WO2022231724A1 - Adipocyte-derived anti-cancer lipid droplets - Google Patents

Adipocyte-derived anti-cancer lipid droplets Download PDF

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WO2022231724A1
WO2022231724A1 PCT/US2022/020817 US2022020817W WO2022231724A1 WO 2022231724 A1 WO2022231724 A1 WO 2022231724A1 US 2022020817 W US2022020817 W US 2022020817W WO 2022231724 A1 WO2022231724 A1 WO 2022231724A1
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lipid
pyrolipid
lipid droplets
droplets
therapeutic material
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PCT/US2022/020817
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French (fr)
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Zhen GU
Tingxizi LIANG
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The Regents Of The University Of California
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Publication of WO2022231724A1 publication Critical patent/WO2022231724A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0076—PDT with expanded (metallo)porphyrins, i.e. having more than 20 ring atoms, e.g. texaphyrins, sapphyrins, hexaphyrins, pentaphyrins, porphocyanines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A61K47/544—Phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors

Definitions

  • the technical field relates to engineered lipid droplets as a therapeutic carrier for enhanced cancer therapy. More specifically, the field of the invention relates to the loading of lipid droplets with a lipid-based photosynthesizer pyrolipid synthesized from pyropheophorbide a (PPa).
  • PPa pyropheophorbide a
  • the drug-loaded lipid droplets (sometimes referred to herein as Pyrolipid@LDs) can then be injected into tumor or cancerous tissue and then subject to illumination from a light source to enhance the therapeutic effect.
  • lipid metabolism plays a fundamental role in the life maintenance of both tumor cells and other non-malignant cells in the tumor microenvironment, such as tumor-associated adipocytes, macrophages and neutrophils that promote tumor growth, invasion and metastasis. Therefore, targeting lipid metabolism in the tumor tissue has become an emerging approach for cancer therapy, where this abnormal tumor metabolism pathway can also be leveraged for the development of new drug delivery strategies.
  • tailored lipid droplets are engineered as a new drug delivery system for cancer therapy.
  • Lipid droplets are abundant in the adipocytes at the nano- and microscale size.
  • the lipid droplet is composed of a hydrophobic core filled with neutral triacylglycerol and a phospholipid monolayer coated with certain proteins as the outer shell as illustrated in FIG. 2A. Due to its unique structure, the lipid droplet can be engineered into a universal drug delivery system for targeted therapy as the lipid core provides an ideal reservoir for lipophilic drugs and the surface proteins on the outer shell retain the capability to interact with other organelles associated with metabolism regulation.
  • the physicochemical and physiological properties of lipid droplets can be leveraged in accommodation with different applications.
  • the engineered lipid droplet could synergistically enhance the therapeutic effect through various pathways.
  • this cell-derived material showed limited side effects, while the physiological stability renders them promising clinical translation potential.
  • a therapeutic material for treating cancer includes a plurality of lipid droplets having contained therein a drug that, in one preferred embodiment, is pyropheophorbide a.
  • the pyropheophorbide a may be conjugated to a lipid (e.g., 1-palmitoyl- 2-hydroxy-sn-glycero-3- phosphocholine) to improve droplet loading.
  • a lipid e.g., 1-palmitoyl- 2-hydroxy-sn-glycero-3- phosphocholine
  • These loaded lipid droplets i.e., Pyrolipid@LDs
  • a method of using the therapeutic material includes injecting the lipid droplets into a tumor or cancerous tissue of a mammalian subject and irradiating the tumor or cancerous tissue with far-red or near-infrared light.
  • the light may be far-red or near-infrared light that is emitted from, for example, a laser, light-emitting diode (LED, laser diode, or the like.
  • one or more light fibers e.g., as part of a catheter, laparoscope or the like may deliver the light internally to the tumor/cancerous tissue.
  • a method of formulating therapeutic lipid droplets includes the operations of synthesizing a lipid-conjugated pyrolipid and incubating the lipid- conjugated pyrolipid with adipocytes. Pyrolipid-loaded lipid droplets are then isolated from the adipocytes. The isolated pyrolipid-loaded lipid droplets can be stored for later use (e.g., freeze dried). The freeze dried Pyrolipid@LDs can be reconstituted in a buffer solution or sera and then injected into the diseased tissue.
  • FIG. 1A illustrates an injection device (e.g., syringe) that contains the therapeutic material for treating cancer.
  • an injection device e.g., syringe
  • a zoomed-in view of the pyrolipid-loaded lipid droplets is also illustrated.
  • FIG. IB illustrates an illumination device that is used to illuminate tissue that has been injected with the therapeutic material.
  • FIG. 2 A illustrates a process of generating or manufacturing pyrolipid-loaded lipid droplets (Pyrolipid@LDs).
  • FIG. 2B shows confocal images of fully differentiated adipocytes, in which lipid droplets were stained with BODIPY® (boron-dipyrromethene) 505/515 (green). Scale bar, 50 pm.
  • BODIPY® boron-dipyrromethene
  • FIG. 2C shows a transmission electron microscopy (TEM) image of isolated lipid droplets. Scale bar, 2 pm.
  • FIG. 2D illustrates the average hydrodynamic size of lipid droplets determined by dynamic light scattering (DLS).
  • FIG. 2E shows the stability of lipid droplet in PBS and DMEM culture medium containing 10% FBS. Diameter was determined by DLS. Data are presented as mean ⁇ s.d.
  • FIG. 2G illustrates the structure of the drug pyrolipid synthesized by conjugating photosensitizer pyropheophorbide a (PPa) with a lipid (l-palmitoyl-2-hydroxy-sn-glycero-3- phosphocholine).
  • FIG. 2H illustrates confocal images of pyrolipid accumulation inside the adipocytes. Photosensitizers were shown in red (middle column). Lipid droplets were shown in green (BODIPY® 505/515 labeled) (left column). Merged images on in the right column. Scale bar, 50 pm.
  • FIG. 3A shows confocal images represent the distribution of fluorescein-labeled lipid droplets in SKOV3 cancer cells. Lysosome, mitochondria and ER were stained with LysoTracker Red DND-99, MitoStatus Red and ER-Tracker Red, respectively. Scale bar, 20 pm. Scale bar for selected area, 10 pm.
  • FIG. 3B illustrates Pearson’s colocalization coefficient between lipid droplet and different organelles.
  • FIG. 3C illustrates the flow cytometric analysis of lipid droplets uptake in SKOV3 cells pretreated with different endocytosis inhibitors.
  • Ctrl control group without any inhibitor pre-treatment.
  • Statistical significance was calculated via one-way ANOVA with a Tukey post-hoc test. * P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.001.
  • FIG. 3D illustrates the flow cytometric analysis of lipid droplet uptake at different temperatures.
  • Ctrl represents the SKOV3 cells without lipid droplet incubation (left peak).
  • Middle peak is 4 °C.
  • Right peak is 37 °C.
  • FIG. 4B illustrates cytotoxicity results (cell viability %) of pyrolipid and Pyrolipid@LDs under normoxic and hypoxic conditions. Data are presented as mean ⁇ s.d.
  • FIG. 4C shows live-dead cell imaging of dark and laser group after incubating cells with Pyrolipid@LDs. Live cells were stained as green color (top image) and dead cells as red color (bottom image). Scale bar, 100 pm.
  • FIG. 4D illustrates flow cytometric analysis of cell apoptosis in different treatment groups determined by Annexin V-FITC/PI staining.
  • GO control group
  • Gl lipid droplet
  • G2 Pyrolipid
  • G3 Pyrolipid@LDs.
  • (-) Dark
  • (+) Laser. Data are presented as mean ⁇ s.d.
  • FIG. 5A illustrates a proposed mechanism for Pyrolipid@LDs-induced phototoxicity.
  • Pyrolipid@LDs can generate ROS upon laser (or light source) irradiation.
  • the generated ROS oxidizes lipid matrix of lipid droplet and increases the oxidative stress.
  • the ER stress caused by excessive accumulation of lipid activates apoptosis signaling pathway, enhancing the metabolic intervention that further promotes the therapeutic effect.
  • FIG. 5B illustrates cytotoxicity results of Pyrolipid@LDs in the presence of ROS scavenger N-acetyl-L-cysteine (NAC, 2 mM), lipid peroxidation inhibitor vitamin E (VE, 250 mM) and ER stress inhibitor 4-Phenylbutyric acid (PBA, 100 mM).
  • FIG. 5C illustrates ROS generation of different formulations under dark and laser conditions detected by fluorescent DCFDA probe.
  • GO control group
  • Gl lipid droplet
  • G2 pyrolipid
  • G3 Pyrolipid@LDs.
  • Laser cells were irradiated with 670 nm light at a power density of 100 mW/cm 2 for 5 min.
  • DCFDA concentration 25 pM.
  • FIG. 5E illustrates Western blot analysis of ER stress-related proteins. Statistical significance was calculated via one-way ANOVA with a Tukey post-hoc test. *P ⁇ 0.05;
  • FIG. 6A illustrates a schematic of Pyrolipid@LDs mediated photodynamic therapy in a mouse model.
  • FIGS. 6B and 6C illustrate the in vivo treatment efficacy of Pyrolipid@LDs.
  • FIG. 6B shows individual tumor growth kinetics while FIG. 6C shows average tumor growth kinetics in different groups.
  • GO PBS; Gl: lipid droplet; G2: pyrolipid; G3: Pyrolipid@LDs.
  • (-): Dark (without laser); (+): Laser. Data are presented as mean ⁇ s.d. (n 8).
  • Statistical significance was calculated via one-way ANOVA with a Tukey post-hoc test for multiple comparisons. * P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.001.
  • FIG. 6D shows representative photographic images of mice and excised tumors in GO and G3 (+) groups.
  • FIG. 6E illustrates a graph showing mouse body weight as a function of time (days).
  • FIG. 7A illustrates a schematic of pre-adipocyte differentiation, maintenance, and lipid droplet isolation.
  • FIG. 7B illustrates representative images at different time points during differentiation. Images were visualized by light microscopy under 20X lens.
  • FIG. 8 illustrates TEM images of lipid droplets in PBS and FBS -containing DMEM culture medium at different time points. Scale bar, 500 nm. The stability of lipid droplet in different mediums is shown.
  • FIG. 10A illustrates the synthesis of pyrolipid.
