Cancer Cell Coating Nanoparticles for Optimal Tumor-Specific Cytokine Delivery

Antonio E Barberio; Sean G Smith; Santiago Correa; Cathy Nguyen; Bang Nhan; Mariane Melo; Talar Tokatlian; Heikyung Suh; Darrell J Irvine; Paula T Hammond

doi:10.1021/acsnano.0c03109

. Author manuscript; available in PMC: 2020 Oct 2.

Published in final edited form as: ACS Nano. 2020 Sep 5;14(9):11238–11253. doi: 10.1021/acsnano.0c03109

Cancer Cell Coating Nanoparticles for Optimal Tumor-Specific Cytokine Delivery

Antonio E Barberio ¹, Sean G Smith ², Santiago Correa ³, Cathy Nguyen ⁴, Bang Nhan ⁵, Mariane Melo ⁶, Talar Tokatlian ⁷, Heikyung Suh ⁸, Darrell J Irvine ^9,^*, Paula T Hammond ^10,^*

¹Department of Chemical Engineering, Massachusetts Institute of Technology, 183 Memorial Drive, Cambridge, MA 02142, USA

²Department of Chemical Engineering, Massachusetts Institute of Technology, 183 Memorial Drive, Cambridge, MA 02142, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02142, USA

³Department of Biological Engineering, Massachusetts Institute of Technology, 21 Ames Street, Cambridge MA 02142, USA

⁴Department of Biological Engineering, Massachusetts Institute of Technology, 21 Ames Street, Cambridge MA 02142, USA

⁵Department of Chemistry, Wellesley College, 106 Central Street, Wellesley, MA 02481

⁶Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02142, USA

⁷Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02142, USA

⁸Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02142, USA

⁹Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02142, USA; Department of Biological Engineering, Massachusetts Institute of Technology, 21 Ames Street, Cambridge MA 02142, USA; Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA

¹⁰Department of Chemical Engineering, Massachusetts Institute of Technology, 183 Memorial Drive, Cambridge, MA 02142, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02142, USA; Institute for Soldier Nanotechnologies;

Author Contributions

AEB and PTH designed the experiments, analyzed the data and wrote the paper with significant aid from SGS. AEB, SGS, SC, CN, BTN, HS conducted the experiments. MM and HS designed and carried out the IL-12 manufacture. SGS and DJI provided advice and support throughout. PTH supervised the study. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

CORRESPONDING AUTHORS: Paula T. Hammond: [email protected], Darrell J. Irvine: [email protected]

PMCID: PMC7530125 NIHMSID: NIHMS1625422 PMID: 32692155

Abstract

Although cytokine therapy is an attractive strategy to build a more robust immune response in tumors, cytokines have faced clinical failures due to toxicity. In particular, interleukin-12 has shown great clinical promise but was limited in translation due to systemic toxicity. In this study, we demonstrate an enhanced ability to reduce toxicity without affecting efficacy of IL-12 therapy. We engineer the material properties of a NP to meet the enhanced demands for optimal cytokine delivery by using the layer-by-layer (LbL) approach. Importantly, using LbL, we demonstrate cell-level trafficking of NPs to preferentially localize to the cell’s outer surface and act as a drug depot, which is required for optimal payload activity on neighboring cytokine membrane receptors. LbL-NPs showed efficacy against a tumor challenge in both colorectal and ovarian tumors at doses that were not tolerated when administered carrier-free.

Keywords: nanomedicine, immuno-oncology, nanoparticle, drug delivery, layer-by-layer, cytokine

Graphical Abstract

graphic file with name nihms-1625422-f0001.jpg

An effective immune response against cancer requires a complex series of steps, eloquently described as the cancer immunity cycle,^1,2 which include tumor antigen release, dendritic cell priming of T cells in tumor-draining lymph nodes, and effector T cell homing to the tumor. These steps of the immune response are in a delicate balance and can either be arrested or driven forward by a variety of therapeutic approaches. Checkpoint inhibitors prevent the arrest of the cycle, which works well for some malignancies that have sufficient pre-existing tumor-specific immune populations present. For example, melanoma and lung cancer have shown some of the greatest success with these therapies; however, many epithelial tumors have been much more difficult to treat using immunotherapies to date. This is particularly true of cancers that are known to have a “cold” immune microenvironment such as advanced serous ovarian cancer, for which the concentration of prognostic leukocytes such as CD8+ T cells is low and the signaling cascade to elicit an immune response is greatly lowered or missing.^3–5 Such “cold” tumor environments need additional therapies to drive the cycle forward, such as proinflammatory cytokines to increase the number and activity level of tumor specific immune cells both locally in the tumor microenvironment and systemically. One promising candidate for driving a proinflammatory response is interleukin-12 (IL-12). An extremely potent cytokine, IL-12 bridges innate and adaptive immunity and drives an antitumoral Th1 type immune response, mediated in large part by inducing the production of interferon-γ (IFN-γ) that acts as the main downstream product of IL-12 signaling.^6–9 IL-12 has been used in multiple preclinical models to great effect against various tumor types,¹⁰ which led to the initiation of multiple clinical trials of this cytokine for cancer therapy.^11–13 Although initial trials found a well-tolerated dose, subsequent trials found high toxicity at the same dose with slightly altered schedule. It was found that these high, schedule-dependent toxicities were linked with the appearance of high IFN-γ levels in blood plasma.

Recently, many delivery methods have been tested to improve IL-12 therapy. These methods have focused largely on local delivery techniques including microparticle delivery^14–17 and chitosan co-formulations^18–20 designed to keep IL-12 in the injected tumor; however, these methods require an accessible tumor to inject and cannot be translated to systemic delivery as is required for metastatic diseases that lack a main tumor such as ovarian cancer. Additional delivery methods have used gene therapy to induce IL-12 production in the tumor.^21,22 Finally, simple nanoparticle delivery vehicles have been tested to deliver IL-12 protein^23,24 relying on passive targeting, and notably lacking in sufficient toxicology studies demonstrating the reduction of toxic side effects for this potent therapy, a critical aspect of bringing the promise of IL-12 back to clinical relevance. All of these approaches lack the ability to spatio-temporally control the delivery of cytokine in the tumor environment from a systemically deliverable package; which is critical for meaningful success in the delivery of a highly potent and systemically toxic therapy such as IL-12 and other cytokines.

Optimally engineered NPs offer an evident opportunity to improve IL-12 therapy as it has been shown that localized IL-12 delivery is of great importance for a successful therapy.^18,20,25 The use of a NP delivery system is particularly attractive because a properly designed NP therapy allows for the possibility of systemic delivery with limited off-target exposure of the IL-12 payload as well as concentration of IL-12 in the tumor microenvironment. Nevertheless, IL-12 poses design challenges for optimal NP delivery vehicles: 1) Cytokines are labile proteins and have been historically difficult to deliver using traditional encapsulation techniques; 2) NPs are often internalized by cells into endosomal compartments; however, IL-12 and other cytokines must engage external receptors to be effective and are rendered useless and often degraded upon internalization; 3) IL-12 is designed to be secreted and act locally in natural immune responses and is very toxic when allowed to circulate systemically, which necessitates a high degree of tumor association and display of IL-12 only within the tumor. Meeting all of these design criteria in the same NP delivery vehicle is paramount to the success of an IL-12 NP therapy.

Layer-by-layer (LbL) assembly is a simple, modular engineering approach for the surface modification of nanoparticles, which provides the means to address each of the design requirements defined above. LbL is a water-based, electrostatic method for layering polymer materials onto surfaces^26,27 to generate a thin film of material that can modulate the material properties of the carrier. This NP design offers many advantages including multiple drug compartments with the potential for sequential cargo release, the ability to tailor surface chemistry with polymer layers to affect targeting and biodistribution, and improved pharmacokinetics.^28–37 In particular, the LbL technique offers the ability to easily alter and screen for surface chemistries that can meet particular design criteria to address the many challenges presented by optimal IL-12 NP construction. In this work, an LbL particle construction approach was used to systematically engineer an optimized NP for IL-12 delivery against cancer. These particles were shown to meet each of the design criteria including 1) high loading and release of active IL-12, 2) localization of NPs on the surface of tumor cells maintaining payload availability to membrane receptors, and 3) high association with tumor cells and decreased systemic exposure. These rationally engineered LbL NPs also crucially demonstrated greatly reduced toxicity of IL-12 therapy while maintaining activity in multiple murine cancer models, thus widening the therapeutic window for this potent therapy. Importantly, these LbL NPs showed efficacy in ovarian cancer, one of the more challenging tumor types to treat with immunotherapy as it often presents as a “cold” tumor that is difficult to treat using current approaches.

