Porphyrin nanovesicles with fatty acid conjugates

Abstract

Described herein are bilayer nanovesicles comprising a porphyrin-phospholipid conjugate and a chelator-fatty acid conjugate, where the chelator-fatty acid conjugate comprises an aminopolycarboxylic acid attached to a single-chain fatty acid, and the porphyrin-phospholipid conjugate comprises one porphyrin, porphyrin derivative or porphyrin analogue covalently attached to the lipid side chain of one phospholipid, preferably at the sn-1 or sn-2 position.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 221212, filed July 13, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to nanovesicles comprising porphyrin and fatty acid conjugates and methods of using said nanovesicles. [Background technology]

[0003] Photodynamic therapy (PDT) involves the combination of a non-toxic photosensitizer with light of an appropriate wavelength, which in the presence of oxygen generates reactive oxygen species for cell death and tissue destruction. PDT is attractive as a cancer treatment because of its dual selectivity, preferential accumulation of the photosensitizer in tumor tissue, and spatially focused light to the target area, confining photosensitizer activation to a localized region. PDT also offers many advantages over conventional cancer treatments due to its minimally invasive nature, little to no scarring, and the ability to treat the same target area multiple times if necessary. 1-3 Most photosensitizers are administered systemically at clinical levels where their accumulation at the tumor site induces a drug-to-light interval (DLI) for treatment, and an extended DLI is used for optimal distribution of the photosensitizer at the cellular level (cell-PDT). 4 .

[0004] Porphyrins are ubiquitous compounds found in nature and are involved in numerous biological processes such as photosynthesis (chlorophyll) and oxygen transport (heme). Porphyrins are tetrapyrrole macrocycles interconnected by methine bridges into a ring structure that provides their stability and optical properties. Porphyrins and their derivatives have been clinically approved as effective PDT agents for the treatment of lung, esophageal, bile duct, bladder, ovarian, and cervical cancers. 5-9However, most porphyrin photosensitizers in clinical use have various drawbacks that limit their application, including low chemical purity, poor tissue permeability, substandard tumor accumulation, and excessive lipophilicity that causes them to aggregate in body fluids, resulting in dark toxicity and prolonged photosensitivity. 10 .

[0005] Porphysomes are self-assembled liposome-like bilayer nanovesicles (approximately 100 nm diameter) composed of porphyrin-lipids, which are well-characterized, biocompatible organic molecules and are enzymatically biodegradable in vivo. Administration of porphyrin-lipids at a dose of 1000 mg / kg elicited minimal acute toxicity in mice. 11,12 Porphysomes contain high-density porphyrin photosensitizers (>80,000 porphyrins / particle) packed into nanostructures, resulting in "ultrafast" quenching of fluorescence and generation of singlet oxygen. 11 , which can effectively convert light of specific wavelengths into heat with extremely high efficiency, resulting in ideal photothermal and photoacoustic properties unprecedented in organic nanoparticles. Upon dissociation of the porphysome nanostructure, the fluorescence and photoactivity of the free porphyrins are restored, enabling imaging with low background fluorescence and activatable photodynamic therapy. In addition, radioisotopes (e.g., 64 Cu) or metals (e.g., manganese) can be robustly chelated to the porphyrin ring, enabling positron emission tomography (PET) and magnetic resonance imaging (MRI), respectively. 13-15 As a result, the simple yet all-encompassing nature of porphysomes represents a novel paradigm in nanomedicine design for multimodal imaging and therapy.

[0006] Porphysomes are potentially well suited for selective PDT because they can transport, preferentially accumulate, and ultimately activate large amounts of porphyrins in tumors under photoreactive quenching conditions, and activation can be tracked by fluorescence unquenching. 11However, the PDT application of conventional porphysomes is limited by their slow cellular uptake and uncontrolled / unpredictable activation in cancer cells, posing challenges in defining the optimal DLI for effective PDT, which is highly dependent on the amount of active photosensitizer localized within the target site. Therefore, it is necessary to find a proactive attempt to control and induce the accumulation and activation of porphysomes in tumors for effective PDT. For example, we reported that the inclusion of an active target such as folate-conjugated PS could significantly enhance porphysome cellular uptake in KB cells after 3 and 24 h of incubation, resulting in significantly enhanced PDT in mouse tumor models. 16 .

[0007] Ethylenediaminetetraacetic acid (EDTA) has had clinical approval for the treatment of heavy metal poisoning since 1953. 17,18 Chelation of divalent metal cations has also been investigated in various antibacterial studies because it increases the membrane permeability of the cell wall of Gram-negative bacteria to exogenous agents. 19 In addition, EDTA is considered an absorption enhancer that enhances the permeation of drugs, proteins, and peptides through the corneal, nasal, and intestinal epithelia. 20 It has been used to improve paracellular permeability by temporarily loosening tight junctions between adjacent epithelial cells. 20 In recent years, EDTA chelation therapy has also been investigated for the treatment of cardiovascular disease, neurodegenerative diseases, and cancer. 21-25 . Summary of the Invention [Means for solving the problem]

[0008] In one embodiment, a bilayer nanovesicle is provided comprising a porphyrin-phospholipid conjugate and a chelator-fatty acid conjugate, wherein the chelator-fatty acid conjugate comprises an aminopolycarboxylic acid attached to a single-chain fatty acid, and the porphyrin-phospholipid conjugate comprises one porphyrin, porphyrin derivative or porphyrin analogue covalently attached to the lipid side chain of one phospholipid, preferably at the sn-1 or sn-2 position.

[0009] In one aspect, there is provided a composition comprising a bilayer nanovesicle as described herein in a buffer solution. In one aspect, a method of performing photodynamic therapy on a target area of ​​a subject is provided, comprising generating a composition described herein, administering the composition to the subject, and irradiating the target area with light of a wavelength that excites the composition to create radicals and / or reactive oxygen species.

[0010] In one aspect, a method is provided for imaging a target area of ​​a subject, the method comprising generating a composition described herein, administering the composition to a subject, and measuring and / or detecting a fluorescent or photoacoustic signal at the target area.

[0011] In one aspect, a method of delivering a radioisotope to a subject is provided, comprising the steps of producing a composition described herein, in which bilayer nanovesicles have a radioisotope chelated therein, and administering the composition to the subject.

[0012] In one aspect, there is provided a use of a composition described herein for performing photodynamic therapy. In one aspect, there is provided a use of a composition described herein for performing imaging.

