Introducing the Low Temperature-Sensitive Liposomal Doxorubicin (LTSL-Dox), invented by David in 1996
The LTSL-Dox Liposome
(Top) Schematic of the LTSL-Dox Liposome consisting of a temperature sensitive lipid bilayer membrane with its permeabilizing (lysolipid) component and a protective polymer layer, encapsulating the crystalline doxorubicin.
(Bottom) Electron micrograph image of an actual LTSL-Dox liposome showing its faceted solid membrane and nano-crystalline doxorubicin.
LTSL-Dox is a liposome that contains the anti-cancer drug doxorubicin. As shown in the schematic, the difference between this and the earlier Doxil (1), is that this new temperature-sensitive lipid bilayer contains a permeabilizing component (10 mol% lysolipid) in the encapsulating membrane that can release the drug. The rationale behind this new design was that, as the ambient temperature is raised to 41-42°C, the lipid membrane melts from its solid phase and the drug is released in 2 seconds (2).
The electron micrograph image shows the solid nature of the membrane indicated by the facetted flat sheets meeting at solid grain boundaries. Doxorubicin is inside the liposome and is actually in a crystalline form that rapidly dissolves when the membrane is permeabilized.
With a diameter of just 100 nanometers (nm), this liposome is just 1/1000th the diameter of a human hair, and 1/100th the diameter of a red blood cell.
Preclinical Evidence Showing that LTSL-Dox Works in Mice
11/11 Implanted Tumors in mice were cured out to 60 days
As published in 2000, (1), the first time we tried it in mice, 11/11 implanted tumors were cured from just a single dose of the LTSL-Dox with the tumors warmed to 42°C for just one hour. The graphs show that the implanted mouse flank tumors normally grew at a fairly rapid rate, reaching 5x initial tumor volume in only 10 days. A single dose of 5mg/kg LTSL-Dox administered via tail-vein injection on Day 0 with the tumor already warmed to 42°C for just one hour resulted in all 11/11 implanted tumors regressing and essentially cured out to 60 days.
(1), Needham, D., et al., A new temperature-sensitive liposome for use with mild hyperthermia: Characterization and testing in a human tumor xenograft model. Cancer Research, 2000. 60(5): p. 1197-1201.
LTSL-Dox at 41oC – 42oC
Mechanistically, LTSL-Dox was designed to rapidly release its encapsulated anti-cancer chemotherapeutic drug, doxorubicin, at mild hyperthermic temperatures (41-42°C )(1, 2). Thus, shown in the schematic is a representation of our initial hypothesis from the year 2000 of how, when a tumor is warmed by hyperthermia and the LTSL-Dox is injected intravenously, it releases the drug from the liposomes in seconds in the tumor blood vessels and drug gets into and throughout the tumor interstitium.
Confocal fluorescent video microscope video of LTSL-Dox injected via the rat tail vein by Manzoor et al (3), and viewed in the micrcoscope window chamber, showing 20 minutes of observation in 20 seconds of video time
Microscopic images of the tumor vasculature in a window chamber by Chen et al, (4) showing the tumor blood vessels before a 1hr treatment with LTSL-Dox + heat, and 24hrs after the treatment, when all the blood vessels had been shut down and all that was left was a small thrombosis.
LTSL-Dox + HT: Confocal MicroscopeVideo
Shown here is a video of a tumor implanted in a microscopic rat window chamber that proves that hypothesis. It was published in 2012, in a paper by AshleyManzoor et al (1) --then graduate student, in Mark Dewhirst's lab and Hyperthermia Center at Duke University. It is a confocal microscopic video taken of a rat tumor warmed to 42°C showing how the LTSL-Dox liposomes (green) release their doxorubicin (red) throughout the whole tumor in only 20 minutes of warming.
Drug also enters the endothelial cells and pericytes that line the blood vessels (red edges to the blood vessels). As a result, as shown in the lower black and white images from Chen et al (2), the blood vessels are shut down within 24 hrs after the 1 hr treatment.
This is why we say: “the only way to get a drug throughout a whole tumor is to release the drug in the blood vessels of the tumor”
(2) Chen, Q., et al., Tumor microvascular permeability is a key determinant for antivascular effects of doxorubicin encapsulated in a temperature sensitive liposome. International Journal of Hyperthermia, 2008. 24(6): p. 475-482
LTSL-Dox + HT: Confocal Sections
In this video, again from the data by Manzoor et al, (1) confocal microscope sections are assembled by scanning through the whole tumor. It shows that every cell is not only loaded with doxorubicin, every "red dot" is doxorubicin in the nucleus of every cell where it kills the cancer.
Back in 2000 we did treat a dog called Tucker in North Carolina using LTSL-Dox and a different heating system (a rotating microwave device). Tucker had a tumor on his thigh, called a sarcoma (a cancer of the soft tissues including connective, muscle, or nervous tissues). Dr Mark Dewhirst and his colleague at NC State Veterinary School, Dr Don Thrall and staff, managed to arrange to treat Tucker with the LTSL-Dox. It was made by the Australian Broadcast Corporation who were visiting the Raleigh-Durham area making local science videos for their Quantum series. The video they made is shown here
Patenting and Licensing
LTSL-Dox was invented by David in 1996 with a submitted Invention Disclosure to the Duke Tech Transfer Office and patented in 2001 (1) and 2004 (2). On the strength of the very impressive data in mice shown above it was licensed to Celsion Corporation by Duke in November 1999. Celsion CMO Nick Borys and Duke's Mark Dewhirst recently wrote up the whole experience in a review, entitled, "Drug development of lyso-thermosensitive liposomal doxorubicin: Combining hyperthermia and thermosensitive drug delivery" (3)
For more information on all aspects of this technology please contact David at David@dnko.net