US20130129636A1 - Novel Liposome Nanoparticles for Tumor Magnetic Resonance Imaging - Google Patents

Novel Liposome Nanoparticles for Tumor Magnetic Resonance Imaging Download PDF

Info

Publication number
US20130129636A1
US20130129636A1 US13/511,126 US201013511126A US2013129636A1 US 20130129636 A1 US20130129636 A1 US 20130129636A1 US 201013511126 A US201013511126 A US 201013511126A US 2013129636 A1 US2013129636 A1 US 2013129636A1
Authority
US
United States
Prior art keywords
liposome
magnetic resonance
tumour
liposomes
folate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/511,126
Other languages
English (en)
Inventor
Nazila Kamaly
Tammy Louise Kalber
Gavin David Kenny
Maya Thanou
Andrew David Miller
Jimmy Bell
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Medical Research Council
Ip2ipo Innovations Ltd
Original Assignee
Medical Research Council
Imperial Innovations Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Medical Research Council, Imperial Innovations Ltd filed Critical Medical Research Council
Publication of US20130129636A1 publication Critical patent/US20130129636A1/en
Assigned to IMPERIAL INNOVATIONS LIMITED reassignment IMPERIAL INNOVATIONS LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MILLER, ANDREW DAVID, THANOU, MAYA
Assigned to MEDICAL RESEARCH COUNCIL reassignment MEDICAL RESEARCH COUNCIL ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KAMALY, NAZILA, KALBER, TAMMY LOUISE, BELL, JIMMY, KENNY, GAVIN DAVID
Abandoned legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1806—Suspensions, emulsions, colloids, dispersions
    • A61K49/1812—Suspensions, emulsions, colloids, dispersions liposomes, polymersomes, e.g. immunoliposomes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00—Nanotechnology
    • Y10S977/70—Nanostructure
    • Y10S977/773—Nanoparticle, i.e. structure having three dimensions of 100 nm or less

Definitions

  • the present invention relates to novel liposomes suitable for use in the preparation of a contrast agent for use in enhancing magnetic resonance imaging (MRI), particularly in enhancing magnetic resonance images of tumours.
  • MRI magnetic resonance imaging
  • Cancer imaging is one of the most important disease areas where molecular imaging is set to play a major role, both in the detection of cancer and subsequent treatment.
  • MRI magnetic resonance imaging
  • nanotechnology has a lot to offer as nanomedicine is set to make considerable contributions in the important areas of drug delivery, disease detection, and therapy.
  • the application of nanotechnology platforms to cancer imaging has opened up opportunities for the use of multifunctional nanoparticle systems such as liposomes, in the study of cancer detection and therapy.
  • MRI Magnetic resonance
  • T 1 longitudinal
  • T 2 transverse
  • Contrast agents incorporating gadolinium increase both 1/T 1 and 1/T 2 but are generally used in T 1 -weighted imaging where their 1/T 1 effect is greater in tissue than their 1/T 2 enhancement.
  • 3 Iron containing agents lead to more substantial increases in 1/T 2 and are therefore visualised with T 2 -weighted images.
  • the use of gadolinium based MRI contrast agents produces a positive image enhancement (bright signal on image) and the use of iron agents leads to a negative image enhancement (darkening of image).
  • FIG. 1 Gd.DTPA [gadolinium(III)-diethylenetrianninepentaacetate complex] ( FIG. 1 ) was the first water soluble, renally excreteable contrast agent approved for clinical use by the FDA since mid 1988, and is currently routinely used under the commercial name Magnevist® 4 .
  • FIG. 1 presents a few examples of the most commonly utilised contrast agents in the clinic.
  • These compounds are generally inert stable complexes where the metal ion is strongly chelated to the poly(aminocarboxylate) ligands.