  • Pyrolipid was prepared from the esterification between pyropheophorbide a (PPa) and l-palmitoyl-2-hydroxy-sn-glycero-3- phosphocholine.
  • FIG. 10B illustrates the 'H NMR spectrum of pyrolipid (in d-DMSO).
  • FIG. 11 A illustrates UV-Vis spectra of PPa and pyrolipid.
  • FIG. 1 IB illustrates fluorescence spectra of photosensitizers under excitation wavelength of 410 nm.
  • FIG. llC illustrates fluorescence spectra of photosensitizer-loaded lipid droplets under excitation wavelength of 410 nm.
  • FIG. 12 includes confocal images of pyrolipid accumulation in the lipid droplets (LDs) at different time points. Fully differentiated adipocytes were incubated with pyrolipid at a concentration of 5 mM. After different incubation time, lipid droplet was stained with BODIPY® 505/515 and visualized by confocal microscopy. Scale bar, 50 pm.
  • FIG. 13A illustrates optimization of pyrolipid concentration for incubation.
  • Cell viability of adipocytes after incubating with different concentrations of pyrolipid was detected by an MTT assay.
  • FIG. 13B is a histogram showing the influence of pyrolipid on the amount of lipids in the lipid droplets.
  • FIG. 15 illustrates images showing the distribution of Pyrolipid@LDs-FITC in SKOV3 cells at different time points (lh, 4h, 12h). Scale bar, 50 pm.
  • FIG. 16 illustrates the dark toxicity of pyrolipid (left data in pairs) and Pyrolipid@LDs (right data in pairs) on SKOV3 cells. Data are presented as mean ⁇ s.d.
  • FIG. 17 illustrates the semi-quantitative analysis of ER stress-related protein expression.
  • the relative protein expressions were normalized to that of b-actin.
  • GO control group
  • Gl lipid droplet
  • G2 pyrolipid
  • G3 Pyrolipid@LDs.
  • FIG. 18 illustrates uncropped Western blots for FIG. 5E. Lanes used for FIG. 5E are indicated by rectangles.
  • FIG. 19 is a graph of relative tumor proliferation rate in different groups.
  • Gl lipid droplet
  • G2 pyrolipid
  • G3 Pyrolipid@LDs.
  • FIG. 20 is a histological analysis of tumors and major organs in different groups.
  • GO PBS; G3 (+): Pyrolipid@LDs with laser irradiation. Scale bar, 100 pm.
  • a therapeutic material 10 for treating cancer (e.g., a tumor or cancerous tissue 100) in a mammal.
  • the therapeutic material 10 includes a plurality of lipid droplets 12 having contained therein a pyrolipid 14 that includes pyropheophorbide a (PPa) conjugated to a lipid (e.g., 1-palmitoyl- 2-hydroxy-sn-glycero-3-phosphocholine) (FIGS. 2G, 10A, 10B).
  • the lipid droplets 12, as described herein, are loaded with pyrolipids in cellulo by incubation with adipocytes.
  • the lipid droplets 12 generated in this manner include a triacylglycerol core and a phospholipid monolayer decorated with proteins. These loaded lipid droplets 12 (i.e., Pyrolipid@LDs) can then be injected into diseased tissue 100 as seen in FIG. IB (e.g., tumor or the cancerous tissue 100) and then subject to illumination from a light source 20 to enhance the therapeutic effect of the therapeutic material 10.
  • diseased tissue 100 e.g., tumor or the cancerous tissue 100
  • the lipid droplets 12 are generally spherical in shape and may have a diameter within the range of about 60 nm to several micrometers in one embodiment. In another embodiment, the lipid droplets 12 have dimeters in the nanometer range and, more specifically, within the range of about 60 nm to about 225 nm. In some embodiments, the lipid droplets 12 within the therapeutic material 10 have a substantially uniform (i.e., same) diameter. In other embodiments, however, the lipid droplets 12 within the therapeutic material 10 may have varied diameters.
  • FIG. IB illustrates an injection device 22 in the form of a syringe that is used to deliver the therapeutic material 10 to the diseased tissue 100.
  • the lipid droplets 12 may be carried in a buffer solution or animal sera as part of the therapeutic material 10.
  • the lipid droplets 12 may be lyophilized or freeze-dried for storage before use.
  • the lyophilized or freeze-dried lipid droplets 12 may then be reconstituted with buffer solution or animal sera for delivery.
  • the tissue 100 is illuminated with far- red or near-infrared light from the light source 20.
  • the light source 20 may include a laser or light-emitting diodes (LEDs), laser diodes, or the like.
  • one or more light fibers may deliver the light internally to the tumor/cancerous tissue 100.
  • ovarian cancer treatment may require laparoscopic visualization and irradiation using one or more light fibers that are disposed in the laparoscopic instrument or other working tool (e.g., separate catheter, endoscope, or the like that delivers light).
  • the light is applied externally and there is no need for a laparoscopic/endoscopic instrument.
  • the light may be applied directly on the tissue 100 using a variety of light sources 20 such as, for instance, guns, wands, lamps, flashlights, etc.
  • a method of using the therapeutic material 10 includes injecting the therapeutic material 10 into a tumor or cancerous tissue 100 of a mammalian subject and irradiating the tumor or cancerous tissue with far-red or near-infrared light from a light source 20.
  • the light source 20 may irradiate the tissue for several seconds to several minutes.
  • the Pyrolipid@LDs 12 can generate reactive oxygen species (ROS) upon irradiation.
  • ROS reactive oxygen species
  • the generated ROS oxidizes lipid matrix of lipid droplet 12 and increases the oxidative stress.
  • the endoplasmic reticulum (ER) stress caused by excessive accumulation of lipid activates apoptosis signaling pathway, enhancing the metabolic intervention that further promotes the therapeutic effect.
  • the therapeutic material 10 may be used alone or in conjunction with other cancer treatments.
  • the therapeutic material 10 may be used together with radiation and/or chemotherapy.
  • a method of formulating a therapeutic material 10 from lipid droplets 12 includes the following operations. This includes synthesizing a lipid- conjugated pyrolipid if not already synthesized or prepared. This may include conjugating pyropheophorbide a to a lipid such as l-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine. This conjugation may occur through esterification reaction between pyropheophorbide a and lipid (l-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine). Differentiated adipocytes are obtained that will be used for the generation of pyrolipid-loaded lipid droplets 12.
  • 3T3-L1 preadipocytes may be exposed to an adipogenic hormone such as insulin that can induced the formation of adipocyte (FIG. 2A).
  • the level of insulin e.g., concentration
  • the differentiated adipocytes are then incubated with the lipid-conjugated pyrolipid. As explained herein, incubation for about 24 hours was found to generate optimal pyrolipid loading. Thus, in one embodiment, incubation may take place for 24 hours or more. After incubation, the pyrolipid-loaded lipid droplets 12 are then isolated from the adipocytes.
  • lipid isolation kits are available.
  • Lipid Droplet Isolation Kit (Abeam, cat no. ab242290). These kits operate by isolating lipid droplets 12 by simple gradient centrifugation. The incubated cells are homogenized and a gradient is created with the homogenate and the material is centrifuged. The lipid droplets 12 float to top of the gradient and may be removed by pipetting or the like. The isolated pyrolipid-loaded lipid droplets 12 can be lyophilized and stored (e.g., -80°C) for later use. The lyophilized Pyrolipid@LDs 12 can be reconstituted in a buffer solution or animal sera and then injected into the diseased tissue 100.
  • Adipocytes were first differentiated from 3T3-L1 preadipocytes in vitro using a 3T3-L1 Differentiation Kit, in which spherical lipid droplets 12 gradually matured (FIGS. 7A-7B). The formation of lipid droplets 12 in adipocytes was also verified by confocal images after staining lipid droplets 12 with a neutral lipid-specific fluorescent dye (FIG. 2B). The lipid droplets 12 were then isolated from adipocytes using a Lipid Droplet Isolation Kit. The transmission electron microscopy (TEM) image revealed that the extracted lipid droplets 12 maintained their morphological integrity and that their sizes ranged from several hundred nanometers to several micrometers (FIG. 2C).
  • TEM transmission electron microscopy
  • Dynamic light scattering (DLS) measurement further showed two peaks, where the major peak was located at -220 nm and a small peak was -1.2 pm (FIG. 2D).
  • FBS fetal bovine serum
  • the lipid droplets 12 remained stable without obvious size change for 4 days (FIG. 2E and FIG. 8).
  • the physiochemical properties (e.g., size) of lipid droplets 12 could also be manipulated during adipocytes differentiation for different applications. Insulin is one adipogenic hormone that can induce formation of mature adipocytes.
  • the diameter of lipid droplets 12 could increase from 63 nm to 225 nm (FIG. 2F).
  • the polydispersity of lipid droplets 12 decreased from 0.52 to 0.34 at the insulin concentration of 10 pg/mL (FIG. 9), which exhibited a more uniform size distribution at nanoscale.
  • lipid droplets 12 The properties of lipid droplets 12 including the size and size distribution could be modulated according to different applications. While a small number of extracted lipid droplets 12 with microscale (e.g., larger) size have a slightly higher drug loading capacity due to their enlarged lipid matrix, nanoscale lipid droplets 12 accounting for the majority of isolated lipid droplets 12 may exert the benefits of nanomedicine for improved therapeutic effects. In addition, a substantially uniform size distribution could facilitate the manufacture of a more stable formulation that has less batch-to-batch variance in therapeutic outcomes. [0065] Preparation of drug-loaded lipid droplets
  • a lipid-conjugated pyrolipid 14 was first synthesized by conjugating PPa with a lipid (1- palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine) through esterification (FIG. 2G and FIGS. 10A-10B).
  • PPa is a hydrophobic photosensitizer commonly used for photodynamic therapy of cancer with high efficacy. Lipid conjugation was hypothesized to increase the loading capacity of PPa into the lipid droplets 12. The synthesized pyrolipid 14 preserved the UV-Vis absorbance and fluorescence spectra of PPa (FIGS. 11 A and 1 IB).
  • Pyrolipids 14 were loaded into the lipid droplet 12 in cellule by incubation with adipocytes.
  • the co- localization between drug (pyrolipid 14 or PPa) and intracellular lipid droplets 12 were studied via confocal microscopy.