Results/Discussion

Engineering layer-by-layer nanoparticles encapsulating IL-12

A single chain version of IL-12 originally described by Lieschke et al³⁸ was used for this work due to its enhanced stability and ease of production. This construct was engineered with a 6x His tag at the C-terminus for purification and encapsulation (Fig S1). The presence of the His tag was further utilized to attach the single chain IL-12 (scIL-12, IL-12) construct onto liposome surfaces via Ni-His interactions with nickel bearing head groups from 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (DOGS-NTA (Ni)) on the liposome surface (Fig 1A).³⁹ This attachment was performed using a simple overnight incubation of formed liposomes with IL-12, preserving the integrity and activity of the labile cytokine by avoiding liposome processing which requires heat, sonication and high pressure with the protein in solution. The IL-12 loaded liposomes were then further modified with a polyelectrolyte bilayer using the well-established LbL process, in which polymer layers of alternating charge are adsorbed to the particle surface and excess polymers removed using tangential flow filtration.³² These polymer layers serve two important purposes. First, the external anionic layer can be altered to tailor the targeting of the particles to particular cell types and cellular compartments. Second, the polymers act as a shield to the underlying IL-12, limiting its systemic exposure and toxicity. The initial cationic polymer layer on the particles was chosen to be poly-L-arginine (PLR) for its low toxicity and well-established use in LbL systems.^35,40 For the external layer, both hyaluronic acid (HA) and poly-L-glutamic acid (PLE) were chosen to test based on their interactions with immune and cancer cells and dense negative charge⁴¹ (Fig 1A).

Figure 1. — ScIL-12 is formulated into monodisperse LbL-NP. A, Schematic of layer-by-layer buildup of particle and cytokine attachment. B, cryoEM image of LbL-NPs shows layered liposome structures 80–120 nm in diameter. C, Dynamic light scattering measurements show effective layering of particles by diameter increase and D, charge reversal through layering process, resulting in ~110nm negatively charged particles. Data presented for PLE terminal polyanion layer particles, other external polymers showed similar results by DLS. E, Encapsulation of scIL-12 in LbL-NPs as measured by ELISA for different encapsulation techniques (passive, heparin layering interaction (HEP), Ni His tag interaction (Ni:His)).

HA was chosen for its known ligand/receptor interaction with CD44 which has been utilized in the past to target cancers that overexpress CD44, such as triple negative breast cancer, non-small cell lung cancer and ovarian cancer.^{29,30,42–45} PLE was chosen based on its strong association and binding to ovarian cancer cells and immune cells of interest, as well as its subcellular localization to cell membranes.⁴¹ The resulting NPs were verified to be monodisperse and successful LbL constructs via cryoEM and dynamic light scattering (DLS) analysis (Fig 1B–D). LbL-NPs showed a number average radius of approximately 110 nm and an approximately −60mV zeta potential for both surface chemistries irrespective of IL-12 loading. These particles showed efficient loading of cytokine, especially when compared to more traditional passive loading or loading via heparin binding of IL-12⁴⁶ in the layers of the particle (Fig 1E), with 90% loading of IL-12 resulting in 13% IL-12 by weight in the final formulation as compared to lipid weight. Using approximations based on lipid head group size, diameter, and monolayer thickness an estimation for number of lipid molecules in a unilamellar liposome can be generated and combined with the loading efficiency of IL-12 from Fig 1 to find that each LbL-NP contains approximately 50 IL-12 molecules (Calculation S1).

Surface chemistry impacts LbL-NPs subcellular localization

It is critical that the NPs associate primarily with tumor cell populations to prevent off-target toxicities; however, the receptor-mediated endocytosis achieved with typical selective binding to cells is not desirable for cytokine delivery, as it prevents the payload’s access to membrane receptors. Thus, we hypothesize that NP delivery vehicles with extended cell surface membrane localization will improve IL-12 activity by allowing for greater interaction with its membrane receptor. To this end, fluorescence microscopy was used to probe the subcellular localization of both PLE and HA terminal layer LbL-NPs in target cells. Fluorescence microscopy showed that HA terminal layer NPs were internalized in both MC38 colon carcinoma and HM-1 ovarian cancer cell lines by 24 hrs, while PLE terminal layer NPs remained bound to the surface membrane of the cells for extended periods without significant internalization in multiple cell lines (Fig 2, S2), consistent with recently reported work.⁴¹ Overall these results show promise for PLE as the terminal layer for IL-12-LbL-NPs as the increased cell surface localization on cancer cells has the potential to prolong IL-12 activity from the particles in the tumor microenvironment as compared to particles that are rapidly internalized.

Figure 2. — LbL-NPs demonstrate differential interactions with cancer cells based on surface chemistry. Fluorescence imaging of MC38 tumor cells incubated with fluorescent carboxy-modified latex core LbL-NPs with either HA or PLE terminal layers for 24 hours. Blue indicates Hoechst nuclear stain, green is LbL-NP fluorescence, red marks wheat germ agglutinin membrane stain.

LbL NPs maintain IL-12 efficacy in vitro

To evaluate the importance of extended membrane localization for IL-12 delivery via the NP carrier, the biological activity of the LbL-IL-12-NP constructs was probed and compared to free IL-12. To demonstrate biological activity, the IL-12 NPs’ efficacy was tested in vitro by measuring their ability to stimulate an IFN-γ response from primary splenocytes⁶ (Fig 3A). In an assay involving direct application to splenocyte culture, the IL-12 NPs showed less activity than an equivalent amount of free IL-12 as measured by the EC50 calculated from dose response curves (Fig 3C, S3). This result is expected, as binding and encapsulation of the cytokine on the NPs should reduce activity, and is indeed relied upon to ameliorate off-target toxicity when the particles are delivered in vivo by preventing systemic IL-12 exposure. However, this experimental design does not fully mimic the tumor microenvironment the NPs experience in vivo upon delivery, which includes many more tumor cells than immune cells.

Figure 3. — LbL-IL-12-NPs demonstrate enhanced efficacy *in vitro*. A, Schematic of *in vitro* efficacy test on splenocyte culture. B, Schematic of *in vitro* efficacy test in tumor mimic co-culture. C, EC50 of IFN-γ response of splenocytes treated with varying IL-12 therapies from A. D, EC50 (+SEM) of IFN-γ response of co-cultured MC38 cells and splenocytes treated with varying IL-12 therapies from B. *** indicated p<.001 calculated by one-way ANOVA with Bonferroni post hoc test across all groups. E, Fold change of activity between C and D.

Having demonstrated activity directly on target immune cells, it is critical to show that the designed IL-12 NPs are able to maintain activity in the event that they are exposed to tumor cells before these target populations, as is likely in vivo. To this end, IL-12 NPs or free cytokine were first incubated with cancer cells before washing off media and adding splenocytes to the culture (Fig 3B). In this way, only therapies bound to cancer cells following the washing step, which mimics clearance, are still available to deliver cytokine to immune cells. PLE terminal layer LbL-IL-12-NPs (henceforth called PLE-IL-12-NPs) maintained the highest activity as measured by the lowest calculated EC50 value (Fig 3D, S3), outperforming both free IL-12 and HA terminal layer LbL-IL-12-NPs. Indeed even in this simulated worst-case scenario, PLE-IL-12-NPs maintained the activity of IL-12 in comparison to their direct activity on the target immune populations approximately 10x more than carrier free IL-12 and 2x more than readily internalized HA-IL-12-NPs (Fig 3E). This is likely due to the extended membrane localization of PLE-IL-12-NPs (Fig 2). Overall, these results indicated PLE-IL-12-NPs were the formulation most likely to maintain the activity of IL-12 within the tumor microenvironment, supporting our hypothesis that extended membrane localization is important for a cytokine delivery vehicle.

In addition, the identified PLE-IL-12-NPs were tested for their selectivity to tumor cells over immune cells as an initial in vitro test for viability of systemic delivery for such a NP system (Fig S4). It is critical that IL-12 be prevented from systemic activity for a successful IL-12 delivery strategy as off target toxicity is the most pronounced limit to IL-12 success in the clinic. To that end, it is paramount that an IL-12 delivery vehicle not only prevent systemic activity by preventing association with immune cells in circulation but also associate with tumor cells to concentrate NPs and their IL-12 payload in the tumor microenvironment. The described PLE-IL-12-NPs demonstrate this selectivity for tumor cell association as measured by flowcytometry using fluorescently tagged NPs dosed on a co-culture of splenocytes and MC38 tumor cells (Fig S4). These data demonstrate that the layering on the PLE-IL-12-NPs enhances NP uptake as compared to an unlayered construct and that NP association is highly (>92%) selective for tumor cells. These data together with the tumor cell membrane association show strong evidence that the described NP meets the design challenges of IL-12 delivery.

IL-12 is exposed and released overtime from particle surface by erosion.

Having demonstrated that the designed PLE-IL-12-NPs maintain pronounced activity in vitro, the mechanism of activity from the NPs was probed. For these studies PLE-IL-12-NPs were created with FRET pairing fluorophores (Fig 4A). The donor fluorophore (Cy3) of the FRET pair was bound to amines on 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) on the liposome surface through NHS-ester chemistry. The acceptor fluorophore (Cy5) was bound to either free amines on IL-12 or the N terminal amine on PLR by NHS-ester chemistry. These NPs were then incubated at different solution conditions and samples were measured for FRET efficiency over a 72 hour period. The conditions tested were water (the storage buffer) and spent media (incubated on MC38 cells for at least 48 hours), which acts as an in vitro mimic to the conditions experienced in vivo, with the inclusion of serum and any factors produced by the tumor cells, such as enzymes. These FRET efficiencies were then normalized to a fully intact particle as defined by FRET efficiency at t=0 and to a fully degraded (using Triton X-100) NP. FRET efficiency showed that the polymer layers undergo erosion of approximately 50% over an initial period of 8 hours followed by a slower erosion for the remainder of the study. IL-12 release showed a lag during the initial time while the polymer layers were still intact, with a matching burst between 8 and 24 hours, followed by a prolonged release over the remainder of the study (Fig 4B). These data demonstrate that the maintained IL-12 activity is achieved via surface erosion, in which the polymer layers first erode over an 8 hour period, subsequently revealing and releasing the underlying IL-12 layer (Fig 4C). Importantly, these data show that a majority of IL-12 is released in the first 24 hours, while the NPs are still localized to tumor surfaces (Fig 2), allowing for maintained IL-12 activity from the delivery vehicle as originally hypothesized.

