Hybrid Nanocrystals for Treatment and Bioimaging of Disease

Hybrid nanocrystals able to reach specific targets in the body for treatment and biological imaging are provided, as well as methods of making and administering same for treatment of disease conditions and for bioimaging and radiotherapy. The hybrid nanocrystals and methods can be used alone or in combination with other treatment and imaging modalities

Fluorescein Dye for Exploring Anti-Cancer Drug Dissolution Kinetics

Nanocrystalline formulations of anti-tumor drugs have been shown to improve desirable characteristics as well as increase the efficacy of delivery. However, the improvements seen are not strong enough to eradicate tumors and there exists a need to better understand how the drug particles move about and release in the body. The use of fluorophores incorporated into substrates allows for highly sensitive and resolute imaging in the body without the need for radioisotopes. This research aims to utilize quenching characteristics of fluorescein in order to provide pharmacokinetic information to aid in the improvement of nanoparticulate delivery formulations. Nanocrystal formulations of paclitaxel were created which included various concentrations of fluorescein and were then analyzed using fluorescent spectroscopy. Experimental results explored the excitation and/or emission wavelength maxima value\u27s dependence on upon the concentration of fluorescein incorporated into paclitaxel nanocrystals. Additional work is needed to validate these conclusions under in vivo conditions but indicate it is possible to gain an understanding of the pharmacokinetics of drug formulations by using a fluorescent agent

Exploring the Effect of Solution Speciation on Crystallization Outcome

In the field of pharmaceuticals, the crystallization process significantly impacts the purity, morphology and polymorphism of active pharmaceutical ingredients (API), all of which are important in drug development. Polymorphism is the existence of more than one solid-state form of the same chemical entity. Tolfenamic acid (TFA), a Non-Steroid Anti-inflammatory drug (NSAID), has been shown to exhibit a unique physical phenomenon called concomitant polymorphism, whereby two polymorphs crystallize from the solution simultaneously. From previous work, it has been established that solution speciation i.e. the presence of monomers or dimers of TFA in solution is concentration and temperature dependent. This study correlates this existing solution speciation with the crystallization outcome by analyzing the ratio of polymorphs obtained at different temperatures at a fixed concentration. Polymorphic purity was examined using powder X-ray diffraction. Crystallization experiments were performed at 10°C, 37°C, and 55°C at a constant supersaturation ratio of 1.95. Samples were taken at various time points in the recrystallization process. These samples were examined using Infrared spectroscopy for solid-state composition and quantified using a calibration curve. Pure polymorphs of TFA have been obtained and a calibration curve to quantify phase composition has been established. The initial recrystallization results suggest that the conformation of the crystals in solution is decided by the composition of the initial crystals formed, however more experimentation is necessary. Ongoing work involving recrystallization experiments under different temperature conditions would lead to a much better understanding of the role of solution speciation in the nucleation and crystallization processes

Cellular Uptake Mechanism of Paclitaxel Nanocrystals

Therapeutic options for metastasized human cancer in current practice remain limited and, sadly, there is no cure for metastatic cancer. The typical approach, chemotherapy, has both low efficacy due to poor drug solubility, and cytotoxic side effects to healthy tissue when delivered indiscriminately. To address both of these issues, we are pursuing the use of nanocrystal formulations of current chemotherapeutic agents as delivery platforms. Herein, we have studied cellular uptake mechanisms in cancer cells of nanocrystals of a chemotherapeutic agent, paclitaxel. Our goal in this study is to determine whether the nanocrystals can be taken up via endocytosis, especially when the surface is conjugated by ligand molecules that target cancer cells. As such, we cultured KB cells with various formulations of paclitaxel nanocrystals, including crystals treated with fluorescent dyes for imaging purposes, and with various polymer coatings for examining their effect on cellular drug uptake. We examined the incubated cells using confocal microscopy in order to identify how the nanocrystals are being taken up. In addition, we measured IC50 values for each nanocrystal formulation. Our results suggest the presence of an endocytotic uptake mechanism, as, in cell samples treated with either of the conjugated nanocrystals, we see some overlap of the nanocrystal and the lysosome imaging agents within the cells. Future work will utilize TEM and SEM imaging to confirm cellular uptake mechanisms identified here. These data will offer important insight for developing novel chemotherapeutic systems that are more effective and safe

