Introducing LTSL-Dox

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

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.

(1) FDA, Doxil. HIGHLIGHTS OF PRESCRIBING INFORMATION 2007.

(2) Anyarambhatla, G.R. and D. Needham, Enhancement of the phase transition permeability of DPPC liposomes by incorporation of MPPC: A new temperature-sensitive liposome for use with mild hyperthermia. Journal of Liposome Research, 1999. 9(4): p. 491-506.

Preclinical Evidence Showing that LTSL-Dox "Cures" Mice with a Single Dose

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, as shown in the schematic is a representation of our initial hypothesis from the year 2000. When a tumor is warmed by hyperthermia and the LTSL-Dox is injected intravenously, LTSL-Dox releases the drug from the liposomes in seconds in the tumor blood vessels and drug gets into and throughout the tumor interstitium.

(1) Mills, J.K. and D. Needham, Targeted drug delivery. Expert Opinion on Therapeutic Patents, 1999. 9(11): p. 1499-1513.

(2) Mills, J.K. and D. Needham, Lysolipid incorporation in dipalmitoylphosphatidyl-choline bilayer membranes enhances the ion permeability and drug release rates at the membrane phase transition. BBA-Biomembranes, 2005. 1716(2): p. 77-96.

Implanted Tumor in Dorsal Skin Flap Window Chamber --means we can See theTumor

(Top) Human mammary carcinoma cells are implanted in this dorsal skin flap window chamber on the back of a rat. It takes 12 days to grow the tumor (the same time it takes to grow a mouse embryo!). This human tumor with mouse blood vessels can now be seen directly through an optical microscope.

(Bottom) While the normal vasculature (top right of image) is well branched with capillaries, the tumor vasculature (left) is heterogeneous with tortuosity, shunts, and was shown to be leaky to proteins like albumin (1) and even liposomes (2).

It is this window chamber model that allowed us ot prove that the LTSL-Dox did in fact release its drug at 41°C-42°C and that this drug permeated the whole tumor, as shown next in the videos

(1) Wu, N.Z., et al., Measurement of material extravasation in microvascular networks using fluorescence video-microscopy. Microvascular Research, 1993. 46(2): p. 231-253.

(2) Wu, N.Z., et al., Increased microvascular permeability contributes to preferential accumulation of stealth liposomes in tumor-tissue. Cancer Research, 1993. 53(16): p. 3765-3770.

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 Microscope Video Proves the Hypothesis

Shown here is a video of a tumor that was implanted in the microscopic rat window chamber that proves that hypothesis. It was published in 2012, in a paper by Ashley Manzoor 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, and 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”

(1) Manzoor, A.A., et al., Overcoming Limitations in Nanoparticle Drug Delivery: Triggered, Intravascular Release to Improve Drug Penetration into Tumors. Cancer Research, 2012. 72(21): p. 5566-5575.

(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.

(1) Manzoor, A.A., et al., Overcoming Limitations in Nanoparticle Drug Delivery: Triggered, Intravascular Release to Improve Drug Penetration into Tumors. Cancer Research, 2012. 72(21): p. 5566-5575.

Treating "Tucker"

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. The video 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 https://youtu.be/aM4GiF_Dy1c

Patenting and Licensing

LTSL-Dox was invented by David in 1996 with a submitted Invention Disclosure to the Duke Tech Transfer Office and was subsequently 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

(1) Needham, D., Temperature-sensitive liposomal formulation U.S. Patent No. 6,200,598 (Filed: June 18, 1998). 2001.

(2) Needham, D., Temperature-sensitive liposomal formulation US Patent Office No. 6,726,925 (Filed: December 9, 1999) Expired. 2004: USA.

(3) Borys, N. and M.W. Dewhirst, Drug development of lyso-thermosensitive liposomal doxorubicin: Combining hyperthermia and thermosensitive drug delivery. Adv Drug Deliv Rev, 2021. 178: p. 113985.