We developed novel intrinsic multifunctional nanovesicles called porphysomes, which have structure-dependent fluorescence self-quenching and unique photothermal properties, thus functioning as potent photothermal therapy agents. We formulated porphysomes using high pressure extrusion and investigated their photothermal therapy efficacy in a xenograft tumor model.
We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide α, was conjugated to a phospholipid and then self-assembled to liposome-like spherical vesicles. Due to the extremely high density of porphyrin in the porphyrin-lipid bilayer, porphysomes generated large extinction coefficients, structure-dependent fluorescence self-quenching, and excellent photothermal efficacy. In our formulation, porphysomes were synthesized using high pressure extrusion, and displayed a mean particle size around 120 nm. Twenty-four hr post-intravenous injection of porphysomes, the local temperature of the tumor increased from 30 °C to 62 °C rapidly upon one minute exposure of 750 mW (1.18 W/cm2), 671 nm laser irradiation. Following the complete thermal ablation of the tumor, eschars formed and healed within 2 weeks, while in the control groups the tumors continued to grow and all reached the defined end point within 3 weeks. These data show how porphysomes can be used as potent photothermal therapy (PTT) agents.
Porphysomes are novel multifunctional nanovesicles that we recently developed which are capable of multimodal imaging and therapy1. They are formed from self-assembled porphyrin bilayers and contain an extremely high density of porphyrin (over 83, 000/porphysome particle), which generates large extinction coefficient and results in unique structure-dependent fluorescence self-quenching. Porphysomes have good in vivo pharmacokinetic and biodistribution properties: they exhibit a blood half-life of 12 hr following systematic administration, and passively accumulate in xenograft tumors with 7.5% ID/g at 24 hr post-injection2.
Their unique structure and physiochemical properties make porphysome a good candidate for multimodal imaging and image-guided therapy. First of all, containing porphyrin, porphysomes are intrinsically suitable for fluorescence imaging of tumors upon the tumor accumulation1. In addition, each porphyrin has a stable site for chelating radioisotopes, therefore, porphysomes can be easily labeled with radioisotopes such as 64Cu for PET imaging3. Furthermore, the absorbed light energy is dissipated thermally under laser irradiation exposure when porphysome structure is intact, so porphysomes also exhibit unique photoacoustic imaging and PTT capabilities. It has been shown that 24 hr after intravenous injection of porphysomes, laser irradiation of the porphysome-accumulated tumor induced a rapid temperature increase and strong photothermal tumor ablation. This demonstrated that porphysomes are efficient photothermal enhancers with extinction coefficient as high as gold nanoparticles (AuNPs)1. On the other hand, in comparison with other inorganic photothermal agents, including AuNPs, porphysomes show an outstanding advantage in biosafety due to their organic nature. Porphysomes are enzymatically biodegradable and induce minimal acute toxicity in mice with intravenous doses as high as 1,000 mg/kg1. Furthermore, similar to liposomes, the large aqueous core of porphysomes could be passively or actively loaded with therapeutic or imaging agents. The optical properties and biocompatibility of porphysomes demonstrate the multimodal potential of organic nanoparticles for biophotonic imaging and therapy.
In this paper, we introduce the synthesis method of pyropheophorbide-lipid conjugates, the manufacturing and the characterization method of porphysomes using high-pressure extrusion. In vivo PTT on mice is conducted as well to demonstrate the efficiency of porphysome-enabled PTT in the tumor treatment using a subcutaneous xenograft tumor model.
1. Synthesis of Pyropheophorbide-lipid
2. Preparation of Porphysomes
3. Preparation of Animal Xenograft Model
4. In vivo Photothermal Therapy
Pyropheophorbide is conjugated to the phospholipid as a stable lipid monomer (Figure 1a) and the conjugates self-assemble to form porphysomes by membrane extrusion method using a high pressure extruder. It is usually difficult to extrude porphysome-lipid suspension during the first 3 cycles of extrusion with relatively slow flow rate. As more extrusions are repeated, the flow rate of extruded solution gradually increases, and pressure can be slightly decreased if needed. Porphysome size, concentration, and quenching efficiency are three important properties to characterize the nanoparticles right after extrusion. Porphysomes with high homogeneity of size distribution (peak around 120 nm) is generated (Figures 1 b and 1c) with two main absorbance peaks (410 nm and 677 nm, Figure 1d) and can remain stable for over 12 months when being stored at 4 °C. The fluorescence of porphyrin is quenched as high as 99.8% when the nanostructure is intact (Figure 1e).
