Siddharth Jhunjhunwala

Delivery of Rapamycin to Dendritic Cells Using Degradable Microparticles

Degradable microparticles have the potential to protect and release drugs over extended periods and, if sized appropriately, can be passively targeted to phagocytic cells in vivo. Dendritic cells (DC) are a class of phagocytic cells known to play important roles in transplant rejection. Previously, we have demonstrated that DC treated with an immunosuppressive drug, rapamycin, have the ability to suppress transplant rejection. Herein, we describe a strategy to deliver an intracellular depot of rapamycin to DC. To achieve this, rapamycin was encapsulated into ~3.4 microm sized poly(lactic-co-glycolic)acid (PLGA) microparticles (rapaMPs), and release behavior was examined under intra-phagosomal (pH=5) and extracellular (pH=7.4) conditions. It was observed that 4 days following phagocytosis of rapaMP, DC have significantly reduced ability to activate T cells, in comparison to DC treated with soluble rapamycin. Hence, we conclude that DC-specific intracellular delivery of rapamycin results in better efficacy of the drug, with respect to its ability to modulate DC function, when compared to treating DC with extracellular rapamycin.

Prevention of inflammation-mediated bone loss in murine and canine periodontal disease via recruitment of regulatory lymphocytes

Significance Periodontal disease (gum disease) is an extremely prevalent inflammatory disease initiated by persistent bacterial insult, leading to the destruction of bone and gingival tissues. Current clinical treatments focus solely on the removal of bacteria. In this study, we put forth a strategy to address the underlying inflammatory imbalance in periodontal disease by harnessing the body’s own sophisticated immunoregulatory mechanisms through the recruitment of regulatory T cells (Tregs). This is accomplished by controllably releasing small quantities (nanogram/kilogram range) of chemokine recognized by Tregs using biodegradable, resorbable polymers with an excellent track record of regulatory approval. Administration of Treg-recruiting treatments to the gingiva of mice and canines reduces clinical scores of disease as well as hard and soft tissue destruction. The hallmark of periodontal disease is the progressive destruction of gingival soft tissue and alveolar bone, which is initiated by inflammation in response to an invasive and persistent bacterial insult. In recent years, it has become apparent that this tissue destruction is associated with a decrease in local regulatory processes, including a decrease of forkhead box P3-expressing regulatory lymphocytes. Accordingly, we developed a controlled release system capable of generating a steady release of a known chemoattractant for regulatory lymphocytes, C-C motif chemokine ligand 22 (CCL22), composed of a degradable polymer with a proven track record of clinical translation, poly(lactic-co-glycolic) acid. We have previously shown that this sustained presentation of CCL22 from a point source effectively recruits regulatory T cells (Tregs) to the site of injection. Following administration of the Treg-recruiting formulation to the gingivae in murine experimental periodontitis, we observed increases in hallmark Treg-associated anti-inflammatory molecules, a decrease of proinflammatory cytokines, and a marked reduction in alveolar bone resorption. Furthermore, application of the Treg-recruiting formulation (fabricated with human CCL22) in ligature-induced periodontitis in beagle dogs leads to reduced clinical measures of inflammation and less alveolar bone loss under severe inflammatory conditions in the presence of a diverse periodontopathogen milieu.

Protein bioactivity and polymer orientation is affected by stabilizer incorporation for double-walled microspheres

Double-walled microspheres present an improved drug delivery technique for sustained release of encapsulated substrates. In this study, the release kinetics and biological activity of lysozyme was analyzed from microspheres comprised of poly(lactic-co-glycolic acid) (PLGA) and poly(L-lactide) (PLLA). In addition, coencapsulation of the anionic surfactant, docusate sodium salt (AOT), was investigated as a method of decreasing protein denaturation during microsphere fabrication. Herein, we show that through the inclusion of AOT, the capacity for two chemically similar polymers to phase separate and form double-walled (DW) microspheres is impaired leading to unique protein release kinetics. Additionally, we present the time period over which our released enzyme, lysozyme, remains biologically active. The consequences of AOT on protein bioactivity are also assessed and provide strong implications for the importance of appropriate stabilizer analysis in future studies involving drug co-encapsulates in polymer based microsphere systems.

Rapamycin ameliorates dystrophic phenotype in mdx mouse skeletal muscle.

