Subcutaneous nanotherapy repurposes the immunosuppressive mechanism of rapamycin to enhance allogeneic islet graft viability

Standard oral rapamycin (i.e. Rapamune®) administration is plagued by poor bioavailability and broad biodistribution. Thus, this pleotropic mTOR inhibitor has a narrow therapeutic window, numerous side effects and provides inadequate protection to transplanted cells and tissues. Furthermore, the hydrophobicity of rapamycin limits its use in parenteral formulations. Here, we demonstrate that subcutaneous delivery via poly(ethylene glycol)-b-poly(propylene sulfide)(PEG-b-PPS) polymersome (PS) nanocarriers significantly alters rapamycin’s cellular biodistribution to repurpose its mechanism of action for tolerance instead of immunosuppression while minimizing side effects. While oral rapamycin inhibits naïve T cell proliferation directly, subcutaneously administered rapamycin-loaded polymersomes (rPS) modulate antigen presenting cells in lieu of T cells significantly improving maintenance of normoglycemia in a clinically relevant, MHC-mismatched, allogeneic, intraportal (liver) islet transplantation model. These results demonstrate the ability of a rationally designed nanocarrier to re-engineer the immunosuppressive mechanism of a drug by controlling cellular biodistribution.

Type 1 diabetes (T1D) is an endocrine disorder that leads to pancreatic β cell destruction and requires management with lifelong exogenous insulin therapy 1 . Islet transplantation has emerged as a promising treatment for T1D by eliminating the need for exogenous insulin 1 . This protocol involves three key components: acquisition of viable insulin-producing cells, surgical transplantation of these cells into a suitable physiological location to maintain glucose sensitivity and responsiveness, and an immunosuppressive regimen to maintain islet viability and protection from the host's immune system 1 . While all three components remain active areas of research, the need for immunosuppression remains the key limitation preventing islet transplantation from becoming the clinical standard of care for all T1D patients 1,2 . A critical advancement in this regard was the advent of orally administered (PO) nanocrystal rapamycin, i.e. Rapamune®. This drug was used in the first non-steroidal immunosuppressive protocol for islet transplantation, known as the Edmonton protocol 3 . Rapamycin inhibits the mammalian target of rapamycin (mTOR) pathway to directly inhibit T cell proliferation by arresting these cells in the G1 phase of the cell cycle and preventing IL-2 secretion 4 . Although more effective than prior immunosuppressive protocols including steroids, patients undergoing transplantation procedures are still plagued by frequent graft rejection and an unpleasant array of side effects 4,5 .
Side effects related to oral rapamycin administration stem primarily from poor and inconsistent bioavailability and the wide cellular biodistribution. Rapamune® has a bioavailability of only 14% in the solution form and 41% in tablet form 4 . The low bioavailability is attributed primarily to the first pass metabolism associated with the oral route of administration, cytochrome P450 elimination and transport by p-glycoprotein efflux pumps. For example, absorption of Rapamune® is significantly affected by fat content in food, and cytochrome P450 isoenzyme CYP3A4 metabolism can cause serious drug-drug interactions 4 . With regards to biodistribution, lipophilic Rapamune® primarily partitions into red blood cells (95%) and then eventually accumulates in off-target organs, including the heart, kidneys, intestines, and testes [6][7][8] , leading to side effects. These side effects occur due to the ubiquitous expression of mTOR in diverse cell types, resulting in unintended cell populations also experiencing cell cycle arrest 2,4 . Adverse effects stated on Rapamune® package insert include malignancy, enhanced susceptibility to infection, impaired wound healing, thrombopenia, alopecia, gastrointestinal distress, gonadal dysfunction, hypertension, hyperlipidemia, nephrotoxicity, and peripheral edema 4,9 . To balance the need to maintain immunosuppression with the avoidance of side effects, patients must undergo frequent blood work to ensure that the rapamycin concentration is within the small therapeutic window of 5 to 15 ng/mL in whole blood 4,6 . Of note, mTOR inhibition can have distinct responses depending on the cell type. For example, rapamycin maintains dendritic cells (DCs) in an immature tolerogenic state that resists coreceptor expression in response to inflammatory stimuli, a process known as costimulation blockade 10 .
Given the plethora of problems associated with oral Rapamune®, an alternative therapy that bypasses the oral route of administration, reduces adverse effects, and improves transplantation outcomes is needed. Subcutaneous administration (SC) would avoid bioavailability issues that plague oral Rapamune® including first pass metabolism, elimination by intestinal cytochrome CYP3A4 and p-glycoprotein, and variability associated with food composition 4 . Importantly, SC administration provides the advantage of targeting lymphatic drainage 11 . Unlike intravenous administration, the SC route would allow patients to take their medication from their own home. The T1D patient population is well versed in the SC method of injection due to the need to inject insulin. Furthermore, the SC route provides access to antigen presenting cells (APCs), including the aforementioned DCs that can elicit potent tolerogenic responses upon modulation by rapamycin. Tolerogenic DCs (tDCs) constitutively generate regulatory T cells (Tregs) as well as express anti-inflammatory cytokines, both of which have been linked to enhanced survival of transplanted islets 11 .
However, due to the lipophilic nature of rapamycin, it is poorly soluble and therefore very difficult to formulate into a parenteral drug formulation for SC administration 12 .
Others have attempted to solve the formulation issues associated with rapamycin by using nanotechnology 13 . Previous studies have fabricated rapamycin nanocarriers from a variety of materials, including lipids, protein, poly(lactic-co-glycolic acid) (PLGA) and other polymers 13 . Due to the ubiquitous nature of rapamycin's mTOR inhibition, these nanocarriers have been researched for a wide array of applications, such as immunomodulation, cancer, cardiovascular diseases, and neurodegenerative diseases [13][14][15] . Of note, literature cites the need for investigation into the use of rapamycin nanocarriers for the treatment of diabetes 13 . To this end, we hypothesized that focusing rapamycin's mTOR inhibition on APCs instead of T cells using engineered nanocarriers could achieve sustained immunosuppression and survival of transplanted islets via the SC route with lower dosage and minimal side effects (Fig. 1). Sustained tolerance to transplanted islet grafts would allow for real-time sensing of glucose and insulin release to modulate blood glucose for the treatment of T1D. To control the biodistribution of rapamycin specifically to target APCs, we generated rapamycin-loaded poly(ethylene glycol)-b-poly(propylene sulfide)) (PEG-b-PPS) polymersomes (rPS). The PEG-b-PPS polymersome (PS) platform allows for efficient loading of lipophilic drugs within the PPS membrane 16 , has been validated to be nontoxic in both mice and nonhuman primates, [17][18][19][20][21] and undergoes uptake by DC and monocyte populations 21 , which are critically responsible for directing T cell activation during immune responses 19,22,23 . Importantly, PEG-b-PPS is non-immunomodulatory relative to other common nanomaterials, with an immunostimulatory profile that is determined almost exclusively by the loaded therapeutic 21 . For example, unloaded blank PEG-b-PPS PS elicit minimal immunomodulatory activity, whereas comparable poly(lactic-co-glycolic acid) (PLGA) nanocarriers cause an extensive immunomodulatory response, including alteration of immune cell populations, changes in coreceptor expression (e.g. CD80, CD86), and modification of the inflammatory status of numerous immune cell subsets 24 . Thus, our mechanistic assessment of rPS-mediated immunosuppression avoids interference from background immunomodulation due to the drug delivery vehicle.
Herein, we present the first application of SC rapamycin nanotherapy for islet transplantation, as well as the first example of reorchestrating the mechanism of an immunosuppressant by rationally controlling its cellular biodistribution. Liquid chromatography with tandem mass spectrometry (LC-MS-MS) was used to assess the biodistribution of the small molecule drug rapamycin delivered within PEG-b-PPS PS at nanogram resolution (ng/mg or ng/mL). Efficacy of this strategy is assessed by high parameter spectral flow cytometry with analysis via T-distributed stochastic neighbor embedding (tSNE), RNA sequencing, and a clinically relevant intraportal fully major histocompatibility complex (MHC) mismatched allogeneic islet transplantation model. Our results provide insight into how nanocarrier-mediated modulation of cellular biodistribution can significantly change the metabolism and therapeutic window, reduce adverse events, and enhance anti-inflammatory efficacy of an immunosuppressant by rationally repurposing its therapeutic mechanism of action.