  • pyrolipid 14 specifically entered into the lipid droplets 12 with higher colocalization efficiency compared to PPa that was mainly present in the cytoplasm of adipocytes, demonstrating the beneficial role of lipid conjugation.
  • Pyrolipid-loaded lipid droplets 12 (Pyrolipid@LDs) were then isolated from the adipocytes using a Lipid Droplet Isolation Kit. As expected, the drug concentration in Pyrolipid@LDs 12 was higher than that of PPa@LDs (FIG. 11C).
  • the drug loading process was further optimized by adjusting incubation time. According to colocalization between pyrolipid 14 and lipid droplets 12 after incubating adipocytes with pyrolipid 14 at the same pyrolipid concentration for varying times, it was found that incubation of 24 h is sufficient for the optimal drug loading (FIG. 12). Pyrolipid 14 did not significantly influence the viability of adipocytes at the concentration of 50 mM (FIG. 13 A). In addition, pyrolipid 14 incubation at all tested doses did not change the lipid amount in the lipid droplets 12 as revealed by Lipid (Oil Red O) Staining Kit (FIG. 13B).
  • singlet oxygen sensor green (SOSG) probe was utilized to estimate the singlet oxygen ( 1 02) generation of Pyrolipid 14 and Pyrolipid@LDs 12.
  • SOSG singlet oxygen sensor green
  • the Pyrolipid@LDs 12 retained the ability of Pyrolipid 14 to generate '0 with a 6.7-fold and 8.2-fold enhancement of relative SOSG intensity compared with the control group, respectively (FIG. 14).
  • the lipid- conjugated drug could be specifically be loaded into the lipid droplet 12 after in vitro formation of fully differentiated adipocytes. Neither lipid content in the lipid droplets 12 nor the ROS generating ability of pyrolipid 14 was influenced by the drug loading process.
  • lipid droplets 12 were stained with corresponding fluorescent probes, followed by colocalization analysis with confocal microscopy. Confocal images indicated that lipid droplets 12 mainly accumulated at the lysosome and mitochondria, with relative lower distribution in the ER (FIGS. 3A and 3B). Because the lipid droplet 12 was derived from the ER and had comprehensive communication with multiple organelles including the lysosome and mitochondria to maintain intracellular metabolic equilibrium. For examples, under starvation conditions, lipid droplets 12 could contact the mitochondria and release fatty acids for b- oxidation.
  • lipid droplets 12 were mostly found to be accumulated at the mitochondria and lysosome, which confirmed the retained intracellular communication of isolated lipid droplets 12. Taking advantage of this property, lipid droplets 12 may acquire the organelle-targeting effect in a drug delivery system if delicately designed.
  • the uptake mechanism of lipid droplet 12 was studied by treating SKOV3 cells with different endocytosis inhibitors before incubation with the fluorescent lipid droplets 12 (LDs-FITC). As revealed by flow cytometric analysis (FIG. 3C), the uptake efficiency was significantly decreased when the cells were pretreated with clathrin-mediated endocytosis inhibitor (chlorpromazine, CPZ) and micropinocytosis inhibitor (amiloride, AMI). On the other hand, the cellular uptake did not significantly change in the cells pre-treated with caveolin-mediated endocytosis inhibitor (nystatin, NYS) and lipid raft inhibitor (methyl-//- cyclodextrin, MCD).
  • nystatin caveolin-mediated endocytosis inhibitor
  • MCD lipid raft inhibitor
  • the Pyrolipid@LDs 12 When irradiated with 670 nm laser (100 mW/cm 2 , 5 min), the Pyrolipid@LDs 12 demonstrated promoted therapeutic effect than pure pyrolipid 14, with a half maximal inhibitory concentration (ICso) of 27.4 nM and 163.6 nM, respectively (FIG. 4A). However, it was found that the cytotoxicity of Pyrolipid@LDs 12 was less influenced by hypoxic conditions compared with that of Pyrolipid 14 (FIG. 4B), indicating that encapsulation with lipid droplet 12 could ameliorate the therapeutic performance of a conventional photosensitizer.
  • ICso half maximal inhibitory concentration
  • the elevated cell killing effect of Pyrolipid@LDs 12 was also confirmed by live/dead cell staining and apoptosis detection (FIG. 4C and FIG. 4D), where Pyrolipid@LDs 12 showed highest cytotoxicity to cancer cells among all the treatment groups.
  • SKOV3 cancer cells were incubated with Pyrolipid@LDs 12 in the presence of an ROS scavenger, /V-acetyl-L-cysteine (NAC), lipid peroxidation inhibitor, vitamin E (VE), or ER stress inhibitor, 4-Phenylbutyric acid (PBA), followed by laser irradiation.
  • ROS scavenger /V-acetyl-L-cysteine
  • VE vitamin E
  • ER stress inhibitor 4-Phenylbutyric acid
  • the viability of cancer cells was significantly recovered when they were incubated with ROS scavenger, lipid peroxidation inhibitor, or ER stress inhibitor compared with the control group, suggesting that the toxicity of Pyrolipid@LDs 12 was related to ROS generation, lipid peroxidation, and ER stress (FIG. 5B).
  • TBARS thiobarbituric acid reactive substances
  • Pyrolipid@LDs 12 with laser irradiation demonstrated upregulation of BiP, p- PERK, p-eIF2a and CHOP with 1.36-, 1.74-, 1.84-, 2.23-fold enhancement, respectively, compared with control groups.
  • Pure drug only showed a slightly higher expression of ER stress-related proteins while drug-loaded lipid droplet 12 (Pyrolipid@LDs) exhibited a more significant increase in ER stress-related proteins than the control group (FIG. 5E).
  • FIG. 5 A The proposed mechanism for Pyrolipid@LDs-induced phototoxicity is summarized in FIG. 5 A.
  • the Pyrolipid@LDs 12 could generate ROS under laser irradiation, which further promoted the oxidative stress by inducing lipid peroxidation.
  • overaccumulation of lipids initiated the apoptosis signaling pathway mediated by ER stress.
  • Pyrolipid@LDs 12 generated ROS under laser irradiation, which further caused lipid peroxidation by oxidizing the lipid matrix of the lipid droplets 12.
  • overaccumulation of lipids and elevated oxidative stress destroyed metabolic equilibrium intracellularly and initiated the apoptosis signaling pathway through ER stress.
  • lipid droplet 12 could enhance performance of pyrolipid 14 and the Pyrolipid@LDs 12 could induce the withdrawal of tumors in a light-controlled manner.
  • Pyrolipid@LDs 12 without laser irradiation and pure lipid droplet with laser irradiation showed no tumor growth inhibition compared with the control group.
  • no detectable body weight changes were observed during treatment (FIG. 6E).
  • Major organs, including the heart, liver, lung and kidney showed little pathological abnormalities in the histological analysis (FIG. 20).
  • a lipid-droplet-based drug delivery system has been demonstrated herein.
  • the lipid droplet 12 can promote anticancer therapy through metabolic intervention.
  • the properties and functions of the lipid droplet 12, such as size, size distribution, and intracellular communication can be manipulated, which may provide a controllable and universal platform to deliver anticancer payloads when appropriately designed.
  • the lipid droplet-based system may be further extended to combine with other treatment modalities such as chemotherapy and immunotherapy.
  • the lipid droplet 12 was validated to be stable and biocompatible.
  • the composition and structure of lipid droplets 12 is much less complex than the whole cell, which may avoid potential side effects of some uncertain ingredients inside the cell.
  • lipid droplets 12 after being freeze dried into powder, lipid droplets 12 could be stably preserved, which may advance its further commercialization.
  • This organelle-based delivery system could prime cell-based therapy to a subcellular level, providing new perspectives on drug delivery and holding promise for clinical translation.
  • Lipid droplets 12 were engineered as a drug delivery system for enhanced photodynamic therapy.
  • Adipocytes were obtained from in vitro differentiation of preadipocytes, where 10-20 passage of 3T3-L1 cells were used to ensure the maximum differentiation capability.
  • Lipid droplets 12 were isolated by a commercialized Lipid Droplet Isolation Kit according to manufacturers’ protocol.
  • In vivo anticancer efficacy was evaluated in SKOV3 tumor model. Animals were randomly divided into each group based on tumor volume. Sample size in each experiment was indicated by n values in the FIG. legends. All the samples were included in the experimental analysis, with no data excluded.
  • SKOV3 cells were cultured with McCoy's 5A Medium (Gibco, Invitrogen) containing 10% fetal bovine serum (FBS) (Invitrogen) and 100 U/mL penicillin/streptomycin (Invitrogen).
  • 3T3-L1 cells were cultured with Dulbecco’s modified Eagle medium (DMEM) (Gibco, Invitrogen) containing 10% bovine calf serum (Thermo Fisher Scientific). Both cells were incubated at 37 °C in 5% CO2.
  • DMEM Dulbecco’s modified Eagle medium
  • Female NSG mice (6-10 weeks) were purchased from the Jackson Lab. All animal studies were correspondent with the protocols approved by the Institutional Animal Care and Use Committee at the University of California, Los Angeles (UCLA).
  • lipid droplets 12 were conducted with Lipid Droplet Isolation Kit (Abeam, cat no. ab242290) with a slight modification. Approximately 3 c 10 7 adipocytes or drug -incubated adipocytes were trypsinized and washed twice with 1 c PBS. Then the cells were resuspended in 200 pL of Reagent A and were placed on ice for 10 min. 800 pL of 1 x Reagent B was added to the cells and mixed thoroughly, followed by incubation on ice for another 10 min. Cells were further homogenized by passing them five times through a one inch 27-gauge needle attached to a 3 mL syringe.
  • Pyrolipid 14 and Pyrolipid@LDs 12 at a pyrolipid concentration of 2 mM were mixed with singlet oxygen sensor green (SOSG) probe (10 pM) in PBS buffer. After being irradiated with a 670 nm light at the power density of 100 mW/cm 2 for predetermined time points, 100 pL sample was pipetted out from each well, and the fluorescence intensity of SOSG at 535 nm under excitation at 488 nm before and after laser irradiation was recorded using a microplate reader.
  • SOSG singlet oxygen sensor green
  • lipid droplets 12 100 pL lipid droplets 12 in 4 mL PBS was mixed with 20 mM NHS-fluorescein (FITC) in 200 pL DMSO. After reacting for 2 h at room temperature, the final product was purified by ultracentrifugation.