[0013] In one aspect, there is provided a use of a composition described herein for delivering a radioisotope to a subject. These and other features of preferred embodiments of the present invention will become more apparent in the following detailed description, in which reference is made to the accompanying drawings. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows a schematic diagram of EDTA-lipid-based novel porphysomes (eNPS). [Diagram 2] A) Representative TEM images of NPS, B) absorbance of intact vs. disrupted NPS, and C) fluorescence of intact vs. disrupted NPS. [Diagram 3] A) Fluorescence microscopy images and B) quantitative fluorescence measurements after cell extraction show eNPS cellular uptake compared to PS. Significant differences (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) were determined using two-way ANOVA with Bonferroni correction. [Figure 4] Showing eNPS uptake by KB epithelial cells versus normal fibroblasts (NFB) under fluorescence microscopy imaging. [Diagram 5] A) Chemical structures of DTPA-lipid W / O Gd3+ chelation and their corresponding TEM images, B) Comparison of cellular uptake of dNPS, eNPS and PS by fluorescence microscopy imaging (scale bar = 20 μm), and C) Quantitative cellular uptake analysis. Significant differences (*p<0.05, **p<0.01, ***p<0.0001) were determined using two-way ANOVA with Bonferroni correction. [Figure 6] Figure 1 shows the effect of metal chelation on eNPS and dNPS uptake. A) Fluorescence microscopy imaging revealed that preincubation with 1 mM Ca2+ or Mg2+ did not affect eNPS-mediated rapid uptake in KB cells. B) Gd3+ chelation did not affect dNPS uptake (DPS vs. DPS(Gd)). Significant differences (*p<0.05) were determined using two-way ANOVA with Bonferroni correction. [Figure 7]Exploring the effect of lipid chains on NPS uptake by replacing single fatty acid DTPA-lipids with various double fatty acid DTPA-lipids. A) Chemical structures of various DTPA-lipids and TEM images of the corresponding NPS formed by these DTPA-lipids. B) Cellular uptake compared to dNPS. [Figure 8] A) Normalized cellular uptake of eNPS, LC-eNPS~PS, *p<0.05, **p<0.0001, two-way ANOVA. B) Serum stability of eNPS (n=3), LC-eNPS (n=3), and PS (n=3) in 50 vol% FBS as tracked by fluorescence quenching efficiency. [Figure 9] Figure 1 shows temperature- and energy-dependent uptake of eNPS and dNPS in KB cells. Fluorescence microscopy images of KB cells incubated with transferrin (Tfn), eNPS, and dNPS: A) for 3 h at different temperatures, B) co-incubated with NaN3 and 2-DG for 1 h. Scale bar = 20 μm. [Figure 10] Subcellular localization of eNPS and dNPS examined in KB cells under confocal microscopy by co-incubation with A) MitoTracker and B) LysoTracker. [Figure 11] Figure 1 shows in vitro PDT evaluation on KB cells after incubation with 5 μM eNPS, PS, and LC-eNPS for different time periods (3, 6, or 24 hours) followed by exposure to different doses of light. The light dose varied with the treatment. Cell viability was evaluated by Alamar Blue assay. [Figure 12] Shows in vivo fluorescence imaging of eNPS vs. PS on a KB subcutaneous mouse model. [Figure 13] Fluorescence imaging of eNPS vs. PS on a hamster cheek carcinogenesis model with a NOVADAQ Pinpoint system: A) Time-dependent fluorescence images and B) tumor fluorescence intensity tracking profiles over time in eNPS and PS tumors. [Figure 14]1 shows the synthesis of EDTA-hexadecylamide (EDTA-lipid). [Figure 15] Fluorescence imaging of cells immediately after removal of incubation medium containing PS, eNPS2, and eNPS3 (1 μM) at 3, 6, and 24 hours postincubation and at 3 and 18 hours postincubation is shown. The results show that no signal enhancement appeared upon postincubation, suggesting that eNPS underwent rapid activation within the intracellular environment. [Figure 16] The results indicate that co-incubation with high concentrations of EDTA can increase PS uptake. Unlike the enhancement of eNPS-mediated transport (Figure 4) (5 μM eNPS contains 7 μM or less EDTA-lipid), 1 mM free EDTA had a negligible effect on PS uptake. [Figure 17] When EDTA-lipids were incorporated into liposomal formulations doped with 1 mol% porphyrin lipids for fluorescence imaging, the resulting 1% pyro-eNPS (1 μM based on pyro concentration) showed similar rapid uptake as eNPS did upon incorporation of EDTA-lipids in Figure 4. EDTA-lipids play the same role in inducing rapid uptake of nanoparticles when EDTA-lipids are incorporated into liposomal formulations containing 1 mol% porphyrin lipids. [Figure 18] Comparison of in vivo PDT efficacy of eNPS, LC-eNPS, and PS at varying drug-light-intervals (DLI) is shown. Both eNPS and LC-eNPS (DLI of 3 hours) demonstrated the greatest in vivo PDT efficacy compared to PS. Significant differences between groups (*p<0.05, **p<0.01, ***p<0.0001) were determined using two-way ANOVA with Tukey post-hoc test. [Figure 19] Figure 1 shows a comparison of in vivo fluorescence activation of eNPS, LC-eNPS, and PS at various time points post-injection. Both eNPS and LC-eNPS demonstrated the greatest in vivo fluorescence activation at all time points compared to PS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be understood that the present invention may be practiced without these specific details.

[0016] In this study, we designed a non-toxic EDTA-hexadecylamide conjugate (EDTA-lipid) for the construction of porphyrin nanovesicles to create next-generation porphysomes (NPS), which showed greatly improved porphysome cellular uptake while maintaining good serum stability. When the NPS contained EDTA-lipid up to 50 mol% of the total lipid content in the porphysome, a remarkable 20-fold enhancement in cancer cell internalization was achieved at 24 h incubation, which consecutively led to a dramatic enhancement of PDT efficacy (cell viability 7%) compared to conventional porphysomes (cell viability 97%) at a concentration of 5 μM. This NPS platform can also be extended from EDTA-lipid to diethylenetriaminepentaacetic acid-hexadecylamide conjugate (DTPA-lipid). As a result, different radioisotopes (e.g., 99m EDTA-lipids or DTPA-lipids enhanced intracellular transport of porphysomes is likely to be driven by Ca transport, as would be expected for typical EDTA-enhanced cell membrane permeability. 2+ or Mg 2+ This finding was supported by several lines of evidence: 1) unlike the NPS-mediated enhanced uptake in epithelial cells, co-incubation of PS with 1 mM EDTA did not enhance intracellular uptake of porphysomes; 2) high concentrations of Ca 2+ and Mg 2+EDTA-lipid-based NPS (eNPS) after preincubation with Gd did not reduce the eNPS-enhanced transport; 3) DTPA-lipid-containing NPS (dNPS) reduced the Gd transport. 3+ Both with and without chelation showed similar enhanced uptake as eNPS. Fluorescence imaging studies in both subcutaneous KB mouse model and in vivo-relevant hamster cheek carcinogenesis further demonstrated rapid and enhanced accumulation / activation of eNPS over PS in tumors. Thus, the NPS platform demonstrates the potential to overcome the limitation of poor intracellular accumulation of porphysomes to advance porphysomes for multimodal imaging and effective PDT of cancer.

[0017] In one embodiment, a bilayer nanovesicle is provided comprising a porphyrin-phospholipid conjugate and a chelator-fatty acid conjugate, wherein the chelator-fatty acid conjugate comprises an aminopolycarboxylic acid attached to a single-chain fatty acid, and the porphyrin-phospholipid conjugate comprises one porphyrin, porphyrin derivative or porphyrin analogue covalently attached to the lipid side chain of one phospholipid, preferably at the sn-1 or sn-2 position.