Figure 4. — Activity mechanism from PLE-IL-12-NPs. A, Schematic of NP structure for FRET kinetic release studies (left). FRET readouts for intact NPs (right, top) and degraded NPs (right, bottom) B, Normalized FRET efficiency overtime for NPs. NPs were tested under two media conditions: water (storage media, dashed curves) and spent media (MC38 media culture on cells for at least 48 hours as an *in vivo* mimic, solid curves). FRET efficiencies were measured for the erosion of the polymer layers as measured by FRET pairing with the acceptor fluorophore on PLR (blue) and release of IL-12 as measured by FRET pairing with the acceptor fluorophore on IL-12 (red). Inset shows the first 24 hours in greater detail. C, schematic of particle break down as indicated by release data from B.

Once full characterization and activity of the PLE-IL-12-NPs was established in vitro, their release and activity in vivo was probed to find an appropriate dosing schedule. C57BL/6 mice bearing MC38 tumors were treated intratumorally with PLE-IL-12-NPs (5 μg IL-12) and sacrificed at different time points. Tumors were then homogenized and assessed for levels of delivered scIL-12, endogenous IL-12, and IFN-γ by ELISA on tumor supernatants (Fig S5). Kinetic release of exogenously delivered scIL-12 matching the release found in Fig 4 was observed, followed by a peak response of endogenous IL-12 and a later peak of IFN-γ. These data suggest an active immune response to PLE-IL-12-NPs over a seven day period, with the expected cascade of activation and interferon generation anticipated for an IL-12 therapy (Fig S5). These data indicate that a dosing schedule of at least weekly or more frequent dosing of PLE-IL-12-NPs may be required to maintain an active immune response in vivo.

PLE-IL-12-NPs reduce the toxicity of IL-12 therapy

The most critical aspect of any IL-12 therapy is the ability to control systemic activity and limit toxicity outside the tumor microenvironment as this has been the largest limitation of this potent class of cytokines in the clinic. PLE-IL-12-NPs were tested for their toxicity in healthy C57Bl/6 mice which were dosed subcutaneously with 5 μg doses of either the free cytokine or the PLE-IL-12-NP, as well as a group treated with 7.5 μg of IL-12 in PLE-IL-12-NPs, each dosed daily for 5 days. These groups were compared to PBS dosing and lipid concentration-matched PLE NPs without IL-12 as controls (Fig 5A). Mice given free IL-12 rapidly lost weight over the dosing period, losing approximately 10% of starting body weight by day 5, indicating a highly toxic therapy. In contrast, mice showed little weight change compared to controls over the dosing period when given PLE-IL-12-NPs, even at 1.5x the dosing of the free IL-12 (Fig 5B). These data demonstrate that PLE-IL-12-NPs reduce the systemic toxicity associated with IL-12 therapy.

Figure 5. — PLE IL-12 NPs reduce toxicity of IL-12 therapy *in vivo*. A, Schematic of experimental design B, Body weight change (Mean+SEM) of healthy animals treated as indicated subcutaneously (PLE IL-12 NPs 5μg n=5, n=3 all other groups). * indicates p<.05, *** indicates p<.001 as measured by 2-way ANOVA with Bonferroni post-hoc test across all groups. C, Cytokine response (Mean+SEM) in serum taken after dose 5 from B as measured by multiplexed assay. * indicates p<.05, ** indicates p<.01, *** indicates p<.001 as measured by One way ANOVA on individual cytokine groups with Bonferroni post-hoc test comparing all groups to IL-12 (5μg) and PBS to PLE NPs.

As an additional test of toxicity, serum was collected from the subjects 3 hours after the final dose and analyzed for a panel of systemic inflammatory cytokines. This panel included IL-12 and IFN-γ which are the direct downstream products of IL-12 signaling and most often associated with toxicity as well as CCL2 which acts as a chemoattractant for T cells, IL-10 which acts as the antithesis of IL-12 and is upregulated to slow down an inflammatory response, and TNF-α which is an immune regulatory molecule. Carrier-free IL-12 therapy showed much higher levels of systemic cytokines IFN-γ and IL-12 than PLE-IL-12-NPs, (Fig 5C), again indicating that PLE-IL-12-NPs are able to reduce the systemic toxicity of IL-12. Of note, when comparing the PLE-IL-12-NPs, specifically at the higher dose of 7.5 μg, to free IL-12 many of the positive aspects of the systemic immune response remain engaged, such as chemoattraction of monocytes via CCL2, while the highly toxic IFN-γ is drastically reduced in systemic circulation by the PLE NP carrier even at the higher dose. This demonstrated reduction of systemic toxicity is critical for a successful IL-12 therapy.

PLE-IL-12-NPs maintain efficacy of IL-12 therapy in multiple tumor models

To determine whether safer IL-12 delivery from the described NP carrier continued to provide antitumor efficacy, PLE-IL-12-NPs were tested for their ability to slow tumor growth and prolong survival in two flank tumor models, MC38 colon cancer and the more difficult to treat HM-1 ovarian cancer, as a test of anti-tumor response. As an initial test, MC38 cells were implanted subcutaneously in C57BL/6 mice and vascularized tumors were allowed to form (6 days). Once tumors were established, mice were treated intratumorally with both free IL-12 and PLE-IL-12-NPs and compared to appropriate controls (PBS and PLE NPs) (Fig 6A). Mice were monitored for tumor response both by growth of tumor size (Fig 6B, C) and overall survival (Fig 6D). Subjects receiving 5 μg weekly injections of both carrier-free IL-12 and PLE-IL-12-NPs showed slowed tumor growth and prolonged survival compared to both the PBS and PLE NP control groups in a statistically significant manner by the thirteenth day of tumor growth. Importantly, subjects treated with free IL-12 and PLE-IL-12-NPs showed no differences in therapeutic response from each other. Also, increasing the dose and dosing frequency of PLE-IL-12-NPs enabled a further improvement in tumor responses, as demonstrated with 7.5 μg dosing of the PLE-IL-12-NPs or increased 2x weekly frequency of dosing; both of these conditions elicited slower tumor growth and a statistically significant survival benefit as compared to 5 μg weekly doses of IL-12 in both the PLE NP encapsulated form and free drug (Fig 6B–D). These data together with the previously discussed reduction in toxicity demonstrate a widening of the therapeutic window for this potent yet toxic therapy.

Figure 6. — PLE IL-12 NPs maintain efficacy against MC38 (A-D) and HM-1 (E-G) tumors *in vivo*. A, Study design. B, MC38 tumor volume (mm³) of indicated treatments. All animals dosed 5 times given weekly intratumorally unless otherwise noted beginning on day 6. (PBS, PLE-NPs, IL-12 n=10; PLE-IL-12-NPs 5 μg weekly and 2x weekly n=8; PLE-IL-12-NPs 7.5 μg n=7 across multiple studies) C, Mean volume (+SEM) from B on day 13 for each group. *** indicates p<.001 as measured by one-way ANOVA using Bonferroni post-hoc test on all pairs of data. D, Survival curves of groups from B and C. * indicates p<.05, *** indicates p<.0001 as measured by Log-rank tests between groups. E, HM-1 tumor volume (mm³) of indicated treatments. All animals dosed 5 times given weekly intratumorally beginning on day 6. (PBS, PLE-NPs, and PLE-IL-12-NPs n=10; IL-12 n=9 across multiple studies) F, Mean volume (+SEM) from E on day 24 for each group. ** indicates p<.01 as measured by one-way ANOVA using Bonferroni post-hoc test on all pairs of data. G, Survival curves of groups from E and F. *** indicates p<.0001 as measured by Log-rank tests between groups.

In addition to the described single tumor efficacy tests, the ability of PLE-IL-12-NPs to provoke an abscopal immune response was tested in a two flank model (Fig S6). Mice were inoculated with MC38 tumor cells subcutaneously on both flanks, with a smaller (5x less inoculant) tumor inoculation on the untreated (left) flank. Tumors on the right flank were treated with PBS, PLE-IL-12-NPs, or free IL-12 at 5 μg weekly doses and both tumors were monitored for response. Treated tumors responded equally well to PLE-IL-12-NPs and free IL-12; however, the abscopal untreated tumor growth was slowed by both PLE-IL-12-NPs and free IL-12 with a slightly better (statistically insignificant) response to the free IL-12. This is likely due to the greater systemic activity of the carrier-free treatment shown in (Fig 5). Notably there is still a trend in abscopal response to the PLE-IL-12-NPs compared to controls suggesting a systemic immune response against the tumor. Coupled with reduced toxicity (Fig 5) the equivalent efficacy of PLE-IL-12-NPs to carrier-free IL-12 demonstrates an improvement in IL-12 therapy using the described PLE-IL-12-NPs.