  • FITC NHS-fluorescein
  • SKOV3 cells were incubated with LD-FITC for 4 h, followed by organelles staining and confocal microscopy imaging. Lysosome was stained with LysoTracker Red DND-99 (500 nM, 1 h); mitochondria was stained with MitoStatus Red (200 nM, 30 min); and ER was stained with ER-Tracker Red (1 pM, 30 min).
  • Laser irradiation for the cellular experiment was set at 100 mW/cm 2 for 5 min on a 670-nm diode laser (Laserglow Technologies).
  • Cell viability was determined by a 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay.
  • MTT 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • Cobalt di chloride (C0CI2, 100 pM) was used as a chemical inducer of hypoxia. Live and dead cell staining using LIVE/DEADTM Cell Imaging Kit (Thermo Fisher, cat no. R37601) and apoptosis detection using Annexin A5 Apoptosis Detection Kit (Biolegend, cat no. 640914) was completed under the guidance of the manufacturer’s protocol. [00102] ROS generation detection
  • TBARS assay kit (Cayman Chemical, cat no. 10009055) was used to detect lipid peroxidation.
  • SKOV3 cells were collected in PBS at 1 x 10 7 cells/mL. The cell solutions were sonicated on ice for 1 min and placed into a 96-well plate. Thiobarbituric acid reagent (13.25 mg/mL) containing different therapeutic formulations was added to the cells (100 pL). After subjected to dark or laser treatment, the 96-well plate was sealed with adhesive tape and was heated at 100 °C for 1 h. Absorbance of TBARS product was measured at 532 nm on a microplate reader after cooling down.
  • SKOV3 cells were applied with different formulations followed by dark or laser treatment. After 12 h, cells were lysed for Western blot analysis.
  • a cell suspension containing 1 x 10 7 SKOV3 cells in 50 pL PBS was inoculated into the right back of NSG mice subcutaneously.
  • the laser condition was set at 150 mW cm 2 for 10 min on a 670-nm laser.
  • the treatment was performed on day 3, 6 and 9 for a total of 3 treatments.

Abstract

Lipid droplets, organelles from adipocytes, were engineered as a therapeutic carrier for enhanced cancer therapy. Lipid droplets were loaded with photosensitizer pyrolipid that is synthesized from pyropheophorbide a (PPa). It was experimentally validated that the engineered lipid droplets maintained their physiological functions to interact with other organelles, and augmented the therapeutic effect through a variety of pathways, including reactive oxygen species (ROS) generation, lipid peroxidation and ER stress. The IC50 of pyrolipid was reduced by 6.0-fold when loaded into the lipid droplet. In vivo results demonstrated that engineered lipid droplets induced significant inhibition effects of tumor growth and improved biocompatibility.

Description

ADIPOCYTE-DERIVED ANTI-CANCER LIPID DROPLETS
Related Application
[0001] This Application claims priority to U.S. Provisional Patent Application No. 63/181,098 filed on April 28, 2021, which is hereby incorporated by reference. Priority is claimed pursuant to 35 U.S.C. § 119 and any other applicable statute.
Technical Field
[0002] The technical field relates to engineered lipid droplets as a therapeutic carrier for enhanced cancer therapy. More specifically, the field of the invention relates to the loading of lipid droplets with a lipid-based photosynthesizer pyrolipid synthesized from pyropheophorbide a (PPa). The drug-loaded lipid droplets (sometimes referred to herein as Pyrolipid@LDs) can then be injected into tumor or cancerous tissue and then subject to illumination from a light source to enhance the therapeutic effect.
Background
[0003] As a hallmark of cancer, abnormal metabolism that sustains unlimited tumor proliferation has gained considerable attentions as the target for cancer therapy. By building up an accommodative tumor microenvironment and reprogramming metabolic pathways, tumor cells can circumvent the restrictions from the body and take a leading role in their progression, during which time multiple nutrients and energy supplies, including glucose, glutamine, lipid, oxygen and ATP, are desperately needed. In particular, lipid metabolism plays a fundamental role in the life maintenance of both tumor cells and other non-malignant cells in the tumor microenvironment, such as tumor-associated adipocytes, macrophages and neutrophils that promote tumor growth, invasion and metastasis. Therefore, targeting lipid metabolism in the tumor tissue has become an emerging approach for cancer therapy, where this abnormal tumor metabolism pathway can also be leveraged for the development of new drug delivery strategies.
[0004] Increasing evidence demonstrates that aggressive cancer cells rely on highly active lipid metabolism, either by gaining lipids from circulation or by upregulating de novo lipid synthesis. Although tumor cells may experience lipotoxicity from excessive lipid accumulation through initiation of endoplasmic reticulum (ER) stress and oxidative stress, these cancerous cells can still survive under such harsh conditions by inducing the formation of lipid droplet, a lipid-rich subcellular compartment that maintains the equilibrium of lipid metabolism. These flexible adaptations in tumor cells in response to internal or external stimuli construct a buffering system to minimize potential damages, which is also one of the major obstacles that dampen or frustrate many therapeutic strategies. The metabolic adaptations and treatment resistance during stress responses pose urgent needs for potent metabolic intervention that can disturb the homeostasis inside the tumor or localized cancer environment.
Summary
[0005] In one embodiment, tailored lipid droplets are engineered as a new drug delivery system for cancer therapy. Lipid droplets are abundant in the adipocytes at the nano- and microscale size. As the main cellular organelle for lipid storage and transport, the lipid droplet is composed of a hydrophobic core filled with neutral triacylglycerol and a phospholipid monolayer coated with certain proteins as the outer shell as illustrated in FIG. 2A. Due to its unique structure, the lipid droplet can be engineered into a universal drug delivery system for targeted therapy as the lipid core provides an ideal reservoir for lipophilic drugs and the surface proteins on the outer shell retain the capability to interact with other organelles associated with metabolism regulation. Here, the physicochemical and physiological properties of lipid droplets can be leveraged in accommodation with different applications. When combined with effective photodynamic therapy by encapsulating lipid- based photosensitizers in the lipid droplets, the engineered lipid droplet could synergistically enhance the therapeutic effect through various pathways. In addition, this cell-derived material showed limited side effects, while the physiological stability renders them promising clinical translation potential.
[0006] In one embodiment, a therapeutic material for treating cancer includes a plurality of lipid droplets having contained therein a drug that, in one preferred embodiment, is pyropheophorbide a. The pyropheophorbide a may be conjugated to a lipid (e.g., 1-palmitoyl- 2-hydroxy-sn-glycero-3- phosphocholine) to improve droplet loading. These loaded lipid droplets (i.e., Pyrolipid@LDs) can then be injected into tumor or cancerous tissue and then subject to illumination from a light source to enhance the therapeutic effect.
[0007] In another embodiment, a method of using the therapeutic material includes injecting the lipid droplets into a tumor or cancerous tissue of a mammalian subject and irradiating the tumor or cancerous tissue with far-red or near-infrared light. The light may be far-red or near-infrared light that is emitted from, for example, a laser, light-emitting diode (LED, laser diode, or the like. In some embodiments, one or more light fibers (e.g., as part of a catheter, laparoscope or the like) may deliver the light internally to the tumor/cancerous tissue.
[0008] In still another embodiment, a method of formulating therapeutic lipid droplets includes the operations of synthesizing a lipid-conjugated pyrolipid and incubating the lipid- conjugated pyrolipid with adipocytes. Pyrolipid-loaded lipid droplets are then isolated from the adipocytes. The isolated pyrolipid-loaded lipid droplets can be stored for later use (e.g., freeze dried). The freeze dried Pyrolipid@LDs can be reconstituted in a buffer solution or sera and then injected into the diseased tissue.
Brief Description of the Drawings
[0009] FIG. 1A illustrates an injection device (e.g., syringe) that contains the therapeutic material for treating cancer. A zoomed-in view of the pyrolipid-loaded lipid droplets (Pyrolipid@LDs) is also illustrated.
[0010] FIG. IB illustrates an illumination device that is used to illuminate tissue that has been injected with the therapeutic material.
[0011] FIG. 2 A illustrates a process of generating or manufacturing pyrolipid-loaded lipid droplets (Pyrolipid@LDs).
[0012] FIG. 2B shows confocal images of fully differentiated adipocytes, in which lipid droplets were stained with BODIPY® (boron-dipyrromethene) 505/515 (green). Scale bar, 50 pm.
[0013] FIG. 2C shows a transmission electron microscopy (TEM) image of isolated lipid droplets. Scale bar, 2 pm.
[0014] FIG. 2D illustrates the average hydrodynamic size of lipid droplets determined by dynamic light scattering (DLS).
[0015] FIG. 2E shows the stability of lipid droplet in PBS and DMEM culture medium containing 10% FBS. Diameter was determined by DLS. Data are presented as mean± s.d.
(n = 3).
[0016] FIG. 2F illustrates a graph showing the size control of lipid droplets after stimulation at different insulin concentrations. Data are presented as mean ± s.d. (n = 3).
[0017] FIG. 2G illustrates the structure of the drug pyrolipid synthesized by conjugating photosensitizer pyropheophorbide a (PPa) with a lipid (l-palmitoyl-2-hydroxy-sn-glycero-3- phosphocholine). [0018] FIG. 2H illustrates confocal images of pyrolipid accumulation inside the adipocytes. Photosensitizers were shown in red (middle column). Lipid droplets were shown in green (BODIPY® 505/515 labeled) (left column). Merged images on in the right column. Scale bar, 50 pm.
[0019] FIG. 3A shows confocal images represent the distribution of fluorescein-labeled lipid droplets in SKOV3 cancer cells. Lysosome, mitochondria and ER were stained with LysoTracker Red DND-99, MitoStatus Red and ER-Tracker Red, respectively. Scale bar, 20 pm. Scale bar for selected area, 10 pm.
[0020] FIG. 3B illustrates Pearson’s colocalization coefficient between lipid droplet and different organelles.
