Research Program
Nanoscale Materials

Project #
NM6

Participating Faculty:             Emmanuel Giannelis, Alex Nikitin              

NBTC Students/Postdocs:     Nikalaos Chalkias, Graduate Student

Other Students/Postdocs:     Jishnu Naskar, Graduate Student

Objectives
The long term goal of this program is to develop nanoparticle-based tools for diagnostic and therapeutic applications.  Our research objectives are:

• to design, synthesize and evaluate the biological efficacy of nanohybrids as diagnostics and therapeutics in cell lines and primary cell cultures. 
• to test the nanohybrids in cancer animal models using genetically modified mouse models

Methods
The nanohybrids are based on specially synthesized layered nanoparticle cores capable of intercalating various biological and therapeutic molecules into the nanometer size galleries between the layers. The outer surface of the nanoparticle can also be treated and conjugated to different molecules allowing for targeted delivery to specific cells. Once uptaken the nanohybrids release their cargo (drug molecules or biomolecules) into the targeted cells.

Toxicity assays were performed on ovarian epithelial cancer cells in 96-well plates.  The plates with the nanohybrids were incubated for 24, 48 and 72h.  For each assay 100μL of fresh media and 20μL of CellTiter96 Aqueous Cell Proliferation Assay reagent was added.  After 1 hr incubation, the plates were read using a UV spectroscopic plate reader.

For the animal studies, one of three different doses, 50, 100, and 200 μl of nanohybrids were administered subcutaneously, SC, intraperitoneally, IP or intraveneously, IV to each mouse. Two mice injected with saline served as controls. Four groups of nine treated mice (each containing mice injected with each dose in the three injection routes) were sacrificed after designated time periods (3, 7, 21 and 35 days post injection).  Tissue samples from brain, lungs, liver, kidney, spleen, pancreas, tight muscle, eye and skin were collected, processed and embedded in paraffin using standard procedures.  Sections stained with hematoxylin and eosin(H&E) were examined under light and the distribution of nanohybrids containing Lucifer Yellow were studied under a fluorescent microscope.

Summary
We have demonstrated that administration of nanohybrids containing Ketoprofen, a nonsteroidal anti-inflammatory drug (NSAID), to cancer cells in vitro results in significantly lower cell survival compared to controls.  Cells exposed to ketoprofen-nanohybrids survive significantly less than untreated cells or cells incubated with only the nanoparticles or neat ketoprofen.  We observed similar results with Ibuprofen- and even Aspirine-nanohybrids.  Recent studies have suggested that anti-inflammatory drugs have a potential for cancer therapy by inhibiting cyclooxygenase-1 and/or cyclooxegenase-2 (COX-1 and COX-2, respectively).  We have also demonstrated that nanohybrids can be used to deliver successfully si-RNA.  The transfection efficiency for the nanohybrids is 85% compared to 65% for liposomes.  In addition to lower efficiency, the high toxicity of liposomes leads to significantly reduced cell count (form 72K to only 16K).  Use of si-RNA nanohybrids may enable a series of gene silencing therapies for a number of diseases.  Furthermore, preliminary MRI experiments show that Gd-based nanohybrids are a promising imaging enabler.  Administration of the nanohybrids into the ovarian bursa of the mouse resulted in increased contrast.  

Lastly, systemic application of nanohybrids did not cause pronounced toxicity in mice as demonstrated by their high survival.  Even the highest possible amount of nanohybrids administered was not sufficient to reach LD₅₀.  The mild pathologic changes suggest that nanohybrids are relatively safe for further animal studies such as delivery of antitumor drugs in mouse models of various cancers.

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