Cellular Uptake of Drug Nanocrystals

Systemic toxicity and poor solubility of existing chemotherapeutic drugs piqued an interest in the use of nanocrystals for chemotherapy. To increase cytotoxicity, surface coating of nanocrystals is of interest to enhance tumor targeting and reduce treatment toxicity. As such, we tested in this project various coated paclitaxel nanocrystals on cancer cells for determining the efficacy of surface coating. An IC50 assay was chosen to determine the cytotoxicity of surface-coated paclitaxel nanocrystals; the lower the IC50 value, the higher the efficacy of the drug. Using the Sulforhodamine B method, paclitaxel, paclitaxel nanocrystals, and polymer coated paclitaxel nanocrystals were tested with regard to the cytotoxicity at various concentrations. The first set of in vitro experiments compared paclitaxel and paclitaxel nanocrystals for varied incubation times. Then, the second set of experiments assessed the efficacy of five different polymer coated paclitaxel nanocrystals for a standard incubation time. Paclitaxel nanocrystals proved to have a higher cytotoxicity than the standard paclitaxel formulation (i.e., Taxol®) at all time increments tested. The data also indicated that coated paclitaxel nanocrystals have a higher cytotoxicity than the paclitaxel nanocrystals. The cytotoxicity data will contribute to the evolving research on improving the efficacy and lowering the toxicity of developing nanocrystal chemotherapeutic formulations

Investigation of Major Intermolecular Interactions in 7,8-dihydrobenzo(k)phenanthridin-6(5H)-one Crystal Using Quantum Calculations and Crystallographic Visualization Programs

Currently, tablets and capsules are the most common ways of delivering drugs. The active pharmaceutical ingredients and excipients used to make those tablets and capsules are in their crystalline form generally. However, a single molecule can form multiple different crystal structures because of different packing arrangements of the molecules. These different crystal structures have identical chemical composition but different properties such as solubility, density, stability, etc. This phenomenon is called polymorphism. Occurrence of polymorphism could be a disaster for both patients and pharmaceutical companies, as the drug could lose its efficacy due to changes in properties. Studying intermolecular interactions in crystals can give us a better understanding of how and why molecules pack together in a certain way. In this research, 7,8-dihydrobenzo(k)phenanthridin-6(5H)-one is the molecule investigated. Its crystal data files were obtained from the Cambridge Crystallographic Data Centre. A crystallographic visualization program called Mercury was used to observe all contact modes and measure distances between atoms. Quantum calculations were performed using Density Functional Theory. Then, Fukui functions and electrostatic potentials for both the crystal and the molecule were calculated. These properties were mapped on the molecule’s Hirshfeld surface and on molecular slices using OpenDX software to help visualize intermolecular interactions. Comparison between crystals and molecules was performed to observe how these properties change when molecules form crystals. These mapped properties were helpful to analyze major intermolecular interactions, but further analysis on other compounds is needed in order to fully explain molecular packing in crystals and predict crystal structures

Investigating Intermolecular Interactions in Crystalline Aspirin Using CDFT

Drugs today are widely administered in their crystalline form, namely via tablets and capsules. The crystal structure of a drug molecule affects important drug qualities such as solubility, bioavailability, shelf life, and compaction properties. In order to form a basis for crystal structure prediction, it is necessary to first understand how intermolecular interactions cause molecules to pack in certain ways. Being able to predict and perhaps even control a drug molecule’s crystal structure will lead to the development of higher quality drugs that perform more consistently. Scientists and engineers do not fully understand the reasons for a molecule assuming a certain crystal structure. Current methods show that many energetically favorable conformations of a specific molecule are possible, but only a small handful of those are actually observed. Aspirin forms I and II were used as the drug molecules of choice for this study. Employing conceptual density functional theory allowed for the calculation of energy, as well as Fukui functions based on charge densities. CRYSTAL09 was used to optimize coordinates and to conduct single point calculations for neutral and charged aspirin for both the crystal and single molecules. Contacts between molecules were found using Mercury and OpenDX. Mapping charge density, Fukui functions, and electrostatic potential were mapped on a molecule’s Hirshfeld surface allowed for the visualization of interactions between molecules in a crystal cell. This was achieved using IBM’s OpenDX. Future work will involve calculating the energies of individual interactions in order to determine how influential they are on crystal structure