[0021] FIG. 3C illustrates the flow cytometric analysis of lipid droplets uptake in SKOV3 cells pretreated with different endocytosis inhibitors. Ctrl: control group without any inhibitor pre-treatment. Chlorpromazine (CPZ), nystatin (NYS), amiloride (AMI), methyl -//- cyclodextrin (MCD), represented the inhibitor of clathrin-mediated endocytosis, caveolin- mediated endocytosis, micropinocytosis, and lipid raft, respectively. Data are presented as mean ± s.d. (n = 3). Statistical significance was calculated via one-way ANOVA with a Tukey post-hoc test. *P< 0.05; **P< 0.01; ***P< 0.001.
[0022] FIG. 3D illustrates the flow cytometric analysis of lipid droplet uptake at different temperatures. Ctrl represents the SKOV3 cells without lipid droplet incubation (left peak). Middle peak is 4 °C. Right peak is 37 °C.
[0023] FIG. 4A illustrates in vitro cytotoxicity and mechanism investigation results with FIG. 4A showing IC50 values of pyrolipid and Pyrolipid@LDs. Data are presented as mean ± s.d. (n = 3).
[0024] FIG. 4B illustrates cytotoxicity results (cell viability %) of pyrolipid and Pyrolipid@LDs under normoxic and hypoxic conditions. Data are presented as mean ± s.d.
(n = 3).
[0025] FIG. 4C shows live-dead cell imaging of dark and laser group after incubating cells with Pyrolipid@LDs. Live cells were stained as green color (top image) and dead cells as red color (bottom image). Scale bar, 100 pm.
[0026] FIG. 4D illustrates flow cytometric analysis of cell apoptosis in different treatment groups determined by Annexin V-FITC/PI staining. GO: control group; Gl: lipid droplet; G2: Pyrolipid; G3: Pyrolipid@LDs. (-): Dark; (+): Laser. Data are presented as mean± s.d.
(n = 3). [0027] FIG. 5A illustrates a proposed mechanism for Pyrolipid@LDs-induced phototoxicity. After entering the SKOV3 cells, Pyrolipid@LDs can generate ROS upon laser (or light source) irradiation. The generated ROS oxidizes lipid matrix of lipid droplet and increases the oxidative stress. In addition, the ER stress caused by excessive accumulation of lipid activates apoptosis signaling pathway, enhancing the metabolic intervention that further promotes the therapeutic effect.
[0028] FIG. 5B illustrates cytotoxicity results of Pyrolipid@LDs in the presence of ROS scavenger N-acetyl-L-cysteine (NAC, 2 mM), lipid peroxidation inhibitor vitamin E (VE, 250 mM) and ER stress inhibitor 4-Phenylbutyric acid (PBA, 100 mM). Ctrl represents control group without any inhibitor addition. Data are presented as mean ± s.d. (n = 3).
[0029] FIG. 5C illustrates ROS generation of different formulations under dark and laser conditions detected by fluorescent DCFDA probe. GO: control group; Gl: lipid droplet; G2: pyrolipid; G3: Pyrolipid@LDs. Laser: cells were irradiated with 670 nm light at a power density of 100 mW/cm2 for 5 min. DCFDA concentration, 25 pM. Data are presented as mean ± s.d. (n = 3).
[0030] FIG. 5D illustrates results of lipid peroxidation detected by TBARS assay kit. Data are presented as mean ± s.d. (n = 3).
[0031] FIG. 5E illustrates Western blot analysis of ER stress-related proteins. Statistical significance was calculated via one-way ANOVA with a Tukey post-hoc test. *P < 0.05;
**P < 0.01; ***P <0.001.
[0032] FIG. 6A illustrates a schematic of Pyrolipid@LDs mediated photodynamic therapy in a mouse model.
[0033] FIGS. 6B and 6C illustrate the in vivo treatment efficacy of Pyrolipid@LDs. FIG. 6B shows individual tumor growth kinetics while FIG. 6C shows average tumor growth kinetics in different groups. GO: PBS; Gl: lipid droplet; G2: pyrolipid; G3: Pyrolipid@LDs. (-): Dark (without laser); (+): Laser. Data are presented as mean± s.d. (n = 8). Statistical significance was calculated via one-way ANOVA with a Tukey post-hoc test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001.
[0034] FIG. 6D shows representative photographic images of mice and excised tumors in GO and G3 (+) groups.
[0035] FIG. 6E illustrates a graph showing mouse body weight as a function of time (days).
[0036] FIG. 7A illustrates a schematic of pre-adipocyte differentiation, maintenance, and lipid droplet isolation. [0037] FIG. 7B illustrates representative images at different time points during differentiation. Images were visualized by light microscopy under 20X lens.
[0038] FIG. 8 illustrates TEM images of lipid droplets in PBS and FBS -containing DMEM culture medium at different time points. Scale bar, 500 nm. The stability of lipid droplet in different mediums is shown.
[0039] FIG. 9 is a graph of the polydispersity index (PDI) of isolated lipid droplets at different time points after stimulation with insulin. The duration of insulin stimulation was compared between 3 and 9 days. Data are presented as mean ± s.d. (n = 3).
[0040] FIG. 10A illustrates the synthesis of pyrolipid. Pyrolipid was prepared from the esterification between pyropheophorbide a (PPa) and l-palmitoyl-2-hydroxy-sn-glycero-3- phosphocholine.
[0041] FIG. 10B illustrates the 'H NMR spectrum of pyrolipid (in d-DMSO).
[0042] FIG. 11 A illustrates UV-Vis spectra of PPa and pyrolipid.
[0043] FIG. 1 IB illustrates fluorescence spectra of photosensitizers under excitation wavelength of 410 nm.
[0044] FIG. llC illustrates fluorescence spectra of photosensitizer-loaded lipid droplets under excitation wavelength of 410 nm.
[0045] FIG. 12 includes confocal images of pyrolipid accumulation in the lipid droplets (LDs) at different time points. Fully differentiated adipocytes were incubated with pyrolipid at a concentration of 5 mM. After different incubation time, lipid droplet was stained with BODIPY® 505/515 and visualized by confocal microscopy. Scale bar, 50 pm.
[0046] FIG. 13A illustrates optimization of pyrolipid concentration for incubation. Cell viability of adipocytes after incubating with different concentrations of pyrolipid was detected by an MTT assay.
[0047] FIG. 13B is a histogram showing the influence of pyrolipid on the amount of lipids in the lipid droplets. The amount of lipid in the lipid droplets was measured by Oil Red O staining and recording OD value at 540 nm through a microplate reader. Data are presented as mean ± s.d. (n = 3).
[0048] FIG. 14 illustrates a graph of singlet oxygen generation abilities of Pyrolipid and Pyrolipid@LDs determined by SOSG probe under laser irradiation. Data are presented as mean ± s.d. (n = 3).
[0049] FIG. 15 illustrates images showing the distribution of Pyrolipid@LDs-FITC in SKOV3 cells at different time points (lh, 4h, 12h). Scale bar, 50 pm. [0050] FIG. 16 illustrates the dark toxicity of pyrolipid (left data in pairs) and Pyrolipid@LDs (right data in pairs) on SKOV3 cells. Data are presented as mean± s.d.
(n = 3).
[0051] FIG. 17 illustrates the semi-quantitative analysis of ER stress-related protein expression. The relative protein expressions were normalized to that of b-actin. GO: control group; Gl: lipid droplet; G2: pyrolipid; G3: Pyrolipid@LDs. (-): Dark (without laser); (+): Laser.
[0052] FIG. 18 illustrates uncropped Western blots for FIG. 5E. Lanes used for FIG. 5E are indicated by rectangles.
[0053] FIG. 19 is a graph of relative tumor proliferation rate in different groups. Relative tumor growth rate (T/C Ratio) was calculated according to T/C Ratio = TRTV/CRTV X 100%, where TRTV and CRTV represent the relative tumor volumes of treatment group and control group, respectively. Gl: lipid droplet; G2: pyrolipid; G3: Pyrolipid@LDs. (-): Dark (without laser); (+): Laser.
[0054] FIG. 20 is a histological analysis of tumors and major organs in different groups. GO: PBS; G3 (+): Pyrolipid@LDs with laser irradiation. Scale bar, 100 pm.
Detailed Description of Illustrated Embodiments [0055] As described herein, and with reference to FIGS. 1 A and IB, a therapeutic material 10 is disclosed for treating cancer (e.g., a tumor or cancerous tissue 100) in a mammal. The therapeutic material 10 includes a plurality of lipid droplets 12 having contained therein a pyrolipid 14 that includes pyropheophorbide a (PPa) conjugated to a lipid (e.g., 1-palmitoyl- 2-hydroxy-sn-glycero-3-phosphocholine) (FIGS. 2G, 10A, 10B). The lipid droplets 12, as described herein, are loaded with pyrolipids in cellulo by incubation with adipocytes. The lipid droplets 12 generated in this manner include a triacylglycerol core and a phospholipid monolayer decorated with proteins. These loaded lipid droplets 12 (i.e., Pyrolipid@LDs) can then be injected into diseased tissue 100 as seen in FIG. IB (e.g., tumor or the cancerous tissue 100) and then subject to illumination from a light source 20 to enhance the therapeutic effect of the therapeutic material 10.
[0056] The lipid droplets 12 are generally spherical in shape and may have a diameter within the range of about 60 nm to several micrometers in one embodiment. In another embodiment, the lipid droplets 12 have dimeters in the nanometer range and, more specifically, within the range of about 60 nm to about 225 nm. In some embodiments, the lipid droplets 12 within the therapeutic material 10 have a substantially uniform (i.e., same) diameter. In other embodiments, however, the lipid droplets 12 within the therapeutic material 10 may have varied diameters.
[0057] FIG. IB illustrates an injection device 22 in the form of a syringe that is used to deliver the therapeutic material 10 to the diseased tissue 100. The lipid droplets 12 may be carried in a buffer solution or animal sera as part of the therapeutic material 10. In some embodiments, the lipid droplets 12 may be lyophilized or freeze-dried for storage before use. The lyophilized or freeze-dried lipid droplets 12 may then be reconstituted with buffer solution or animal sera for delivery. In one embodiment, once the therapeutic material 10 has been injected or otherwise delivered to the tissue 100, the tissue 100 is illuminated with far- red or near-infrared light from the light source 20. Far-red and near-infrared spans wavelengths from generally about 600 nm to about 1,000 nm. The light source 20 may include a laser or light-emitting diodes (LEDs), laser diodes, or the like. In some embodiments, one or more light fibers (e.g., as part of a catheter, laparoscope or the like) may deliver the light internally to the tumor/cancerous tissue 100. For example, ovarian cancer treatment may require laparoscopic visualization and irradiation using one or more light fibers that are disposed in the laparoscopic instrument or other working tool (e.g., separate catheter, endoscope, or the like that delivers light). In still other applications (e.g., treatment of skin cancer), the light is applied externally and there is no need for a laparoscopic/endoscopic instrument. The light may be applied directly on the tissue 100 using a variety of light sources 20 such as, for instance, guns, wands, lamps, flashlights, etc.