  • These types of agents are non-specific, mainly reside within the blood stream and also accumulate in the kidneys due to their glomerular filtration and are generally excreted un-metabolised. Nevertheless, their use in clinical MR imaging has great value as anatomical abnormalities such as gliomas and lesions within the brain can be visualised, since under normal physiological conditions these agents do not cross an intact blood brain barrier. Pathologies within the liver and other organs can also be visualised since these contrast agents rapidly accumulate into interstitial spaces and can therefore increase the signal to noise ratio, in such regions of increased fluid volume.
  • Liposomes are composed of lipid constituents, with hydrophilic head groups and hydrophobic tail groups ( FIG. 2 ). When hydrated, these lipids aggregate together to form self-assembled bilayer vesicles that enclose an aqueous compartment. Due to this aqueous cavity, they have traditionally been used as drug delivery vehicles, encapsulating water soluble drugs in order to improve drug pharmacokinetics. In addition, nucleic acids have also been condensed into liposome formulations for effective transfection and gene delivery. Despite these additional functionalities, liposomes were originally studied as models of biological membranes, and this is a key concept in the realisation of their biocompatibility.
  • liposomes can be altered to change their interaction with various molecules or even larger structures such as cells. This can be done by altering the overall charge of the liposome surface by incorporating lipids with highly charged polar head groups in the liposome formulation, e.g. the incorporation of cationic lipids in the formulation produces cationic liposomes.
  • Cationic lipids have been used to formulate liposome/DNA complexes (lipoplexes) used as gene delivery systems in vitro and in vivo.
  • Liposomes are typically characterised by their size, shape and lamellarity. They may be composed of a single bilayer (unilamellar), a few bilayers (oligolamellar), or multiple bilayers (multilamellar).
  • the rigidity of the membrane can be modified with the use of suitable lipids; and the fluidity of the membrane may be varied by using phospholipids with high or low phase transition temperatures.
  • lipid derivatives of stearic acids (fully saturated C18 lipidic chains) bestow rigidity and impermeability to the membrane
  • lipid derivatives of oleic acid unsaturated C18 lipidic chains
  • gadolinium lipids By incorporating gadolinium lipids into the membranes of liposomes they can be rendered MRI visible and systems with a better control of size can be obtained. 5 Liposomes are well suited as carriers of a high payload of gadolinium into cells. The incorporation of amphipathic gadolinium complexes into liposomal membranes has yielded paramagnetically labelled liposomes which significantly enhance proton relaxivity. These paramagnetic liposomes have been used in a number of investigations including that of cellular labelling and tracking. 6 The incorporation of gadolinium lipids into liposome formulations was demonstrated by Kabalka et al.
  • Gd.DTPA.BSA gadolinium(III).diethylenetriaminopentaacetic acid-bis(stearylamide)
  • liposome size, surface charge and specificity allows for potential pathological imaging such as the imaging of solid tumours in vivo.
  • This tuning of liposomes is made possible by adjusting the composition of the liposome formulation.
  • Surface charges tending to neutrality are best suited for in vivo purposes in order to reduce the recognition of liposome particles by plasma proteins and the reticuloendothelial system (RES). This can be achieved through the inclusion of charge neutral lipids in the liposome formulation.
  • RES reticuloendothelial system
  • Gd.DOTA.Chol gadolinium(III).1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetate-cholesterol
  • FIG. 3 MAGfect a liposome formulation containing this gadolinium lipid to be an efficient cellular dual labelling and transfection vehicle.
  • a paramagnetic lipid Gd.DOTA.DSA (gadolinium(III)2- ⁇ 4,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N′-amido-methyl]-1,4,7,10-tetra-azacyclododec-1-yl ⁇ -acetic acid) (see FIG. 4 ) was synthesised using a combination of solution and solid phase chemistries.
  • the DOTA chelate was conjugated to the lipid via a stable yet biodegradable amide functional group.
  • This gadolinium lipid was also designed with a five-atom spacer between the gadolinium chelator and lipidic alkyl chain moieties. This spacing between the head group and the lipidic alkyl tail was considered to be optimal in order to ensure maximum exposure of the gadolinium chelate to water on the hydrophilic surface of the liposome particles.
