Maat ontworpen laser gebaseerde Verwarming Apparaten voor gestuurde release van cisplatine van temperatuurgevoelige liposomen met Magnetic Resonance Image Guidance

The overall goal of this procedure is to demonstrate the operation of a custom built, laser-based heating apparatus that can be used to heat subcutaneous tumors locally for delivery of a heat activated liposome formulation. This is accomplished by first preparing the HTLC formulation through lipid film formation, hydration extrusion, and dialysis. In the second step, a donor mouse is inoculated with cervical carcinoma cells.

After four to five weeks, the tumor is excised cut into fragments and individually implanted into a recipient mouse. In the final step, the recipient mouse tumor is locally heated to 42 degrees Celsius using the laser-based heating system. The laser-based heating apparatus is MR Compatible, which enables monitoring of the changes in tissue temperature during light delivery, and a small and portable allowing for simple and effective heating of the tumor in the bore of the small animal mr.

Ultimately, the heat activated release of the thermo sensitive HTLC can be monitored by real-time magnetic resonance imaging. The main advantages of this technique over existing method, like using a water bath and a heating catheter, is that our technique provides a conformal method of delivering heat to the tumor within one to two minutes of initiating the heat treatment and allows real time adjustment of the laser power minimizing the temperature fluctuations during the procedure. We first had the idea for this method when we started to conduct in vivo studies with our HTLC thermo sensitive liposome formulation.

We found that the existing methods were not able to efficiently provide local heating of a subcutaneous tumor. The liposome preparation procedures will be demonstrated by myung, a PhD student from my lab To prepare the liposomes begin by combining freshly prepared lipid mixture with freshly prepared lipid drug mixture, followed by a one hour hydration with vortexing every 15 to 20 minutes while the lipid mixture is hydrating. Assemble the 10 milliliter extruder with two stacks of 200 nanometer polycarbonate filters thermo barrel of the extruder to a circulating water bath set to 70 degrees Celsius, and connect the device to a compressed nitrogen tank.

Immediately following the hydration, transfer the lipids to the extruder chamber and open the nitrogen flow. Then at a pressure of 200 PSI extrude the liposomes through the membranes, collecting the liposomes in a 50 milliliter conical tube set in the 70 degree Celsius water bath. After extruding the liposomes five times, disassemble the device and reassemble it with two stacks of 100 nanometer polycarbonate filters.

Set the pressure to 400 PSI and extrude the liposomes 10 times as just demonstrated. Collecting the liposomes from the final extrusion in a 15 milliliter conical tube allow the liposomes to cool to room temperature, followed by precipitation of the insoluble cisplatin by centrifugation, then dialyze the liposomes overnight against 0.9%saline in dialysis tubing with a 15, 000 molecular weight cutoff under sterile conditions. To implant the cervical tumor xenograft harvest me 180 cells from a 90%confluent culture and count the number of viable cervical carcinoma cells by hemo.

Cytometer then dilute the cells to one times 10 to the six me 180 cells per 100 microliters. All animal work was performed at Star Innovation Center, inoculate female skit mice by intramuscular injection into the gastroc muscle of the hind limb using calipers, measured the resulting tumor size until the tumors have reached nine to 12 millimeters in their longest dimension. Then excise the tumor from the donor mouse and mince it into two to three cubic millimeter fragments.

Now shave the left hind limb of the recipient animal and make an incision in the bare skin. Insert one piece of the donor tumor subcutaneously through the incision and use one to two wound clips to close the skin three to five days after the implantation. Remove the clips and allow the tumor to grow for two to three weeks to perform the heat treatment.

First, connect one end of a laser fiber to the laser device and the other end to the illuminator. Next, insert a 27 gauge injection catheter into the lateral tail vein of an anesthetized recipient mouse, followed by the insertion of a 22 gauge catheter into the center of the tumor. Then place a fiber optic temperature probe into the hollow catheter to monitor any temperature changes and cover the entire tumor with the illuminator.Final.

Set the power to 0.8 to one watt, then turn on the laser and wait for the temperature to rise manually adjusting the laser power throughout the treatment between 0.1 to 0.8 watts to maintain the temperature of the tumor at 42 degrees Celsius as illustrated in this image of a cross-section through a heat treated tumor, assuming a total power of one watt, the maximum fluence rate at any point in the tumor is 70 milliwatts per square centimeter with the fluence rate in the tumor at the skin depth, measuring 50%of the maximum fluence rate as confirmed by the proton resonance frequency shift to magnetic resonance thermometry heating. Using the laser-based heating setup generates a relatively uniform temperature distribution map shown in the image which tracks the relative temperature change from an absolute baseline measurement acquired before heating by the point source. The heated tumor displays the highest relative signal increase compared to the unheated turt muscle after administration of gad HTLC as determined by magnetic resonance signal analysis, maintaining the signal until the end of the heating period Once mastered, this technique can be set up in less than 15 minutes if it is performed properly, providing a valuable tool for preclinical evaluation of thermo sensitive liposome formulations.

Don't forget that working with a Class four laser can be extremely dangerous, and that precautions such as wearing safety goggles should always be taken while performing this procedure.