[0018] In some embodiments, the aminopolycarboxylic acid is glycinate, IDA, NTA, EDTA, DTPA, EGTA, BAPTA, NOTA, DOTA, nicotianamine, EDDHA, or EDDS.Preferably, the aminopolycarboxylic acid is EDTA or DTPA.

[0019] In some embodiments, the single chain fatty acid contains between 10 and 26 carbons. In some embodiments, the single chain fatty acid comprises 12-22 carbons. Preferably, the single chain fatty acid comprises 14-18 carbons. More preferably, the single chain fatty acid comprises 16 carbons. In one embodiment, the single chain fatty acid is hexadecylamide.

[0020] In some embodiments, the chelator-fatty acid conjugate is EDTA-hexadecylamide or DTPA-hexadecylamide. In some embodiments, the bilayer nanovesicles comprise 15%-60% chelator-fatty acid conjugate, preferably 25%-50% chelator-fatty acid conjugate, more preferably 30%-40% chelator-fatty acid conjugate, and even more preferably about 30% chelator-fatty acid conjugate.

[0021] In some embodiments, the bilayer nanovesicles comprise 1-60 mol % porphyrin-phospholipid conjugate, preferably 20-40 mol % porphyrin-phospholipid conjugate, and more preferably about 27 mol % porphyrin-phospholipid conjugate.

[0022] In some embodiments, the porphyrin, porphyrin derivative or porphyrin analog in the porphyrin-phospholipid conjugate is selected from the group consisting of hematoporphyrin, protoporphyrin, tetraphenylporphyrin, pyropheophorbide, bacteriochlorophyll, chlorofer a, benzoporphyrin derivatives, tetrahydroxyphenylchlorins, purpurins, benzochlorins, naphthochlorins, verdin, rosin, ketochlorins, azachlorins, bacteriochlorins, triporphyrins, benzobacteriochlorins, extended porphyrins and porphyrin isomers.

[0023] Preferably, the expanded porphyrin is a texaphyrin, sapphyrin or hexaphyrin and the porphyrin isomer is a porphycene, an inverted porphyrin, a phthalocyanine or a naphthalocyanine.

[0024] In some embodiments, the phospholipid in the porphyrin-phospholipid conjugate comprises phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol. Preferably, the phospholipid comprises an acyl side chain of 12 to 22 carbons.

[0025] In some embodiments, the porphyrin in the porphyrin-phospholipid conjugate is pyropheophorbide-a acid. In some embodiments, the porphyrin in the porphyrin-phospholipid conjugate is a bacteriochlorophyll derivative.

[0026] In some embodiments, the phospholipid in the porphyrin-phospholipid conjugate is 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine or 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine.

[0027] In some embodiments, the porphyrin-phospholipid conjugate is a pyro-lipid. In some embodiments, the porphyrin-phospholipid conjugate is an oxy-bacteriochlorophyll-lipid, a texaphyrin-phospholipid conjugate, or an aza-boron dipyrromethene (BODIPY)-phospholipid conjugate.

[0028] In some embodiments, the porphyrin is attached to a glycerol group on the phospholipid by a carbon chain linker of 0-20 carbons. In some embodiments, the bilayer nanovesicles further comprise a PEGylated emulsifier. Preferably, the PEGylated emulsifier has a molecular weight ranging from about 1000 to about 5000. More preferably, the PEGylated emulsifier is selected from the group consisting of N-(methoxypolyethylene glycol 5000 carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (MPEG5000-DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)-2000 (DMPE-PEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)-2000 (DSPE-PEG2000), polyoxyethylene 40 stearate (PEG40S), and combinations thereof.

[0029] In some embodiments, the PEG or PEG-lipid is present in an amount of 1-10 mol %. Preferably, the PEG or PEG-lipid is present in an amount of 2-7 mol %. In some embodiments, the bilayer nanovesicles further comprise cholesterol.

[0030] In some embodiments, the remaining composition of the bilayer nanovesicles substantially comprises cholesterol. In some embodiments, cholesterol is present in an amount of 1-60 mole %.

[0031] In some embodiments, the bilayer nanovesicles are substantially spherical. In some embodiments, the bilayer nanovesicles are about 70-120 nm in diameter.Preferably, the bilayer nanovesicles are about 90-100 nm in diameter.

[0032] In some embodiments, the porphyrin-phospholipid conjugate contains a metal chelated therein, and optionally a radioisotope of the metal. In one aspect, there is provided a composition comprising a bilayer nanovesicle as described herein in a buffer solution.

[0033] In one aspect, a method of performing photodynamic therapy on a target area of ​​a subject is provided, comprising generating a composition described herein, administering the composition to the subject, and irradiating the target area with light of a wavelength that excites the composition to create radicals and / or reactive oxygen species.

[0034] In one aspect, a method is provided for imaging a target area of ​​a subject, the method comprising generating a composition described herein, administering the composition to a subject, and measuring and / or detecting a fluorescent or photoacoustic signal at the target area.

[0035] In one aspect, a method of delivering a radioisotope to a subject is provided, comprising the steps of producing a composition described herein, in which bilayer nanovesicles have a radioisotope chelated therein, and administering the composition to the subject.

[0036] In one aspect, there is provided a use of a composition described herein for performing photodynamic therapy. In one aspect, there is provided a use of a composition described herein for performing imaging.

[0037] In one aspect, there is provided a use of a composition described herein for delivering a radioisotope to a subject. As used herein, "pharmaceutically acceptable carrier" refers to any and all physiologically compatible solvents, dispersion media, coatings, antibiotics and antifungals, isotonic and absorption delaying agents, etc. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. In many cases, it is preferred to include isotonic agents, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, or sodium chloride, in the composition. Pharmaceutically acceptable carriers may further include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the drug.

[0038] As used herein, "therapeutically effective amount" refers to an amount that is effective at a dosage and for a certain period of time necessary to achieve a desired therapeutic result.The therapeutically effective amount of a drug may vary according to factors such as the individual's medical condition, age, sex, and weight, and the drug's ability to elicit a desired response in an individual.A therapeutically effective amount is also an amount in which any toxic or adverse effects of the drug are outweighed by the therapeutically beneficial effects.

[0039] The advantages of the present invention are further illustrated by the following examples. The examples and specific details thereof described herein are provided for illustrative purposes only and should not be construed as limiting the claims of the present invention. EXAMPLES

[0040] Methods and Materials Materials and Reagents Cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (16:0PE-DTPA), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (gadolinium salt) (16:0PE-DTPA(Gd)) and diethylenetriaminepentaacetic acid-bis(stearylamide) (gadolinium salt) (DTPA-BSA(Gd)) were purchased from Avanti Polar Lipids (USA). All other solvents and reagents were obtained from Sigma Aldrich. Porphyrin-lipids were synthesized according to previously published methods. 11 EDTA-hexadecylamide conjugate (EDTA-lipid) and DTPA-hexadecylamide conjugate (DTPA-lipid) were synthesized (see Supporting Information). Hoechst 33258 and LIVE / DEAD® Viability / Cytotoxicity Kit were purchased from Invitrogen Corporation (Carlsbad, CA, USA). Mitotracker Green FM or Lysotracker Red DND-99 were obtained from Invitrogen / Thermo Fisher.