[0058] In another embodiment, a method of using the therapeutic material 10 includes injecting the therapeutic material 10 into a tumor or cancerous tissue 100 of a mammalian subject and irradiating the tumor or cancerous tissue with far-red or near-infrared light from a light source 20. The light source 20 may irradiate the tissue for several seconds to several minutes. The Pyrolipid@LDs 12 can generate reactive oxygen species (ROS) upon irradiation. The generated ROS oxidizes lipid matrix of lipid droplet 12 and increases the oxidative stress. In addition, the endoplasmic reticulum (ER) stress caused by excessive accumulation of lipid activates apoptosis signaling pathway, enhancing the metabolic intervention that further promotes the therapeutic effect. The therapeutic material 10 may be used alone or in conjunction with other cancer treatments. For example, the therapeutic material 10 may be used together with radiation and/or chemotherapy.
[0059] In still another embodiment, a method of formulating a therapeutic material 10 from lipid droplets 12 includes the following operations. This includes synthesizing a lipid- conjugated pyrolipid if not already synthesized or prepared. This may include conjugating pyropheophorbide a to a lipid such as l-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine. This conjugation may occur through esterification reaction between pyropheophorbide a and lipid (l-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine). Differentiated adipocytes are obtained that will be used for the generation of pyrolipid-loaded lipid droplets 12. 3T3-L1 preadipocytes may be exposed to an adipogenic hormone such as insulin that can induced the formation of adipocyte (FIG. 2A). The level of insulin (e.g., concentration) may be used to control the degree of differentiation (and diameter polydispersity of the lipid droplets 12). The differentiated adipocytes are then incubated with the lipid-conjugated pyrolipid. As explained herein, incubation for about 24 hours was found to generate optimal pyrolipid loading. Thus, in one embodiment, incubation may take place for 24 hours or more. After incubation, the pyrolipid-loaded lipid droplets 12 are then isolated from the adipocytes. Commercially available lipid isolation kits are available. An example includes the Lipid Droplet Isolation Kit (Abeam, cat no. ab242290). These kits operate by isolating lipid droplets 12 by simple gradient centrifugation. The incubated cells are homogenized and a gradient is created with the homogenate and the material is centrifuged. The lipid droplets 12 float to top of the gradient and may be removed by pipetting or the like. The isolated pyrolipid-loaded lipid droplets 12 can be lyophilized and stored (e.g., -80°C) for later use. The lyophilized Pyrolipid@LDs 12 can be reconstituted in a buffer solution or animal sera and then injected into the diseased tissue 100.
[0060] Results
[0061] Engineering and characterization of lipid droplet
[0062] Adipocytes were first differentiated from 3T3-L1 preadipocytes in vitro using a 3T3-L1 Differentiation Kit, in which spherical lipid droplets 12 gradually matured (FIGS. 7A-7B). The formation of lipid droplets 12 in adipocytes was also verified by confocal images after staining lipid droplets 12 with a neutral lipid-specific fluorescent dye (FIG. 2B). The lipid droplets 12 were then isolated from adipocytes using a Lipid Droplet Isolation Kit. The transmission electron microscopy (TEM) image revealed that the extracted lipid droplets 12 maintained their morphological integrity and that their sizes ranged from several hundred nanometers to several micrometers (FIG. 2C). Dynamic light scattering (DLS) measurement further showed two peaks, where the major peak was located at -220 nm and a small peak was -1.2 pm (FIG. 2D). When dispersed in PBS or fetal bovine serum (FBS)-containing medium, the lipid droplets 12 remained stable without obvious size change for 4 days (FIG. 2E and FIG. 8). [0063] The physiochemical properties (e.g., size) of lipid droplets 12 could also be manipulated during adipocytes differentiation for different applications. Insulin is one adipogenic hormone that can induce formation of mature adipocytes. By elevating the insulin concentration during preadipocyte differentiation from 0.1 to 10 pg/mL, the diameter of lipid droplets 12 could increase from 63 nm to 225 nm (FIG. 2F). By reducing the duration of insulin stimulation, the polydispersity of lipid droplets 12 decreased from 0.52 to 0.34 at the insulin concentration of 10 pg/mL (FIG. 9), which exhibited a more uniform size distribution at nanoscale.
[0064] The properties of lipid droplets 12 including the size and size distribution could be modulated according to different applications. While a small number of extracted lipid droplets 12 with microscale (e.g., larger) size have a slightly higher drug loading capacity due to their enlarged lipid matrix, nanoscale lipid droplets 12 accounting for the majority of isolated lipid droplets 12 may exert the benefits of nanomedicine for improved therapeutic effects. In addition, a substantially uniform size distribution could facilitate the manufacture of a more stable formulation that has less batch-to-batch variance in therapeutic outcomes. [0065] Preparation of drug-loaded lipid droplets
[0066] To demonstrate that the lipid droplet could be employed as a drug delivery system, a lipid-conjugated pyrolipid 14 was first synthesized by conjugating PPa with a lipid (1- palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine) through esterification (FIG. 2G and FIGS. 10A-10B). PPa is a hydrophobic photosensitizer commonly used for photodynamic therapy of cancer with high efficacy. Lipid conjugation was hypothesized to increase the loading capacity of PPa into the lipid droplets 12. The synthesized pyrolipid 14 preserved the UV-Vis absorbance and fluorescence spectra of PPa (FIGS. 11 A and 1 IB). Pyrolipids 14 were loaded into the lipid droplet 12 in cellule by incubation with adipocytes. The co- localization between drug (pyrolipid 14 or PPa) and intracellular lipid droplets 12 were studied via confocal microscopy. As shown in FIG. 2H, pyrolipid 14 specifically entered into the lipid droplets 12 with higher colocalization efficiency compared to PPa that was mainly present in the cytoplasm of adipocytes, demonstrating the beneficial role of lipid conjugation. Pyrolipid-loaded lipid droplets 12 (Pyrolipid@LDs) were then isolated from the adipocytes using a Lipid Droplet Isolation Kit. As expected, the drug concentration in Pyrolipid@LDs 12 was higher than that of PPa@LDs (FIG. 11C).
[0067] The drug loading process was further optimized by adjusting incubation time. According to colocalization between pyrolipid 14 and lipid droplets 12 after incubating adipocytes with pyrolipid 14 at the same pyrolipid concentration for varying times, it was found that incubation of 24 h is sufficient for the optimal drug loading (FIG. 12). Pyrolipid 14 did not significantly influence the viability of adipocytes at the concentration of 50 mM (FIG. 13 A). In addition, pyrolipid 14 incubation at all tested doses did not change the lipid amount in the lipid droplets 12 as revealed by Lipid (Oil Red O) Staining Kit (FIG. 13B).
[0068] In order to verify the in vitro PDT effect, singlet oxygen sensor green (SOSG) probe was utilized to estimate the singlet oxygen ( 102) generation of Pyrolipid 14 and Pyrolipid@LDs 12. After exposure to a 670 nm laser, the Pyrolipid@LDs 12 retained the ability of Pyrolipid 14 to generate '0 with a 6.7-fold and 8.2-fold enhancement of relative SOSG intensity compared with the control group, respectively (FIG. 14). Thus, the lipid- conjugated drug could be specifically be loaded into the lipid droplet 12 after in vitro formation of fully differentiated adipocytes. Neither lipid content in the lipid droplets 12 nor the ROS generating ability of pyrolipid 14 was influenced by the drug loading process.
[0069] Uptake and distribution of the lipid droplets in cancer cells [0070] Before validating the therapeutic effect of Pyrolipid@LDs 12, the uptake mechanism and distribution of lipid droplets 12 in cancer cells were investigated. Lipid droplets 12 were first labeled with fluorescein (FITC)-NHS. Then, the fluorescently labeled Pyrolipid@LDs-FITC were incubated with SKOV3 cancer cells. The conjugation of FITC to the Pyrolipid@LDs 12 was validated by confocal images, exhibiting the overlaid color from red pyrolipid and green LDs-FICT (FIG. 15). The increasing intracellular fluorescence intensity against time indicated that the drug-loaded lipid droplet 12 could indeed enter into cancer cells.
[0071] To study the distribution of lipid droplets 12 within cancer cells, different intracellular compartments, including the lysosome, mitochondria and endoplasmic reticulum (ER), were stained with corresponding fluorescent probes, followed by colocalization analysis with confocal microscopy. Confocal images indicated that lipid droplets 12 mainly accumulated at the lysosome and mitochondria, with relative lower distribution in the ER (FIGS. 3A and 3B). Because the lipid droplet 12 was derived from the ER and had comprehensive communication with multiple organelles including the lysosome and mitochondria to maintain intracellular metabolic equilibrium. For examples, under starvation conditions, lipid droplets 12 could contact the mitochondria and release fatty acids for b- oxidation. Lipid droplet shuttling to the lysosome driven by autophagy was closely correlated with lipid hydrolysis and recycling. Here, when co-localized with fluorescent dye-labeled organelles, lipid droplets 12 were mostly found to be accumulated at the mitochondria and lysosome, which confirmed the retained intracellular communication of isolated lipid droplets 12. Taking advantage of this property, lipid droplets 12 may acquire the organelle-targeting effect in a drug delivery system if delicately designed.