Showing efficacy in the MC38 model is an excellent indicator of the viability of the described PLE-IL-12-NPs as an effective cytokine delivery vehicle. However, the MC38 cell line is known to be an immune infiltrated or “hot” tumor model. Ovarian tumors have proven much more difficult in their response to immunotherapies.^3–5 As a further measure of IL-12 efficacy and test for viability of cytokine delivery via PLE-NPs, their efficacy in HM-1 tumors (murine ovarian tumors known to be heavily infiltrated with myeloid derived suppressor cells and poorly infiltrated with T-cells) was tested.^47,48 This tumor model also showed improvement in tumor responses both by slowing of tumor growth (Fig 6 E, F) and survival (Fig 6G) when treated with IL-12, with PLE-IL-12-NPs achieving equivalent efficacy to the more toxic carrier-free therapy. This model was further tested using the same in vitro assays described for the MC38 model in Fig 3. Similar responses in the in vitro efficacy tests in both the splenocyte only and co-culture models (Fig S7) were obtained with HM-1 cells as well as 4T1 breast cancer cells as compared to the MC38 experiments, establishing PLE-IL-12-NPs as a diversely applicable therapeutic strategy. These data together with toxicity reduction suggest that PLE-IL-12-NPs show promise for treatment of multiple tumor types, including those traditionally less responsive to immunotherapy such as ovarian cancer.

Ovarian cancer generally has not benefitted from the clinically available checkpoint inhibitors, and the exploration of inflammatory molecules such as IL-12 and some other cytokines as a supplement to checkpoint inhibiters has been limited by toxicity concerns. Ovarian cancer usually does not present with a single injectable tumor but instead with many metastatic nodules spread throughout the abdomen, making intratumoral treatment an impractical option clinically. The delivery system described in the current study demonstrates promise for systemic or intraperitoneal application of toxic cytokines in these difficult tumors; polymer layers not only reduce the toxicity of the cytokine as described herein, but also may provide a significant targeting effect to ovarian tumors in vivo that can effectively concentrate the delivery of cytokines to the tumor nodules.⁴¹ Indeed, systemic immunotherapy via cytokine delivery offers promise for widely disseminated metastatic disease such as ovarian cancer as developing a local immune response in some tumors can lead to systemic immunity against the cancer at other metastatic sites. Our PLE-IL-12-NPs demonstrate this systemic effect in their efficacy against abscopal tumors. Expanding the applications of the described PLE-IL-12-NPs to systemic delivery poses the promise of bringing immunotherapy advancements to the ovarian treatment space.

As a further test of the LbL-NP’s ability to improve IL-12 therapy, PLE-IL-12-NPs were tested against carrier-free delivery for both toxicity and efficacy at greatly increased dosing levels to further demonstrate the described widening of the therapeutic window. For these tests, subjects were treated with PLE-IL-12-NP doses increased to 25 μg (5x the normal dose from Fig 6) and 50 μg (10x the normal dose) given twice weekly (as opposed to weekly dosing in Fig 6) for five doses and compared to 25 μg and 50 μg free IL-12 given twice weekly. Tumor growth and survival was analyzed for efficacy and weight change over the dosing period and serum cytokine levels after the first and last treatments were analyzed for toxicity (Fig 7A). Groups treated with free IL-12 lost weight after each dose, with the 50 μg treated mice losing significant weight (~10% body weight) following the first treatment, thus reaching the level of weight loss that exceeds that of the maximum tolerated dose as defined for these studies, evidencing a highly toxic therapy. Conversely, groups treated with PLE-IL-12-NPs continued to gain weight with the controls (Fig 7B) at the same dosing levels. In addition, both IL-12 and IFN-γ levels in the serum were higher in the free IL-12 treated mice after the first and last dose, particularly at the higher dosing level of 50 μg. At this dose the free IL-12 treated groups showed significant differences to the PLE-IL-12-NP groups throughout the dosing period, especially in the toxic systemic IFN-γ levels (Fig 7C), again demonstrating the systemic toxicity of free cytokine in these subjects. These data suggest that PLE-IL-12-NPs greatly reduce the toxicity of IL-12 treatment even at very high doses. Taking this study further, the group treated with the 25 μg dosing level of the PLE-IL-12-NPs showed equivalent efficacy compared to the group dosed with free IL-12 in terms of both tumor growth arrest and survival (Fig 7D, E). Moreover, the group dosed with the 50 μg PLE-IL-12-NPs showed significantly longer survival compared to the group dosed with 25 μg of free IL-12, which was the highest well-tolerated dose of free cytokine tested, at reduced toxicity. Furthermore, the PLE-IL-12-NP (50 μg) resulted in a cured mouse which rejected a rechallenge with 1 × 10⁶ tumor cells 85 days after treatment, indicating a memory response. Combining these data with Fig 5, the dose response achieved by IL-12 therapy both in free and PLE NP form is evident (Fig 7F). These data show that the rationally engineered PLE-IL-12-NPs make a significant improvement for IL-12 therapy by allowing for increased dosing levels with reduced toxicity over what is achievable with carrier-free cytokine with no loss in efficacy of the IL-12 treatment. The ability to effectively treat epithelial cancers including ovarian cancers at tolerated doses with proinflammatory cytokines such as IL-12 is critical in potentiating the promise of immunotherapies in these tumors, as these therapies are capable of driving immune infiltration and activity in these difficult to treat tumors.

Figure 7. — Combined toxicity and efficacy at high doses demonstrates PLE IL-12 NPs are a safer, more effective IL-12 therapy. A, Schematic of study. C57Bl/6 mice are inoculated with 5E06 MC38 cells on day 0. Mice are treated twice weekly for 5 doses with serum collected 24 hrs after the first and last dose. Animals are monitored throughout for tumor burden and weight change. (PLE-NPs n=3; 25 μg PLE-IL-12-NP, 25 μg IL-12, 50 μg IL-12 n=5; 50 μg PLE-IL-12-NPs n=4) B, Weight change (Mean+SEM) over initial dosing period,* indicates p<.05, ** indicates p<.01, *** indicates p<.001 compared to PBS as measured by 2-way ANOVA with Bonferroni post-hoc tests comparing all groups to PBS. C, Cytokine levels (Mean +SEM) in serum at indicated time points as measured by ELISA. * indicates p<.05, *** indicates p<.001 as measured by 2way ANOVA with Bonferroni post-hoc test comparing all pairs of groups. Comparisons are to PBS where not indicated otherwise. D, Tumor volumes of individual animals of indicated treatment groups. E, Survival curves of animals from treated and control groups (PBS and PLE NP combined over all studies). ** indicates p<.01, *** indicates p<.001 as measured by Log-rank tests between groups. F, Average tumor volumes (mean +SEM) over time for animals from figure 5B low dose experiment (IL-12 5μg, PLE IL-12 NPs 5μg, PLE IL-12 NPs 7.5μg, PLE IL-12 NPs 5μg 2xweekly) and figure 6D high dose study (IL-12 25μg 2xweekly, PLE IL-12 NPs 25μg 2xweekly, PLE IL-12 NPs 50μg 2xweekly).

One limitation of the current study is that it relies on an intratumoral approach in in vivo studies to deliver the therapeutic. However, overcoming IL-12 based toxicity even from a local injection has remained a challenge clinically and preclinically with several investigations taking a local delivery approach still failing to reduce systemic toxicity when the IL-12 is given at therapeutically relevant dose. Indeed, even in the current study, locally delivered free IL-12 showed significant toxicity at relevant doses. Therefore, even though the reduction in toxicity shown in the current study is thus far only demonstrated for intratumoral administration, this result still stands as a relevant advance for IL-12 delivery. Nonetheless, ongoing and future work will explore the application of this delivery system for systemic administration, focusing on ovarian tumor models to bring the promise of immunotherapy to this difficult to treat malignancy. Indeed the in vitro work here shows promise for a successful systemic cytokine delivery platform. Firstly, the described PLE-IL-12-NPs showed selective association with tumor cells both in this work (Fig S4) and previous studies,⁴¹ which is critical for concentrating NPs and payload in the tumor microenvironment. In addition, mechanistic studies showed the majority of the IL-12 remains attached to the particle for greater than 8 hours (Fig 4), which is longer than similar actively targeted NPs take to concentrate in the tumor microenvironment upon systemic delivery. This delayed IL-12 release is critical for potential success in systemic delivery as it prevents off-tissue exposure of potent cytokine payloads. Equally important, the IL-12 payload is rapidly released thereafter while the NPs remain localized to the cell surfaces, maintaining the cytokine payload activity once in the tumor microenvironment. Taken together, the in vitro characterizations of IL-12 loading (Fig 1), subcellular localization (Fig 2), tumor cell association selectivity (Fig S4), biological activity (Fig 3), and kinetic release of IL-12 (Fig 4) demonstrate a NP platform that meets all the criteria for a successful systemically deliverable cytokine therapy.

PLE-IL-12-NPs enhance lymphocyte activity in tumors and tumor draining lymph nodes

After showing that the described PLE-IL-12-NPs were capable of reducing toxicity with comparable anti-tumor efficacy compared to free cytokine, flowcytometry was used to probe the immunological mechanisms of the treatments in the MC38 model. As described above, vascularized tumors were allowed to establish before starting therapy (6 days). Tumors were then treated intratumorally with biweekly doses of 5 μg IL-12 either in PLE-IL-12-NP form or carrier-free, PLE-NPs with matched lipid dose, or PBS. Tumors, spleens, and tumor draining lymph nodes (TDLNs) were prepared for flowcytometry 24 hours after the third dose which was chosen as an endpoint based on significant differences in tumor sizes occurring between treatment groups at that time as shown in Fig 6. Flowcytometry was used to distinguish different cell populations in the isolated tissues (gating strategy Fig S8, all data Fig S9).