[0041] Chromatographic purification was performed using flash chromatography (Biotage, Isolera™). UPLC-MS was performed using a Waters Acuity UPLC(C)Peptide BEH C18 column (130 Å, 1.7 μm, 2.1 mm×50 mm) (Waters Canada, Ontario, Canada) equipped with a Waters 2695 controller, a 2996 photodiode array detector, and a Waters triple quadrupole (TQ) mass detector. UPLC conditions were as follows: Solvent A) 0.1% TFA and B) acetonitrile; column temperature: 60° C.; flow rate: 0.6 mL / min; gradient from 80% A+20% B to 0% A+100% B in 5 min, held at 100% B for 2 min, followed by a 1 min jump back to 80% A+20% B. NMR spectra were recorded on a Bruker Ultrashield 400 Plus NMR spectrometer measuring 1D 1H NMR, 2D COSY 1H NMR 400.18 MHz. All measurements were referenced to the internal standard tetramethylsilane (TMS).

[0042] Synthesis of EDTA monoanhydride Ethylenediaminetetraacetic dianhydride (3.2 g, 0.0125 mol) was added followed by dry DMF (20 mL) under Ar and heated to 80° C. After 15 min of stirring, water (0.225 mL) was added slowly and the reaction mixture was refluxed under Ar for 1 h 45 min. The crude product was filtered and washed with DMF and ether to give a white solid (1.95 g, 61%). The molecular structure is 1 Characterized by H NMR (400 MHz, DMSO-d6) δ 12.32 (s, 2H, CO2H), 3.72 (s, 4H), 3.43 (s, 4H), 2.83 - 2.74 (m, 2H), 2.58 (t, J = 6.1 Hz, 2H).

[0043] EDTA-Hexadecylamide Lipid (EDTA-Lipid) EDTA monoanhydride (1.42 g, 5.2 mmol) and hexadecylamine (1.2 g, 5 mmol) were dissolved in dry DMF (60 mL) and refluxed at 100° C. under Ar for 10 h. The reaction mixture was cooled to room temperature and precipitated by pouring into water and filtering. The crude product was washed with water and diethyl ether to give a white solid (1.25 g, 49%). The molecular structure is 1 H NMR (400 MHz, DMSO-d6) δ 8.01 (s, 1H, NH), 3.42 (s, 4H), 3.36 (s, 2H), 3.18 (s, 2H), 3.05 (d, J = 7.0 Hz, 2H), 2.79 - 2.63 (m, 4H), 1.38 (s, 2H), 1.30 - 1.14 (m, 26H), 0.85 (t, J = 6.6 Hz, 3H). UPLC-MS(ESI):516m / z[M+H]+.tr=1.3min. See Figure 14.

[0044] NPS synthesis and characterization NPS was synthesized according to a previously reported protocol for formulating porphysomes. 11Various molar ratios of lipid components consisting of porphyrin-lipid (pyropheophorbide-lipid), cholesterol (Avanti Polar Lipids, Alabaster, AL), distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethene glycol) (PEG2000-DSPE, Avanti Polar Lipids), and EDTA / DTPA lipid were thoroughly mixed and dissolved in chloroform. The lipid mixture was dried under a gentle stream of nitrogen gas and further placed under vacuum for 1 h. The dried lipid film was then rehydrated (concentration 3 mg / mL) with PBS buffer (150 mM, pH 7.5), subjected to a freeze-thaw process 9 times, and extruded 10 times through a polycarbonate membrane (pore size = 100 nm). To determine the morphology of NPS, the particles were stained with 2% uranyl acetate counterstaining and then scanned with a Hitachi H-7000 electron microscope (Hitachi High Technologies America, Inc., Illinois, USA). Size and distribution were measured by dynamic light scattering (ZS90 Nanosizer, Malvern Instruments). In spectroscopic studies, PLPs were diluted either in PBS as intact / quenched samples or in PBS containing 0.5% Triton X-100 as disrupted / unquenched samples. Absorption and fluorescence spectra of intact and disrupted NPS were measured by UV / Vis spectrophotometers Cary 50 (Agilent, Mississauga, ON) and Fluoromax-4 (Horiba Jobin Yvon, USA) (excitation: 420 nm, emission: 630–800 nm, slit width: 5 nm), respectively. Porphyrin fluorescence quenching efficiency was calculated using the following equation: Quenching efficiency = (1-F i / F d )×100% F i and F d means the fluorescence intensity of intact and the corresponding disrupted NPS, the concentration of porphyrin is 1 µM.

[0045] Intracellular uptake assessment First, we used fluorescence microscopy to track the intracellular uptake of particles on KB cells cultured in RPMI-1640 medium with 10% FBS. Briefly, 24 h prior to incubation, 5 × 10 4 Cells / well were seeded in 8-well chamber slides. Cells were incubated with NPS and PS at a porphyrin concentration of 1 μM for 4 h at 37°C, rinsed three times with PBS, and then recultured in fresh medium. An Olympus FV1000 laser fluorescence scanning microscope (Olympus, Tokyo, Japan) was used to monitor the porphyrin fluorescence changes of cells over time (immediately after removal of the incubation medium, 3 h and 18 h later). Fluorescence microscopy was also used to examine eNPS uptake in KB epithelial cells versus normal fibroblasts (NFB).

[0046] To further compare the cellular uptake of NPS versus PS, quantitative cellular uptake studies were performed. KB cells were incubated with 10 6 Cells were seeded in 12-well plates at 1000 x 1000 cells / well. The cells were then incubated with various NPS and PS with varying porphyrin concentrations for different times at 37°C. After rinsing three times with PBS, the cells were trypsinized and the suspension was centrifuged at 4000 rpm for 5 min. 2.5 x 10 5 The cell pellet containing the cells was resuspended in 500 μL of lysis buffer (DMSO) and incubated for 1 h. The solution was centrifuged at 10,000 rpm for 10 min and the supernatant was collected for porphyrin fluorescence measurement using a Fluoromax-4 fluorometer to quantify the uptake of porphyrin molecules in the cells.

[0047] Serum stability evaluation The serum stability of various eNPS, LC-eNPS and PS (1 μM) was assessed by incubation with 50 vol% FBS in PBS for various times at 37°C and determining the change in fluorescence quenching efficiency of the samples using a CLARIOstar microplate reader (BMG LABTECH) (excitation: 410 / 8 nm, emission: 671 / 8 nm, gain = 2500).

[0048] Evaluation of intracellular uptake pathways of NPS The uptake of eNPS and dNPS in KB cells at various temperatures was assessed using fluorescence microscopy. After 3 h of incubation at 4, 18, and 37 °C with eNPS and dNPS, cells were imaged and the fluorescence signals at each temperature condition were compared to determine whether eNPS and dNPS were actively or passively taken up by cancer cells. These uptakes were further assessed under adenosine-triphosphate (ATP)-depleted cell conditions by using sodium azide (NaN3) and 2-deoxy-D-glucose (2-DG). KB cells were preincubated with NaN3 and 2-DG for 30 min prior to 1 h of co-incubation with eNPS and dNPS followed by fluorescence microscopy. The changes in fluorescence between normal and ATP-depleted cell conditions were assessed.