[0072] The uptake mechanism of lipid droplet 12 was studied by treating SKOV3 cells with different endocytosis inhibitors before incubation with the fluorescent lipid droplets 12 (LDs-FITC). As revealed by flow cytometric analysis (FIG. 3C), the uptake efficiency was significantly decreased when the cells were pretreated with clathrin-mediated endocytosis inhibitor (chlorpromazine, CPZ) and micropinocytosis inhibitor (amiloride, AMI). On the other hand, the cellular uptake did not significantly change in the cells pre-treated with caveolin-mediated endocytosis inhibitor (nystatin, NYS) and lipid raft inhibitor (methyl-//- cyclodextrin, MCD). Moreover, the uptake rate was also impeded at low temperature (FIG. 3D), indicating that the process was energy -dependent. Taken together, these results suggested that the lipid droplet 12 was capable of entering into cancer cells via clathrin- mediated endocytosis and micropinocytosis, as well as maintaining multiple interactions with intracellular organelles, which could be favorable to control the intracellular fate of therapeutics.
[0073] In vitro cytotoxicity of Pyrolipid@LDs and mechanism investigation [0074] After confirming that the isolated lipid droplet 12 could not only function as a reservoir for lipophilic drugs, but also maintain interactions with other organelles, the in vitro therapeutic effect of Pyrolipid@LDs 12 was evaluated using SKOV3 cancer cells. Neither pyrolipid 14 nor Pyrolipid@LDs 12 exhibited obvious dark toxicity (without laser irradiation) toward cancer cells when the drug concentration was below 500 nM (FIG. 16). When irradiated with 670 nm laser (100 mW/cm2, 5 min), the Pyrolipid@LDs 12 demonstrated promoted therapeutic effect than pure pyrolipid 14, with a half maximal inhibitory concentration (ICso) of 27.4 nM and 163.6 nM, respectively (FIG. 4A). However, it was found that the cytotoxicity of Pyrolipid@LDs 12 was less influenced by hypoxic conditions compared with that of Pyrolipid 14 (FIG. 4B), indicating that encapsulation with lipid droplet 12 could ameliorate the therapeutic performance of a conventional photosensitizer. The elevated cell killing effect of Pyrolipid@LDs 12 was also confirmed by live/dead cell staining and apoptosis detection (FIG. 4C and FIG. 4D), where Pyrolipid@LDs 12 showed highest cytotoxicity to cancer cells among all the treatment groups.
[0075] Based on the fact that the Pyrolipid@LDs 12 exhibited augmented anticancer effect and less dependence on the normoxic condition compared to pure pyrolipid 14, it is reasonable to hypothesize that the lipid droplet 12 also plays an important role in the therapy. It was assumed that ROS generated by pyrolipid 14 could oxidize the massive amounts of lipid inside the lipid droplet 12, resulting in lipid peroxidation which further promoted oxidative stress to cause cell death. In addition, overaccumulation of lipids within cells caused lipotoxity and disturbed the homeostasis of intracellular lipid metabolism that was beyond the affordability of cancer cells, thereby initiating ER stress and the subsequent apoptosis signaling pathway.
[0076] To substantiate this hypothesis, SKOV3 cancer cells were incubated with Pyrolipid@LDs 12 in the presence of an ROS scavenger, /V-acetyl-L-cysteine (NAC), lipid peroxidation inhibitor, vitamin E (VE), or ER stress inhibitor, 4-Phenylbutyric acid (PBA), followed by laser irradiation. The viability of cancer cells was significantly recovered when they were incubated with ROS scavenger, lipid peroxidation inhibitor, or ER stress inhibitor compared with the control group, suggesting that the toxicity of Pyrolipid@LDs 12 was related to ROS generation, lipid peroxidation, and ER stress (FIG. 5B).
[0077] ROS generation detected by 2', 7'-dichlorofluorescin diacetate (DCFDA) probe indicated that control group (no lipid droplet 12 or drug treatment) and pure lipid droplet both had relatively low ROS generation, regardless of the laser irradiation (FIG. 5C). While Pyrolipid@LDs 12 maintained the ability of pyrolipid 14 to generate ROS under irradiation, the total amount of generated ROS was compromised compared to that of pure pyrolipid 14. [0078] As one of the byproducts of lipid peroxidation, thiobarbituric acid reactive substances (TBARS) were detected by a TBARS assay kit (FIG. 5D) to reflect the level of lipid peroxidation. After cancer cells were incubated with Pyrolipid@LDs 12 followed by laser irradiation, the degree of lipid peroxidation was much higher than the control group. Once in the presence of ROS scavenger or lipid peroxidation inhibitor, alleviated level of TBARS demonstrated that the lipid peroxidation during cell death was mediated by light- triggered ROS generation (FIG. 5D).
[0079] Additionally, the expression of lipotoxicity- and ER stress-related proteins was detected by Western blot analysis (FIGS. 5E, 17, and 18). In order to adapt to the ER stress, cancer cells promote detachment between BiP and PERK proteins and upregulate the phosphorylation of PERK, followed by the phosphorylation of eIF2a that can induce the upregulation of apoptotic protein CHOP. During treatment with different formulations, pure lipid droplet treatment showed no obvious change in the expression of ER stress-related proteins. Pyrolipid@LDs 12 with laser irradiation demonstrated upregulation of BiP, p- PERK, p-eIF2a and CHOP with 1.36-, 1.74-, 1.84-, 2.23-fold enhancement, respectively, compared with control groups. Pure drug only showed a slightly higher expression of ER stress-related proteins while drug-loaded lipid droplet 12 (Pyrolipid@LDs) exhibited a more significant increase in ER stress-related proteins than the control group (FIG. 5E). It is believed that the synergistic effect between drug and lipid droplet 12 resulted in the elevation of ER stress, where ROS generated by pyrolipid 14 under laser irradiation functioned as a trigger and lipid droplet 12 provided substrate to boost ER stress.
[0080] The proposed mechanism for Pyrolipid@LDs-induced phototoxicity is summarized in FIG. 5 A. In brief, after endocytosis into cancer cells, the Pyrolipid@LDs 12 could generate ROS under laser irradiation, which further promoted the oxidative stress by inducing lipid peroxidation. Moreover, overaccumulation of lipids initiated the apoptosis signaling pathway mediated by ER stress. Taken together, after endocytosis into cancer cells, Pyrolipid@LDs 12 generated ROS under laser irradiation, which further caused lipid peroxidation by oxidizing the lipid matrix of the lipid droplets 12. Moreover, overaccumulation of lipids and elevated oxidative stress destroyed metabolic equilibrium intracellularly and initiated the apoptosis signaling pathway through ER stress.
[0081] In vivo tumor growth inhibition
[0082] In vivo anti-cancer efficacy of Pyrolipid@LDs 12 was evaluated in the SKOV3- tumor mouse model. After the volume of subcutaneously injected tumor reached 50-100 mm3, 40 mice were randomly divided into five groups: PBS, Pyrolipid@LDs 12 without laser irradiation, lipid droplet with laser irradiation, pyrolipid with laser irradiation and Pyrolipid@LDs 12 with laser irradiation. The laser irradiation was completed on a 670 nm- light at the power density of 150 mW/cm2 for 10 min. Tumor growth and body weight of mice in different groups were monitored every three days. After three treatments at the day 3, 6, and 9 (FIG. 6A), individual and average tumor growth kinetics (FIGS. 6B-6D) showed that, tumor growth in the Pyrolipid@LDs group with laser irradiation was significantly inhibited compared with the control group or pyrolipid with laser irradiation. Specifically, the relative tumor proliferation rate of mice in Pyrolipid@LDs group with laser irradiation was reduced to 8.0 % (FIG. 19), which was 10.0-fold lower than that in the Pyrolipid@LDs group without laser irradiation and 5.4-fold lower than that in pyrolipid group with laser irradiation. Of note, complete tumor regression was found in 3 of 8 mice receiving Pyrolipid@LDs 12 with laser irradiation. These results demonstrated that the lipid droplet 12 could enhance performance of pyrolipid 14 and the Pyrolipid@LDs 12 could induce the withdrawal of tumors in a light-controlled manner. Moreover, Pyrolipid@LDs 12 without laser irradiation and pure lipid droplet with laser irradiation showed no tumor growth inhibition compared with the control group. Meanwhile, no detectable body weight changes were observed during treatment (FIG. 6E). Major organs, including the heart, liver, lung and kidney showed little pathological abnormalities in the histological analysis (FIG. 20). These in vivo results along with the in vitro evaluation verified that the Pyrolipid@LDs-mediated photodynamic therapy showed improved anti-tumor efficacy with minimal systemic toxicity.
[0083] A lipid-droplet-based drug delivery system has been demonstrated herein. Instead of being a mere drug carrier, the lipid droplet 12 can promote anticancer therapy through metabolic intervention. The properties and functions of the lipid droplet 12, such as size, size distribution, and intracellular communication can be manipulated, which may provide a controllable and universal platform to deliver anticancer payloads when appropriately designed. The lipid droplet-based system may be further extended to combine with other treatment modalities such as chemotherapy and immunotherapy. In the meantime, the lipid droplet 12 was validated to be stable and biocompatible. The composition and structure of lipid droplets 12 is much less complex than the whole cell, which may avoid potential side effects of some uncertain ingredients inside the cell. Moreover, after being freeze dried into powder, lipid droplets 12 could be stably preserved, which may advance its further commercialization. This organelle-based delivery system could prime cell-based therapy to a subcellular level, providing new perspectives on drug delivery and holding promise for clinical translation.
[0084] Materials and Methods
[0085] Lipid droplets 12 were engineered as a drug delivery system for enhanced photodynamic therapy. Adipocytes were obtained from in vitro differentiation of preadipocytes, where 10-20 passage of 3T3-L1 cells were used to ensure the maximum differentiation capability. Lipid droplets 12 were isolated by a commercialized Lipid Droplet Isolation Kit according to manufacturers’ protocol. In vivo anticancer efficacy was evaluated in SKOV3 tumor model. Animals were randomly divided into each group based on tumor volume. Sample size in each experiment was indicated by n values in the FIG. legends. All the samples were included in the experimental analysis, with no data excluded.