As expected, both IL-12 therapies caused large shifts in lymphocyte populations indicative of a more active antitumor immune response in the tumor microenvironment. The main impact from IL-12 therapy both in carrier-free and PLE-IL-12-NPs was a shift towards higher CD8+ T cell populations in the tumor (Fig 8A). This is evidenced not only by a higher CD8+ fraction of T cells in the tumor, but also by a higher ratio of CD8+/CD4+ T cells in the tumor (Fig 8E). Moreover, IL-12 therapy yielded more active CD8+ T cells in the tumor, as measured by degranulation markers (CD69+) (Fig 8B,E). Importantly, the PLE-IL-12-NPs showed no difference in CD8+ T cell or degranulated CD8+ T cell populations compared to free IL-12, indicating the same gains in immune activation state in the tumor microenvironment as the free drug, despite the therapy’s encapsulation. These T cell shifts are expected from an IL-12 therapy and indicative of a shift toward a more active, antitumoral Th1 type immune response in the tumor. In addition, the monocyte populations in the tumor undergo a shift from more monocytic (Ly6C+) to more granulocytic (Ly6G+) (Fig S9) indicating a polarization of monocytes to neutrophils and a more active local immune response.

Figure 8. — Flowcytometry analysis shows IL-12 shifts immune populations towards an antitumoral response. A. CD3+ T cell gating for CD4 and CD8 T cells in different treatment groups (CD4 y-axis, CD8 x-axis). B. Degranulation of CD8+ T cells as measured by CD69 staining in the tumor. C. F4/80 staining of CD45+ cells in TDLNs. D. DC content as measured by CD11c+ staining in CD45+ cells in TDLNs. E. Quantification of chosen T cell populations from A and B. * indicates p<.05 as measured by one way ANOVA with Dunnett posthoc test F. Quantification of chosen APC populations flowcytometry. (T=tumor, S=spleen, LN=TDLN)

In addition to these intratumoral T cell shifts, the IL-12 therapies showed a shift towards greater APC migration to TDLNs. Both DC and macrophage populations were increased in TDLNs with both the PLE-IL-12-NPs and free IL-12 (Fig 8C,D,F). This migration of APCs to the TDLNs is again indicative of a more active immune response to the tumor. Migration of APCs to the lymph node is an expected step in the immunity cycle in response to an IL-12 therapy driving the cycle forward.^1,2

Little changes were seen in the spleen of treated mice, with the sole differences between IL-12 therapy and the control being an increase in neutrophil populations and a slight decrease in CD45+ leukocyte fractions in the treated groups (Fig S9). Taken together, these immune population changes upon treatment with IL-12 both within PLE-IL-12-NPs and carrier-free show a shift towards a more active and antitumoral Th1 type immune response in the tumor microenvironment and local lymphoid tissue, as is expected from an IL-12 therapy. These data demonstrate that PLE-IL-12-NPs are able to trigger a potent immune response and drive the cancer immunity cycle forward equivalently to carrier free IL-12 at significantly reduced toxicity.

Additional leukocyte population shifts indicate a shift towards a less suppressive immune microenvironment with IL-12 therapy. The PLE-IL-12-NP treatment showed a reduced number of regulatory T cells (Tregs) in the tumor coupled with a higher ratio of CD8/Treg cells in the TDLN. In addition, PLE-IL-12-NPs showed a trend towards reduced expression of PD-L1 (CD274) on CD45+ cells as compared to free IL-12. This difference is mainly from macrophage expression differences in PD-L1, as there is little difference in the DC population PD-L1 expression (Fig S9). Together, these data indicate that PLE-IL-12-NPs are capable of reducing the immune suppressive tumor microenvironment, particularly in the macrophage population, potentially beyond what is achievable with free cytokine, which could prove to be a potentially helpful enhancement in treatment.

Conclusions

In this work we have shown the rational engineering of an LbL NP delivery vehicle for the potent but toxic cytokine IL-12. The PLE-IL-12-NP met all of the key design criteria for cytokine delivery including efficiently encapsulating and releasing the cytokine, maintaining cytokine activity on external cell receptors, selectively interacting with tumor cells, achieving anti-tumor efficacy in multiple tumor models at reduced systemic toxicity compared to carrier free cytokine, and engaging local and systemic immunity. We have demonstrated that the described PLE-IL-12-NPs have a pronounced ability to encapsulate IL-12, showing a 90% encapsulation at 13% by weight in stable particles (Fig 1). We have shown that PLE-IL-12-NPs maintain the efficacy of IL-12 in its ability to stimulate IFN-γ production from target cells, and indeed exceed the activity of free IL-12 in a worst-case tumor mimic in vitro model, likely due to the distinctive ability of PLE-IL-12-NPs to localize on tumor cell membranes and act as a drug depot (Fig 2,3,4). We have also demonstrated that PLE-IL-12-NPs are capable of releasing active IL-12 and triggering an immune response in vivo, and that this immune response is capable of slowing tumor growth in multiple cancer models as a monotherapy, notably including more difficult to treat ovarian cancer (Fig 5,6).

Most importantly for IL-12 therapy, we have demonstrated in multiple studies the ability of the described PLE-IL-12-NPs to reduce the systemic toxicity of IL-12 at multiple schedules and doses. Indeed, we have demonstrated that the PLE-IL-12-NPs offer a safer therapy, even at higher doses than the free cytokine (Fig 5,7), which in turn yields a more effective therapy due to the ability to access higher dosing levels as compared to carrier free cytokine with the two delivery strategies showing equivalent efficacy at equivalent dosing. This is of utmost importance for any IL-12 therapy as toxicity has been the largest factor in preventing IL-12 from progressing in the clinic as evidenced by the severe toxicity, including two deaths in previous trials.^11–13

Finally, while these PLE-IL-12-NPs make use of the potent cytokine IL-12, no IL-12 specific techniques are used for their generation, making them adaptable to many other cytokines or proteins of interest for delivery. Moving forward, these particles can be utilized to deliver any protein with an appropriate binding ligand, or indeed a combination of such molecules. The general design is also easily adaptable to many other conjugation strategies for linking the cytokines to liposomes that can be used to modulate the release rate of delivered proteins. Accordingly, another advantage of the described LbL NP system is easy adaptability to combination therapy. Additional therapeutics can be added to the particle in multiple ways, including adding additional cytokines on the liposome surface, adding small molecules to the liposome core as described previously,^29,36 or including therapeutics such as siRNA within the layers. These particles can also be used to incorporate checkpoint inhibitors such as anti PD-1 or anti CTLA4 in a similar manner to cytokines on the liposome surface or attached to the layers. These combinations could prove to be most effective, because for many malignancies, a combination of immunotherapies is viewed as the most promising path forward.^49–51 As this approach progresses, the need to deliver combinations of immunotherapeutics in precise ratios and schedules⁵² will also need to be controlled - another potential benefit of using the described LbL-NPs, which can be designed to stage release of combination therapies.³¹

Methods/Experimental

Materials

1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (DGS-NTA (Ni)), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), Cholesterol, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (sodium salt) (POPG) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were purchased from Avanti Polar Lipids and used without modification. PLR and PLE were purchased from Alamanda Polymers and used without modification. Single chain IL-12 was produced in house from HEK-293 cells.

Protein Synthesis and Purification

Single chain IL-12 sequence³⁸ was synthesized as a genomic block (Integrated DNA Technologies) and cloned into gWIZ expression vector (Genlantis). Plasmids were transiently transfected into Expi293 cells (ThermoFisher Scientific). After 5 days, cell culture supernatants were collected and protein was purified in an ÄKTA pure chromatography system using HiTrap HP Niquel sepharose affinity column, followed by size exclusion using Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences). Endotoxin levels in purified protein was measured using Endosafe Nexgen-PTS system (Charles River) and assured to be < 5EU/mg protein.

Particle Formulation and Characterization

LbL nanoparticle assembly was performed as described previously³² with minor modifications. Briefly, liposomes were prepared by the rehydration/extrusion method. A lipid solution containing 5% DGS-NTA (Ni), 65% DSPC, 23.9% Cholesterol, and 6.1% POPG by mole in chloroform was dried at 20 mbar for 1 hr by rotovap and desiccated under vacuum overnight. The lipid film was then reconstituted in PBS to a concentration of 1 mg/mL under sonication at 65°C for 30 minutes. Rehydrated liposomes were extruded through 50 nm filters (Whatman) using Avestin Liposofast-50 pressure driven extruder at 65°C until they reached a size of appx 60 nm as measured by number average diameter by DLS (Malvern ZS90). Single chain IL-12 was added to liposomes in a 28:1 Ni:His ratio by mole overnight at 4C. Particle buffer was switched to water by tangential flow filtration (TFF) by 5x washing through a 100 kDa membrane (Spectrum Labs). Particles were added to a bath of PLR solution in glass vial under sonication at 0.1 weight equivalent of polymer compared to lipid and allowed to equilibrate on ice for 1–2 hours. Excess polymer was purified by TFF through a 100 kDa membrane (Spectrum Labs) and characterized for size and charge by DLS. Similarly, for terminal layer polyanion, particles were then added to a bath of polymer in glass vial under sonication at 1 weight equivalent of polymer compared to lipid and allowed to equilibrate on ice for 1–2 hours. Particles were purified by TFF and characterized for size and charge by DLS.

