Oxaliplatin-resistant colorectal cancer models for nanoparticle hyperthermia.

INTRODUCTION: Metastatic colorectal cancer (CRC) is complicated by chemotherapy-resistant cell populations. Oxaliplatin is used in heated intraperitoneal hyperthermic chemoperfusion (HIPEC) for treatment of disseminated CRC. Photothermal nanoparticles can provide focal heating to improve the response of CRC cells to oxaliplatin, by confining heating near individual cells. Reduction in cellular luciferase signal may allow single-cell-resolution recording of thermal dosimetry. METHODS: Oxaliplatin resistant (OxR) variants of luciferase-expressing CT26.WT-Fluc-Neo CRC cells were developed and their sensitivity to hyperthermia was evaluated. Polymer-based photothermal nanoparticles were developed, characterized and used to explore their potential for imparting a thermal dose to improve cell response to oxaliplatin. A correlation of thermal dose to intracellular luciferase activity was established using quantitative luminescence monitoring and microscopy. RESULTS: Luciferase-based monitoring of thermal dose within CT26 cell lines was validated within the ranges of 0.04-8.33 CEM43 for parental cells and 0.05-9.74 CEM43 for OxR CT26 cells. This was further confirmed using nanoparticle-induced hyperthermia, where the single-cell resolution of the thermal dose can be achieved. The nanoparticles enhance cell killing of resistant cells when combined with oxaliplatin and stimulated to generate heat. CONCLUSION: Nanoparticle-based hyperthermia is effective for augmenting chemotherapy and can be coupled with reductions in CT26 luciferase expression to monitor thermal dose at single-cell resolution. The development of OxR CT26.WT-Fluc-Neo CRC cells sets the stage for pre-clinical evaluations to measure nanoparticle-induced hyperthermia to augment chemotherapy (Nano-HIPEC) in a chemotherapy-resistant model of disseminated CRC.

Semiconducting polymer nanoparticles for photothermal ablation of colorectal cancer organoids.

Colorectal cancer (CRC) treatment is currently hindered by micrometastatic relapse that cannot be removed completely during surgery and is often chemotherapy resistant. Targeted theranostic nanoparticles (NPs) that can produce heat for ablation and enable tumor visualization via their fluorescence offer advantages for detection and treatment of disseminated small nodules. A major hurdle in clinical translation of nanoparticles is their interaction with the 3D tumor microenvironment. To address this problem tumor organoid technology was used to evaluate the ablative potential of CD44-targeted polymer nanoparticles using hyaluronic acid (HA) as the targeting agent and coating it onto hybrid donor acceptor polymer particles (HDAPPs) to form HA-HDAPPs. Additionally, nanoparticles composed from only the photothermal polymer, poly[4,4-bis(2-ethylhexyl)-cyclopenta[2,1-b;3,4-b']dithiophene-2,6-diyl-alt-2,1,3-benzoselenadiazole-4,7-diyl] (PCPDTBSe), were also coated with HA, to form HA-BSe NPs, and evaluated in 3D. Monitoring of nanoparticle transport in 3D organoids revealed uniform diffusion of non-targeted HDAPPs in comparison to attenuated diffusion of HA-HDAPPs due to nanoparticle-matrix interactions. Computational diffusion profiles suggested that HA-HDAPPs transport may not be accounted for by diffusion alone, which is indicative of nanoparticle/cell matrix interactions. Photothermal activation revealed that only HA-BSe NPs were able to significantly reduce tumor cell viability in the organoids. Despite limited transport of the CD44-targeted theranostic nanoparticles, their targeted retention provides increased heat for enhanced photothermal ablation in 3D, which is beneficial for assessing nanoparticle therapies prior to in vivo testing.

Binding of Targeted Semiconducting Photothermal Polymer Nanoparticles for Intraperitoneal Detection and Treatment of Colorectal Cancer.