  • this gadolinium lipid was also designed with the DOTA ligand rather than the more frequently used DTPA [diethylenetriaminopentaacetic acid], since the former macrocyclic ligand is considered to be a more effective chelator of gadolinium, able to retain the metal ion even in the acidic environment of the endosome.
  • the FDA approved Gd.DOTA chelate was chosen, since due to their higher stability constants, DOTA based conjugates are known to be more stable in vivo in comparison to DTPA ligands.
  • Gd.DOTA.DSA In order to establish the relaxation properties of Gd.DOTA.DSA, MRI studies of the lipid in aqueous solution were performed and T 1 values and relaxivity parameters generated in milliseconds. The efficacy of gadolinium lipid Gd.DOTA.DSA was compared to the clinical contrast agent Magnevist® (Schering AG) and Gd.DTPA.BSA (see Table 1), and was found to compare favourably at the clinically relevant dose. These data also showed Gd.DOTA.DSA to have a comparable, if slightly better, T 1 relaxation than the widely used Gd.DTPA.BSA lipid. A standard T 1 saturation recovery method (spin echo sequence) was used to determine T 1 values (according to Eq. 1), where x is TR (time to repeat), and Si is the measured signal for a given TR.
  • T 1 saturation recovery method spin echo sequence
  • Equation 1 T 1 saturation recovery equation used to determine T 1 values.
  • Table 1 presents T 1 relaxation values for the synthesised Gd lipids in addition to relevant controls.
  • a liposome comprising Gd.DOTA.DSA (gadolinium(III)2-14,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N′-amido-methyl]-1,4,7,10-tetra-azacyclododec-1-yl ⁇ -acetic acid), characterised in that said liposome further comprises a neutral, fully saturated phospholipid component.
  • Preferred aspects of the liposome of this first aspect of the invention include: (2) a liposome according to (1), wherein said fully saturated phospholipid component is a 1,2-di(C 12 -C 20 saturated lipid)-sn-glycero-3-phosphocholine, wherein the saturated lipid groups can be the same or different from each other. (3) a liposome according to (1), wherein said fully saturated phospholipid component is DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine). (4) a liposome according to any one of (1) to (3), wherein said liposome further comprises cholesterol.
  • said polyethylene glycol-phospholipid is DSPE-PEG(2000) [distearoylphosphatidylethanolamine-polyethylene glycol (2000)].
  • (11) a liposome according to anyone of (1) to (10), wherein the amount of cholesterol in said liposome is from 29 to 31 mol % of the total liposome formulation.
  • Gd.DOTA.DSA, cholesterol, DSPC and DSPE-PEG(2000) are present in the ratio 30:33:30:7 mol % respectively in said liposome formulation.
  • Preferred liposomes comprising a tumour targeting agent include: (20) a liposome according to (19), wherein said tumour targeting agent comprises a ligand for a receptor that is over-expressed in tumour cells relative to the expression of said receptors in the cells of non-tumourous tissue of mammals. (21) a liposome according to (20), wherein said tumour targeting agent comprises a folate moiety. (22) a liposome according to (20), wherein said tumour targeting agent is a phospholipid-polyethylene glycol-folate compound.
  • a magnetic resonance contrast agent comprising liposomes according to any one of (1) to (26) and a pharmaceutically acceptable carrier.
  • said pharmaceutically acceptable carrier is an aqueous carrier.
  • a magnetic resonance contrast agent according to (27) or (28) for use in medicine e.g. in diagnosis.
  • a magnetic resonance contrast agent according to (27) or (28) for use in medicine e.g. in diagnosis.
  • a liposome according to any one of (1) to (26) in the preparation of a magnetic resonance contrast agent for enhancing magnetic resonance images of organs and organ structures in a mammal.