Characterization of rapamycin-loaded polymersomes
PEG-b-PPS PS were characterized to assess encapsulation efficiency and retention of their vesicular nanostructure following the loading of rapamycin to form rPS. Rapamycin encapsulation efficiency was found to be greater than 55% for rPS following self-assembly and therapeutic loading via thin film hydration of desiccated PEG-b-PPS films. Neither the PS vesicular nanostructure nor the polydispersity were significantly modulated by rapamycin loading as assessed by dynamic light scattering (DLS), cryogenic transmission electron micrograph (cryoTEM) and small angle x-ray scattering (SAXS) (Fig. 2a-c). The stability of rapamycin loading was assessed in 1X phosphate buffered saline (PBS) at 4 °C, finding approximately 94% of the drug was retained over the course of 1 month (Fig. S1). Rapamycin is relatively lipophilic with a logP of 4.3 12 , and thus these results were consistent with past attempts to load molecules of low water solubility into PEG-b-PPS nanostructures.
To demonstrate that PEG-b-PPS PS can alter the biodistribution of a small molecule following SC administration, indocyanine green dye (ICG-PS) was loaded into PS to serve as a traceable model payload. C57BL/6 mice were administered ICG via oral gavage (PO), ICG via SC injection or ICG-PS via SC, sacrificed animals at 2, 24, and 48 h post administration and analyzed organs via IVIS (Fig. S2). We show that SC of ICG-PS allowed for sustained residence within the superficial axillary/brachial lymph nodes at 24 and 48 h post-injection, whereas free form ICG dye had been cleared at these later time points (Fig. 2d). To confirm that this effect holds true for rapamycin, a single dose (1 mg per kg body weight, 0.125 mg rapamycin per mL) of Rapamune® PO, rapamycin (in 0.2% carboxymethyl cellulose) SC or rPS (6.7 mg polymer per mL) SC was administered to C57BL/6 mice. Animals were sacrificed at 0. 5,2,8,18,24, and 48 h post-injection to assess rapamycin content in blood and various organs. We found that delivery of rapamycin via rPS increases rapamycin concentration in immune cell-rich tissues, such as the blood, liver, axillary lymph center (deep axillary/axillary/axial and superficial axillary/brachial lymph nodes; AX LN), subiliac lymph center (subiliac/inguinal lymph nodes; IN LN) and spleen (Fig. 2e, S3). Regarding elimination, as expected, oral Rapamune® was found in the feces due to the established route of biliary elimination. SC rapamycin also resulted in fecal elimination. Surprisingly, when SC rPS was administered, rapamycin was primarily found in the urine relative to the feces, indicating renal elimination (Fig. 2e, S3).
To assess both the organ and cellular effects of rapamycin delivery via PEG-b-PPS PS, immune cell populations from various tissues were isolated after repeated doses (11 doses, over 11 days, 1 mg rapamycin per kg body weight per dose or equivalent volume (Table S1), formulated at 0.125 mg rapamycin per mL) of unloaded PS SC, Rapamune® PO, rapamycin SC or rPS SC (PS and rPS formulations contained 6.7 mg polymer per mL). This "standard dosage" immunosuppressive protocol is accepted to provided similar immunosuppressive effects in mice as compared to clinical immunosuppressive protocols, such as the Edmonton protocol, used in humans 25,26 . When unloaded PS were injected, very little immunomodulation was observed via flow cytometry (Fig. 2f, S4, Tables S2-26). However, when rapamycin was loaded within PS, potent immunomodulation occurred (Fig.  2f, Tables S2-26). The relative immunologically inert status of the unloaded PS allowed for the majority of the effects of rPS to be attributed to the altered biodistribution of the drug, as opposed to the nanocarrier itself. A significant change in immunomodulation is observed for PS-mediated rapamycin delivery (Fig. 2f, S4, Tables S2-26). Taken in combination with the inert nature of PEG-b-PPS, our results demonstrate that rapamycin's organ and cellular biodistribution have a strong influence on the resulting immunological effect.