Duchenne muscular dystrophy (DMD) Is an X-linked, lethal, degenerative disease that results from mutations In the dystrophin gene, causing necrosis and inflammation in skeletal muscle tissue. Treatments that reduce muscle fiber destruction and immune cell infiltration can ameliorate DMD pathology. We treated the mdx mouse, a model for DMD, with the immunosuppressant drug rapamycin (RAPA) both locally and systemically to examine its effects on dystrophic mdx muscles. We observed a significant reduction of muscle fiber necrosis in treated mdx mouse tibialis anterior (TA) and diaphragm (Dia) muscles 6 wks post-treatment. This effect was associated with a significant reduction in infiltration of effector CD4+ and CD8+ T cells in skeletal muscle tissue, while Foxp3+ regulatory T cells were preserved. Because RAPA exerts its effects through the mammalian target of RAPA (mTOR), we studied the activation of mTOR in mdx TA and Dia with and without RAPA treatment. Surprisingly, mTOR activation levels in mdx TA were not different from control C57BL/10 (B10). However, mTOR activation was different in Dia between mdx and B10; mTOR activation levels did not rise between 6 and 12 wks of age in mdx Dia muscle, whereas a rise in mTOR activation level was observed in B10 Dia muscle. Furthermore, mdx Dia, but not TA, muscle mTOR activation was responsive to RAPA treatment.

Controlled release formulations of IL-2, TGF-β1 and rapamycin for the induction of regulatory T cells

The absence of regulatory T cells (Treg) is a hallmark for a wide variety of disorders such as autoimmunity, dermatitis, periodontitis and even transplant rejection. A potential treatment option for these disorders is to increase local Treg numbers. Enhancing local numbers of Treg through in situ Treg expansion or induction could be a potential treatment option for these disorders. Current methods for in vivo Treg expansion rely on biologic therapies, which are not Treg-specific and are associated with many adverse side-effects. Synthetic formulations capable of inducing Treg could be an alternative strategy to achieve in situ increase in Treg numbers. Here we report the development and in vitro testing of a Treg-inducing synthetic formulation that consists of controlled release vehicles for IL-2, TGF-β and rapamycin (a combination of cytokines and drugs that have previously been reported to induce Treg). We demonstrate that IL-2, TGF-β and rapamycin (rapa) are released over 3-4weeks from these formulations. Additionally, Treg induced in the presence of these formulations expressed the canonical markers for Treg (phenotype) and suppressed naïve T cell proliferation (function) at levels similar to soluble factor induced Treg as well as naturally occurring Treg. Most importantly, we show that these release formulations are capable of inducing FoxP3(+) Treg in human cells in vitro. In conclusion, our data suggest that controlled release formulations of IL-2, TGF-β and rapa can induce functional Treg in vitro with the potential to be developed into an in vivo Treg induction and expansion therapy.

