Therapeutic Nanodevices: Drug Delivery

For the paper click here.
For the slides click here.
December 18, 2008

Integrated molecular targeting of IGR1R and HER2 surface receptors and destruction of breast cancer cells using single wall carbon nanotubes

Ning Shao, Shaoxin Lu, Eric Wickstrom, Balaji Panchapakesan, Nanotechnology 18 (2007) 315101 (9pp).

Purpose of the Study:   

Demonstrate the in vitro ability of single walled carbon nanotubes (SWCNT) functionalized with anti-IGF1R and anti-HER2 monoclonal antibodies to destroy breast cancer cells upon excitation with near infrared light (808nm). 

Methods:

 

•  SWCNTs having an average diameter of 1.4nm and lengths ranging from 500nm to 1mm were fabricated with a self-assembled monolayer of polyethylene glycol (PEG) to prevent undesirable binding with other biomolecules.
• Transmission electron micrography was used to characterize the SWCNTs.
• SWCNTs were divided into three groups and functionalized with a non-specific antibody (control), anti-IGF1R antibody, or anti HER2 antibody.
• Cytotoxicity was minimized, consistent with published research, by chemically modifying SWCNT surface with –OH and –COOH groups in addition to the PEG conjugates.
• SWCNTs were mixed with fluorescent dyes (phalloidin 488 & 555) to form fluorescent SWCNT-antibody hybrids.
• SWCNTs were incubated with two different human breast cancer cell lines:  (a) BT474, which exhibit high expression of HER2 receptors and lower expression of IGF1R receptors, and (b) MCF7, which exhibit high expression of IGF1R receptors and lower expression of HER2 receptors.  Each cell line was incubated with both anti-HER2 SWCNTs and anti-IGF1R SWCNTs.
• Confocal fluorescent microscopy was used to characterize the fluorescent SWCNT-antibody hybrids and the cellular uptake thereof.
• An 808nm laser at 800mW cm^-2 and 3 min exposure time was used for the photo-thermal therapy regimen. 
• Trypan blue assay was used to verify and quantify cell death (i.e. cell viability).  

Key Findings:

 

1. SWCNT-receptor specific antibody conjugates were readily internalized into the cells over large areas while the SWCNTs with the non-specific antibody were not.  The authors hypothesize the following mechanism (consistent w/ previous research):  upon receptor-specific attachment, stresses are generated due to free energy release which generates a pressure differential across membrane pores, thereby facilitating endocytosis.
2. Cells receiving only the NIR light without internalized SWCNT survived the photo-thermal therapy whereas those incubated with the SWCNT anti-HER2 and anti-IGF1R conjugates did not survive (100% kill rate).  Those cells to which SWCNT-non specific antibody conjugates were attached, 50% survived (most likely due to fewer attached).
3. Cells were killed due to the localized increase in temperature resulting from high absorption by the SWCNT that occurs with NIR light while normal cells remain transparent to NIR.   
4. The energy used to destroy the cancer cells was estimated to be ~200nW per cell, too low to create any damage to the normal cells.  Due to the high specificity, antibody-directed targeting, the authors were able to use half the laser power of past nanotube based cell killing techniques. 

Definitions:

Her2:  human epidermal growth factor receptor (often over expressed by breast cancer cells)

IGF1R:  insulin-like growth factor receptor (often over expressed by breast cancer cells)

In vivo Imaging and Drug Storage by Quantum-Dot-Connugated Carbon Nanotubes

Yan Guo, Donglu Shi, Hoonsung Cho, Zhongyun Dong, Amit Kulkarni, Gioanni Pauletti, Wei Wang, Jie Lian, Wen Liu, Lei Ren, Qiping Zhang, Guokui Liu, Christopher Huth, Lumin Wang, and Rodney Ewing; Advanced Functional Materials, DOI: 10.1002/adfm.200800406, August 2008.

 

Purpose of the Study:   

 

Evalute the in vitro anti-cancer activity and in vivo imaging capabilities of a novel carbon nanotubule (CNT) functionalized with paclitaxel-loaded polymer and quantum dots (QD).

 

Methods:

 

The CNTs were coated with poly lactic-co-glycolic acid (PLGA), a biodegradable and FDA approved drug delivery polymer, by plasma polymerization in which monomers (acrylic acid, lactic acid, and glycolic acid) were introduced to the plasma reactor.  HRTEM images reveal a PLGA film was deposited (~ 7nm thick) with an acrylic acid film on top (~ 3nm).

 

Amine-functionalized quantum dots were conjugated to the CNTs via a covalent bond between the amine group and carboxyl group in the acrylic acid film on the CNTs.  HRTEM imaging and energy dispersive spectroscopy (EDS) confirmed conjugation.  TEM imaging shows QDs were not evenly distributed – preferential conjugation of QD probably due to inhomogenous CNT dispersions).  Fluorescence spectra shows emission wavelength of 73nm (shifted from 795nm with QD alone – mechanism not understood).

 

A well known chemotherapeutic agent, paclitaxel, was added by mixing PLGA-coated CNT with methanolic solution, evaporated, and then washed with distilled water. 

 

Therapeutic efficacy was estimated in vitro by adding the QD-CNTs and free paclitaxel at varying doses to human PC-3MM2 prostate cancer cells and evaluating cell viability via MTT assay.

 

In vivo imaging capabilities were evaluated by injecting the QD-CNTs in the tail vein of mice and then pictures were taken with a Kodak 4000MM whole mouse imaging system at time intervals over 6 days.

Key Findings:

 

1. HRTEM and EDS revealed that the novel CNT was fabricated as planned with a paclitaxel-loaded polymer and conjugated QDs.