Polymersome-mediated costimulation blockade induces CD4+ T cell anergy
To characterize changes more deeply in immune cell populations in response to rPS delivery, we dosed healthy mice with unloaded blank PS SC, Rapamune® PO, rapamycin SC, or rPS SC (11 doses, over 11 days, 1 mg rapamycin per kg body weight equivalent, formulated at 0.125 mg rapamycin per mL, polymersome formulations contained 6.7 mg polymer per mL). Subsequently, organs (blood, liver, AX LN, IN LN, and spleen) were extracted for assessment via high-parameter spectral flow cytometry. To further understand the changes in immune cell populations as a result of rPS treatment, the inflammatory state of APC populations was assessed via receptor expression. Specifically, CD40, CD80 and CD86 coreceptor presentation on DCs (Fig. 3a-c,g) and monocyte-and-macrophage-linage (M/ Ms) 27 ( Fig. 3d-f,h) was analyzed. MHC II presentation was assessed on DCs and M/Ms (Fig. 3g,h, S4). With rPS, costimulation blockade is observed as indicated by the significant downregulation of CD40, CD80, and CD86 ( Fig. 3a-f) 28 in AX LN. Furthermore, rPS enhances MHC II+ APCs (Fig. 3g,h). Opposing expression by MHC and coreceptors causes depletion of the CD4+ T cell population (Fig. 3i, S4). Any remaining CD4+ T cells are left in a state of anergy as indicated by the significant decrease in CD4 expression (Fig. 3j). These effects are most potent in the AX LN near the site of SC injection, but also occur to various lesser extents in blood, liver, IN LN, and spleen (Tables S2-26). rPS treatment causes a significant increase in DCs within AX LN and IN LN (Fig. 3k). More specifically, an increase in novel CD8+ CD11b+ double positive (DP) conventional DCs (cDCs) is observed (Fig. 3l). Despite the overall increase in the DC population, plasmacytoid DC (pDC) are significantly reduced (Fig. 3m). A significant decrease in the overall T cell population was observed due to a significant decrease in CD4+ T cells (Fig.  3i). As a result, CD8+ T cells take over a larger portion of the T cell population (Fig. 3n), accompanied by a significant upregulation of CD8+ Tregs (Fig. 3o). Interestingly, with rPS treatment, DP CD4+ CD8+ T cells have a significantly larger population in the AX LN ( Fig. 5e,g). tSNE with Barnes-Hut approximations was used to visualize the data after gating, down sampling, and concatenation of treatment groups. From, tSNE visualization, it is observed that DP T cells cluster within the CD4+ CD8− T cell group, rather than within the CD4− CD8+ T cells (Fig. 5e). Using expression level analysis, CD4 expression by DP T cells in the rPS treatment group is similar to that of CD4+ CD8− T cells, whereas CD8 expression by DP T cells is significantly reduced as compared to CD4− CD8+ T cells (Fig. 5f,h). The relationship between the expression of the DP and single positive (SP) T cells can be quantified as a DP:SP expression ratio. Thus, the rPS treated DP T cell population is deemed CD4 bright CD8 dim .

Elicitation of antigen-specific allogeneic islet graft tolerance
In vivo assessment of rapamycin redistribution via rPS was conducted using a clinically relevant intraportal (liver) fully-MHC mismatched allogeneic islet transplantation model. Diabetes was induced in C57BL/6 mice via streptozotocin injection. To ensure the most stringent and severe model of T1D, diabetes was defined by blood glucose over 400 mg/ dl 29 . A standard dosage protocol known to allow for fully-MHC mismatched allogeneic islet graft viability for more than 100 days was compared to a low dosage protocol (Fig.  6a). The standard dosage protocol consisted of 11 injections given daily. The low-dosage protocol consisted of 6 doses given every 3 days (Fig. 6a). Each dose, regardless of protocol, consists of 1 mg rapamycin per kg body weight, formulated at 0.125 mg rapamycin per mL. rPS formulations contained 6.7 mg polymer per mL. Diabetic C57BL/6 mice received approximately 200 islets from fully MHC mismatched Balb/c mice in the liver via the portal vein (175 IEQ). Efficacy of the dosing regimen was confirmed by the restoration and maintenance of normoglycemia (blood glucose concentration < 200 mg/dL), confirming survival of the islet graft. As expected, mice that did not receive treatment all experienced graft rejection within 10 days of transplantation ( Fig. 6b, S5-7). 67% of mice treated with the standard SC rapamycin protocol and only 8% of the mice treated with Rapamune® remained normoglycemic 100 days post transplantation ( Fig. S5-7). When the low-dosage protocol was used, only 25% of the mice treated with Rapamune® and 58% of the mice treated with SC rapamycin remained normoglycemic 100 days post-transplantation, whereas 83% of mice treated with low-dosage rPS had normal blood glucose concentrations (Fig.  6b, S5-7). Furthermore, intraperitoneal glucose tolerance test (IPGTT), conducted at 30 days post-transplantation showed no difference in islet responsiveness with low dosage rPS treatment as compared to standard dosage rapamycin (Fig. S5,6).
A MLR was performed to assess antigen-specific tolerance induction. At 100 days posttransplantation, normoglycemic mice were sacrificed and splenic T cells were isolated. Recipient B6 T cells were cultured with donor, T cell depleted, mitomycin-c-treated, Balb/c splenocytes (Fig. 6d). T cells from recipients treated with low dosage rPS showed significantly less proliferation relative to those that achieved normoglycemia with low dosage rapamycin treatment (Fig. 6d).
We observed that mice treated with SC free form rapamycin controls experienced injection site alopecia (Fig. 6e). Alopecia is a known side effect of rapamycin, impacting approximately 10% of patients 30 . While alopecia was reduced in the low dosage SC rapamycin group (Fig. 6e, S8), no alopecia was observed in the low dosage rPS group (Fig. 6e, S8). Histological analysis confirms our gross observations (Fig. 6e). Only immature follicles were identified in the standard SC rapamycin group (Fig. 6e), with some mature follicles present in the low dosage SC rapamycin group (Fig. 6e). Organized mature follicles were identified in the low dosage rPS group (Fig. 6e). Additionally, mice treated with standard dosage rPS had no significant alteration in albumin:globulin ratio (A/G) relative to control and PS treated mice. Both Rapamune® PO and rapamycin SC treated mice showed elevation in A/G (Fig. S9).