Bioinspired controlled release of CCL22 recruits regulatory T cells in vivo

Deficiency of regulatory T cells (Treg), a type of lymphocyte that promotes immunological homeostasis in the healthy steady state, [1–3] is a causative factor for destructive inflammation and autoimmune disease. [2,4] Accordingly, increasing the presence of Treg at the site of autoimmunity has been suggested as a potential method to treat these types of disorders. [3,4] However, to our knowledge, techniques to increase the local presence of specific immune cell types in vivo do not yet exist. Herein, we develop and test a microscale controlled-release system for the recruitment of Treg that draws inspiration from a specific biological system: tumors. Specifically, a wide variety of tumors release the chemokine CCL22, [5,6] which is responsible for tumor-directed migration of Treg and corresponding tumor-specific immune evasion. Our hypothesis was that if a steady CCL22 release could be maintained, by tuning the material properties of a controlled-release system, Treg may be preferentially recruited to a local site in vivo. n nThe first step towards testing this hypothesis was to design a release vehicle suitable for the steady release of CCL22. Although polymeric-microparticle-based controlled-release systems for proteins (including chemokines) have previously been developed, [7,8] the release of hydrophilic proteins from these particles typically follows a triphasic release profile. [9,10] Our goal was to achieve release of CCL22 without any periods of lag in order to produce a corresponding steady gradient of chemokine originating from a point source. To this end, new mechanistic descriptions of how controlled release from degradable materials [9,11] suggest that one of the major factors affecting the rate of release is the erosion of a dense polymer matrix into a porous macrostructure that allows for protein egress. The timing of this process and, in turn, the period of lag is determined by the type of polymer, its molecular weight (inherent viscosity) and the degree of porosity in the matrix. Accordingly, we hypothesized that addition of contiguous pores to the microparticles during fabrication would pre-establish pathways for diffusive protein egress and should bypass the need for erosion as a requisite for release. Thus, we used the polymer poly(lactic-co-glycolic acid) (PLGA) of a specific inherent viscosity (0.16–0.24 dl/g in 0.1% chloroform; in order to achieve release within a 3–8 week period) and modified the surface porosity using an osmotic gradient between the inner aqueous emulsion and the outer bulk aqueous phase (Supporting Information, Figure S1). We observed that porous microparticles prepared using a specific osmotic gradient (corresponding to 15 × 10−3 m NaCl in inner aqueous emulsion), in contrast to unmodified degradable particles ( Figure 1a), did not display the standard lag phase that would be expected with release of proteins and released CCL22 continuously over a 4-week period (Figure 1b). As an additional design parameter, the particles were made to be large enough to avoid uptake by phagocytic cells and to prohibit their movement across vascular endothelium (Supporting Information, Figure S2). As a consequence, particles would remain immobilized at the site of injection. n n n nFigure 1 n nCCL22MP Characteristics. a) Scanning electron micrographs of nonporous (left; top and bottom) and porous (right; top and bottom) CCL22 microparticles (CCL22MP). The top images were taken at 500× magnification, while the bottom images were taken ... n n n nIn order to test the ability of rationally designed CCL22MP to attract Treg, an in vivo adoptive transfer model coupled with non-invasive live-animal imaging was used. Specifically, fluorescently labeled CCL22MP were injected into the triceps surae of normal FVB mice followed by intravenous (i.v.) infusion of ex vivo-alloactivated Treg [12] (AATreg) that constitutively expresses the luciferase gene. The hind limb muscles were chosen as the site for MP injections as these distal sites are not expected to produce large quantities of CCL22. The migration pattern of these bioluminescent AATreg was studied following the injection of non-labeled mature allogeneic dendritic cells (DC), which provide an activation stimulus (Supporting Information, Figure S3). Soon after DC stimulation, a significantly greater number of AATreg was recruited to the site of CCL22MP injection compared to an internal control of microparticles lacking CCL22 ( Figure 2a and Supporting Information Figure S4). The adoptively transferred Treg are expected to translocate to sites in the body (e.g., lungs, gut etc.) that secrete CCL22, as observed in Figure S4, but not sites such as the hind limb (as confirmed by our preliminary experiments (data not shown)). However, a significant number of Treg do migrate towards the hind limb muscles upon injection of CCL22MP but not upon addition of blank particles ( p < 0.05), suggesting that the migration must be due to the release of CCL22 from these particles. Importantly, several reports suggest that small changes in Treg numbers at local sites (leading to a change in the ratio of Treg to effector T cells) is sufficient to dramatically alter local immune responses. [13] In line with these reports, and as a test for the ability of CCL22MP to replace the function of CCL22 secreting cells in vivo (e.g., site-specific recruitment of regulatory cells), we also demonstrate that CCL22MP are effectively able to delay the rejection of transplanted allogeneic cells. Specifically, allogeneic luciferase-expressing Lewis lung carcinoma cells (which do not endogenously produce CCL22) were implanted subcutaneously into mice at the site of CCL22MP injections (or for comparison BlankMP or Bolus CCL22 as controls), and the time to rejection was recorded using non-invasive live imaging (Figure S3). We observed that the rejection rates were significantly slower in the CCL22MP group when compared to both bolus CCL22 and BlankMP controls (Figure 2b and Figure S5), supporting the hypothesis that establishing a CCL22 gradient in vivo using synthetic systems can help modulate local immune respons1es. n n n nFigure 2 n nCCL22MP in vivo. a) Representative fluorescence (red-gold) and luminescence images (blue-yellow) showing localization of particles and Treg, respectively; fluorescence images were used to outline (red-line) areas of particle localization (Igor Pro Living ... n n n nPotential therapeutic implications of such a bioinspired degradable controlled-release formulation capable of recruiting Treg in vivo are manifold. One obvious application is the use of CCL22MP in combination with an infusion of Treg expanded ex vivo. Current pre-clinical data suggest that freshly-isolated or ex vivo-expanded Treg infusion can prevent organ transplant rejection or suppress autoimmune diseases. [2,14,15] However, challenges such as obtaining adequate numbers and highly-purified populations of Treg have hindered progress into clinical trials. [14,15] Using formulations that release CCL22, it may be possible to lower the numbers of injected Treg, or potentially use populations with lower purity. Another use of CCL22MP would be to attract endogenous Treg populations, wherein these formulations have the potential to function as “off-the-shelf” therapeutics for the treatment of a wide variety of disorders associated with unrestrained immune reactivity. n nA potential drawback of using a CCL22 sustained release vehicle is that the receptor for this chemokine (CCR4) is expressed on both activated Treg and activated effector T cells, [16] suggesting that both these cell populations would be attracted towards CCL22MP. Yet in vivo studies show that CCL22 production associated with tumors [5,17] or long-surviving allografts [18] result in preferential recruitment of Treg leading to local immunosuppression. One possible explanation for these results is that Treg could express significantly more CCR4 than effector T cells [17] (Supporting Information, Figure S6). Further, it has been suggested that an optimal ratio of Treg to effector T cells, and not a complete absence of effector T cells, is necessary for effective local suppression of immunity. [14,19] Regardless, if local effector T cells abrogate a suppressive environment, CCL22MP can easily be modified to simultaneously release immunosuppressive agents (such as rapamycin) [20] to inhibit these effector T cells in situ, thereby assisting Treg to control adverse immune responses. n nIn conclusion, we demonstrate that site-specific attraction of Treg leading to local immunomodulation can be achieved in vivo using CCL22MP. These bioinspired controlled-release formulations are particularly attractive as modular platforms for therapeutic development, as well as tools to study Treg-dependent modulation of immune responses in situ.

