Nanocapsule Delivery of IL-12.

Interleukin(IL)-12 is a protein that activates T cells and macrophages to kill tumor cells. However, despite this cytokine showing strong antitumor activity in preclinical settings, translation to patients has been slowed by toxic side effects, poor distribution to peripheral tissues, and improper dosing regimens. Osteosarcoma (OS) is an aggressive primary tumor of bone that has shown particular responsiveness to recombinant (r)IL-12 in preclinical models. Poly(lactic-co-glycolic) acid (PLGA) nanospheres, an FDA-approved drug delivery vector, may be a viable delivery vector for transporting biologically active IL-12 to tissues without disturbing normal homeostasis. In this chapter, we explore the potential for using IL-12-loaded nanospheres (IL-12-NS, <1 µm in diameter) to treat cancer, describe the synthesis process, and examine a typical protein release profile while providing insight and future directions of nanoscale tumor immunotherapeutics.

Nanosphere pharmacodynamics improves safety of immunostimulatory cytokine therapy.

Systemic administration of interleukin (IL)-12 induces potent anti-tumor immune responses in preclinical cancer models through the systemic activation of effector immune cells and release of proinflammatory cytokines. IL-12-loaded PLGA nanospheres (IL12ns) are hypothesized to improve therapeutic efficacy and thwart unwanted side effects observed in previous human clinical trials. Through the investigation of peripheral blood and local tissue immune responses in healthy BALB/c mice, the immune-protective pharmacodynamics of IL12ns were suggested. Nanospheres increased pro-inflammatory plasma cytokines/chemokines (IFN-γ, IL-6, TNF-α, and CXCL10) without inducing maladaptive transcriptomic signatures in circulating peripheral immune cells. Gene expression profiling revealed activation of pro-inflammatory signaling pathways in systemic tissues, the likely source of these effector cytokines. These data support that nanosphere pharmacodynamics, including shielding IL-12 from circulating immune cells, depositing peripherally in systemic immune tissues, and then slowly eluting bioactive cytokine, thereafter, are essential to safe immunostimulatory therapy.

Single-cell RNA-seq reveals intratumoral heterogeneity in osteosarcoma patients: A review.

While primary bone malignancies make up just 0.2% of all cancers, osteosarcoma (OS) is the third most common cancer in adolescents. Due to its highly complex and heterogeneous tumor microenvironment (TME), OS has proven difficult to treat. There has been little to no improvement in therapy for this disease over the last 40 years. Even the recent success of immunotherapies in other blood-borne and solid malignancies has not translated to OS. With frequent recurrence and lung metastases continuing to pose a challenge in the clinic, recent advancements in molecular profiling, such as single-cell RNA sequencing (scRNA-seq), have proven useful in identifying novel biomarkers of OS tumors while providing new insight into this TME that could potentially lead to new therapeutic options. This review combines the analyses of over 150,000 cells from 18 lesions ranging from primary, recurrent, and metastatic OS lesions, revealing distinct cellular populations and gene signatures that exist between them. Here, we detail these previous findings and ultimately convey the intratumoral heterogeneity that exists within OS tumor specimens.

Optimizing the synthesis of interleukin-12-loaded PLGA nanospheres (rmIL-12ns) via ultrasonication for treatment of metastatic osteosarcoma.

Clinical trials exploring bolus intravenous delivery of interleukin-12 (IL-12) for treatment of solid tumors ultimately failed due to lack of clinical response and severe dose-limiting toxicities. The present study was conducted to evaluate whether recombinant murine IL-12 (rmIL-12) could be successfully encapsulated within Poly (D, l-lactide-co-glycolide) (PLGA) nanospheres (rmIL-12ns) for safe and effective systemic delivery at pharmacologic scale. Optimal fabrication of rmIL-12ns occurs with dichloromethane as the organic solvent and emulsion formation via ultrasonication at 50% power (250 W sonicator) for 10 s (50W10s). We then determined whether utilization of synthesis modifiers including fetal bovine serum (FBS), magnesium hydroxide [Mg(OH)2 ], trehalose, or the surfactants polysorbate 80 and Span 60 alone or in combination could increase the encapsulation efficiency (EE) and/or modify the burst elution profile characteristic of the 50W10s rmIL-12ns formulation. The greatest EEs compared to the unmodified formulation were measured with modifications containing the surfactants polysorbate 80 and Span 60 (surfactant: 28.3 ± 6.10%, p = 0.29 and Surf/FBS: 85.4 ± 2.19%, p = 0.039). The Surf/FBS formulation was further modified for in vivo murine injection by substituting FBS with mouse serum albumin (MSA). The resulting Surf/MSA rmIL-12ns were then characterized before delivery at three doses (0.1, 1, and 10 mg rmIL-12ns) in our established murine model of metastatic osteosarcoma to assess efficacy. Preliminary results suggested no evidence of disease with delivery of the 0.1 mg dose in 75% of mice (3 of 4) versus a nontreated historical control (2 of 34).

Synthesis, Characterization, and In Vivo Cytokinome Profile of IL-12-Loaded PLGA Nanospheres.

We report the successful encapsulation and elution of recombinant murine IL-12 (rmIL-12) from poly(lactide-co-glycolic) acid (PLGA) nanospheres (IL-12-NS) synthesized using the double emulsion/solvent evaporation (DESE) technique with microsphere depletion through ultracentrifugation. Images obtained with scanning electron microscopy (SEM) showcased a characteristic spherical shape with a mean particle diameter of 138.1 ± 10.8 nm and zeta potential of -15.1 ± 1.249 mV. These values suggest minimal flocculation when in solution, which was reflected in an in vivo biodistribution study that reported no observed morbidity/mortality. Encapsulation efficiency (EE) was determined to be 0.101 ± 0.009% with average particle concentration obtained per batch of 1.66 × 109 ± 4.45 × 108 particles/mL. Disparate zeta (ζ) potentials obtained from both protein-loaded and protein-unloaded batches suggested surface adsorption of protein, and confocal microscopy of BSA-FITC-loaded nanospheres confirmed the presence of protein within the polymeric shell. Furthermore, elution of rmIL-12 from IL-12-NS at a concentration of 500 million particles/mL was characterized using enzyme-linked immunosorbent assay (ELISA). When IL-12-NS was administered in vivo to female BALB/c mice through retroorbital injection, IL-12-NS produced a favorable systemic cytokine profile for tumoricidal activity within the peripheral blood. Whereas IFN-γ nadir occurred at 72 hours, levels recovered quickly and displayed positive correlations postburst out to 25 days postinjection. IL-12-NS administration induced proinflammatory changes while prompting minimal counterregulatory increases in anti-inflammatory IL-10 and IL-4 cytokine levels. Further, while IL-6 levels increased to 30 folds of the baseline during the burst phase, they normalized by 72 hours and trended negatively throughout the sill phase. Similar trends were observed with IL-1ß and CXCL-1, suggesting a decreased likelihood of progression to a systemic inflammatory response syndrome-like state. As IL-12-NS delivers logarithmically lower amounts of IL-12 than previously administered during human clinical trials, our data reflect the importance of IL-12-NS which safely create a systemic immunostimulatory environment.