 

1. In vitro dose dependence curves indicate the QD-CNTs exhibited a five-fold increase in the loading capacity when the PLGA polymer is deposited on the CNTs versus no PLGA. 

 

1. The QD-CNTs had equivalent anti-cancer effects to free paclitaxel at a concentration of 100ng/ml.

 

1. The QD’s emitted enough fluorescence for effective in vivo imaging.  After six days, the QD-CNTs were concentrated in certain organ systems (particularly liver, kidney, stomach, and intestine.

 

Definitions:

 

MTT Assay – common assay performed to test cell viability.

“Optical characterization of quantum dots entrained in microstructured optical fibers”, Kenith E. Meissner, Carvel Holton, William B. Spillman Jr., Physica E26(2005) 377-381.

Purpose:  To investigate the optical properties of a microstructured optical fiber infused with colloidal CdSe/ZnS quantum dots (QD).

 

Methods:

 

·      CdSe/ZnS quantum dots were synthesized using trioctylphosphine as the coordinating solvent and following established methods.  The resulting nanocrystals were precipitated using methanol and then suspended in heptane.  The suspension exhibited peak luminescence at approximately 573nm.

·      The optical fibers were created from standard, optical-fiber grade silica using a rod and tube fabrication process.  Cross section shows each fiber consists of thirty-six capillary holes in the cladding material forming a ‘web’ region surrounding a solid core.  Each core and capillary has a diameter of approximately 12.7microns and 14.6microns, respectively.

·      Optical fiber distal ends were immersed in the colloidal QD suspension with QD’s distributing throughout the capillaries by way of capillary effect. 

·      An argon laser (488nm) served as a high power pump laser to photoexcite the QD’s and a HeNe laser (594nm) served as a low power probe laser.

 

Key Findings:

 

·      Upon excitation by the high-power (10mW) argon laser, QD luminescence from the capillaries was captured and propagated down the core of the fiber as a result of tight evanescent field coupling and scattering.  Emission spectrum exhibits no phase shift from excitation of QD’s in suspension alone.

·      When the low power (20microW) laser is applied to the fiber core, images show light becomes coupled to both the core and capillary clad region of the fiber microstructure.

·      When both the probe and pump lasers are applied to the fiber, significantly more probe light is observed at the distal end, representing an optical gain.  This gain is represented by both the spectral output as well as a CCD camera image.

·      The experiment was repeated many times with the same optical gain result. 

·      The mechanism explaining the optical gain is unexplained and warrants further investigation.

 

Definitions:

 

Luminescence:  here the term is used broadly and refers to fluorescence – photons emitted (at a wavelength higher than the excitation wavelength) from a chemical substrate undergoing an internal energy transition resulting from excitation photons.

Manipulation and light-induced agglomeration of carbon nanotubes through optical trapping of attached silver nanoparticles

Chao Shi, Yi Zhang, Claire Gu, Leo Seballos and Jin Z Zhang

Nanotechnology 19 (2008).  p. 215304-1-4.

 Purpose of the study

 Evaluate a method of using lasers to control carbon nanotube position and agglomeration through the use of silver nanoparticles.

 Methods

 Silver nanoparticles with an average diameter of 25nm were fabricated by reducing silver nitrate with sodium citrate.  Size was determined using transmission electron microscopy (TEM).  Silver was chosen as the base metal because it has a surface plasmon resonance (SPR) in the visible region thereby simplifying the technique.  Multi-walled carbon nanotubes (MWCNTs) were synthesized by thermal enchanced chemical vapor deposition with Al₂O₃ -coated (200nm) silicon wafers as the substrate.  MWCNTs were subsequently scraped off the silicon substrate for mixture with SNPs.  High resolution TEM and Raman scattering were used to measure dimensions (30micron length; 20nm diameter) and multi-walled structure.  To verify adhesion of SNPs to MWCNTs, a TEM image was taken before and after adding SNPs to MWCNTs in an isopropyl alcohol solvent.  Optical trapping was achieved by applying a TEM₀₀ mode laser (532nm wavelength) to a sample (water-based solution of SNP-attached MWCNTs) placed on a glass slide.  Trapping was confirmed by taking TEM images before and after moving the slide.  An un-trapped MWCNT bundle is used as a reference point on the slide demonstrating relative movement of the trapped bundle to the un-trapped bundle. 

 Key Findings

1. SNPs appear on or at junctions of MWCNT implying binding; most likely due to electrostatic and/or van der Waals forces.
2. Optical trapping was not observed with MWCNTs when SNPs were not present.
3. Two-dimensional optical trapping was observed when SNPs were bound.
4. The minimum volume ratio of SNPs/CNTs to achieve optical trapping is 1:4.
5. Hydrodynamic drag force determines the maximum speed at which a CNT bundle can remain optically trapped:     F_drag = hdu    (h = viscosity; d = longest dimension of the moving object; u = object’s velocity relative to the fluid)
6. MWCNT cannot exceed 24.8micron s-1 and remain optically trapped all else remaining equal (water solution, size/shape of the MWCNT, fixed laser power).
7. MWCNT agglomeration was observed under the laser beam in a donut-shaped disk with the outer diameter reaching 100micron and center hole diameter reaching 20micron.  Size of the microstructure as well as formation rate increased with increased laser power.

Definitions

Surface plasmon resonance:  Light-induced excitation of surface electron gas surrounding lattice metallic structures.

Optical trapping:  method of stabilizing and manipulating position of nanoparticles through use of laser-induced gradient forces.

Transverse ElectroMagnetic Mode (TEM₀₀ mode laser): type of laser in which there are no electric or magnetic fields in the direction of propagation.