Porous calcium phosphate-poly (lactic-co-glycolic) acid composite bone cement: A viable tunable drug delivery system.

Calcium phosphate based cements (CPCs) are frequently used as bone void fillers for non-load bearing segmental bone defects due to their clinically relevant handling characteristics and ability to promote natural bone growth. Macroporous CPC scaffolds with interconnected pores are preferred for their ability to degrade faster and enable accelerated bone regeneration. Herein, a composite CPC scaffold is developed using newly developed resorbable calcium phosphate cement (ReCaPP) formulation containing degradable microspheres of bio-compatible poly (lactic-co-glycolic acid) (PLGA) serving as porogen. The present study is aimed at characterizing the effect of in-vitro degradation of PLGA microspheres on the physical, chemical and structural characteristics of the composite cements. The porosity measurements results reveal the formation of highly interconnected macroporous scaffolds after degradation of PLGA microspheres. The in-vitro characterizations also suggest that the degradation by products of PLGA reduces the pH of the local environment thereby increasing the dissolution rate of the cement. In addition, the in-vitro vancomycin release from the composite CPC scaffold suggests that the drug association with the composite scaffolds can be tuned to achieve control release kinetics. Further, the study demonstrates control release lasting for longer than 10weeks from the composite cements in which vancomycin is encapsulated in PLGA microspheres.

All-trans retinoic acid and rapamycin synergize with transforming growth factor-β1 to induce regulatory T cells but confer different migratory capacities

Tregs play important roles in maintaining immune homeostasis, and thus, therapies based on Treg are promising candidates for the treatment for a variety of immune‐mediated disorders. These therapies, however, face the significant challenge of obtaining adequate numbers of Tregs from peripheral blood that maintains suppressive function following extensive expansion. Inducing Tregs from non‐Tregs offers a viable alternative. Different methods to induce Tregs have been proposed and involve mainly treating cells with TGF‐β‐iTreg. However, use of TGF‐β alone is not sufficient to induce stable Tregs. ATRA or rapa has been shown to synergize with TGF‐β to induce stable Tregs. Whereas TGF‐β plus RA‐iTregs have been well‐described in the literature, the phenotype, function, and migratory characteristics of TGF‐β plus rapa‐iTreg have yet to be elucidated. Herein, we describe the phenotype and function of mouse rapa‐iTreg and reveal that these cells differ in their in vivo homing capacity when compared with mouse RA‐iTreg and mouse TGF‐β‐iTreg. This difference in migratory activity significantly affects the therapeutic capacity of each subset in a mouse model of colitis. We also describe the characteristics of iTreg generated in the presence of TGF‐β, RA, and rapa.

Effect of rapamycin on immunity induced by vector-mediated dystrophin expression in mdx skeletal muscle

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. Therapeutic gene replacement of a dystrophin cDNA into dystrophic muscle can provide functional dystrophin protein to the tissue. However, vector-mediated gene transfer is limited by anti-vector and anti-transgene host immunity that causes rejection of the therapeutic protein. We hypothesized that rapamycin (RAPA) would diminish immunity due to vector-delivered recombinant dystrophin in the adult mdx mouse model for DMD. To test this hypothesis, we injected limb muscle of mdx mice with RAPA-containing, poly-lactic-co-glycolic acid (PLGA) microparticles prior to dystrophin gene transfer and analyzed treated tissue after 6 weeks. RAPA decreased host immunity against vector-mediated dystrophin protein, as demonstrated by decreased cellular infiltrates and decreased anti-dystrophin antibody production. The interpretation of the effect of RAPA on recombinant dystrophin expression was complex because of an effect of PLGA microparticles.

Microparticulate systems for targeted drug delivery to phagocytes

Comment on: Jhunjhunwala S, et al. J Control Release 2009;133:191-7.