Conclusions
A grand challenge of pharmaceutical development is to harness the rational engineering of nanoscale drug carriers (i.e. nanocarriers) to selectively modify target cells while minimizing uptake by cells and organs responsible for side effects 31 . By controlling delivery kinetics and target specificity, nanocarriers can alter the interconnected network of cells contributing to observed therapeutic effects, thus significantly changing the therapeutic window and reducing both the dosage and adverse events of a drug during treatment 31 . Effects of changing the network of targeted cells is particularly evident during immunotherapy, where small subsets of immune cells can elicit potent cytokine and T cell responses that can propagate into unique systemic responses. With these concepts in mind, we investigated whether SC delivery and nanocarrier-directed changes in the cellular biodistribution of rapamycin, a common therapeutic that elicits diverse cell-specific effects, can repurpose its mechanism of action at the cellular level to decrease side effects and enhance efficacy.
The targeted cell population and amount of delivered drug are critical considerations for targeted therapies. Rapamycin achieves immunosuppression by directly acting on T cells 4 . However, when given clinically via standard oral administration, the resulting broad biodistribution of rapamycin influences numerous off-target cells and reduces the dose that reaches T cells for desired effects 4,6,13 . Lack of specificity cannot be overcome with increased dosage given that rapamycin is associated with dose-dependent toxicity 6,13 . We have previously shown that giving drugs, including rapamycin, via PS, allows for selective uptake by APCs while avoiding T cells 16,21 . We hypothesized that switching the target cell population from T cells to APCs would change the immunosuppressive mechanism of rapamycin to reduce both dosage and side effects.
Route of administration is another tool that is employed to impact biodistribution and overcome drug-specific barriers to delivery. For example, SC injection could avoid diet-dependent bioavailability and variable metabolism via CYP3A4 and P-glycoprotein that are associated with orally administered rapamycin 4 . Using these tools-cellular targeting and route of administration-the temporal and both organ and cellular biodistribution of a drug can be precisely manipulated for the desired effect. Herein, we show that SC delivery of rapamycin via PS creates a rapamycin biodistribution that perturbs the network of inflammatory cells in a manner that supports the survival of transplanted allogeneic islets. While others have attempted to use nanocarriers for delivery of rapamycin 13 , to the best of our knowledge, we showcase the first use of SC rapamycin nanotherapy for a transplantation application.
Although drug-loaded PS primarily target APCs within lymphoid organs, as we have previously shown 21 , the downstream effects of SC rPS modulate a diverse network of immune cells. The most profound cellular effects of rPS were observed in the draining AX LN and included an upregulation of APCs and a down regulation of CD4+ T cells. These rPS-induced modulations of immune cells provide a foundation for an inflammatory environment that is amenable to allogenic islet transplantation. Importantly, we show a downregulation of T cells in immunomodulatory organs and at the site of intraportal islet transplantation-the liver-a key objective of immunosuppressive rapamycin therapy 4 . This was achieved without directly targeting T cells, and instead via enhanced targeting of APCs that dictate T cell function during inflammatory responses 28 . Thus, redistribution of our cellular network via rPS treatment establishes a foundation for cellular immunomodulation.
While direct donor antigen recognition by both CD4+ or CD8+ T cells and indirect presentation of donor antigen to CD8+ T cells contribute to a rejection response, only indirect donor antigen presentation to CD4+ T cells is required for rejection 32 . Therefore, rPS cause deletion and/or anergy in CD4+ T cells as indicated by the significant reduction in the CD4+ T cell population and reduction in expression of CD3 and CD4 28,33 . The unique combination of costimulation blockade as evidenced by reduced CD40/80/86 expression and enhanced MHC II presentation by APCs may account for the observed CD4+ T cell demise.
In the lymph nodes, rPS induces phenotypic changes in the DC population which are amenable to islet transplantation tolerance. Phenotypic changes are enhanced by symbiotic relationships between these DCs and CD8+ T cells to promote a quiescent environment. Along with the overall significant increase in DCs, a significant increase in novel DP CD8+ CD11b+ cDCs was observed. CD11b+ cDCs cross-present antigens to CD4+ T cells and CD8+ cDCs cross-present antigens to CD8+ T cells to induce tolerogenic behavior 10 . The presence of DP cDCs suggests that these cells may have the ability to cross-present donor antigens to both CD4+ and CD8+ T cells or DP CD4+ CD8+ T cells, which are also significantly upregulated in the lymph nodes. Furthermore, tolerogenic tDCs can cause CD8+ T cells to become CD8+ CD25+ FoxP3+ Tregs. CD8+ Tregs have enhanced suppressor capabilities relative to their CD4+ counterparts 34 . The tolerogenic properties of CD8+ Tregs have been shown to prevent graft-versus-host disease and autoimmune diseases 34 . Despite their tolerized state, CD8+ Tregs confer immunoprotection against pathogens 34 . In addition, rPS causes a significant downregulation of pDCs, which are known to secrete interferon-gamma and activate cytotoxic CD8+ T cells 35 . Both interferon-gamma secretion and cytotoxic CD8+ T cells are known to damage islet grafts, thus pDC-mediated reduction boosts potential for graft survival 36 .
In addition to DCs, rPS treatment induces suppressor phenotypes in M/Ms. Suppressor M/Ms are notable in blood lymph nodes and spleen. While other treatments confer a M/M population that is dividing between activator and suppressor phenotypes, rPS treatment promotes a single phenotype characterized by its MHC II+ Ly-6C Lo status. Mature MHC II+ Ly-6C Lo M/Ms are a type of patrolling cell that is able to penetrate tissue during steady state conditions. Ly-6C Lo monocytes have the ability to phagocytose both nanoparticles and apoptotic debris 23 . This non-classical monocyte population has a dual-fold advantage for transplantation applications, in which it supports an anti-inflammatory phenotype amenable to graft tolerance 37 and it has been shown to aid in the prevention of viral infections 38 . These monocytes have the ability to cross present the apoptotic debris to CD8+ T cells and tolerize the CD8+ T cell, suppressing antigen specific responses 23 , in a similar manner to that of CD8+ cDCs 10 .
The tolerogenic effects of rPS-treated APCs go beyond CD4+ and CD8+ T cells to create a hospitable environment for the islet graft. Niche T cell populations also make an important contribution to the congenial environment observed with rPS immunomodulatory therapy. For example, the upregulation of DP CD4+ CD8+ T cells in the lymph nodes is observed. Controversy has surrounded DP T cells as both suppressive and cytotoxic functions have been demonstrated 39,40 . This is because while CD4 dim CD8 bright cells are cytotoxic 39 , CD4 bright CD8 dim DP T cells are anti-inflammatory 40,41 . tSNE visualization in combination with CD4 and CD8 expression level analysis helped to revel that rPS confer CD4 bright CD8 dim DP T cells with known suppressor function, such as secreting anti-inflammatory cytokines 41 . Interestingly, these cells also show enhanced responsiveness during infection, for example activating effector cells in the case of human immunodeficiency virus 41 .
rPS treatment confers antigen-specific tolerance. At 100 days after successful transplantation, T cells from low dosage rPS treated mice show restraint in ex vivo proliferation in response to a donor antigen challenge. However, these T cells still respond to a foreign (non-donor) antigen with proliferation. Achieving antigen specific tolerance as opposed to immunosuppression may allow patients to only take a short course of immunomodulatory therapy to maintain the viability of their grafts for the long term. Short course therapy may be particularly advantageous for the transplant field, a common cause of graft rejection (36 patients per 100 patients per year for kidney transplant) is patient nonadherence with long term immunosuppressive therapy 42 . Furthermore, side effects, including progressive cancers and infections, common to drugs that induce nonspecific tolerance, such as Nulojix® (belatacept), can be avoided 43 . In addition to its responsiveness to foreign antigens, rPS treatment demonstrates maintenance of immune function as indicated by unaltered A/G.
Our use of the liver transplantation site is critical for translation of murine studies as the commonly used kidney capsule is not a feasible site for human islet transplantation 29 . Kidney capsule transplantation fails to expose the islets to the immune environment of the liver 29 . For example, islets transplanted to the kidney capsule are not exposed to blood to induce the instant blood-mediate inflammatory reaction (IBMIR) 29 . Additionally, the exposure of the islets to immunosuppressive drugs differs between the liver and kidney capsule transplantation site 29 . When islets are infused into the vasculature of the liver, they first encounter neutrophils. Furthermore, rPS treatment significantly reduces the neutrophil population in blood and liver and downregulates expression of CD11b (Fig. S10). CD11b is critical for neutrophil migration 44 . Graft infiltrating neutrophils have been shown to cause transplant failure 45 , thus reduction in this cell type and reduced mobility may contribute to enhanced graft survival. With reduced CD11b expression, neutrophils may show decreased ability to reach islets and infiltrate the graft. Furthermore, MHC molecules are the most significant alloantigens involved in graft rejection, thus using a fully MHC-mismatched model is critical for rigorous assessment of allogeneic transplantation. It is important to note that all combinations of fully mismatched mouse models confer the same potency and kinetics of allo-immune response. We utilized the combination of Balb/c islets transplanted into C57BL/6 recipient mice, which provides the greatest challenge to islet survival and normoglycemia restoration 29 . Utilizing excess islets can delay the graft rejection, giving a false sense of maintained normoglycemia and immunosuppression. While other models use up to 1000 islet equivalents (IEQ) 2,46 , our model uses a minimal islet mass of only ~200 murine islets (~175 IEQ).
Subcutaneous rPS injection engages lymphatic drainage, simplifies therapeutic administration, 11 and changes the method of elimination from biliary to renal. Changing the routes of administration and elimination overcomes several challenges that have historically plagued oral rapamycin regimen 13 . The SC route of administration avoids interactions in the intestine as well as variability due to food intake. Furthermore, rPS formulation favors renal over biliary elimination of rapamycin, thus reducing interaction with the liver. Specifically, SC rapamycin delivery via rPS may improve bioavailability over oral delivery by circumventing first pass metabolism and p-glycoprotein efflux 13 . Many murine studies involving rapamycin use intraperitoneal injection 2 , however this route is not easily translatable to humans. Finally, SC injection is advantageous over infusion as patients can perform the injection themselves, as opposed to requiring the services of a health care professional. In summary, this study demonstrates how the rational delivery of engineered nanoparticles can repurpose the biochemical mechanism of action of a drug by targeting specific immune cell types, laying the foundation for methods of rationally enhancing therapeutic efficacy while mitigating adverse effects.

