Efficient magnetic enrichment of antigen-specific T cells by engineering particle properties.

Magnetic particles can enrich desired cell populations to aid in understanding cell-type functions and mechanisms, diagnosis, and therapy. As cells are heterogeneous in ligand type, location, expression, and density, careful consideration of magnetic particle design for positive isolation is necessary. Antigen-specific immune cells have low frequencies, which has made studying, identifying, and utilizing these cells for therapy a challenge. Here we demonstrate the importance of magnetic particle design based on the biology of T cells. We create magnetic particles which recognize rare antigen-specific T cells and quantitatively investigate important particle properties including size, concentration, ligand density, and ligand choice in enriching these rare cells. We observe competing optima among particle parameters, with 300 nm particles functionalized with a high density of antigen-specific ligand achieving the highest enrichment and recovery of target cells. In enriching and then activating an endogenous response, 300 nm aAPCs generate nearly 65% antigen-specific T cells with at least 450-fold expansion from endogenous precursors and a 5-fold increase in numbers of antigen-specific cells after only seven days. This systematic study of particle properties in magnetic enrichment provides a case study for the engineering design principles of particles for the isolation of rare cells through biological ligands.

Controlling release from 3D printed medical devices using CLIP and drug-loaded liquid resins.

Mass customization along with the ability to generate designs using medical imaging data makes 3D printing an attractive method for the fabrication of patient-tailored drug and medical devices. Herein we describe the application of Continuous Liquid Interface Production (CLIP) as a method to fabricate biocompatible and drug-loaded devices with controlled release properties, using liquid resins containing active pharmaceutical ingredients (API). In this work, we characterize how the release kinetics of a model small molecule, rhodamine B-base (RhB), are affected by device geometry, network crosslink density, and the polymer composition of polycaprolactone- and poly (ethylene glycol)-based networks. To demonstrate the applicability of using API-loaded liquid resins with CLIP, the UV stability was evaluated for a panel of clinically-relevant small molecule drugs. Finally, select formulations were tested for biocompatibility, degradation and encapsulation of docetaxel (DTXL) and dexamethasone-acetate (DexAc). Formulations were shown to be biocompatible over the course of 175 days of in vitro degradation and the clinically-relevant drugs could be encapsulated and released in a controlled fashion. This study reveals the potential of the CLIP manufacturing platform to serve as a method for the fabrication of patient-specific medical and drug-delivery devices for personalized medicine.

Organ-specific metastases obtained by culturing colorectal cancer cells on tissue-specific decellularized scaffolds.

Metastatic disease remains the primary cause of mortality in cancer patients. Yet the number of available in vitro models to study metastasis is limited by challenges in the recapitulation of the metastatic microenvironment in vitro, and by difficulties in maintaining colonized-tissue specificity in the expansion and maintenance of metastatic cells. Here, we show that decellularized scaffolds that retain tissue-specific extracellular-matrix components and bound signalling molecules enable, when seeded with colorectal cancer cells, the spontaneous formation of three-dimensional cell colonies that histologically, molecularly and phenotypically resemble in vivo metastases. Lung and liver metastases obtained by culturing colorectal cancer cells on, respectively, lung and liver decellularized scaffolds retained their tissue-specific tropism when injected in mice. We also found that the engineered metastases contained signet ring cells, which has not previously been observed ex vivo. A culture system with tissue-specific decellularized scaffolds represents a simple and powerful approach for the study of organ-specific cancer metastases.

Effect of particle size on the biodistribution, toxicity, and efficacy of drug-loaded polymeric nanoparticles in chemoradiotherapy.

Nanoparticle (NP) chemotherapeutics can improve the therapeutic index of chemoradiotherapy (CRT). However, the effect of NP physical properties, such particle size, on CRT is unknown. To address this, we examined the effects of NP size on biodistribution, efficacy and toxicity in CRT. PEG-PLGA NPs (50, 100, 150 nm mean diameters) encapsulating wotrmannin (wtmn) or KU50019 were formulated. These NP formulations were potent radiosensitizers in vitro in HT29, SW480, and lovo rectal cancer lines. In vivo, the smallest particles avoided hepatic and splenic accumulation while more homogeneously penetrating tumor xenografts than larger particles. However, smaller particles were no more effective in vivo. Instead, there was a trend toward enhanced efficacy with medium sized NPs. The smallest KU60019 particles caused more small bowel toxicity than larger particles. Our results showed that particle size significantly affects nanotherapeutics' biodistrubtion and toxicity but does not support the conclusion that smaller particles are better for this clinical application.

Preclinical Evaluation of Promitil, a Radiation-Responsive Liposomal Formulation of Mitomycin C Prodrug, in Chemoradiotherapy.

PURPOSE: To examine the effect of radiation on in vitro drug activation and release of Promitil, a pegylated liposomal formulation of a mitomycin C (MMC) lipid-based prodrug; and examine the efficacy and toxicity of Promitil with concurrent radiation in colorectal cancer models. METHODS AND MATERIALS: Promitil was obtained from Lipomedix Pharmaceuticals (Jerusalem, Israel). We tested the effects of radiation on release of active MMC from Promitil in vitro. We next examined the radiosensitization effect of Promitil in vitro. We further evaluated the toxicity of a single injection of free MMC or Promitil when combined with radiation by assessing the effects on blood counts and in-field skin and hair toxicity. Finally, we compared the efficacy of MMC and Promitil in chemoradiotherapy using mouse xenograft models. RESULTS: Mitomycin C was activated and released from Promitil in a controlled-release profile, and the rate of release was significantly increased in medium from previously irradiated cells. Both Promitil and MMC potently radiosensitized HT-29 cells in vitro. Toxicity of MMC (8.4 mg/kg) was substantially greater than with equivalent doses of Promitil (30 mg/kg). Mice treated with human-equivalent doses of MMC (3.3 mg/kg) experienced comparable levels of toxicity as Promitil-treated mice at 30 mg/kg. Promitil improved the antitumor efficacy of 5-fluorouracil-based chemoradiotherapy in mouse xenograft models of colorectal cancer, while equitoxic doses of MMC did not. CONCLUSIONS: We demonstrated that Promitil is an attractive agent for chemoradiotherapy because it demonstrates a radiation-triggered release of active drug. We further demonstrated that Promitil is a well-tolerated and potent radiosensitizer at doses not achievable with free MMC. These results support clinical investigations using Promitil in chemoradiotherapy.