Abstract The human epidermal growth factor receptor (EGFR) is expressed at high levels in at least 20% of breast cancers overall, but in 60-70% of patients with triple-negative breast cancer (TNBC), which makes EGFR a potential treatment target. Herein we developed a novel therapy for TNBC that utilizes anti-EGFR monoclonal antibody (mAb)-conjugated nanodiamonds (NDs) to specifically deliver paclitaxel (PTX) to breast cancer cells over-expressing EGFR. NDs, 2-8 nm diameter carbon nanoparticles, have recently emerged as promising drug carriers. In this study, we have synthesized a versatile ND construct that incorporates anti-EGFR mAb, fluorescent imaging agent and paclitaxel for multimodal imaging, targeting and treatment of TNBC. Sulfo-LC-SPDP was attached on to an aminated ND surface (ND-NH2), yielding sulfhydryl-reactive NDs (ND-SPDP). Thiolated paclitaxel-oligonucleotide (PTX-DNA) conjugates and thiolated mAbs were then simultaneously attached to ND-SPDPs, forming PTX-DNA/mAb@NDs. For the PTX-DNA conjugate, a fluorescein-labeled dT-nucleotide was inserted within the thiolated 20-mer poly-dT strand for cellular imaging and PTX loading quantification. The resultant nanoparticles could form highly stable suspensions in aqueous solutions. Dithiothreitol treatment detaches PTX-DNA and thiolated mAbs from the ND surface. About 2.2 ± 0.4 nmol of PTX and 53.5 ± 2.1 µg of mAb were loaded on per mg of PTX-DNA/mAb@NDs, while 3.1 ± 0.7 nmol of PTX for per mg of PTX-DNA@NDs. The targeting capacity of PTX-DNA/mAb@NDs was tested through flow cytometry analysis and confocal microscopy within 2 breast cancer cells of contrasting EGFR expression. Internalization of PTX-DNA@NDs and PTX-DNA/mAb@NDs was nearly equivalent within basal EGFR expressing MCF7 cells. However, when EGFR-overexpressing MDA-MB-231 cells were treated with PTX-DNA/mAb@NDs, there was a nearly 150% enhancement in the fluorescence signal as compared to that of PTX-DNA@NDs. Internalization of anti-EGFR-conjugated NDs within MDA-MB-231 cells was competitively inhibited by the addition of EGF. MTT assays were performed comparing free PTX with untargeted and targeted versions of PTX-DNA@NDs at equivalent PTX dosages in both cells. Enhanced cytotoxicity was observed in escalating-dose studies after 48 h treatment of PTX-DNA@NDs and PTX-DNA/mAb@NDs across all concentrations. Within MDA-MB-231 cells, anti-EGFR linked conjugates had an IC50 value of 43.8 nM, a nearly two-fold increase in efficacy compared to untargeted conjugates (83.3 nM). The data suggests that targeting through the mAb moiety increases specificity and internalization, which subsequently enhances therapeutic activity. Intravenous injection of NDs (up to 1.2 mg) didn't induce any toxicity and systemic immune response in mice, attesting to NDs as a biocompatible nanocarrier platform. In vivo efficacy of PTX-DNA/mAb@NDs will be further investigated in tumor-bearing mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1962. doi:1538-7445.AM2012-1962

The versatility of nanodiamonds (NDs) as a polymer-embedded therapeutic vehicle was demonstrated by the development of a hybrid material consisting of a poly(ethylene glycol) diacrylate (PEGDA) hydrogel embedded with ND-drug complexes. A wide range of PEGDA structures, including single and multiple drug reservoirs, exhibited high loading capabilities and stable slow-release, augmented with the addition of ND conjugates. ND-PEGDA hydrogels were compared to unmodified, drug-embedded PEGDA hydrogels to demonstrate their robust drug sequestering capacity in vitro. Remarkably, ND-embedded hydrogels were capable of mitigating the harmful effects of burst-release by displaying slow and controlled administration of therapeutic compounds, an improvement over standard diffusion-based release profiles. The tunable customization of PEGDA hydrogel, both in structure and content, as well as its facile methods of synthesis, make this material an effective multi-therapeutic complement to the drug delivery abilities of NDs. A hybrid material that utilizes the synergy of PEGDA and ND components has the potential for applications toward a diverse array of biomedical challenges. An artistic rendering of a multi-layered PEGDA patch. The center of the patch (purple) contains drug-bound ND particles that are being released (gray truncated octahedra with purple glow), while the outer ring depicts the molecule Temozolomide (green). This image illustrates the ability for tiered patches to simultaneously administer multiple drugs, as well as the drug-sequestering ability of NDs that mediates sustained delivery and reduced burst release. The nested structure of the two drugs was designed so that the therapeutics could be released sequentially, at different peak-elution time intervals.