Animals
8 to 12-week-old, male C57BL/6, Balb/c, and C3H mice were purchased from Jackson Labs. Mice were housed in the Center for Comparative Medicine at Northwestern University. All animal protocols were approved by Northwestern University's Institutional Animal Care and Use Committee (IACUC).

Materials
Unless explicitly stated below, all reagents and chemicals were purchased from Sigma-Aldrich.

Polymer Synthesis
PEG-b-PPS was synthesized as previously described by us 16 . In brief, methyl ether PEG (MW 750) was functionalized with mesylate. The mesylate was reacted with thioacetic acid to form PEG-thioacetate and then base activating the thioacetate to form a thiolate anion and initiate ring opening polymerization of propylene sulfide. Benzyl bromide was used as an end-capping agent to form PEG 17 -b-PPS 30 -Bz or the thiolate anion was protonated to form PEG 17 -b-PPS 30 -SH. The polymer was characterized by H-NMR and gel permeation chromatography (GPC).

Nanocarrier Formulation
PS were formed via thin film hydration, as previously described 16,21 . In brief, 20 mg of PEG 17 -b-PPS 30 -Bz was weighted in a sterilized 1.8 mL glass HPLC vial. 750 ul of dichloromethane (DCM) was added to the vial. To form, rPS 0.5 mg of rapamycin (Selleckchem), dissolved at 25 mg/mL in ethanol, was also added. The vial was desiccated to remove the DCM. Next, 1 mL of PBS was added to the vial. The vials were shaken at 1500 rpm overnight. PS were extruded multiple times first via 0.2 um and then 0.1 um syringe filters. Excess rapamycin was removed via size exclusion chromatography using a Sephadex LH-20 column with PBS.

Nanocarrier Characterization
DLS: DLS measurements were performed on a Nano 300 ZS Zetasizer (Malvern) and were used to determine nanocarrier diameter distribution and corresponding polydispersity index. cryoTEM: 200-mesh lacey carbon grids were glow-discharged for 30 seconds in a Pelco easiGlow glow-discharger at 15mA with a chamber pressure of 0.24 mBar. 4 μL of sample was then pipetted onto the grid and plunge-frozen into liquid ethane in a FEI Vitrobot Mark III cryo plunge freezing device for 5 seconds with a blot offset of 0.5mm. Grids were then loaded into a Gatan 626.5 cryo transfer holder, imaged at −172 °C in a JEOL JEM1230 LaB6 emission TEM at 100kV, and the data was collected on a Gatan Orius 2k × 2k camera.