Nanoparticles offer many promising advantages for improving current surgical regimens through their ability to detect and treat disseminated colorectal cancer (CRC). Hybrid Donor-Acceptor Polymer Particles (HDAPPs) have recently been shown to fluorescently detect and thermally ablate tumors in a murine model. Here, HDAPPS were functionalized with hyaluronic acid (HA) to improve their binding specificity to CT26 mouse CRC cells using HA to target the cancer stem cell marker CD44. In this work, we compared the binding of HA functionalized HDAPPs (HA-HDAPPs) in in vitro, ex vivo, and in vivo environments. The HA-HDAPPs bound to CT26 cells 2-fold more in vitro and 2.3-fold higher than un-functionalized HDAPPs ex vivo. Compared to intraoperative abdominal perfusion, intraperitoneal injection prior to laser stimulation for nanoparticle heat generation provides a superior modality of HA-HDAPPs delivery for CRC tumor selectivity. Photothermal treatment of disseminated CRC showed that only HA-HDAPPs delivered via intraperitoneal injection had a reduction in the tumor burden, and these nanoparticles also remained in the abdomen following resolution of the tumor. The results of this work confirm that HA-HDAPPs selectively bind to disseminated CRC, with ex vivo tumors having bound HA-HDAPPs capable of photothermal ablation. HA-HDAPPs demonstrated superior binding to tumor regions compared to HDAPPs. Overall, this study displays the theranostic potential of HDAPPs, emphasizing their capacity to detect and photothermally treat disseminated CRC tumors.

Murine Models of Intraperitoneal Perfusion for Disseminated Colorectal Cancer.

BACKGROUND: Reproduction of the perfusion used in therapy (hyperthermic intraperitoneal chemotherapy) procedures preclinically represents a valuable asset for investigating new therapeutic agents that may improve patient outcomes. This article provides technical descriptions of our execution of closed and open "coliseum" abdominal perfusion techniques in a mouse model of peritoneal carcinomatosis of colorectal cancer. MATERIALS AND METHODS: BALB/c mice presenting with disseminated colorectal cancer (CT26-luciferin cells) underwent 30-min perfusions mimicking either the closed perfusion or the coliseum perfusion technique. Disease burden was monitored by bioluminescence signaling using an in vivo imaging system. Perfusion circuits consisted of single inflow lines with either a single or dual outflow line. RESULTS: Twelve mice presenting with disseminated disease underwent the closed perfusion technique. Surgical complications included perfusate leakage and organ constriction/suction into the outflow line(s). Nine mice underwent the coliseum perfusion technique with surgical debulking, using bipolar cauterization to remove tumors attached to the peritoneum. All mice survived the coliseum perfusion with limited intraoperative complications. CONCLUSIONS: Fewer intraoperative complications were experienced with our coliseum perfusion technique than the closed perfusion. The methods described here can be used as a guideline for developing future perfusion murine models for investigating perfusion models useful for delivery of chemotherapy or other tumor-sensitization agents, including selective targeted agents, nanoparticles, and heat.

Elastin-like-polypeptide based fusion proteins for osteogenic factor delivery in bone healing.

Modern treatments of bone injuries and diseases are becoming increasingly dependent on the usage of growth factors to stimulate bone growth. Bone morphogenetic protein-2 (BMP-2), a potent osteogenic inductive protein, exhibits promising results in treatment models, but recently has had its practical efficacy questioned due to the lack of local retention, ectopic bone formation, and potentially lethal inflammation. Where a new delivery technique of the BMP-2 is necessary, here we demonstrate the viability of an elastin-like peptide (ELP) fusion protein containing BMP-2 for delivery of the BMP-2. This fusion protein retains the performance characteristics of both the BMP-2 and ELP. The fusion protein was found to induce osteogenic differentiation of mesenchymal stem cells as evidenced by the production of alkaline phosphatase and extracellular calcium deposits in response to treatment by the fusion protein. Retention of the ELPs inverse phase transition property has allowed for expression of the fusion protein within a bacterial host (such as Escherichia coli) and easy and rapid purification using inverse transition cycling. The fusion protein formed self-aggregating nanoparticles at human-body temperature. The data collected suggests the viability of these fusion protein nanoparticles as a dosage-efficient and location-precise noncytotoxic delivery vehicle for BMP-2 in bone treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1029-1037, 2016.