  • Preferred aspects of this fifth embodiment include: (31) use according to (30) in the preparation of a magnetic resonance contrast agent for enhancing a magnetic resonance image of a tumour in a mammal.
  • (32) use according to (30) or (31), wherein the concentration of said liposomes in said magnetic resonance contrast agent is 1-50 mg/mL, more preferably 1-30 mg/mL.
  • (33) a method of magnetic resonance imaging of an organ or organ structure in a mammal, comprising the steps of: (a) administering the magnetic resonance contrast agent according to (27) or (28) to a patient; and (b) taking images of the organ of interest in the patient.
  • Preferred aspects of this sixth embodiment include: (34) a method according to (33), wherein said magnetic resonance contrast agent is used for enhancing a magnetic resonance image of a tumour in a mammal.
  • 35) a method according to (33) or (34), wherein the concentration of liposomes in said magnetic resonance contrast agent is 1-50 mg/mL, more preferably 1-30 mg/mL.
  • 36) a method of magnetic resonance imaging of an organ or organ structure in a mammal pre-administered with the magnetic contrast agent according to (27) or (28) comprising the step of: (i) taking images of the organ of interest in the patient.
  • a method of making a liposome according to (1) to (26) comprising mixing a solution of Gd.DOTA.DSA (gadolinium(III)2-14,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N′-amido-methyl]-1,4,7,10-tetra-azacyclododec-1-yl ⁇ -acetic acid) and a solution of a neutral, fully saturated phospholipid.
  • a preferred aspect of the eighth embodiment includes: (38) drying the mixture (e.g. in vacuo) and optionally rehydrating the resulting liposome.
  • a ninth aspect of the present invention there is provided: (39) a method of making a magnetic contrast agent according to (27) or (28) comprising mixing a liposome of (1) to (26) and a pharmaceutically acceptable carrier.
  • Preferred aspects of the eighth and ninth embodiments are the same as those listed above in relation to the first, second and third aspects.
  • FIGS. 1 to 29 wherein:
  • FIG. 1 shows Gadolinium based clinical contrast agents approved by the FDA
  • FIG. 2 shows liposome formation from amphipathic lipids
  • FIG. 3 shows Gd.DOTA.Chol, a T 1 lipidic contrast agent component of MAGfect
  • FIG. 4 shows the paramagnetic gadolinium lipid target, Gd.DOTA.DSA;
  • FIG. 5 shows an electrospray mass spectrum of Gd.DOTA.DSA, m/z: 1117.2 (M ⁇ H), the isotopic peaks of Gd are visible in the top right corner;
  • FIG. 7 depicts the EPR, where normal tissue does not have endothelial gaps wide enough to allow for macromolecular or nanoparticulate agents to leak into the extracellular tissue lining blood vessels, whereas tumour tissue has a disrupted endothelial layer, allowing for larger particles to “seep” into the tumour extracellular domain;
  • FIG. 8 provides a depiction of one of the preferred liposomes of the invention, liposome A, a novel MRI active liposome with tumour imaging utility;
  • FIG. 9 depicts the structures of lipids forming one of the preferred liposomes of the invention, liposome A;
  • FIG. 10 shows the influence of cholesterol lipid on liposome bilayer permeability and rigidity
  • FIG. 11 shows the size distribution of the Liposome A particles
  • FIG. 12 shows the results of the MTT cell viability assay on kidney LCC PK1 cells using the Liposome A particles of the present invention at various doses and three incubation periods;
  • FIG. 13 shows the results of the MTT cell viability assay on HepG2 liver cells using the Lipsome A particles of the present invention at various doses and three incubation periods;
  • FIG. 14 shows the results obtained in the LDH assay of on kidney LCC PK1 cells using the Liposome A particles of the present invention at various doses and three incubation periods;
  • FIG. 15 shows the results obtained in the LDH assay on HepG2 liver cells using the Lipsome A particles of the present invention at various doses and three incubation periods;
  • FIG. 16 presents magnetic resonance images of tumour bearing mice at various periods after injection with a preparation comprising Liposome A, with the dotted white circles marking the tumour area and the white arrow pointing to the tumour location;