SAXS:
SAXS was performed at Argonne National Laboratory's Advanced Photo Source with collimated X-rays (10 keV; 1.24 Å). Data reduction was performed using Primus software and modeling was performed using SASView.

Quantification of Rapamycin Loading 16
rPS nanocarriers (50 ul) were lyophilized and re-dissolved in HPLC grade dimethylformamide (DMF). Salts were removed via centrifugation at 17,000 g for 10 minutes. Rapamycin content of the nanocarriers was characterized via HPLC (Thermo Fisher Dionex UltiMate 3000) using an Agilent Polypore 7.5 × 300 mm column and an Agilent Polypore 7.5 × 50 mm guard column. The system was housed at 60°C. DMF (0.5 mL/minute) was used as the mobile phase. Rapamycin was detected at 270 nm. Thermo Scientific Chromeleon software was used for analysis. The concentration of rapamycin was characterized via the area under the curve in comparison to a standard curve of rapamycin concentrations.

Rapamycin Stability in Nanocarrier
rPS formulations were fabricated as previously described. Formulations were stored at 4°C in glass scintillation vials. At various time points, the formulations were vortexed, 1 mL samples were transferred to Millipore Amicon Ultra Centrifuge 10,000 NMWL Tubes and centrifuged at 4000 g in a swinging bucket rotor to remove unloaded drug. The retentate was brought back up to its original volume using PBS. Quantification of rapamycin was performed as previously described.

ICG Biodistribution
ICG PS were formed using thin film rehydration, as previously described 21 . In brief, 20 mg of PEG 17 -b-PPS 30 -Bz was weighted in a sterilized 1.8 mL glass HPLC vial. 750 ul of DCM was added to the vial. The vial was desiccated to remove the DCM. Next, 1 mL of 0.258 mM ICG in PBS was added to the vial. The vials were shaken at 1500 rpm overnight. PS were extruded multiple times first via 0.2 um and then 0.1 um syringe filters. Float-A Lyzer G2 Dialysis devices (Fisher) were used to remove unloaded ICG. ICG loading was quantified relative to standards composed of known amounts of polymer and ICG in a 1:33 molar ratio using absorbance at 820 nm as previously described by our group 21 . ICG concentration was matched at 50 ug/mL. C57BL/6 mice received ICG PO, ICG SC or ICG-PS SC. The injection volume was 150 ul. At 2, 24-and 48-h post-injection, the mice were sacrificed, blood was collected via cardiac puncture, and perfusion was performed using heparinized PBS. Liver, spleen, kidneys, heart, and lung were harvested and imaged via IVIS Lumina with an excitation wavelength of 745 nm, an emission wavelength of 810 nm, an exposure time of 2 seconds and a f/stop of 2.

Rapamycin Biodistribution
Healthy C57BL/6 mice were administered a single 1 mg per KG body weight dose of Rapamune® oral solution (Pfizer) or generic equivalent (VistaPharm) PO, rapamycin (in 0.2% CMC) via SC or rPS SC. All formulations were at a concentration 0.125 mg rapamycin per mL or equivalent volume; polymersome formulations contained 6.7 mg polymer per mL (Table S1). Mice were sacrificed at the following time points: 0.5, 2, 8, 16, 24, and 48 h. Urine and feces were collected via metabolic cages during the duration between injection and sacrifice for the 8, 16, 24 and 48-h timepoints. The following tissues and/or organs were collected: blood, brain, fat pad, heart, kidneys, liver, lungs, AX LN, IN LN, spleen. Rapamycin was extracted from blood and urine using a solution of methanol and acetonitrile (50:50 v/v) doped with rapamycin-D3 (Cambridge Isotope Laboratories) as an internal standard. Tissue samples were homogenized in homogenization tubes prefilled with stainless steel ball bearings (Sigma) using a solution of phosphoric acid (8%), acetonitrile and acetic acid (30:67.2:2.8 v/v/v). After homogenization, tissue samples were also doped with rapamycin-D3. All samples were precipitated via incubation at −20 °C, followed by centrifugation. The supernatant was collected and LC-MS/MS (Shimadzu LC-30AD pumps; SIL-30ACMP autosampler; CBM-20A oven; Sciex Qtrap 6500) was used to determine rapamycin concentration. Rapamycin had a retention time of 2.7 minutes. Rapamycin-D3 had a retention time of 3.0 minutes.

Immunomodulation Study
Healthy C57BL/6 mice were subjected to a "standard dosage regime." Animals were administered with blank PS SC, Rapamune® oral solution (Pfizer) or generic equivalent (VistaPharm) PO, rapamycin (in 0.2% CMC) via SC or rPS via SC at a dose of 1 mg/kg (Table S1). All formulations were at a concentration 0.125 mg rapamycin per mL or equivalent volume; polymersome formulations contained 6.7 mg polymer per mL (Table  S1). A cohort of mice were left as an untreated control. After 11 days, the mice were sacrificed. Blood, liver, AX LN, IN LN and spleen were collected and processed for flow cytometry.

tSNE
For each analysis, FlowJo's DownSample plugin was used to randomly select an equal number of events from the T cell population for every sample. The purpose of DownSample was to both normalize the contribution of each mouse replicate and reduce computational burden. Next, samples from mice that underwent the same treatment and same cell population were concatenated. The tSNE plugin was run on concatenated samples using the Auto opt-SNE learning configuration with 3000 iterations, a perplexity of 50 and a learning rate equivalent to 7% of the number of events 49 . The KNN algorithm was set to exact (vantage point tree), and the Barnes-Hut gradient algorithm was employed.

