resistance caused by RRM1 overexpression.

Chemoresistance is a major issue for most gemcitabine-related chemotherapies. The overexpression of ribonucleotide reductase subunit M1 (RRM1) plays a key role in gemcitabine resistance. In this study, we synthesized a new highly acid-sensitive amphiphilic micelle material by conjugating hydrophilic polyethylene glycol with a hydrophobic stearic acid derivative (C18) using a hydrazone bond, which was named as PHC-2. A lipophilic prodrug of gemcitabine, 4-(N)-stearoyl gemcitabine (GemC18), was loaded into micelles prepared with PHC-2, a previously synthesized less acid-sensitive PHC-1, and their acid-insensitive counterpart, PAC. GemC18 loaded in acid-sensitive micelles can overcome gemcitabine resistance, and GemC18 in the highly acid-sensitive PHC-2 micelles was more cytotoxic than in the less acid-sensitive PHC-1 micelles. Mechanistic studies revealed that upon cellular uptake and lysosomal delivery, GemC18 in the acid-sensitive micelles was released and hydrolyzed more efficiently. Furthermore, GemC18 loaded in the highly acid-sensitive PHC-2 micelles inhibited the expression of RRM1 and increased the level of gemcitabine triphosphate (dFdCTP) in gemcitabine resistant tumor cells. The strategy of delivering lipophilized nucleoside analogs using highly acid-sensitive micelles may represent a new platform technology to increase the antitumor activity of nucleoside analogs and to overcome tumor cell resistance to them.