  • FIG. 17 shows a graph of % tumour signal intensity at various TR time points post administration of Liposome A over the 24 hour MRI experiment in which the images of FIG. 16 were obtained;
  • FIG. 18 shows a graph of tumour signal intensity increase over time, post-administration of Liposome A over the 24 hour MRI experiment in which the images of FIG. 16 were obtained;
  • FIG. 19 shows the results of fluorescence microscopy on sectioned IGROV-1 tumours post Liposome A administration; in the left panel a fluorescence image is shown while in the right panel an H&E stain (X 400) is depicted);
  • FIG. 20 provides a depiction of one of the preferred liposomes of the invention, liposome B, a novel MRI active liposome which has a folate receptor moiety and which has tumour imaging utility;
  • FIG. 21 depicts the structures of lipids forming one of the preferred liposomes of the invention, liposome B;
  • FIG. 22 shows the results of FACS analysis of four cell lines for ⁇ -FR expression
  • FIG. 23 is a graph setting out the amount of Gd taken up by IGROV-1 cells post incubation with Liposome B with varying mol % of the folate targeting lipid;
  • FIG. 24 presents data on the size and polydispersity distributions of Liposome B
  • FIG. 25 is a graph showing the results of a MTT assay with Liposome B particles at various doses and three incubation periods in LCC PK1 cells;
  • FIG. 26 is a graph showing the results of an LDH cytotoxicity assay with Liposome B particles at various doses and three incubation periods in LCC PK1 cells;
  • FIG. 27 presents magnetic resonance images of tumour bearing mice at various periods after injection with a preparation comprising Liposome B, with dotted white circles marking the tumour location;
  • FIG. 29 shows the results of fluorescence microscopy on sectioned IGROV-1 tumours post Liposome B administration; in the left panel a fluorescence image is shown while in the right panel an H&E stain (X 400) is depicted);
  • Appropriate neutral, fully saturated phospholipids suitable for use in the construction of Gd.DOTA.DSA liposomes of the present invention are typically 1,2-di(C 12 -C 20 saturated lipid)-sn-glycero-3-phosphocholines. More preferred examples include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids. 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) is most preferred.
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • the amount of fully saturated phospholipid component in said liposome is from 32 to 34 mol % of the total liposome formulation, and most preferably it is 33 mol %.
  • the amount of Gd.DOTA.DSA component in said liposome is from 29 to 31 mol % of the total liposome formulation, and most preferably it is 30 mol %.
  • the liposomes typically have a size of 100 nm or less. By carefully nanoengineering the liposomes in this way to ensure that their size remains below 100 nm, this size range is considered optimal for the accumulation of liposomes in solid tumours due to the characteristics of tumour tissue.
  • Tumour tissue is considered to possess a universal affinity for macromolecular agents, termed the enhanced permeation and retention effect (EPR), whereby macromolecular agents accumulate in tumour tissue.
  • EPR enhanced permeation and retention effect
  • tumour properties such as increased angiogenesis, a heterogeneous and destructive vascular infrastructure, impaired lymphatic drainage and a “leaky” endothelial layer are all factors that contribute to the accumulation of macromolecular structures within tumour tissue (see FIG. 7 ).
  • this provides particular, substantial advantages over the prior art MRI active liposomes and non-liposome paramagnetic contrasting agents as a result.
  • the EPR effect has become a standard model for the targeting of macromolecular drugs and polymeric or liposomal macromolecules to tumours. These agents are easily adapted for the imaging of tumours through their modification to include an imaging probe or moiety for signal localisation.
  • the key mechanism here being the retention of macromolecules in solid tumours, in contrast to low-molecular weight agents, such as Gd.DTPA (MagnevistTM) which are re-circulated into blood through diffusion and cleared through the kidneys in relatively short periods post injection.