Allogeneic Islet Transplantation
Diabetes was induced via streptozotocin (IP; 190 mg/kg) injection five days prior to transplantation and confirmed via hyperglycemia (blood glucose > 400 mg/dl). Starting the day prior to transplantation, mice were treated with: PS SC, Rapamune® PO, rapamycin (in 0.2% carboxymethyl cellulose) SC, or rPS SC at 1 mg rapamycin per kg body weight (Table S1) in accordance with a standard dosage (11 doses, given daily) or a low dosage (6 doses, given every 3 rd day). All drugs were formulated at 0.125 mg rapamycin per mL. rPS formulations contained 6.7 mg polymer per mL. On the day of transplantation, islets were isolated from Balb/c mice via common bile duct cannulation and pancreas distension with collagenase. Islets isolated from two donors (~200 mouse islets, ~175 IEQ) were transplanted to C57BL/6 recipients via the portal vein. Body weight and blood glucose concentration were monitored closely for 100 days post-transplantation. Intraperitoneal glucose tolerance test (IPGTT) was performed one-month post transplantation. The animals were fasted for 16 h before being injected intraperitoneally with 2 g dextrose (200 g/L; Gibco) per kg body weight. Blood glucose concentrations were measured at 0, 15, 30, 60and 120-minutes post-injection.

Alopecia Assessment
Dorsal photos were taken weekly to assess for alopecia. At 100-days post-transplantation, the mice were euthanized, and skin samples were excised in the dorsal region at the SC injection site. Skin samples were placed in cassettes, fixed in 4% paraformaldehyde, and embedded in paraffin. Tissue blocks were sectioned at a thickness of 5 nm and stained with hematoxylin and eosin (H&E). Digital images were taken on a Nikon microscope.

T Cell RNA Sequencing
Healthy C57BL/6 mice were treated with: PS SC, Rapamune® PO, rapamycin (in 0.2% carboxymethyl cellulose) SC, or rPS SC, using the standard dosage protocol (11 doses, over 11 days, 1 mg rapamycin per kg body weight per dose or equivalent volume (Table  S1), formulated at 0.125 mg rapamycin per mL, PS and rPS formulations contained 6.7 mg polymer per mL). A cohort of mice were left as an untreated control. After 11 days, the mice were sacrificed, and the spleen were excised. The organs were processed as was done for flow cytometry. T cells were isolated via nanobead incubation and magnetic sorting (BioLegend). RNA was isolated from T cells using RNeasy Mini Kit with DNase digestion (Qiagen). RNA-seq was conducted at the Northwestern University NUSeq Core Facility. Total RNA will be quantified with Qubit and quality assessed with Agilent Bioanalyzer. The NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) was used for library prep following the manufacturer's protocol using the NEBNext rRNA depletion option. Briefly, rRNA was first depleted from the total RNA samples. Then after fragmentation, reverse transcription was performed to convert RNA to cDNA, followed by end repair, adaptor ligation, and PCR amplification of libraries. Prior to sequencing, the prepared libraries were checked for fragment sizing on a Bioanalyzer using High Sensitivity DNA chip and quantified with Qubit. The sequencing of the libraries was conducted on an Illumina NovaSeq 6000 NGS System, using an SP flow cell to generate paired-end 150 bp reads. Sequencing quality was analyzed with FastQC v0.11.5 50 and reads were trimmed and filtered with Trimmomatic v0.39 51 . One sample from each treatment group was discarded due to low sequencing and/or alignment quality. Reads were aligned with STAR v2.7.6a 52 to the GENCODE M27 GRCm39 mouse reference genome primary assembly using the GRCm39 mouse reference primary comprehensive gene annotation (https://www.gencodegenes.org/mouse/). Quantification and differential expression were performed with Cuffdiff from Cufflinks v2.2.1 [53][54][55] , again using the GENCODE GRCm39 mouse reference primary comprehensive gene annotation and a 0.05 FDR. Detailed settings for each software are included in Table S27. The raw data displayed in Table S28.

A:G Ratio
Healthy C57BL/6 mice were treated with: PS SC, Rapamune® PO, rapamycin (in 0.2% carboxymethyl cellulose) SC, or rPS SC, using the standard dosage protocol (11 doses, over 11 days, 1 mg rapamycin per kg body weight per dose or equivalent volume (Table  S1), formulated at 0.125 mg rapamycin per mL, PS and rPS formulations contained 6.7 mg polymer per mL). A cohort of mice were left as an untreated control. After 11 days, the mice were sacrificed, and cardiac puncture was used for blood collection. Blood samples were allowed to clot and then spun down at 2000 g for 10 minutes to obtain serum. Pierce 660 nm Protein Assay (ThermoFisher) was used to quantify serum protein concentration. Samples were diluted to fall within the range of the mouse albumin ELISA kit (abcam) using 1X PBS. Albumin concentration was analyzed using the ELISA assay. Globulin concertation was determined by subtracting the albumin concentration from the total protein concentration in each sample.

Data availability
The main data supporting the results in this study are available within the paper and Supplementary Information. The raw and analyzed datasets generated during the study are available for research purposes from the corresponding author on reasonable request.

