Research Article Controlled Release of Second Generation mTOR Inhibitors to Restrain Inflammation in Primary Immune Cells Emily A. Gosselin, 1 Lisa H. Tostanoski, 1 and Christopher M. Jewell 1,2,3,4,5 Received 28 January 2017; accepted 14 April 2017 Abstract. Autoimmune disease occurs when the immune system incorrectly targets the body’ s own tissue. Inflammatory CD4 + T cell phenotypes, such as T H 1 and T H 17, are key drivers of this attack. Recent studies demonstrate treatment with rapamycin—a key inhibitor of the mTOR pathway—can skew T cell development, moving T cell responses away from inflammatory phenotypes and toward regulatory T cells (T REGS ). T REGS are important in inducing and maintaining tolerance to self-antigens, creating new potential to treat autoimmune diseases more effectively and specifically. Next generation analogs of rapamycin, such as everolimus and temsirolimus, confer increased potency with reduced toxicity, but are understudied in the context of autoimmunity. Further, these drugs are still broadly-acting and require frequent treatment due to short half-lives. Thus, there is strong interest in harnessing the unique properties of biomaterials—controlled drug release and targeting, for example, to improve autoimmune therapies. Using second generation mTOR inhibitors and rapamycin, we prepared sets of degradable polymer particles from poly(lactide-co-glycolide). We then used these materials to assess physicochemical properties and the ability to control autoimmune inflammation in a primary cell co-culture model. Treatment with particle formulations resulted in significant dose-dependent decreases in dendritic cell activation, T cell proliferation, inflammatory cytokines, and frequencies of inflammatory T H 1 phenotypes. Considering the current limitations of rapamycin, and the potential of next-generation analogs, this work provides a screening platform for biomaterials and sets the stage for in vivo evaluation, where delivery kinetics, stability, and targeting could improve autoimmune therapies through biomaterial-enabled delivery. KEY WORDS: controlled release; immune tolerance and autoimmunity; microparticle and nanoparticle; rapamycin, everolimus, and temsirolimus; vaccine and immunotherapy. INTRODUCTION The molecular target of rapamycin (mTOR) pathway is a central pathway regulating cell growth and metabolism (1,2). Not surprisingly, the mTOR pathway has been studied fundamentally and exploited translationally in areas from human development, to cancer, transplantation, and autoimmunity (1,3–6). One of the central inhibitors of mTOR, rapamycin (Rapa) is approved, as a long-standing immunosuppressant to limit transplant rejection, and as a coating for coronary stents (3). Rapa is also in clinical trials for use as a cancer therapeutic (4), and in clinical and preclinical trials as a treatment for autoimmune diseases (5,7–12). Newer generations of Rapa, Brapalogs,^ have been developed to improve efficacy through increased potency, improved solubility, and altered pharmacokinetics (13). Some of these rapalogs, including everolimus (Evero) and temsirolimus (Tems), have been approved for use as cancer therapeutics, and others are in clinical or pre-clinical trials (13–18). In several of these cases, rapalogs work as well as or better than Rapa to reduce cell proliferation, and range in application from preventing stent failure in coronary artery disease (19), to enhancing allograft survival after transplant (20), treating nephropathy (21), and reducing prostate cancer cell proliferation (22). One area where rapalogs have great potential, but where clinical applications lag behind the areas above, is autoimmunity. Autoimmune diseases occur when the immune system incorrectly Electronic supplementary material The online version of this article (doi:10.1208/s12248-017-0089-1) contains supplementary material, which is available to authorized users. 1 Fischell Department of Bioengineering, University of Maryland, 2212 Jeong H. Kim Engineering Building, 8228 Paint Branch Drive, College Park, Maryland 20742, USA. 2 Department of Microbiology and Immunology, University of Maryland Medical School, Baltimore, Maryland, USA. 3 Marlene and Stewart Greenebaum Cancer Center, Baltimore, Maryland, USA. 4 United States Department of Veterans Affairs, Baltimore, Mary- land, USA. 5 To whom correspondence should be addressed. (e-mail: cmjewell@umd.edu) The AAPS Journal ( # 2017) DOI: 10.1208/s12248-017-0089-1 1550-7416/17/0000-0001/0 # 2017 American Association of Pharmaceutical Scientists