  • Gd.DTPA MagneticnevistTM
  • This retention effect or particle accumulation within tumour tissue is also referred to as passive-targeting, and it has been shown that due to this effective phenomena very high levels (10-50 fold) of polymeric drugs can accumulate at tumour sites within a few days.
  • tumour accumulation of nanoparticles in tumour tissue has been established as the extravasation of large molecules through the disrupted endothelium lining tumour blood vessels.
  • tumour extravasation size threshold a further reason for liposome size to remain within the 100 nm range for in vivo injections is due to clearance of large liposomes through the liver.
  • Large liposomes are taken up by liver cells which include hepatocytes and Kupffer cells, liposomal particles may accumulate in liver or spleen tissue due to the larger endothelial lining in these organs.
  • Cholesterol may preferably be incorporated into the formulation since this lipid induces diverse effects on the liposomal bilayer. Cholesterol has been shown to increase the head group spacing in liposome formulations and stabilise the resulting bilayer membranes. 9 Here, cholesterol presence in the liposome formulation controls membrane permeability of both fluid and rigid bilayers by inducing conformational ordering of the lipid chains ( FIG. 10 ). In addition, cholesterol can reduce serum induced aggregation as a direct result of its neutral charge. 10 Typically, the amount of cholesterol component in said liposome is from 29 to 31 mol % of the total liposome formulation, and most preferably it is 30 mol %.
  • polyethylene glycol may also be anchored into the liposome bilayer using a polyethylene glycol-phospholipid tethered construct.
  • preferred polyethylene glycol-phospholipids for use in the liposomes of the invention include DSPE-PEG(2000) [distearoylphosphatidylethanolamine-polyethylene glycol (2000)]. It has been shown that liposomes bearing a surface decorated with the neutral hydrophilic PEG polymer benefit from prolonged circulation times with half lives reported from 2 to 24 h in rodents, and as high as 45 h in humans.
  • the theory here is that surface-grafted PEG liposomes have reduced uptake by liver cells as the liposomes are not effectively bound by plasma proteins. 12 These liposomes are also referred to as sterically stabilised liposomes.
  • the PEG layer sterically inhibits both electrostatic and hydrophobic interactions of plasma components with the liposome bilayer.
  • the amount of polyethylene glycol-phospholipid component in said liposome is from 5 to 8 mol % of the total liposome formulation, and most preferably it is 7 mol %.
  • phospholipids with neutral head groups have been incorporated in the liposome formulation; as described above these include but are not limited to; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids.
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine
  • the utilisation of neutral lipids in addition to the incorporation of between 5-10 molar ratios of a PEGylated lipid in the liposome formulation provides steric stabilisation and protection from blood plasma proteins such as opsonins, and leads to the reduction of Kupffer cell uptake. It is thought that stabilisation occurs by the formation of highly hydrated shields of polymer molecules around the liposome surface. Due to this “shielding” characteristic, these types of lipo
  • the liposomes of the present invention may further incorporate a tumour targeting agent.
  • Liposomes of the present invention comprising a tumour targeting agent typically comprise a ligand for a receptor that is over-expressed in tumour cells relative to the expression of said receptors in the cells of non-tumourous tissue of mammals.
  • tumour targeting agent is one which comprises a folate moiety.
  • the tumour targeting agent is a phospholipid-polyethylene glycol-folate compound. More preferably the phospholipid-polyethylene glycol-folate compound is DSPE-PEG(2000)-Folate [distearoylphosphatidylethanolamine-polyethylene glycol (2000)-folate].
  • the amount of the folate moiety present in the liposome is 1-2 mol % of the total liposome formulation.
  • tumour targeting agents folate is a good example of such a targeting moiety; as folate-based targeting systems present an effective means of selectively delivering therapeutic or imaging agents to tumours.
  • FR folate receptor
  • the FR is over-expressed in several cancer types, such as brain, kidney, lung and breast cancers and in particular, in epithelial carcinomas such as ovarian cancers.