Paclitaxel, a potent chemotherapeutic utilized in a variety of cancers, can be limited In its effectiveness due to inherent insolubility in aqueous media and acquired chemoresistance within certain cells. An approach has been developed for increasing Paclitaxel solubility and effectiveness by covalent attachment to gold nano particles via DNA linkers. The resulting conjugates are highly soluble in aqueous buffer, exhibiting greater than a 50-fold increase in solubility over the unconjugated drug. DNA linkers are labeled with a fluorophore, which affords a convenient means of visualizing resultant conjugates within cells. Internalized conjugates demonstrate increased activity as compared with free drug across a variety of cell types, including a Paclitaxel-resistant cell line. Attachment to DNA-nanoparticle conjugates may become a general strategy for solubilizing and enhancing a wide variety of therapeutic agents in aqueous media.

In this work, we have combined constant-pH molecular dynamics simulations and experiments to provide a quantitative analysis of pH dependent interactions between doxorubicin hydrochloride (DOX) cancer therapeutic and faceted nanodiamond (ND) nanoparticle carriers. Our study suggests that when a mixture of faceted ND and DOX is dissolved in a solvent, the pH of this solvent plays a controlling role in the adsorption of DOX molecules on the ND. We find that the binding of DOX molecules on ND occurs only at high pH and requires at least ∼10% of ND surface area to be fully titrated for binding to occur. As such, this study reveals important mechanistic insight underlying an ND-based pH-controlled therapeutic platform.

Multimodal nanodiamonds (NDs) were prepared by attaching fluorescently labeled drug-oligonucleotide conjugates and monoclonal antibodies onto the ND surface. Fluorescently labeled oligonucleotide linkers enabled the intracellular observation and quantification of resultant ND conjugates. The covalent attachment of the chemotherapeutic and targeting moiety to the ND surface significantly enhanced cellular internalization and therapeutic activity.

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.

The enormous therapeutic potential of RNA interference (RNAi) has long been recognized. While efficient small interfering RNA (siRNA) delivery vectors exist, many sacrifice biocompatibility, which can challenge their applicability as a therapeutic agent. Nanodiamonds (NDs) represent promising strategies for efficient siRNA delivery given the multitude of beneficial properties integrated into one platform that include uniform particle sizes, material scalability, the ability to carry nearly any type of therapeutic, and preserved biocompatibility, among others. Here we present a broadly applicable ND delivery platform that demonstrates biocompatible siRNA delivery with enhanced efficacy in media, signifying the translational potential of this approach.

The purpose of gene therapy is to introduce foreign genetic material into host cells to either supplement aberrant genes or to endow additional biological functions. To date, however, there has been only modest progress towards this goal, mainly due to the lack of safe, effective and broadly applicable delivery methods. Functional nanodiamonds (NDs) are rapidly emerging as promising platform carriers for next-generation therapeutics due to their innate biocompatibility, scalability, precise particle distribution, high surface area-to-volume ratio, near-spherical aspect ratio, and easily adaptable carbon surface for bioagent attachment. NDs have been functionalized with a range of therapeutics, proteins, antibodies, DNA, polymers, and other assorted biological agents. Furthermore, NDs are stable and dispersible in water, making them a promising and clinically important modality in improving the efficacy of the treatment of diseases and even some cancers at the molecular level. Mitochondrial function (MTT) and luminescent ATP production assays have demonstrated that NDs are not toxic to a wide variety of cell types. In this study, we functionalized NDs with amine groups via either covalent attachment of (3-aminopropyl) trimethoxysilane or surface immobilization of 800 Da low molecular weight polyethyleneimine (LMW PEI800) for plasmid DNA delivery. The latter delivery approach combines complementary characteristics of PEI800 and NDs to create a hybrid material that exhibits the high transfection efficiency of high molecular weight PEI, but without the inherent high cytotoxicity.

