Mark Chen

Nanodiamond Therapeutic Delivery Agents Mediate Enhanced Chemoresistant Tumor Treatment

Nanodiamond-based drug delivery significantly enhanced treatment efficacy and safety in multiple chemoresistant cancer models. Nanodiamonds Are a Girl’s Best Friend When it comes to diamonds on the finger or ear lobes, bigger is better. However, for drug delivery, a small diamond may be the key to overcoming drug resistance in cancer. Nanodiamonds—tiny carbon particles—are biocompatible, can be scalably synthesized, and can bind therapeutic agents, features that make them a promising platform for drug delivery. Now, Chow et al. have found that binding nanodiamonds to the anticancer drug doxorubicin (Dox) improved therapeutic response and overcame chemoresistance in mouse models of mammary and liver cancer. Believed to act by interfering with DNA synthesis, Dox is commonly used to treat a wide variety of cancers; however, many cancers become resistant to Dox during treatment due in part to efflux of the drug from the tumor cells. In an attempt to overcome tumor chemoresistance, Chow et al. conjugated Dox to nanodiamonds as a possible means of preventing the drug from being pumped out of cells. Indeed, the nanodiamond-Dox complexes were retained better by cancer cells, decreased tumor growth, and displayed less toxicity in mammary and liver cancer mouse models when compared with unconjugated Dox. The gradual release of Dox from the nanodiamonds allowed for enhanced tumor retention and efficacy, but the small size allowed for clearance before toxicity occurred in slower-dividing healthy tissues. Thus, nanodiamonds may provide a drug delivery platform that has it all—improved safety profiles and enhanced efficacy. Like their much larger predecessor, nanodiamonds are truly diamonds of Hope. Enhancing chemotherapeutic efficiency through improved drug delivery would facilitate treatment of chemoresistant cancers, such as recurrent mammary tumors and liver cancer. One way to improve drug delivery is through the use of nanodiamond (ND) therapies, which are both scalable and biocompatible. Here, we examined the efficacy of an ND-conjugated chemotherapeutic in mouse models of liver and mammary cancer. A complex (NDX) of ND and doxorubicin (Dox) overcame drug efflux and significantly increased apoptosis and tumor growth inhibition beyond conventional Dox treatment in both murine liver tumor and mammary carcinoma models. Unmodified Dox treatment represents the clinical standard for most cancer treatment regimens, and NDX had significantly decreased toxicity in vivo compared to standard Dox treatment. Thus, ND-conjugated chemotherapy represents a promising, biocompatible strategy for overcoming chemoresistance and enhancing chemotherapy efficacy and safety.

Polymer-functionalized nanodiamond platforms as vehicles for gene delivery.

Gene therapy holds great promise for treating diseases ranging from inherited disorders to acquired conditions and cancers. Nonetheless, because a method of gene delivery that is both effective and safe has remained elusive, these successes were limited. Functional nanodiamonds (NDs) are rapidly emerging as promising carriers for next-generation therapeutics with demonstrated potential. Here we introduce NDs as vectors for in vitro gene delivery via surface-immobilization with 800 Da polyethyleneimine (PEI800) and covalent conjugation with amine groups. We designed PEI800-modified NDs exhibiting the high transfection efficiency of high molecular weight PEI (PEI25K), but without the high cytotoxicity inherent to PEI25K. Additionally, we demonstrated that the enhanced delivery properties were exclusively mediated by the hybrid ND-PEI800 material and not exhibited by any of the materials alone. This platform approach represents an efficient avenue toward gene delivery via DNA-functionalized NDs, and serves as a rapid, scalable, and broadly applicable gene therapy strategy.

Nanodiamond-mediated delivery of water-insoluble therapeutics.

A broad array of water-insoluble compounds has displayed therapeutically relevant properties toward a spectrum of medical and physiological disorders, including cancer and inflammation. However, the continued search for scalable, facile, and biocompatible routes toward mediating the dispersal of these compounds in water has limited their widespread application in medicine. Here we demonstrate a platform approach of water-dispersible, nanodiamond cluster-mediated interactions with several therapeutics to enhance their suspension in water with preserved functionality, thereby enabling novel treatment paradigms that were previously unrealized. These therapeutics include Purvalanol A, a highly promising compound for hepatocarcinoma (liver cancer) treatment, 4-hydroxytamoxifen (4-OHT), an emerging drug for the treatment of breast cancer, as well as dexamethasone, a clinically relevant anti-inflammatory that has addressed an entire spectrum of diseases that span complications from blood and brain cancers to rheumatic and renal disorders. Given the scalability of nanodiamond processing and functionalization, this novel approach serves as a facile, broadly impacting and significant route to translate water-insoluble compounds toward treatment-relevant scenarios.