Macrophage Uptake and Cytotoxicity of Paclitaxel Nanocrystals

Effective drug delivery remains one of the most challenging tasks in combatting cancer cells. Anti-cancer drugs such as Paclitaxel (PTX) often struggle in having a high drug effect because they are often phagocytized by macrophages in Reticuloendothelial System (RES) before reaching the cancer cells. To combat this problem, PTX was inserted in a nanocrystal, coated with non-ionic surfactant F68; it is speculated that this formulation will minimize the particle aggregation and decrease the cellular uptake in the RES, which will increase the overall efficacy of the drug in the target areas. The aim of this project was to compare the cellular uptake and the toxicity of commercially produced PTX, PTX-loaded nanocrystals and F68-PTX-loaded nanocrystals, specifically in the RES macrophages. Confocal imaging was used to visualize the cellular uptake of the nanocrystals, and SRB assay was used to measure in-vitro cytotoxicity. The imaging and the assay suggest that while both surface coated formulation and pure paclitaxel nanocrystal are uptaken by the RES macrophages, the surface coating formulation in the nanocrystal reduces the cytotoxicity in the RES. Based on the results, the formulations show the potential to improve pharmacokinetics and biodistribution of PTX. This work can be used to seek ways to reduce toxicity in RES cells and increase the drug potency in the targeting cells

Preparation and Characterization of Multimodal Hybrid Organic and Inorganic Nanocrystals of Camptothecin and Gold

We demonstrate a novel inorganic-organic crystalline nanoconstruct, where gold atoms were imbedded in the crystal lattices as defects of camptothecin nanocrystals, suggesting its potential use as simultaneous agents for cancer therapy and bioimaging. The incorporation of gold, a potential computed tomography (CT) contrast agent, in the nanocrystals of camptothecin was detected by transmission electron microscope (TEM) and further quantified by energy dispersive X-ray spectrometry (EDS) and inductively coupled plasma-optical emission spectrometers (ICP-OES). Due to gold\u27s high attenuation coefficient, only a relatively small amount needs to be present in order to create a good noise-to-contrast ratio in CT imaging. The imbedded gold atoms and clusters are expected to share the same biological fate as the camptothecin nanocrystals, reaching and accumulating in tumor site due to the enhanced permeation and retention (EPR) effect

Development and Evaluation of Transferrin-Stabilized Paclitaxel Nanocrystal Formulation

The aim of the present study was to prepare and evaluate a paclitaxel nanocrystal-based formulation stabilized by serum protein transferrin in a non-covalent manner. The pure paclitaxel nanocrystals were first prepared using an antisolvent precipitation method augmented by sonication. The serum protein transferrin was selected for use after evaluating the stabilizing effect of several serum proteins including albumin and immunoglobulin G. The formulation contained approximately 55~60% drug and was stable for at least 3 months at 4 °C. In vivo antitumor efficacy studies using mice inoculated with KB cells demonstrate significantly higher tumor inhibition rate of 45.1% for paclitaxel-transferrin formulation compared to 28.8% for paclitaxel nanosuspension treatment alone. Interestingly, the Taxol® formulation showed higher antitumor activity than the paclitaxel-transferrin formulation, achieving a 93.3% tumor inhibition rate 12 days post initial dosing. However, the paclitaxel-transferrin formulation showed a lower level of toxicity, which is indicated by steady increase in body weight of mice over the treatment period. In comparison, treatment with Taxol® resulted in toxicity issues as body weight decreased. These results suggest the potential benefit of using a serum protein in a non-covalent manner in conjunction with paclitaxel nanocrystals as a promising drug delivery model for anticancer therapy