For in vivo PTT, heat can be generated rapidly upon the laser irradiation at 24 hr post-porphysome administration (Figure 2). Porphysomes with laser irradiation cause the temperature to increase by over 35 °C after 1 min of irradiation, while the laser alone induces a temperature increase of less than 10 °C. It is important that the final tumor temperature reaches 55 °C to ensure complete tumor ablation. Following the PTT irradiation, the tumor usually turns whitish because of the potent thermal effect. The mouse leg becomes a little swollen at the tumor area for around 2 days following PTT. Thermal ablation results in dark brownish eschar on tumors and 100% reduction of tumor volume. Eschars can be observed obviously at 24 hr post-treatment and they gradually recover during the following 2 weeks (Figure 3a). Using porphysomes, subcutaneous tumors can be completely eliminated without recurrence (Figure 3b), while the tumors in porphysome alone and laser alone control continue to grow (Figure 3b), and all reach end point in 2 weeks.
Figure 1. Structure of porphysome nanovesicles and characterizations. a. Schematic representation of a pyropheophorbide-lipid porphysome. b. corresponding TEM image. c. size distribution of porphysomes after high-pressure extrusion. d. Absorbance of the pyropheophorbide-lipid in methanol, normalized to the peak at 410 nm. e. fluorescence emission (em) of quenched (red dash line) and unquenched (blue solid line) porphysomes, normalized to maximum fluorescence. Click here to view larger figure.
Figure 2. Thermal response during PTT. a. laser set up for transdermal light irradiation. b and c. Temperature of tumor upon laser irradiation in KB tumor-bearing mice at 24 hr post-injection of porphysomes or PBS. d, Maximum tumor temperature during 60 sec laser irradiation (mean +/- SD for 5 mice in each group). Click here to view larger figure.
Figure 3. a. Photographs showing therapeutic response to PTT1, including three groups: porphysomes injection with laser irradiation, PBS injection with laser irradiation, and porphysomes injection alone. b. Average tumor volume after each treatment (n=5, with * represents p<0.0005). Click here to view larger figure.
In the development of drug delivery technologies, multifunctional nanoparticles are currently under wide investigations for accurate tracking of the drug delivery vehicle while maintaining a high drug payload. Porphyrin-loaded liposomes have been developed for better pharmacokinetic properties and more efficient delivery than direct administration of porphyrin, but obstacles exist, including the restricted loading amount of porphyrin and rapid redistribution of porphyrin from liposomes to plasma proteins5,6. In contrast to conventional liposomal delivery systems, where free porphyrin are inserted into either the core or lipid-bilayer of the liposome, pyropheophorbide (pyro) is now directly and stably conjugated to the phospholipid and self-assemble into liposome-like nanostructures to form porphysomes. As a result, the porphysome bilayer achieves extremely high porphyrin packing density, which leads to structure-dependent self-quenching of pyro fluorescence and high extinction coefficient.