[0049] The subcellular localization of eNPS and dNPS was examined on KB cells under confocal microscopy. The subcellular localization of eNPS and dNPS in mitochondria and lysosomes was evaluated by colocalization assessment with the mitochondrial tracker Mitotracker Green FM and the lysosomal tracker Lysotracker Red DND-99. KB cells were incubated with nanoparticles for 24 h before incubation with the organelle trackers. The concentrations and incubation times used for these trackers were optimized according to the manufacturer's recommendations. Confocal image settings: Porphyrin channel: excitation: 633 nm / emission: 670-740 nm; Lysotracker channel: excitation: 552 / emission: 573-620 nm, and Mitotracker channel (excitation: 488 nm / emission: 510-600 nm).

[0050] In vitro PDT assay The in vitro PDT activation of eNPS, LC-eNPS, and PS was determined by measuring the viability of KB cells after treatment. KB cells were incubated with 5 μM eNPS, LC-eNPS, and PS for 3, 6, and 24 h, and then exposed to light irradiation using a homemade LED light box at 660 nm (660 nm; irradiance: 30 mW / cm). 2 ;Dose: 5 and 10J / cm 2 ) was performed. Cell viability was assessed 24 hours after light treatment using the alamarblue assay, where cells were incubated with 50 μg / mL alamarblue for 2 hours prior to fluorescence measurement. Alamarblue fluorescence was then measured using a CLARIOstar microplate reader by exciting at 540 nm and collecting emission at 590 nm. Cells cultured in normal medium were used as untreated controls. Cells treated with porphysomes but cultured in the dark were used to assess dark toxicity of each formulation. Cell viability of all samples was normalized to the untreated control.

[0051] In vivo fluorescence imaging All animals received humane care according to the University Health Network (UHN) Animal Care and Use Committee, the Ontario Animal for Research Act, and policies devised by the Canadian Society for Animal Care. All animal experiments were approved by UHN Animal Care and Use Committee protocols. The KB subcutaneous mouse model and the hamster cheek carcinogenesis model were used throughout this study.

[0052] To develop the KB xenograft model, 8-week-old female athymic nude mice were implanted with 2 × 10 HBs cells in 200 µL of PBS medium into the right flank under general anesthesia (isoflurane in oxygen). 6KB was inoculated subcutaneously. Animals were maintained in pathogen-free conditions in autoclaved microisolator cages at the UHN Animal Resource Center. When tumor size reached an average size of 4.0-5.0 mm in diameter, mice were randomized (n=3 per group) and intravenously injected with PS and eNPS at a dose of 4 mg / kg porphyrin. In vivo whole-body imaging was performed at various time points using a Maestro imaging system (CRI, USA) with 575-605 nm excitation / 645 nm long-pass emission filter to compare tumor accumulation and activation while mice were anesthetized with 2% (v / v) isoflurane. Low-fluorescence diet (Harlan Tekland®, product no. TD.97184) was fed to animals 3 days before NPS administration.

[0053] In the hamster study, 6- to 8-week-old male Syrian hamsters (Harian, Indianapolis, USA) were used to develop a model that mimics the clinical symptoms of oral cancer in humans. 26 Briefly, while animals were anesthetized with isoflurane, 0.5% DMBA (7,12-dimethylbenz(a)anthracene) in DMSO was applied to both cheeks along with a non-absorbent material. Before application, Kimwipes were stuffed into the oral cavity to minimize running down the throat and were removed after application was completed. This procedure was repeated three times a week for 16–20 weeks. Generally, visible tumors were observed 10 weeks after DMBA application and could reach a maximum size of 5–10 mm. PS and eNPS were administered to hamsters via the cephalic vein at a dose of 4 mg / kg porphyrin concentration and subjected to a preclinical fluorescence endoscope (PINPOINT Imaging System, Novadaq Technologies Inc., Mississauga, ON, Canada) with a laser excitation wavelength of 665 nm for in vivo fluorescence imaging, and real-time videos were recorded for fluorescence intensity analysis.

[0054] statistical analysis Two-way analysis of variance with Bonferroni correction was used to determine statistical significance between experimental groups. A P value of <0.05 was considered significant.

[0055] Results and Discussion Design and synthesis of EDTA-lipid-based novel porphysomes (eNPS) To incorporate EDTA moieties into the nanostructure of porphysomes, an amphiphilic EDTA-lipid was designed and synthesized by coupling EDTA monoanhydride with hexadecylamine to obtain an EDTA-monoC16 lipid conjugate (EDTA-lipid) (Figure 14). The chemical structure was identified by NMR and uPLC-MS (see synthesis and characterization in the Supporting Information).

[0056] Various eNPSs were then prepared by varying the ratio of EDTA-lipid in the porphysome formulation (18 mol%-50 mol%). A schematic diagram of the eNPSs is shown in Figure 1, and the contents of the components in each formulation are listed in Table 1. The abbreviation PS stands for conventional porphysome formulation.

[0057] [Table 1]

[0058] eNPS showed similar structural and optical properties to porphysomes Incorporation of EDTA-lipids (18-50 mol%) into the porphysome formulations had negligible effects on particle morphology (liposome-like nanovesicular structures shown in the TEM images in Figure 2A), particle size (Z-average: 90-100 nm) and monodispersity (PDI: 0.14-0.18) (Table 2), indicating the compatibility of EDTA-lipids in the porphysome formulations. In addition, eNPS exhibited similar absorption spectra as PS (Figure 2B) and a high degree of fluorescence self-quenching (>150-fold) (Figure 2C and Table 2), indicating minimal impact on the optical properties of intact porphysomes.

[0059] [Table 2]

[0060] eNPS exhibited rapid and enhanced cellular uptake in KB cells The cellular uptake of various eNPS and PS was monitored by fluorescence microscopy, where KB cells were incubated with 1 μM particles for 4 h prior to imaging. As shown in Figure 3A, eNPS2 and eNPS3 containing more than 30 mol% EDTA-lipids showed rapid uptake and significantly enhanced fluorescence in KB cells when compared to PS. Since the fluorescence is highly quenched in intact eNPS, cells were continuously cultured in fresh medium after incubation to monitor whether eNPS dissociation / activation after internalization is a time-consuming process. As shown in Figure 15, the whole-cell fluorescence signal after incubation with eNPS2 and eNPS3 showed a slow fluorescence fade with increasing post-incubation time (3 h, 18 h), while no enhancement of the signal appeared upon post-incubation, suggesting that eNPS undergoes rapid activation within the intracellular environment.