Nanodiamond-Embedded Microfilm Devices for Localized Chemotherapeutic Elution

Nanodiamonds (NDs) of 2-8 nm diameters physically bound with the chemotherapeutic agent doxorubicin hydrochloride (DOX) were embedded within a parylene C polymer microfilm through a facile and scalable process. The microfilm architecture consists of DOX-ND conjugates sandwiched between a base and thin variable layer of parylene C which allows for modulation of release. Successive layers of parylene and the DOX-ND conjugates were characterized through atomic force microscopy (AFM) images and drug release assays. Elution rates were tested separately over a period of 8 days and up to one month in order to illustrate the release characteristics of the microfilms. The microfilms displayed the stable and continuous slow-release of drug for at least one month due to the powerful sequestration abilities of the DOX-ND complex and the release-modulating nature of the thin parylene layer. Since the fabrication process is devoid of any destructive steps, the DOX-ND conjugates are unaffected and unaltered. A DNA fragmentation assay was performed to illustrate this retained activity of DOX under biological conditions. Specifically, in this work we have conferred the ability to tangibly manipulate the NDs in a polymer-packaged microfilm format for directed placement over diseased areas. By harnessing the innate ND benefits in a biostable patch platform, extended targeted and controlled release, possibly relevant toward conditions such as cancer, viral infection, and inflammation, where complementary alternatives to systemic drug release enabled by the microfilm devices, can allow for enhanced treatment efficacy.

Nanofountain-probe-based high-resolution patterning and single-cell injection of functionalized nanodiamonds.

Nanodiamonds are rapidly emerging as promising carriers for next-generation therapeutics and drug delivery. However, developing future nanoscale devices and arrays that harness these nanoparticles will require unrealized spatial control. Furthermore, single-cell in vitro transfection methods lack an instrument that simultaneously offers the advantages of having nanoscale dimensions and control and continuous delivery via microfluidic components. To address this, two modes of controlled delivery of functionalized diamond nanoparticles are demonstrated using a broadly applicable nanofountain probe, a tool for direct-write nanopatterning with sub-100-nm resolution and direct in vitro single-cell injection. This study demonstrates the versatility of the nanofountain probe as a tool for high-fidelity delivery of functionalized nanodiamonds and other agents in nanomanufacturing and single-cell biological studies. These initial demonstrations of controlled delivery open the door to future studies examining the nanofountain probes potential in delivering specific doses of DNA, viruses, and other therapeutically relevant biomolecules.

Ultrananocrystalline Diamond Thin Films Functionalized with Therapeutically Active Collagen Networks

The fabrication of biologically amenable interfaces in medicine bridges translational technologies with their surrounding biological environment. Functionalized nanomaterials catalyze this coalescence through the creation of biomimetic and active substrates upon which a spectrum of therapeutic elements can be delivered to adherent cells to address biomolecular processes in cancer, inflammation, etc. Here, we demonstrate the robust functionalization of ultrananocrystalline diamond (UNCD) with type I collagen and dexamethasone (Dex), an anti-inflammatory drug, to fabricate a hybrid therapeutically active substrate for localized drug delivery. UNCD oxidation coupled with a pH-mediated collagen adsorption process generated a comprehensive interface between the two materials, and subsequent Dex integration, activity, and elution were confirmed through inflammatory gene expression assays. These studies confer a translational relevance to the biofunctionalized UNCD in its role as an active therapeutic network for potent regulation of cellular activity toward applications in nanomedicine.

Generation and comparison of CRISPR-Cas9 and Cre-mediated genetically engineered mouse models of sarcoma

Genetically engineered mouse models that employ site-specific recombinase technology are important tools for cancer research but can be costly and time-consuming. The CRISPR-Cas9 system has been adapted to generate autochthonous tumours in mice, but how these tumours compare to tumours generated by conventional recombinase technology remains to be fully explored. Here we use CRISPR-Cas9 to generate multiple subtypes of primary sarcomas efficiently in wild type and genetically engineered mice. These data demonstrate that CRISPR-Cas9 can be used to generate multiple subtypes of soft tissue sarcomas in mice. Primary sarcomas generated with CRISPR-Cas9 and Cre recombinase technology had similar histology, growth kinetics, copy number variation and mutational load as assessed by whole exome sequencing. These results show that sarcomas generated with CRISPR-Cas9 technology are similar to sarcomas generated with conventional modelling techniques and suggest that CRISPR-Cas9 can be used to more rapidly generate genotypically and phenotypically similar cancers.