[0086] Statistical analysis
[0087] All results are presented as the mean ± standard deviation mean (s.d.). Tukey post- hoc tests and one-way ANOVA were used for multiple comparisons (when more than two groups were compared). All statistical analyses were carried out with Prism software package (PRISM 8.0; GraphPad Software). The threshold for statistical significance was P<0.05. [0088] Materials, cell lines and animals
[0089] All the materials other than indicated were purchased from Sigma- Aldrich Corp. (St. Louis, MO, USA). Pyropheophorbide a (PPa) was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). l-Palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine was obtained from Avanti Polar Lipids Inc. (Alabama, USA). LysoTracker Red DND-99 and ER- Tracker Red were obtained from Thermo Fisher Scientific Inc. MitoStatus Red was from BD Biosciences Pharmingen (Milan, Italy). SKOV3 and 3T3-L1 cell lines were purchased from American Type Culture Collection. SKOV3 cells were cultured with McCoy's 5A Medium (Gibco, Invitrogen) containing 10% fetal bovine serum (FBS) (Invitrogen) and 100 U/mL penicillin/streptomycin (Invitrogen). 3T3-L1 cells were cultured with Dulbecco’s modified Eagle medium (DMEM) (Gibco, Invitrogen) containing 10% bovine calf serum (Thermo Fisher Scientific). Both cells were incubated at 37 °C in 5% CO2. Female NSG mice (6-10 weeks) were purchased from the Jackson Lab. All animal studies were correspondent with the protocols approved by the Institutional Animal Care and Use Committee at the University of California, Los Angeles (UCLA).
[0090] 3T3-L1 adipocytes differentiation
[0091] In vitro formation of adipocytes was carried out with 3T3-L1 Differentiation Kit (Sigma- Aldrich, cat no. DIF001). Briefly, when 3T3-L1 preadipocytes became confluent, culture medium was replaced with Differentiation Medium (DMEM/F12 (1:1) medium containing 10% FBS, 10 pg/mL insulin, 1 mM dexamethasone, 500 pM IBMX, and 1 pM rosiglitazone). After three days, culture medium was replaced with Maintenance Medium (DMEM/F12 (1:1) containing 10% FBS and insulin) that was renewed every two days. Fully differentiated adipocytes could be obtained 10 days after differentiation.
[0092] Lipid droplet isolation
[0093] The isolation of lipid droplets 12 was conducted with Lipid Droplet Isolation Kit (Abeam, cat no. ab242290) with a slight modification. Approximately 3 c 107 adipocytes or drug -incubated adipocytes were trypsinized and washed twice with 1 c PBS. Then the cells were resuspended in 200 pL of Reagent A and were placed on ice for 10 min. 800 pL of 1 x Reagent B was added to the cells and mixed thoroughly, followed by incubation on ice for another 10 min. Cells were further homogenized by passing them five times through a one inch 27-gauge needle attached to a 3 mL syringe. The homogenate was centrifuged at 100 c g for 5 seconds and carefully layered with 600 pL of 1 x Reagent B. After centrifugation for 3 hours at 20,000 c g at 4°C, lipid droplets 12 were collected and stored at -80°C after lyophilization. [0094] Synthesis of pyrolipid
[0095] 107 mg PPa, 98.7 mg lipid, 76.3 mg l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), 48.7 mg 4-(dimethylamino) pyridine (DMAP) and 100 pL N,N- diisopropylethylamine (DIPEA) were dissolved in 10 mL anhydrous dichloromethane (DCM). The reaction mixture was stirred for 48 hours under dark at room temperature. The resulting pyrolipid was purified by column chromatography.
[0096] Singlet oxygen detection
[0097] Pyrolipid 14 and Pyrolipid@LDs 12 at a pyrolipid concentration of 2 mM were mixed with singlet oxygen sensor green (SOSG) probe (10 pM) in PBS buffer. After being irradiated with a 670 nm light at the power density of 100 mW/cm2 for predetermined time points, 100 pL sample was pipetted out from each well, and the fluorescence intensity of SOSG at 535 nm under excitation at 488 nm before and after laser irradiation was recorded using a microplate reader.
[0098] Cellular uptake and distribution
[0099] To obtain fluorescently labeled lipid droplets 12 (LDs-FITC), 100 pL lipid droplets 12 in 4 mL PBS was mixed with 20 mM NHS-fluorescein (FITC) in 200 pL DMSO. After reacting for 2 h at room temperature, the final product was purified by ultracentrifugation.
For the intracellular distribution experiment, SKOV3 cells were incubated with LD-FITC for 4 h, followed by organelles staining and confocal microscopy imaging. Lysosome was stained with LysoTracker Red DND-99 (500 nM, 1 h); mitochondria was stained with MitoStatus Red (200 nM, 30 min); and ER was stained with ER-Tracker Red (1 pM, 30 min). For the endocytosis pathway study, cells were preincubated with different endocytosis inhibitors including Chlorpromazine (CPZ, 10 pM), Nystatin (NYS, 25 pg/mL), Amiloride (AMI, 1 mM) and Methyl- -cyclodextrin (MCD, 3 mM) for 2 h. After adding LDs-FITC, flow cytometry was used to analyze the cellular uptake.
[00100] In vitro cytotoxicity
[00101] Laser irradiation for the cellular experiment was set at 100 mW/cm2 for 5 min on a 670-nm diode laser (Laserglow Technologies). Cell viability was determined by a 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay.
Cobalt di chloride (C0CI2, 100 pM) was used as a chemical inducer of hypoxia. Live and dead cell staining using LIVE/DEAD™ Cell Imaging Kit (Thermo Fisher, cat no. R37601) and apoptosis detection using Annexin A5 Apoptosis Detection Kit (Biolegend, cat no. 640914) was completed under the guidance of the manufacturer’s protocol. [00102] ROS generation detection
[00103] SKOV3 cells were pretreated with DCFDA probe (20 mM). After washing with IX PBS, the cells were treated with different formulations in PBS and incubated for 2 h in the dark. After dark or laser treatment, the fluorescence intensity was recorded on a microplate reader at Ex/Em = 485/535 nm.
[00104] Lipid peroxidation detection
[00105] TBARS assay kit (Cayman Chemical, cat no. 10009055) was used to detect lipid peroxidation. SKOV3 cells were collected in PBS at 1 x 107 cells/mL. The cell solutions were sonicated on ice for 1 min and placed into a 96-well plate. Thiobarbituric acid reagent (13.25 mg/mL) containing different therapeutic formulations was added to the cells (100 pL). After subjected to dark or laser treatment, the 96-well plate was sealed with adhesive tape and was heated at 100 °C for 1 h. Absorbance of TBARS product was measured at 532 nm on a microplate reader after cooling down.
[00106] Western blot analysis
[00107] SKOV3 cells were applied with different formulations followed by dark or laser treatment. After 12 h, cells were lysed for Western blot analysis. Primary antibodies: Anti- GRP78 BiP antibody (1:1000, Abeam, cat no. ab21685); Anti-eIF2a antibody (1:1000, Abeam, cat no. ab26197); Anti-eIF2a (phospho S51) antibody (1:1000, Abeam, catno. ab32157); Anti-PERK antibody (1:1000, Abeam, cat no. ab65142); Anti-PERK (phospho T982) antibody (1:1000, Abeam, catno. abl92591); Anti-DDIT3 antibody (1:1000, Abeam, cat no. abl79823); Anti-beta Actin antibody (1:1000, Abeam, cat no. ab8224).
[00108] In vivo anti-tumor model
[00109] A cell suspension containing 1 x 107 SKOV3 cells in 50 pL PBS was inoculated into the right back of NSG mice subcutaneously. When the tumor size reached 50-100 mm3, mice were randomly divided into five groups (n=8) and were treated with PBS, Pyrobpid@LDs 12 without laser (0.2 mg kg 1 pyrobpid-equiv. dose), lipid droplet with laser, pyrobpid 14 with laser (0.2 mg kg 1), or Pyrobpid@LDs 12 with laser (0.2 mg kg-1 pyrolipid-equiv. dose). The laser condition was set at 150 mW cm2 for 10 min on a 670-nm laser. The treatment was performed on day 3, 6 and 9 for a total of 3 treatments. Tumor sizes and body weights were monitored every 3 days. The tumor volume (V) was calculated according to V (mm3) = L (mm) *W2 (mm2) x 0.5, where L represents tumor length and W represents tumor width. After mice were sacrificed, tumor and major organs were collected for histological analysis. [00110] While embodiments of the present invention have been shown and described, various modifications may be made without departing from the scope of the present invention.

Claims

What is claimed is:
1. A therapeutic material for treating cancer comprising a plurality of isolated lipid droplets having contained therein a pyrolipid comprising pyropheophorbide a conjugated to a lipid.
2. The therapeutic material of claim 1, wherein the lipid comprises 1-palmitoyl- 2-hydroxy-sn-glycero-3-phosphocholine.
3. The therapeutic material of claim 1, wherein the plurality of lipid droplets comprise a triacylglycerol core and a phospholipid monolayer decorated with proteins.
4. The therapeutic material of claim 1, wherein the lipid droplets are contained in a buffer solution or animal sera.
5. The therapeutic material of claim 1, wherein the lipid droplets have a diameter within the range of about 60 nm to several micrometers.
6. The therapeutic material of claim 1, wherein the lipid droplets have a diameter within the range of about 60 nm to about 225 nm.
7. The therapeutic material of claim 1, wherein the lipid droplets have a substantially uniform diameter.
8. The therapeutic material of claim 1, wherein the lipid droplets have varied diameters.
9. A method of using the therapeutic material of any of claims 1-8, comprising injecting the therapeutic material into a tumor or cancerous tissue of a mammalian subject and irradiating the tumor or cancerous tissue with far-red or near-infrared light.
10. The method of claim 9, wherein the far-red or near-infrared light is emitted from a laser, light-emitting diode (LED), or laser diode.
11. The method of claim 9, wherein the far-red or near-infrared light is emitted from a light fiber or catheter.
12. A method of formulating therapeutic lipid droplets comprising: synthesizing a lipid-conjugated pyrolipid; incubating the lipid-conjugated pyrolipid with adipocytes to generate pyrolipid- loaded lipid droplets; and isolating pyrolipid-loaded lipid droplets from the adipocytes.
13. The method of claim 12, wherein the incubation is at least 24 hours.
14. The method of claim 12, further comprising freeze drying the pyrolipid-loaded lipid droplets.
15. The method of claim, 14 further comprising suspending the freeze dried pyrolipid-loaded lipid droplets in a buffer or animal sera.
16. The method of claim 12, where the pyrolipid comprises pyropheophorbide a and the lipid comprises l-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine.
17. The method of claim 12, wherein the adipocytes are generated from preadipocytes exposed to insulin.
18. The method of claim 12, wherein the pyrolipid-loaded lipid droplets have a substantially uniform diameter.
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