[0061] Although eNPS showed rapid activation in cells, to quantify the eNPS-enhanced uptake over PS, a quantitative cell extraction method was further applied to determine the time-dependent cellular uptake profile of various particles. Using this method, internalized eNPS and PS were completely destroyed in DMSO lysate, thereby not quenching the porphyrin fluorescence for uptake quantification. As shown in Figure 3B, a clear trend of enhanced uptake was observed with increasing ratio of EDTA-lipid in the nanoparticles. eNPS3, which has the highest EDTA-lipid content (50 mol%), demonstrated the greatest uptake at all time points (p<0.05). At a concentration of 5 μM, 2.1-, 5.5-, 33.7-, and 26.1-fold enhanced uptake compared to PS was observed at 3-, 6-, 18-, and 24-h incubation, respectively. eNPS2, which contained 30 mol% EDTA-lipid, also showed significantly enhanced transport with longer incubation times, e.g., 12.3- and 11.3-fold enhanced uptake (p<0.05) after 18 and 24 h of incubation with 5 μM particles, respectively. We selected eNPS3 as the eNPS formulation of choice for evaluation of enhanced transport in all subsequent studies.

[0062] eNPS induced selective uptake in epithelial cells but not in fibroblasts Fluorescence microscopy was used to assess the uptake of eNPS in epithelial KB cells versus normal human fibroblasts. Interestingly, eNPS significantly enhanced particle uptake in epithelial cells but not in fibroblasts (Figure 4). It is possible that eNPS-enhanced transport in epithelial cells may be due to increased uptake of divalent cations (e.g., Ca) that may increase the permeability of the cell membrane. 2+ , Mg 2+To investigate whether the effect of EDTA on the uptake of PS was caused by the interaction of EDTA head groups (5 μM eNPS contains 7 μM EDTA-lipid) with EDTA-lipid, a high concentration of free EDTA (1000 μM) was added and co-incubated with plain PS (5 μM) to examine any effect on PS uptake. The results (Figure 16) revealed that 1 mM free EDTA provided negligible enhancement on PS uptake. In addition, EDTA-lipid plays the same role in inducing rapid internalization of liposomes in cells when EDTA-lipid was incorporated into liposomal formulation (HSPC:EDTA-lipid:pyrolipid:cholesterol:DSPE-PEG2000=32:49:1:15:2 (mol / mol)) (Figure 17).

[0063] DTPA-lipid-based novel porphysomes (dNPS) To investigate whether NPS platforms could be constructed by replacing the tetraacetic acid head group of EDTA-lipids with other hydrophilic head groups, diethylenetriaminepentaacetic acid-hexadecylamide (DTPA-lipid) was synthesized for NPS construction. The resulting dNPS (see details of the formulation components in Table 3 and TEM images in Figure 5A) showed enhanced transport behavior similar to eNPS. As shown in Figure 5B, dNPS and eNPS containing either 50 mol% DTPA-lipid or EDTA-lipid showed similar enhanced fluorescent signals compared to PS under fluorescence microscopy imaging at both 3 and 6 h incubation times, suggesting that the addition of carboxylic acid groups (five groups in DTPA vs. four groups in EDTA) to the EDTA-lipid head group did not affect their uptake. Quantitative cellular uptake studies further confirmed non-significant differences between the intracellular signals of dNPS and eNPS at each time point (6, 18, or 24 hours) (p>0.05), suggesting that the cellular uptake profiles of the two nanoparticles were similar (Figure 5C). Similarly, both eNPS and dNPS had significantly higher intracellular accumulation compared to PS at all time points (p<0.05). Collectively, these data suggest that both the tetraacetate group of the EDTA-lipid and the pentaacetate head group of the DTPA-lipid in the NPS platform are responsible for the greatly improved PS uptake.

[0064] Ca 2+ or Mg 2+ To investigate whether EDTA chelation with eNPS plays a role in the enhanced PS uptake, eNPS was treated with high concentrations of Ca prior to cell incubation. 2+ or Mg 2+ As shown in Figure 6A, the Ca 2+ / Mg 2+Treated eNPS showed enhanced uptake similar to untreated eNPS when compared to PS. In addition, dNPS(Gd) produced by Gd-chelating DTPA-lipids (Figure 5A) showed similar cellular uptake as metal-free dNPS (Figure 6B). These results, taken together with the previous observation in Figure 16 that there was no enhancement of PS uptake caused by 1 mM free EDTA, suggest that the mechanism of eNPS-enhanced transport is Ca 2+ or Mg 2+ This confirms that the mechanism of action is not dependent on EDTA chelation with riboflavin, thus suggesting a previously unknown mechanism of action.

[0065] [Table 3]

[0066] Assessment of the effect of the lipid chain of the chelator-lipid on NPS uptake To examine the effect of the lipid chain of the chelator-lipid on NPS uptake, DTPA-lipids with various double lipid chains, including 16:0PE-DTPA, 16:0PE-DTPA(Gd), and DTPA-BSA(Gd) (their chemical structures are shown in Figure 7A), were incorporated into porphysomes and allowed to uptake of DPS. 16PE , D.P.S. 16PE (Gd) and DPS BSA The components and characterization of these formulations are summarized in Table 3, and representative TEM images of these are shown in Figure 7A. eNPS and dNPS (containing a single fatty acid lipid chain) were 16PE , dNPS 16PE (Gd) and dNPS BSA We demonstrated significantly higher uptake compared to the (Gd) (containing double fatty acid lipid chains) variation (Figure 7B), indicating that a single fatty acid lipid chain in eNPS or dNPS results in maximal uptake / non-quenching of NPS.

[0067] Characterization of the effect of cholesterol and PEG content on the cellular uptake and serum stability of NPS To further increase the serum stability and possible blood circulation time of NPS, a novel eNPS formulation, designated LC-eNPS, was developed. LC-eNPS contains the same ratio of porphyrin-lipid:EDTA-lipid as NPS, but with increased cholesterol (30 mol%) and DSPE-PEG2000 (5 mol%) content (Table 3). After 24 h of incubation with 5 M nanoparticles, LC-eNPS maintained 10-fold enhanced cellular uptake over PS, but showed less than 50% cellular uptake when compared to eNPS (Figure 8A). Because the fluorescence is highly quenched in intact porphysomes but not when the porphysome nanostructures are dissociated, the change in the fluorescence quenching efficiency of NPS was used to track the stability of the particles. After incubation with PBS containing 50 vol% FBS, LC-eNPS appeared to be more stable compared to eNPS, with higher fluorescence quenching at all time points and maintaining 85% quenching over 24 h (Figure 8B), suggesting that increasing the cholesterol and PEG content to 30% and 5%, respectively, could improve the stability of eNPS in serum conditions with a gradual decrease in the cellular uptake efficiency.

[0068] Study of the mechanism of cellular uptake of NPS The general uptake mechanism (passive or active) of NPS in vitro was then examined by temperature and energy modulation studies. Fluorescence microscopy was used to monitor their uptake. After incubation of KB cells with eNPS and dNPS at various temperature conditions, stronger fluorescent signals were observed at 37°C compared to 18°C ​​and 4°C, suggesting that the rapid uptake of eNPS and dNPS was mostly via an active process (Figure 9A). By creating ATP-depleted cell conditions using NaN3 and 2-DG, it was observed that there was minimal fluorescent signal of eNPS and dNPS in the cells compared to normal culture conditions (Figure 9B). This suggests that eNPS and dNPS are mainly actively transported into the cells.