Encapsulation characterization

Encapsulation of scIL-12 in particles at various stages of the particle manufacture process was characterized by breaking up particles in 1% triton-100 (Sigma), 0.1% BSA (Sigma) under vortex for 1 min. IL-12 content was then measured by ELISA (Peprotech) and compared to initial amount of IL-12 added to the NPs (measured by nanodrop at time of addition) for encapsulation efficiency. Final particle lipid concentration was measured by Stewart assay⁵³ using chloroform dissolved lipid mixture from liposome formulation to produce a standard curve and weight percent encapsulation was calculated using IL-12 and lipid concentrations (mg IL-12/(mg lipid+mg IL-12)).

Flowcytometry

Antibodies used for immunostaining were against CD69 (biolegend 104545), CD25 (biolegend 102041), NK-1.1 (biolegend 108753), CD3 (biolegend 100232), CD4 (biolegend 100423), CD8a (BD biosciences 566410), FoxP3 (biolegend 126404), CD45 (biolegend 103112), Ly-6C (biolegend 128032), Ly-6G (biolegend 127633), CD274 (biolegend 124331), F4/80 (biolegend 123110), CD11c (BD biosciences 566504), CD11b (biolegend 101217), CD86 (biolegend 105037) CD103 (biolegend 562722). FoxP3 intracellular staining was carried out using FoxP3 intracellular staining kit (Thermo 00-5523-00) following manufacture protocol. Immunostained cells were run on an LSR Fortessa HTS with FACSDIVA software and analyzed using FlowJo V10.5.3.

Fluorescence Imaging

Chambered cover slips (LabTek) were coated with 50 μg/mL rat tail collagen (corning). Cells were seeded on coverslips at 5000 cells per well and allowed to establish for 24 hours. Cells were treated at different time points at 6.2 μg/mL particles. Fluorescent core carboxy modified latex particles (Thermo Fisher F8803) layered with PLR and indicated terminal layer polyanions were used in place of scIL-12 liposome cores for visualization. Cells were incubated for 4 or 24 hours after particle treatment. Cells were fixed with 4% formaldehyde for 15 minutes and stained with wheat germ agglutinin-AF647. Cells were then fixed again for 2 minutes and permeabilized with 0.2% triton x-100 and stained with Hoechst solution at 1.25 μg/mL. After staining, wells were protected with Vectashield and imaged on an Applied Precision DeltaVision Spectris Imaging System with Softworx deconvolution software. Images were further analyzed in FIJI.

Cell Culture

MC38 cells were a gift from the laboratory of Darrell Irvine. HM-1 cells were acquired through Riken BRC. Cells were cultured in DMEM or α-MEM respectively, supplemented with 10% FBS and penicillin/streptomycin or as recommended by the supplier in a 5% CO₂ humidified atmosphere at 37C. All cell lines were murine pathogen tested and confirmed mycoplasma negative by Lonza MycoAlert™ Mycoplasma Detection Kit.

In vitro activity experiments

Constructed scIL-12 LbL NPs were tested for bioactivity by ability to stimulate an IFN-γ response. Briefly, splenocytes were isolated from appropriate background strain mice (based on the tumor cell line being used; MC38 paired with C57Bl/6 mice and HM-1 paired with B6C3F1) by pushing spleens through 70 μm strainers, lysing red blood cells via ACK lysis buffer and resuspending splenocytes in RPMI containing 10% FBS, 1% penicillin/streptomycin, 1% sodium pyruvate, and 0.0005% β-mercaptoethanol. Splenocytes were cultured for 24 hours or less to prevent changes in populations from culture. Splenocytes were either dosed with varying doses of scIL-12 in particles or soluble form or added to cancer cell cultures that had been dosed for 6 hrs previously with varying doses of scIL-12 in particle or soluble form for 18 hours. Supernatants were then tested for IFN-γ content by ELISA (peprotech). Data were analyzed in Graph Pad PRISM 5 to find EC50 values based on dose response curves.

Tumor selectivity studies

MC38 cells were seeded in 96 well plates at 50000 cells per well and cultured for 24 hours. Splenocytes were harvested similar to in vitro activity experiments and added to wells (1:1 ratio with MC38 cells) containing subcultured MC38 cells for a total volume of 90 μl. Wells were then dosed with 10 μl of 0.1 mg/mL or 0.01 mg/mL Cy5 NPs at varying timepoints. Cy5 NPs were made by addition of 5% DOPE by mol replacing 5% by mol DSPC in original liposome formulation. These liposomes were then tagged with sulfo-Cy5 NHS ester (Lumiprobe 13320) following manufacture protocol. Briefly, 8 fold molar excess of dye was added to NP solution titrated to pH 8.5 and allowed to react at 4C overnight under agitation. Excess dye was purified off the NPs using TFF. NP solution was washed until the permeate from TFF showed <5% fluorescence as compared to the start of purification. IL-12 and polymer layering was then carried out similar to previous formulations. After NP incubation cultures were processed and stained for live/healthy cells (Biolegend 423114) and CD45 (BD 564279). Flowcytometry was carried out similar to previous description.

FRET studies

NPs were formulated similar to the above description. For these studies liposomes were made following the same protocol as described previously with the exception that 5% DOPE was added and DSPC mol% was reduced to 60%. Once liposomes were extruded and buffer exchanged to water, Sulfo-Cy3 NHS-ester (Lumiprobe 11320) was added to the liposome surface following manufacture protocol for NHS-ester amine reaction with DOPE head groups. Briefly, 8 fold molar excess of dye was added to NP solution titrated to pH 8.5 and allowed to react at 4C overnight under agitation. Excess dye was purified off the NPs using TFF. NP solution was washed until the permeate from TFF showed <5% fluorescence as compared to the start of purification. IL-12 was then added to the liposome surface as described previously. For IL-12 release studies Sulfo-Cy5 NHS ester was added to the IL-12 similar to Sulfo-Cy3 NHS ester addition to DOPE described above. NPs were then layered with polymer layers similar to previous experiments. For NP erosion FRET particles Sulfo-Cy5 NHS ester was added to PLR similar to addition to IL-12. NPs were made in two groups, IL-12 tagged and PLR tagged. Each group contained NPs with only Sulfo-Cy3 on the liposome surface, NPs with only Sulfo-Cy5 either on the IL-12 or PLR respectively, and FRET pairing NPs with both dyes present. NPs were then added at 10% by volume to different media conditions. Media conditions tested included water (storage condition) and cell media that had been incubated with active cultures of MC38 cells for no less than 48 hours (spent media) as a mimic of an in vivo environment. Samples were taken at 1, 2, 4, 8, 24, 48 and 72 hour timepoints and measured for fluorescence. Two measurements were taken, 1) 540 excitation and 570 emission reflecting excitation and emission of Cy3 and 2) 540 excitation and 680 emission reflecting excitation of Cy3 and emission of Cy5. FRET efficiency was then calculated based on equation 1.

FRET efficiency = \frac{D_{D} - F r e t_{D}}{D_{D}};

(1)

D_D=fluorescence intensity of NP with donor fluorophore only in the donor channel

Fret_D= fluorescence intensity of NP with both fret fluorophores in the donor channel

FRET efficiency was normalized to 100%=FRET efficiency at t=0 and 0%=FRET efficiency of NPs degraded with triton x-100 similar to encapsulation experiments described above.

Animal Studies.

All animal experiments were approved by the Massachusetts Institute of Technology Committee on Animal Care (CAC) and were conducted under the oversight of the Division of Comparative Medicine (DCM).

In vivo toxicity tests

To test toxicity, C57Bl/6 mice (Taconic) were injected subcutaneously (healthy mice) on the flank with varying doses as indicated of PLE-IL-12-NPs, lipid dose matched LbL-NPs without IL-12, dose matched soluble IL-12, or PBS and monitored daily for weight change. Serum was collected after 5 days and assayed using multiplex inflammatory cytokine assay (done by Abcam). (PLE IL-12 NPs 5μg n=5, n=3 all other groups)

In vivo efficacy tests

C57Bl/6 (Taconic) or B6C3F1 (Jackson Labs) mice as appropriate for tumor type were injected subcutaneously with 5E05 (MC38) or 1E06 (HM-1) cells in 1:1 PBS:Matrigel (Corning) mixture. Tumors were allowed to establish for 6 days prior to treatment. Mice were treated intratumorally with scIL-12 LbL-NPs, lipid dose matched LbL-NPs without IL-12, dose matched soluble scIL-12, or PBS either weekly or 2x weekly for a maximum of 5 doses. Mice were monitored 2x weekly for tumor growth as measured by digital calipers (Vernier) taking tumor volume to be L*Ŵ2*1/2 where L is the longest diameter and W the shortest diameter of the tumor. Mice were euthanized when volume exceeded 1000 mm³. For combined toxicity/efficacy studies mice were monitored daily for weight changes over the dosing period and serum was collected 24 hours after the first and last dose and analyzed for IFN-γ and IL-12 content via ELISA (Peprotech) in addition to the above.