A simple method for rapid detection of melamine in the food is presented. Citrate-coated gold nanoparticles (AuNPs) and cyanuric acid were utilized to generate melamine-concentration dependent colorimetric and turbidometric signals, respectively. As low as 0.4 ppm of melamine can be detected from the whole milk within 15 minutes with this new detection method and a simple acid precipitation based milk pretreatment. This melamine detection method provides a time- and cost- effective alternative to conventional methods.

Several reports have described the relationship between size, aspect ratio, surface modification and internalization for a variety of nanoparticles (i.e. gold, polymer, carbon nanotubes). Nanodiamonds (NDs) in particular have recently been implicated in a variety of biomedical applications. One of the most promising is in utilizing NDs as drug delivery carriers where successful internalization is of utmost importance. A few reports recently have demonstrated the energy dependent internalization of bare NDs. In this report, we investigate the internalization mechanism and kinetics of functional ND-conjugate translocation.

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.

A strategy for rapid and facile detection of melamine in food and other substances is described. Unmodified gold nanoparticles enable a colorimetric signal output that is amplified through integration by precipitation formation via a chemical component specific to melamine. The sensitivity was determined to be approximately 40 ppb with a two standard deviation cutoff.

A diverse range of synthetic and natural nanoscale carriers in both particle and film/scaffold formats have been developed to enable controlled therapeutic release. Examples of these systems include metallic nanoparticles, polymer-protein conjugates, liposomes, micelles, dendrimers, polyelectrolyte films, copolypeptides, carbon nanotubes, etc. Nanodiamonds (NDs), in particular, possess several advantageous properties that make them suitable for advanced drug delivery while also remaining biocompatible. We have previously developed a method of functionalizing aqueous solubilized NDs of diameter 2-8 nm with doxorubicin (DOX), a clinically relevant chemotherapeutic capable of inducing potent DNA fragmentation and cellular apoptosis. This work has realized a scalable approach toward the fabrication of ND-embedded polymer microfilms for localized and sustained drug elution for post-operative chemotherapy. Due to their high surface-area-to-volume ratio and noninvasive dimensions, NDs are capable of extremely high loading capacities of therapeutic compounds. In addition, we have demonstrated the capability of ND binding with a broad range of charged therapeutic molecules via physical interactions due to their inherent surface charge properties. NDs are also biologically stable and appear to be non-toxic, which prevents adverse stressful/inflammation-inducing cellular reactions in the event that they are dispersed throughout the body for either systemic or more localized release activity. The combination of these properties in one system makes the NDs promising platforms for slow-release therapeutics to treat a broad array of physiological disorders (e.g. cancer, heart disease, wound healing, etc.).

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.

Enhanced specificity in drug delivery aims to improve upon systemic elution methods by locally concentrating therapeutic agents and reducing negative side effects. Due to their robust physical properties, biocompatibility and drug loading capabilities, nanodiamonds serve as drug delivery platforms that can be applied towards the elution of a broad range of therapeutically-active compounds. In this work, bovine insulin was non-covalently bound to detonated nanodiamonds via physical adsorption in an aqueous solution and demonstrated pH-dependent desorption in alkaline environments of sodium hydroxide. Insulin adsorption to NDs was confirmed by FT-IR spectroscopy and zeta potential measurements, while both adsorption and desorption were visualized with TEM imaging, quantified using protein detection assays and protein function demonstrated by MTT and RT-PCR. NDs combined with insulin at a 4:1 ratio showed 79.8±4.3% adsorption and 31.3±1.6% desorption in pH-neutral and alkaline solutions, respectively. Additionally, a 5-day desorption assay in NaOH (pH 10.5) and neutral solution resulted in 45.8±3.8% and 2.2±1.2% desorption, respectively. MTT viability assays and quantitative RT-PCR (expression of Ins1 and Csf3/G-csf genes) reveal bound insulin remains inactive until alkaline-mediated desorption. For applications in sustained drug delivery and therapy we have developed a therapeutic protein–ND complex with demonstrated tunable release and preserved activity.

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 probe's potential in delivering specific doses of DNA, viruses, and other therapeutically relevant biomolecules.