[0069] To gain insight into eNPS on the PDT mechanism, the subcellular localization of eNPS and dNPS was examined on KB cells under confocal microscopy imaging. A punctate pattern of eNPS and dNPS signals was clearly observed, suggesting that both nanoparticles were not dispersed throughout the cytosol. By colocalization with mitochondrial and lysosomal trackers, eNPS and dNPS did not appear to colocalize with either Mitotracker Green FM or Lysotracker Red DND-99 (Figure 10). Further colocalization studies with other organelle trackers such as endoplasmic reticulum, Golgi, and endosome trackers will be examined.

[0070] Evaluation of NPS for in vitro PDT The in vivo PDT activation of eNPS, LC-eNPS, and PS was evaluated in KB cells. The results are shown in Figure 11 and demonstrate that 5 μM eNPS, PS, and LC-eNPS had minimal dark toxicity to cells after 24 h of incubation. Upon light treatment, eNPS induced the greatest decrease in cell viability at all time points when compared to the other groups, suggesting that it had the greatest PDT efficacy. LC-eNPS also caused a greater decrease in cell viability, and the resulting cell death was highly proportional to the concentration of LC-eNPS particles and the dose of light. After 24 h of cell incubation, both 5 μM eNPS and LC-eNPS induced minimal dark toxicity to cells after 24 h of incubation at 5 or 10 J / cm. 2 A dose of 100 μg / kg caused >95% cell death. However, PS-treated cells showed minimal cytotoxicity under all experimental conditions (all concentrations, incubation times, and light doses). The in vitro PDT efficacy trends of eNPS, LC-eNPS, and PS correlated well with their uptake efficiencies (Figure 8A). Thus, the enhanced transport of eNPS and LC-eNPS ultimately induced effective PDT.

[0071] Assessment of NPS-enhanced transport and activation in vivo To evaluate the effect of NPS on in vivo tumor accumulation and activation, equivalent doses of eNPS and PS (4 mg / kg) were administered intravenously to mice bearing KB subcutaneous tumors, followed by in vivo fluorescence imaging of all animals over a 24-h period using a Maestro system (CRI, USA). Images were acquired using a 575-605 nm excitation filter and a 645 nm long-pass emission filter with an exposure time of 200 ms. As shown in Figure 12, eNPS demonstrated significant accumulation and activation in tumors 30 min after injection and remained stable for 24 h (n=5), whereas PS showed negligible fluorescence signal in tumors within 5 h and even lower signal at 24 h, indicating that eNPS significantly enhanced porphysome accumulation and activation in tumors for successful tumor fluorescence imaging.

[0072] We then applied NPS-enhanced fluorescence imaging to more biologically relevant hamster cheek carcinogenesis, which occurs with repeated DMBA treatment and closely mimics the clinical manifestations of human oral cancer. 26After intravenous injection of eNPS and PS, in vivo fluorescence imaging was performed using a NOVADAQ Pinpoint system (Mississauga, Ontario) to visualize porphyrin fluorescence in hamster cheek tumors. As shown in Figure 13A, strong tumor-specific fluorescence was detected 15 min after injection of eNPS, peaked at 3 h, and remained stable for 24 h, while the PS group showed a much weaker tumor signal at all time points. The tumor fluorescence intensity at various time points was quantified by the Pinpoint system and profiled in Figure 13B. It was clear that enhanced fluorescence signals were observed in the tumors of the eNPS group (n=12) versus the PS group (n=2) throughout all time points, specifically, a 4.4-fold enhanced signal was observed at the early time point of 3 h and a 1.7-fold enhancement at 24 h. These data further demonstrated the rapid and enhanced accumulation and activation of eNPS over PS in tumors. Thus, the NPS platform offers the potential to overcome the limitations of poor tumor accumulation of PS to advance porphysomes for cancer imaging and effective PDT.

[0073] In vivo PDT Referring to FIG. 18, after preliminary in vivo PDT experiments to determine optimal laser treatment parameters, an evaluation of the in vivo PDT efficacy of eNPS, LC-eNPS, and PS at various drug-light-intervals was performed. Athymic nude mice bearing subcutaneous KB tumors were intravenously injected with eNPS, LC-eNPS, or PS (10 mg / kg porphyrin concentration), followed by 671 nm laser irradiation (50 mW, 115 J / cm) at 1, 3, and 6 hours after injection. 2) was performed. PDT efficacy was evaluated by monitoring the survival rate of mice and changes in tumor size after treatment for up to 4 weeks. Tumor sizes in all PDT treatment groups were found to be statistically lower compared to those in the control group. Both eNPS and LC-eNPS (3 h DLI) demonstrated enhanced PDT efficacy compared to PS. ePS and LC-ePS PDT treatments with 3 h DLI showed significantly higher rates of complete tumor resection (100% and 80%, respectively) compared to 20% for PS with 3 h DLI.

[0074] In vivo fluorescence activation With reference to FIG. 19, to assess the extent to which unquenched or "active" eNPS and LC-eNPS, compared with PS, are available for PDT at the tumor site, tumor fluorescence activation following injection of these nanoparticles was monitored. Athymic nude mice bearing subcutaneous KB tumors were intravenously injected with eNPS, LC-eNPS or PS (10 mg / kg porphyrin concentration), followed by in vivo tumor fluorescence imaging at 1, 3, and 6 hours post-injection. Both eNPS and LC-eNPS demonstrated the greatest in vivo fluorescence activation at all time points compared with PS.

[0075] In this study, we developed a next-generation porphysome platform (NPS) by introducing single-chain fatty acid EDTA-lipids into porphysome formulations, which demonstrated significantly enhanced intracellular tumor cell accumulation, resulting in efficacious PDT. Enhanced tumor accumulation and activation of NPS has also been validated in both subcutaneous mouse tumor models and biorelevant hamster buccal carcinogenesis models. To our knowledge, this is the first report to incorporate EDTA-lipids into nanoparticle formulations to improve their biological and therapeutic properties. Importantly, the enhanced transport of nanoparticles by EDTA-lipids is via a previously unknown mechanism and can be extended to single fatty acid DTPA-lipid conjugates, but not to other dual fatty acid DTPA-lipids. Apart from their phototherapeutic potential, NPSs show great promise for the application of fluorescence-guided surgery due to their preferential rapid uptake and induction of activated fluorescence. The additional metal / radioisotope chelating capacity of EDTA-lipid / DTPA-lipid complements the inherent metal chelating properties of porphysomes, allowing NPS to transport a wide range of radioisotopes for multimodal imaging and radiotherapy. Thus, the NPS platform demonstrates the potential to overcome the limitation of poor intracellular accumulation of porphysomes to advance porphysomes for multimodal imaging and effective PDT of cancer.

[0076] Although preferred embodiments of the invention have been described herein, those skilled in the art will recognize that modifications may be made without departing from the spirit of the invention or the scope of the appended claims. All documents disclosed herein, including the following reference list, are incorporated by reference.