MC38 single tumor studies (Fig 6): (PBS, PLE-NPs, IL-12 n=10; PLE-IL-12-NPs 5 μg weekly and 2x weekly n=8; PLE-IL-12-NPs 7.5 μg n=7 across multiple studies)

HM-1 single tumor studies (Fig 6): (PBS, PLE-NPs, and PLE-IL-12-NPs n=10; IL-12 n=9 across multiple studies)

High dose MC38 single tumor studies (Fig 7): (PLE-NPs n=3; 25 μg PLE-IL-12-NP, 25 μg IL-12, 50 μg IL-12 n=5; 50 μg PLE-IL-12-NPs n=4)

Abscopal Response

C57Bl/6 mice were injected subcutaneously with 5E05 cells in 1:1 PBS:matrigel mixture on the right flank and 1E05 cells in 1:1 PBS:matrigel mixture of the left flank. Tumors were allowed to establish for 6 days prior to treatment. Mice were treated intratumorally with PLE-IL-12-NPs, dose matched soluble IL-12, or PBS weekly for 5 doses in the right tumor only. Mice were monitored 2x weekly for tumor growth as measured by digital calipers (Vernier) taking tumor volume to be L*Ŵ2*1/2 where L is the longest diameter and W the shortest diameter of the tumor. Mice were euthanized when volume exceeded 1000 mm³. (PBS, PLE-IL-12-NPs n=9; IL-12 n=8)

Statistical Analysis

GraphPad PRISM 5 was used to perform statistical analyses. Multiple comparisons were performed using multiple t tests, one-way ANOVA, or two-way ANOVA followed by post-hoc tests as indicated in figures.

Data Availability

The data for this study are available within the article, with additional data available in the Supporting Information.

Supplementary Material

Supplemental information

NIHMS1625422-supplement-Supplemental_information.pdf^{(3.6MB, pdf)}

Figures

NIHMS1625422-supplement-Figures.pdf^{(678.7KB, pdf)}

ACKNOWLEDGEMENTS

The authors also thank the MIT Koch Institute Swanson Biotechnology Center, which is supported by the Koch Institute Core Grant P30-CA14051 from the NCI, for the use of facilities and specifically the High Throughput Screening, Flowcytometry, Microscopy, and Animal Imaging and Preclinical Testing core facilities. We thank E. Dreaden for the helpful discussions and advice.

This work was supported by the US Department of Defense Congressionally Directed Medical Research Programs (PTH, Teal Innovator Award W81XWH-13-1-0151), the Bridge Project a partnership between the Koch Institute for Integrative Cancer Research at MIT and the Dana-Farber/Harvard Cancer Center, NIH (1-R01-CA235375-01A1), the Marble Center for Cancer Nanomedicine, and a Cancer Center Support (core) Grant P30-CA14051 from the National Cancer Institute, the Koch Institute’s Marble Center for Cancer Nanomedicine Fellowship (SGS), Sloan UCEM Fellowship (SC), the Siebel Scholars Fellowship (SC), the National Science Foundation (SC, GFRP 1122374), the MIT Undergraduate Research Opportunity Office (CN), the Frost Environmental Science/Studies Fund EN34770 (BTN), Ronald E. McNair Post-Baccalaureate Achievement Program GR26419 (BTN), and NIH interdepartmental biotechnology training program 5T32GM008334-30 (AEB). Resources were provided in part by the Koch Institute Support Grant (P30-CA14051) from the National Cancer Institute and the MIT MRSEC Shared Experimental Facilities Grant (DMR-0819762) from the National Science Foundation.

Footnotes

Supporting Information Available

The supporting information includes calculations for number of IL-12 per NP, scIL-12 protein construct, additional microscopy images tracking subcellular localization, dose response curves for in vitro studies, in vitro tumor selectivity, temporal in vivo immune response studies, abscopal response studies, in vitro activity in additional models, flow cytometry gating strategies, and full detailed immune profiling results. This material is available free of charge via the internet at http://pubs.acs.org.

Contributor Information

Antonio E. Barberio, Department of Chemical Engineering, Massachusetts Institute of Technology, 183 Memorial Drive, Cambridge, MA 02142, USA

Sean G Smith, Department of Chemical Engineering, Massachusetts Institute of Technology, 183 Memorial Drive, Cambridge, MA 02142, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02142, USA.

Santiago Correa, Department of Biological Engineering, Massachusetts Institute of Technology, 21 Ames Street, Cambridge MA 02142, USA.

Cathy Nguyen, Department of Biological Engineering, Massachusetts Institute of Technology, 21 Ames Street, Cambridge MA 02142, USA.

Bang Nhan, Department of Chemistry, Wellesley College, 106 Central Street, Wellesley, MA 02481.

Mariane Melo, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02142, USA.

Talar Tokatlian, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02142, USA.

Heikyung Suh, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02142, USA.

Darrell J. Irvine, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02142, USA; Department of Biological Engineering, Massachusetts Institute of Technology, 21 Ames Street, Cambridge MA 02142, USA; Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.

Paula T. Hammond, Department of Chemical Engineering, Massachusetts Institute of Technology, 183 Memorial Drive, Cambridge, MA 02142, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02142, USA; Institute for Soldier Nanotechnologies;.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental information

NIHMS1625422-supplement-Supplemental_information.pdf^{(3.6MB, pdf)}

Figures

NIHMS1625422-supplement-Figures.pdf^{(678.7KB, pdf)}

Data Availability Statement

The data for this study are available within the article, with additional data available in the Supporting Information.

[R1] 1.Chen DS, Mellman I. Oncology Meets Immunology: The Cancer-Immunity Cycle. Immunity. 2013,39,1–10. [DOI] [PubMed] [Google Scholar]

[R2] 2.Chen DS, Mellman I. Elements of Cancer Immunity and the Cancer-Immune Set Point. Nature. 2017,541,321–30. [DOI] [PubMed] [Google Scholar]

[R3] 3.Bindea G, Mlecnik B, Angell HK, Galon J. The Immune Landscape of Human Tumors: Implications for Cancer Immunotherapy. OncoImmunology. 2014,3,e27456. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] 4.Galon J, Pages F, Marincola FM, Angell HK, Thurin M, Lugli A, Zlobec I, Berger A, Bifulco C, Botti G, Tatangelo F, Britten CM, Kreiter S, Chouchane L, Delrio P, Arndt H, Asslaber M, Maio M, Masucci GV, Mihm M, et al. Cancer Classification Using the Immunoscore: A Worldwide Task Force. J. Transl. Med 2012,10,1–10. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R5] 5.Pages F, Mlecnik B, Marliot F, Bindea G, Ou FS, Bifulco C, Lugli A, Zlobec I, Rau TT, Berger MD, Nagtegaal ID, Vink-Borger E, Hartmann A, Geppert C, Kolwelter J, Merkel S, Grutzmann R, Van den Eynde M, Jouret-Mourin A, Kartheuser A, et al. International Validation of the Consensus Immunoscore for the Classification of Colon Cancer: A Prognostic and Accuracy Study. Lancet. 2018,391,2128–2139. [DOI] [PubMed] [Google Scholar]

[R6] 6.Trinchieri G Interleukin-12: A Proinflammatory Cytokine With Immunoregulatory Functions That Bridge Innate Resistance and Antigen-Specific Adaptive Immunity. Annu. Rev. Immunol 1995,13,251–276. [DOI] [PubMed] [Google Scholar]

[R7] 7.Robertson MJ, Ritz J. Interleukin 12: Basic Biology and Potential Applications in Cancer Treatment. Oncologist. 1996,1,88–97. [PubMed] [Google Scholar]

[R8] 8.Trinchieri G; Scott P. Interleukin-12: Basic Principles and Clinical Applications In Redirection of Th1 and Th2 Responses; Coffman RL, Romagnani S, Eds; Springer: Berlin, Heidelberg, 1999; p57–78. [DOI] [PubMed] [Google Scholar]

[R9] 9.Colombo MP, Trinchieri G. Interleukin-12 in Anti-Tumor Immunity and Immunotherapy. Cytokine Growth Factor Rev. 2002,13,155–168. [DOI] [PubMed] [Google Scholar]

[R10] 10.Brunda MJ, Luistro L, Warrier RR, Wright RB, Hubbard BR, Murphy M, Wolf SF, Gately M. Antitumor and Antimetastatic Activity of Interleukin 12 against Murine Tumors. J. Exp. Med 1993,178,1223–1230. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] 11.Atkins MB, Robertson MJ, Gordon M, Lotze MT, DeCoste M, DuBois JS, Ritz J, Sandler AB, Edington HD, Garzone PD. Phase I Evaluation of Intravenous Recombinant Human Interleukin 12 in Patients with Advanced Malignancies. Clin. Cancer Res 1997,3,409–417. [PubMed] [Google Scholar]

[R12] 12.van Herpen CM, Huijbens R, Looman M, de Vries J, Marres H, van de Ven J, Hermsen R, Adema GJ, De Mulder PH. Pharmacokinetics and Immunological Aspects of a Phase Ib Study with Intratumoral Administration of Recombinant Human Interleukin-12 in Patients With Head and Neck Squamous Cell Carcinoma: A Decrease of T-Bet in Peripheral Blood Mononuclear Cells. Clin. Cancer Res 2003,9,2950–2956. [PubMed] [Google Scholar]