These pyro-lipid conjugates self-assemble into porphysomes in aqueous buffer via similar preparation method of liposomes, such as membrane extrusion method that is commonly used to generate unilamellar vesicles of well-defined size7-9. The extrusion starts from a dry lipid film that is hydrated in the aqueous solution and subjected to several freeze-thaw cycles to form multilamellar vesicles with broad size distribution10. When the hydrated porphysome lipid film reaches more than 1 mg/ml, manual extrusion becomes physically challenging, so high pressure extrusion is chosen for scaled up production of porphysomes, such as for animal studies when at least 5 mg/ml of porphysome is required8,9. To achieve even higher final pyro concentration, porphysome lipid-film can be prepared with higher initial amount. But concentration higher than 10 mg/ml is not recommended, due to the difficulty of extrusion with the 10 ml high pressure extruder. During the extrusion, if the flow rate of the extruded solution coming out is extremely slow (e.g. 30 min/ml) even with very high pressure (≤800 psig for safety issue), the filter can be changed to a new set, and the temperature of water-circulation can be increased from 65 °C-75 °C. Size homogeneity of liposomal nanovesicles has been shown to help decrease liver and spleen uptake in vivo 11. Therefore, to obtain a homogeneous and narrow size distribution, the extrusion of porphysome lipid suspension through filter membrane (100 nm) is repeated at least ten times or even more.
We found porphysomes are very stable after extrusion when stored at 4 °C, with negligible change in size or quenching properties for at least one year in our studies (data not shown). It is still recommended to test the porphysome size and quenching efficiency before each animal injection to ensure porphysome intactness.
Porphysomes are the first organic nanoparticles to serve as efficient photothermal enhancers while maintaining the drug delivery capacity and biocompatibility of conventional liposomes. They are based on porphyrins, which have an excellent record for theranostic applications.12 They have high extinction coefficient comparative to gold nanoparticles (GNPs) for rapid heat generation and efficient thermal ablation1. The in vivo studies have shown the advantages of porphysome-enabled PTT as an alternative method to conventional cancer treatment. PTT is very simple, with laser treatment relatively localized to the selected irradiation area (9 mm diameter), and very short treatment period (1 min) required to achieve tumor temperature higher than 55 °C for effective thermal ablation. Both the simplicity and high selectivity can help to improve recovery time and reduce the risk of complications for translational therapeutic applications. If other xenograft types that have less tumor accumulation than KB tumor (7.5% ID/g) is used, higher porphysome dose can be IV injected for efficient heat generation upon laser irradiation. During the irradiation process, it is important that laser spot covers the whole tumor area, and tumor temperature is increased to 55 °C or higher for quick and complete thermal ablation at tumors.
Studies now are ongoing to explore the multimodal imaging and therapeutic applications of porphysomes as potent PTT agents in more clinically relevant animal models for future translational study.
The authors have nothing to disclose.
This work was supported by the Princess Margaret Hospital Foundation, the National Science and Engineering Research Council of Canada, the Canadian Institute for Health Research, the Canadian Foundation of Innovation, and the Joey and Toby Tanenbaum/Brazilian Ball Chair in Prostate Cancer Research, a grant from the SUNY Research Foundation and startup support from University at Buffalo.
Name of Equipment | Company | Catalog Number | Comments (optional) |
Rotary evaporator | Thermo-Savant | SPD131DDA | |
Votex | Scientific Industries | SI-0236 | Model number: G560 |
High pressure extruder | LIPEX, Northern Lipids Inc. | T.001 | 10 ml Thermobarrel Extruder |
Heated bath circulators (Thermostatted circulator) | Thermo SCIENTIFIC | SC100-S5P | |
Polycarbonate filters | Avanti Polar Lipids | 610005 | Pore size 100 nm, and membrane diameter 19 mm |
Zetasizer | Malvern Instruments | ZS90 | |
UV/Visible Spectrophototmeter | Varian Australia | Cary 50 Bio UV/ Visible Spectrophotometer | |
Spectrofluorometer | HORIBA Scientific | FluoroMax-4 | |
Transmission Electron Microscopy (TEM) | Hitachi | H-7000 | |
Cell culture incubator | SANYO | MCO-18AIC | |
Powermeter | Thorlabs | PM100D | with sensor S142C |
671nm Laser | LaserGlow Technologies | LRS-0671_PFN-02000-05 | S/N:10097270 |
Infrared Thermometer | Mikroshot, LUMASENSE Technologies | 9102409 | |
Laser protective eye goggles | LaserGlow Technologies | AGF6602XX | Optical Density: 1.5+ at 630-700 nm |