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Claims

1. A bilayer nanovesicle comprising a porphyrin-phospholipid conjugate and a chelator-fatty acid conjugate, wherein the chelator-fatty acid conjugate comprises an aminopolycarboxylic acid bound to a single-chain fatty acid, and the porphyrin-phospholipid conjugate preferably comprises one porphyrin, porphyrin derivative or porphyrin analog covalently bound to the lipid side chain of one phospholipid at the sn-1 or sn-2 position.

2. The bilayer nanovesicle according to claim 1, wherein the aminopolycarboxylic acid is glycinate, IDA, NTA, EDTA, DTPA, EGTA, BAPTA, NOTA, DOTA, nicotianamine, EDDHA, or EDDS.

3. The bilayer nanovesicle according to claim 2, wherein the aminopolycarboxylic acid is EDTA or DTPA.

4. The bilayer nanovesicle according to any one of claims 1 to 3, wherein the single-chain fatty acid contains 10 to 26 carbons.

5. The bilayer nanovesicle according to claim 4, wherein the single-chain fatty acid contains 12 to 22 carbons.

6. The bilayer nanovesicle according to claim 5, wherein the single-chain fatty acid contains 14 to 18 carbons.

7. The bilayer nanovesicle according to claim 6, wherein the single-chain fatty acid contains 16 carbons.

8. The bilayer nanovesicle according to claim 7, wherein the single-chain fatty acid is hexadecylamide.

9. The bilayer nanovesicle according to claim 1, wherein the chelator-fatty acid conjugate is EDTA-hexadecylamide or DTPA-hexadecylamide.

10. The bilayer nanovesicle according to any one of claims 1 to 3, comprising 15% to 60% of the chelator-fatty acid conjugate.

11. The bilayer nanovesicle according to claim 10, comprising 25% to 50% of the chelator-fatty acid conjugate.

12. The bilayer nanovesicle according to claim 11, comprising 30% to 40% of the chelator-fatty acid conjugate.

13. The bilayer nanovesicle according to claim 12, comprising about 30% of the chelator-fatty acid conjugate.

14. The bilayer nanovesicle according to any one of claims 1 to 3, comprising 1 to 60 mol% of the porphyrin-phospholipid conjugate.

15. The bilayer nanovesicle according to claim 14, comprising 20 to 40 mol% of the porphyrin-phospholipid conjugate.

16. The bilayer nanovesicle according to claim 1, comprising about 27 mol% of the porphyrin-phospholipid conjugate.

17. The porphyrin, porphyrin derivative or porphyrin analog in the porphyrin-phospholipid conjugate is selected from the group consisting of hematoporphyrin, protoporphyrin, tetraphenylporphyrin, pyropheophorbide, bacteriochlorophyll, chlorophyll a, benzoporphyrin derivative, tetrahydroxyphenylchlorin, purpurin, benzochlorin, naphthochlorin, verdin, rhodin, ketochlorin, azachlorin, bacteriochlorin, triporphyrin, benzobacteriochlorin, expanded porphyrin and porphyrin isomer, the bilayer nanovesicle according to any one of claims 1 to 3.

18. The expanded porphyrin is texaphyrin, sapphyrin or hexaphyrin, and the porphyrin isomer is porphycene, inverted porphyrin, phthalocyanine, or naphthalocyanine, the bilayer nanovesicle according to claim 17.

19. The phospholipid in the porphyrin-phospholipid conjugate contains phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine or phosphatidylinositol, the bilayer nanovesicle according to any one of claims 1 to 3.

20. The phospholipid contains an acyl side chain of 12 to 22 carbons, the bilayer nanovesicle according to claim 19.

21. The porphyrin in the porphyrin-phospholipid conjugate is pyropheophorbide-a acid, the bilayer nanovesicle according to any one of claims 1 to 3.

22. The porphyrin in the porphyrin-phospholipid conjugate is a bacteriochlorophyll derivative, the bilayer nanovesicle according to any one of claims 1 to 3.

23. The phospholipid in the porphyrin-phospholipid conjugate is 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine or 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine, the bilayer nanovesicle according to any one of claims 1 to 3.

24. The porphyrin-phospholipid conjugate is pyro lipid, the bilayer nanovesicle according to any one of claims 1 to 3.

25. The bilayer nanovesicle according to any one of claims 1 to 3, wherein the porphyrin-phospholipid conjugate is an oxy-bacteriochlorophyll-lipid, a texaphyrin-phospholipid conjugate, or an aza-boron dipyrromethene (BODIPY)-phospholipid conjugate.

26. The bilayer nanovesicle according to any one of claims 1 to 3, wherein the porphyrin is bound to the glycerol group on the phospholipid by a carbon chain linker having 0 to 20 carbons.

27. The bilayer nanovesicle according to any one of claims 1 to 3, further comprising a PEGylated emulsifier.

28. The bilayer nanovesicle according to claim 27, wherein the PEGylated emulsifier has a molecular weight in the range of about 1000 to about 5000.

29. The bilayer nanovesicle according to claim 27, wherein the PEGylated emulsifier is selected from the group consisting of N-(methoxypolyethylene glycol 5000 carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (MPEG5000-DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)-2000 (DMPE-PEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)-2000 (DSPE-PEG2000), polyoxyethylene 40 stearate (PEG40S), and combinations thereof.

30. The bilayer nanovesicle according to claim 28, wherein PEG or PEG-lipid is present in an amount of 1 to 10 mol%.

31. The bilayer nanovesicle according to claim 28, wherein PEG or PEG-lipid is present in an amount of 2 to 7 mol%.

32. The bilayer nanovesicle according to any one of claims 1 to 3, further comprising cholesterol.

33. The bilayer nanovesicle according to claim 32, wherein the remaining composition of the bilayer nanovesicle consists essentially of cholesterol.

34. The bilayer nanovesicle according to claim 32, wherein the cholesterol is present in an amount of 1 to 60 mol%.

35. The bilayer nanovesicle according to any one of claims 1 to 3, which is substantially spherical.

36. The bilayer nanovesicle according to any one of claims 1 to 3, having a diameter of about 70 to 120 nm.

37. The bilayer nanovesicle according to claim 36, having a diameter of about 90 to 100 nm.

38. The bilayer nanovesicle according to any one of claims 1 to 3, wherein the porphyrin-phospholipid conjugate contains a metal chelated therein and optionally a radioisotope of the metal.

39. A composition comprising the bilayer nanovesicle according to any one of claims 1 to 3 in a buffer solution.

40. The composition according to claim 39 for performing photodynamic therapy on a target region of a subject, wherein the target region is used to irradiate the composition with light having a wavelength that excites the composition to produce radicals and / or reactive oxygen species.

41. The composition according to claim 39 for imaging a target region of a subject, wherein the composition is used to measure and / or detect a fluorescence or photoacoustic signal in the target region.

42. The composition according to claim 39 for transporting a radioisotope to a subject, wherein the bilayer nanovesicle in the composition has a radioisotope chelated therein.

43. The composition according to claim 39 for performing photodynamic therapy.

44. The composition according to claim 39 for performing imaging.

45. The composition according to claim 39 for transporting a radioisotope to a subject.

Images