[R13] 13.Portielje JE, Kruit WH, Schuler M, Beck J, Lamers CH, Stoter G, Huber C, de Boer-Dennert M, Rakhit A, Bolhuis RL. Phase I Study of Subcutaneously Administered Recombinant Human Interleukin 12 in Patients with Advanced Renal Cell Cancer. Clin. Cancer Res 1999,5,3983–3989. [PubMed] [Google Scholar]

[R14] 14.Egilmez NK, Jong YS, Sabel MS, Jacob JS, Mathiowitz E, Bankert RB. In Situ Tumor Vaccination with Interleukin-12-Encapsulated Biodegradable Microspheres: Induction of Tumor Regression and Potent Antitumor Immunity. Cancer Res. 2000,60,3832–3837. [PubMed] [Google Scholar]

[R15] 15.Hill HC, Conway TF, Sabel MS, Jong YS, Mathiowitz E, Bankert RB, Egilmez NK. Cancer Immunotherapy with Interleukin 12 and Granulocyte-Macrophage Colony-Stimulating Factor-Encapsulated Microspheres: Coinduction of Innate and Adaptive Antitumor Immunity and Cure of Disseminated Disease. Cancer Res. 2002,62,7254–7263. [PubMed] [Google Scholar]

[R16] 16.Sabel MS, Hill H, Jong YS, Mathiowitz E, Bankert RB, Egilmez NK. Neoadjuvant Therapy with Interleukin-12–Loaded Polylactic Acid Microspheres Reduces Local Recurrence and Distant Metastases. Surgery. 2001,130,470–478. [DOI] [PubMed] [Google Scholar]

[R17] 17.Sabel MS, Arora A, Su G, Mathiowitz E, Reineke JJ, Chang AE. Synergistic Effect of Intratumoral IL-12 and TNF-α Microspheres: Systemic Anti-Tumor Immunity Is Mediated by Both CD8+ CTL and NK Cells. Surgery. 2007,142,749–760. [DOI] [PubMed] [Google Scholar]

[R18] 18.Smith SG, Koppolu Bp, Ravindranathan S, Kurtz SL, Yang L, Katz MD, Zaharoff DA. Intravesical Chitosan/Interleukin-12 Immunotherapy Induces Tumor-Specific Systemic Immunity against Murine Bladder Cancer. Cancer Immunol. Immunother 2015,64,689–696. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R19] 19.Vo JL, Yang L, Kurtz SL, Smith SG, Koppolu Bp, Ravindranathan S, Zaharoff DA. Neoadjuvant Immunotherapy with Chitosan and Interleukin-12 to Control Breast Cancer Metastasis. OncoImmunology. 2014,3,e968001. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R20] 20.Yang L, Zaharoff DA. Role of Chitosan Co-Formulation in Enhancing Interleukin-12 Delivery and Antitumor Activity. Biomaterials. 2013,34,3828–3836. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] 21.Lai I, Swaminathan S, Baylot V, Mosley A, Dhanasekaran R, Gabay M, Felsher DW. Lipid Nanoparticles That Deliver IL-12 Messenger RNA Suppress Tumorigenesis in MYC Oncogene-Driven Hepatocellular Carcinoma. J. Immunother. Cancer 2018,6,125. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] 22.Thaker PH, Bradley WH, Leath CA, Gunderson CC, Borys N, Musso L, Anwer K, Alvarez RD. Phase I Study of the Safety and Activity of Formulated IL-12 Plasmid Administered Intraperitoneally in Combination with Neoadjuvant Chemotherapy in Patients With Newly Diagnosed Advanced-Stage Ovarian Cancer. J. Clin. Oncol 2019,37,2–2. [Google Scholar]

[R23] 23.Xu Q, Guo L, Gu X, Zhang B, Hu X, Zhang J, Chen J, Wang Y, Chen C, Gao B. Prevention of Colorectal Cancer Liver Metastasis by Exploiting Liver Immunity via Chitosan-TPP/Nanoparticles Formulated with IL-12. Biomaterials. 2012,33,3909–3918. [DOI] [PubMed] [Google Scholar]

[R24] 24.Shimizu T, Kishida T, Hasegawa U, Ueda Y, Imanishi J, Yamagishi H, Akiyoshi K, Otsuji E, Mazda O. Nanogel DDS Enables Sustained Release of IL-12 for Tumor Immunotherapy. Biochem. Biophys. Res. Commun 2008,367,330–335. [DOI] [PubMed] [Google Scholar]

[R25] 25.Colombo MP, Vagliani M, Spreafico F, Parenza M, Chiodoni C, Melani C, Stoppacciaro A. Amount of Interleukin 12 Available at the Tumor Site Is Critical for Tumor Regression. Cancer Res. 1996,56,2531–2534. [PubMed] [Google Scholar]

[R26] 26.Decher G Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites. Science. 1997,277,1232–1237. [Google Scholar]

[R27] 27.Hammond PT. Form and Function in Multilayer Assembly: New Applications at the Nanoscale. Adv. Mater 2004,16,1271–1293. [Google Scholar]

[R28] 28.Shaillender M, Luo RC, Venkatraman SS, Neu B. Layer-by-Layer Microcapsules Templated on Erythrocyte Ghost Carriers. Int. J. Pharm 2011,415,211–217. [DOI] [PubMed] [Google Scholar]

[R29] 29.Dreaden EC, Kong YW, Morton SW, Correa S, Choi KY, Shopsowitz KE, Renggli K, Drapkin R, Yaffe MB, Hammond PT. Tumor-Targeted Synergistic Blockade of MAPK and PI3K from a Layer-by-Layer Nanoparticle. Clin. Cancer Res 2015,21,4410–4419. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R30] 30.Dreaden EC, Morton SW, Shopsowitz KE, Choi J-H, Deng ZJ, Cho N-J, Hammond PT. Bimodal Tumor-Targeting from Microenvironment Responsive Hyaluronan Layer-by-Layer (LbL) Nanoparticles. ACS Nano. 2014,8,8374–8382. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] 31.Hammond PT. Engineering Materials Layer-by-Layer: Challenges and Opportunities in Multilayer Assembly. AIChE J. 2011,57,2928–2940. [Google Scholar]

[R32] 32.Correa S, Choi KY, Dreaden EC, Renggli K, Shi A, Gu L, Shopsowitz KE, Quadir MA, Ben‐Akiva E, Hammond PT. Highly Scalable, Closed-Loop Synthesis of Drug-Loaded, Layer‐by-Layer Nanoparticles. Adv. Funct. Mater 2016,26,991–1003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R33] 33.Correa S, Dreaden EC, Gu L, Hammond PT. Engineering Nanolayered Particles for Modular Drug Delivery. J. Controlled Release 2016,240,364–386. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R34] 34.Dang X, Gu L, Qi J, Correa S, Zhang G, Belcher AM, Hammond PT. Layer-by-Layer Assembled Fluorescent Probes in the Second Near-Infrared Window for Systemic Delivery and Detection of Ovarian Cancer. Proc. Natl. Acad. Sci. U. S. A 2016,113,5179–5184. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R35] 35.Deng ZJ, Morton SW, Ben-Akiva E, Dreaden EC, Shopsowitz KE, Hammond PT. Layer-by-Layer Nanoparticles for Systemic Codelivery of an Anticancer Drug and siRNA for Potential Triple-Negative Breast Cancer Treatment. ACS Nano. 2013,7,9571–9584. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R36] 36.Morton SW, Lee MJ, Deng ZJ, Dreaden EC, Siouve E, Shopsowitz KE, Shah NJ, Yaffe MB, Hammond PT. A Nanoparticle-Based Combination Chemotherapy Delivery System for Enhanced Tumor Killing by Dynamic Rewiring of Signaling Pathways. Sci. Signaling 2014;7,ra44. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Cancer Cell Coating Nanoparticles for Optimal Tumor-Specific Cytokine Delivery

Antonio E Barberio

Sean G Smith

Santiago Correa

Cathy Nguyen

Bang Nhan

Mariane Melo

Talar Tokatlian

Heikyung Suh

Darrell J Irvine

Paula T Hammond

Abstract

Graphical Abstract

Results/Discussion

Engineering layer-by-layer nanoparticles encapsulating IL-12

Figure 1.

Surface chemistry impacts LbL-NPs subcellular localization

Figure 2.

LbL NPs maintain IL-12 efficacy in vitro

Figure 3.

IL-12 is exposed and released overtime from particle surface by erosion.

Figure 4.

PLE-IL-12-NPs reduce the toxicity of IL-12 therapy

Figure 5.

PLE-IL-12-NPs maintain efficacy of IL-12 therapy in multiple tumor models

Figure 6.

Figure 7.

PLE-IL-12-NPs enhance lymphocyte activity in tumors and tumor draining lymph nodes

Figure 8.

Conclusions

Methods/Experimental

Materials

Protein Synthesis and Purification

Particle Formulation and Characterization

Encapsulation characterization

Flowcytometry

Fluorescence Imaging

Cell Culture

In vitro activity experiments

Tumor selectivity studies

FRET studies

Animal Studies.

In vivo toxicity tests

In vivo efficacy tests

Abscopal Response

Statistical Analysis

Data Availability

Supplementary Material

ACKNOWLEDGEMENTS

Footnotes

Contributor Information

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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