Engineering Multifunctional Biologically-Amenable Nanomaterials for Interfacial Therapeutic Delivery and Substrate-Based Cellular Interrogation

The advent of materials that can enhance the interfaces between biological tissue and engineered devices will enable unprecedented medical capabilities in the context of prolonged implantation, and novel information gleaned from cellular interrogation, etc. This work addresses a spectrum of novel technologies that can serve a broad range of therapeutically relevant scenarios ranging from inflammation attenuation to stand-alone chemotherapeutic delivery systems. They include copolymer-based multifunctional platforms that can be applied towards dynamic cell adhesion/patterning, drug delivery, and localized manipulation of key cyto-regulatory networks for clinical applications. In addition, soluble nanodiamond platforms in free-floating or thin film platforms will be addressed as next generation therapeutic vehicles. In addition to cytokine expression knockdown studies as well as in vivo validation of their efficacy, this suite of modalities successfully addresses a key element of optimized interfacing based upon innate biocompatibility which has been verified at the genetic level, confirming their potential clinical significance.

Genomic Status of MET Potentiates Sensitivity to MET and MEK Inhibition in NF1-Related Malignant Peripheral Nerve Sheath Tumors

Malignant peripheral nerve sheath tumors (MPNST) are highly resistant sarcomas that occur in up to 13% of individuals with neurofibromatosis type I (NF1). Genomic analysis of longitudinally collected tumor samples in a case of MPNST disease progression revealed early hemizygous microdeletions in NF1 and TP53, with progressive amplifications of MET, HGF, and EGFR To examine the role of MET in MPNST progression, we developed mice with enhanced MET expression and Nf1 ablation (Nf1fl/ko;lox-stop-loxMETtg/+;Plp-creERTtg/+ ; referred to as NF1-MET). NF1-MET mice express a robust MPNST phenotype in the absence of additional mutations. A comparison of NF1-MET MPNSTs with MPNSTs derived from Nf1ko/+;p53R172H;Plp-creERTtg/+ (NF1-P53) and Nf1ko/+;Plp-creERTtg/+ (NF1) mice revealed unique Met, Ras, and PI3K signaling patterns. NF1-MET MPNSTs were uniformly sensitive to the highly selective MET inhibitor, capmatinib, whereas a heterogeneous response to MET inhibition was observed in NF1-P53 and NF1 MPNSTs. Combination therapy of capmatinib and the MEK inhibitor trametinib resulted in reduced response variability, enhanced suppression of tumor growth, and suppressed RAS/ERK and PI3K/AKT signaling. These results highlight the influence of concurrent genomic alterations on RAS effector signaling and therapy response to tyrosine kinase inhibitors. Moreover, these findings expand our current understanding of the role of MET signaling in MPNST progression and identify a potential therapeutic niche for NF1-related MPNSTs.Significance: Longitudinal genomic analysis reveals a positive selection for MET and HGF copy number gain early in malignant peripheral nerve sheath tumor progression. Cancer Res; 78(13); 3672-87. ©2018 AACR.

Microfluidic Platforms for Nanoparticle Delivery and Nanomanufacturing in Biology and Medicine

Nanoparticles are rapidly emerging as promising vehicles for next-generation therapeutic delivery. These highly mobile nanomaterials exhibit large carrier capacity and excellent stability which, when combined with innate biocompatibility, have captured the focus of numerous research efforts. As such, the ability to deliver well-controlled subcellular doses of these functional nanoparticles, both for fundamental research at the single cell level and in related device manufacturing, remains a challenge. Patterning these nanomaterials on biologically compatible substrates enables both novel biological studies and nanomanufacturing avenues through precise spatial control of dosing. Delivering them directly to live cells enables further studies where transfection remains a challenge. This chapter describes a unique tool for functional nanoparticle delivery, called the Nanofountain Probe. The Nanofountain Probe is capable of both direct-write nanopatterning of these materials with sub-100-nm resolution and targeted in vitro injection to individual cells. To motivate the discussion, a brief overview of microfluidic tools developed to deliver nanoparticles is presented. We then focus on the function of the Nanofountain Probe and its application to functional nanodiamond-based biological studies and nanomanufacturing. Development and application of the Nanofountain Probe and other nanomaterial delivery systems will be critical in developing future nanoscale devices and arrays that harness these nanoparticles.