Statistical analysis
Statistical analysis was performed using one-or two-way ANOVA with Tukey's multiple comparisons test or paired two-tailed t-test. Significant P-values relative to rPS treatment are displayed in the figures. Non-diabetic survival data was assessed using the Mantel-Cox Log-rank test. All statistical analyses were performed using GraphPad Prism software.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Fig. 1 |. Subcutaneous rapamycin delivery via polymersomes (rPS) tolerizes intraportal islet grafts via direct modulation of APCs instead of T cells.
a, Rapamycin is a hydrophobic mTOR inhibitor that is used as an immunosuppressive drug. b, Clinically, rapamycin is given orally. Oral administration (i.e. Rapamune®) results in a wide biodistribution and low bioavailability, as it is a substrate for CYP3A4 and p-glycoprotein and s cleared via biliary elimination. c, Oral rapamycin primarily acts on T cells to prevent cytotoxic CD8+ T cell proliferation. d, Alternately, rapamycin can be easily loaded into the hydrophobic membrane of polymersomes (PS) to form rapamycin-loaded polymersomes (rPS). e, When injected subcutaneously (SC) into mice, rPS drain into the brachial lymph nodes where they are uptaken by antigen-presenting cells (APCs). As a result, APCs develop an anti-inflammatory, semi-mature phenotype, in which they express high levels of MHC II to present to CD4+ T cell receptors, but they do not express costimulatory molecules. Without activation from costimulation, acute rejection causing CD4+ T cells go into a state of anergy or become tolerogenic CD8+ regulatory T cells (Tregs). g, To assess the ability of SC rPS to provide a tolerogenic state that allows for fully major histocompatibility complex (MHC) mismatched allogeneic graft survival, islet transplantation was performed in diabetic mice at the clinically relevant intraportal (liver) transplantation site and graft viability was assessed by the restoration and maintenance of normoglycemia.  (Table S1) of Rapamune® via oral gavage (PO), rapamycin (in 0.2% carboxymethyl cellulose) via SC or rapamycin-loaded polymersomes (rPS) via SC. All formulations were at a concentration of 0.125 mg/mL rapamycin. rPS formulations contained 6.7 mg polymer per mL. Rapamycin concentration (ng/mL or ng mg) in various tissues the over time (0.5 h, 2 h, 8 h, 16 h, 24 h and 48 h) as assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Assessed tissues included blood, liver, axillary lymphocenter (deep axillary/axillary/axial and superficial axillary/brachial lymph nodes; AX LN), subiliac lymphocenter (subiliac/inguinal lymph nodes; IN LN), spleen, urine, and feces. (n = 6 mice/group). f, Flow cytometry analysis of CD45+ cell populations from mice administered with PS SC, Rapamune® PO, rapamycin SC or rPS SC, using the standard dosage protocol (11 doses, over 11 days, 1 mg rapamycin per kg body weight per dose or equivalent volume (Table S1), formulated at 0.125 mg rapamycin per mL, PS and rPS formulations contained 6.7 mg polymer per mL). A cohort of mice were left untreated as a control. Macrophages were not assessed in blood as indicated by a black box" All data is presented as mean percentage change relative to the untreated control cohort. (n = 6 mice/group). Mice were treated with: polymersomes (PS; ▲) subcutaneous injection (SC), Rapamune® (•) oral gavage (PO), rapamycin (in 0.2% carboxymethyl cellulose; ■) SC, or rapamycinloaded polymersomes (rPS; ▼) SC, using the standard dosage protocol (11 doses, over 11 days, 1 mg rapamycin per kg body weight per dose or equivalent volume (Table  S1), formulated at 0.125 mg rapamycin per mL, PS and rPS formulations contained 6.7 mg polymer per mL). A cohort of mice were left as an untreated control (♦).

cells of T cells (n) and percentage of CD8+
Tregs of T cells (o). Data are from the axillary lymphocenter (deep axillary/axillary/axial and superficial axillary/brachial lymph nodes; AX LN). All data are presented as mean percentage or median florescent intensity (MFI) ± SD. Significant p-values relative to rPS treatment are displayed on the graphs. Statistical significance was determined by one-way ANOVA with Tukey's multiple comparisons test. (n = 6 mice/group). Mice were treated with: polymersomes (PS; ▲) subcutaneous injection (SC), Rapamune® (•) oral gavage (PO), rapamycin (in 0.2% carboxymethyl cellulose; ■) SC, or rapamycinloaded polymersomes (rPS; ▼) SC, using the standard dosage protocol (11 doses, over 11 days, 1 mg rapamycin per kg body weight per dose or equivalent volume (Table S1), formulated at 0.125 mg rapamycin per mL, PS and rPS formulations contained 6.7 mg polymer per mL). A cohort of mice were left as an untreated control (♦). A cohort of mice were left as an untreated control (♦). Cell populations were analyzed by flow cytometry. . All data are presented as mean percentage ± SD. Significant p-values relative to rPS treatment are displayed on graphs. Statistical significance was determined by one-way ANOVA with Tukey's multiple comparisons test. (n = 6 mice/group). d) Analysis of M/M populations by phenotype with consideration for Ly-6C, macrophage markers (F4/80 and/or CD169; except for blood where macrophages were not considered), CD40, CD80, and CD86, MHC II. Data from blood, AX LN and spleen are shown in heatmap form. (n = 6 mice/group). Mice were treated with: polymersomes (PS; ▲) subcutaneous injection (SC), Rapamune® (•) oral gavage (PO), rapamycin (in 0.2% carboxymethyl cellulose; ■) SC, or rapamycinloaded polymersomes (rPS; ▼) SC, using the standard dosage protocol (11 doses, over 11 days, 1 mg rapamycin per kg body weight per dose or equivalent volume (Table S1), formulated at 0.125 mg rapamycin per mL, PS and rPS formulations contained 6.7 mg polymer per mL). A cohort of mice were left as an untreated control (♦). Cell populations were analyzed by flow cytometry. a-e, tSNE visualization of CD3+ immune cell populations from the axillary lymphocenter (deep axillary/axillary/axial and superficial axillary/brachial lymph nodes; AX LN) with color-coded gated overlays of the previously described cell populations: CD4+ CD8− (orange), CD4− CD8+ (blue), CD4+ CD8+ double positive (DP; black), CD4+ regulatory (CD4+ Treg; green) and CD8+ regulatory (CD8+ Treg; magenta). Solid line outlines DP T cell populations for rPS treated cohort (e). f, tSNE heatmap statistic of CD4 (left) and CD8 (right) expression from rPS treated group. g, Percentage of DP T cells in AX LN. All data are presented as mean percentage (of T cells) ± SD. Significant p-values relative to rPS treatment are displayed on graphs. Statistical significance was determined by one-way ANOVA with Tukey's multiple comparisons test. h, Ratio of DP:SP (single positive CD4+ CD8− or CD4− CD8+) T cell CD4 and CD8 expression in the AX LN for rPS treated mice. The significant p-value is displayed on the graph. Statistical significance was determined by paired two-tailed t-test. (n = 6 mice/group). a, Standard dosage and low dosage schemes for rapamycin during fully major histocompatibility complex (MHC) mismatched allogeneic islet transplantation (day 0) experiment. Diabetes was induced (day −5) via streptozotocin (STZ) injection. The standard dosage protocol consists of 11 doses, given daily starting at day −1. The low dosage protocol consists of 6 doses, given every 3 days, starting at day −1. Mice were treated with: Rapamune® oral gavage (PO), rapamycin (in 0.2% carboxymethyl cellulose) subcutaneous injection (SC) or rapamycin-loaded polymersomes (rPS) SC. Regardless of protocol, each dose consisted of 1 mg rapamycin per kg body weight per dose, formulated at a concentration of 0.125 mg rapamycin per mL (Table S1). PS formulations contained 6.7 mg polymer per mL. A cohort of mice were left as an untreated control. b, Post-transplantation normoglycemia (%) (blood glucose < 200 mg/dl) following islet transplantation for low dosage protocol. No treatment (♦); Rapamune® PO (•); rapamycin SC (■); rPS SC (n ≥