  • the FR ligand, folate (or folic acid) is a vitamin that is used for the biosynthesis of nucleotides and is utilized in high levels to meet the needs of proliferating cancer cells.
  • folate-targeted technology has been successfully applied to radio-imaging of therapeutic agents, 19 fluorescence imaging of cancer cells, 20 MRI contrast agents, and gadolinium liposomes. 22 Choi et al., have demonstrated the use of folate-targeted iron oxide nanoparticles for the imaging of induced KB tumours and showed these particles to have a 38% signal intensity increase compared to controls. 23 Successful tumour MRI with a non-targeted bimodal liposomal contrast agent was shown recently, whereby bimodal paramagnetic and fluorescent liposomes of ⁇ 100 nm in size were seen to accumulate in a mouse xenograft model of ovarian cancer.
  • Liposomes are able to accumulate within tumour tissue due to the widely reported enhanced permeation and retention effect (EPR) which relies on the passive accumulation of colloidal macromolecules of ⁇ 40 kDa and above in tumours. 25
  • EPR enhanced permeation and retention effect
  • the EPR effect arises due to aberrant tumour endothelium, which as a result of its “leakiness” allows the penetration of nanoparticles into tumour tissue.
  • Liposome accumulation in tumour tissue could be improved through the use of receptor targeting moieties that are either post-conjugated to the surface of liposomes, or are attached to lipids that become incorporated within the liposomal bilayer.
  • the human nasopharyngeal KB carcinoma cell line is considered to have the highest level of FR expression, yet the number of cases for this cancer are low in comparison to ovarian cancer which has the highest frequency (>90% of cases).
  • the ⁇ -FR isoform which is a glycosyl phosphatidylinositol (GPI)-anchored membrane protein is highly expressed in ovarian carcinoma.
  • the ⁇ -FR isoform has also been shown to have specific biomarker value, aiding in the identification of metastatic tumour site origin. 29 Therefore, we were interested in using this receptor in order to test the efficacy of folate targeted bimodal liposomes for the imaging of ovarian tumours using MRI.
  • FR targeted bimodal fluorescent and paramagnetic liposomes have been formulated and compared to non-targeted liposomes by both MRI and fluorescence microscopy. We have found that they give remarkably good results with low toxicity, excellent targeted MR signal enhancement and, after rapid accumulation in the tumour initially, a quick and natural clearance of the contrast agents from the body thereafter.
  • a magnetic resonance contrast agent which comprises liposomes according to any one of the first and second aspects of the present invention and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is an aqueous carrier such as a HEPES [(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] buffered solution.
  • a magnetic resonance agent according to the third aspect for use in medicine, preferably for use in diagnosis and particularly preferably for use in imaging organs and organ structures (e.g. tumours).
  • a liposome according to any one of the first and second aspects of the invention in the preparation of a magnetic resonance contrast agent for enhancing magnetic resonance images of organs and organ structures in a mammal.
  • the liposomes of the present invention are of particular use in the preparation of a magnetic resonance contrast agent for enhancing a magnetic resonance image of a tumour in a mammal.
  • the paramagnetic liposomes of the present invention have superior properties due to their optimal size (increased accumulation in tumours due to the EPR effect and reduced liver toxicity due to reduced uptake by Kupffer cells), greater stability, stronger gadolinium chelation while their non-ionic nature reduces the physichochemical consequences that have previously been observed with ionic gadolinium contrast agents wherein an excess of negative charge leads to competitive reactions in vivo and displacement of Gd 3+ .
  • the magnetic resonance contrast agents of the present invention provide substantial and surprising advantages over the prior art paramagnetic gadolinium contrast agents as they have excellent image enhancement ability while at the same time showing a much improved safety profile owing to the reduced dose of gadolinium that is required as the gadolinium liposomes of the present invention gradually accumulate in tumour tissues without accumulating in other organs, particularly the liver.
  • the contrast agents of the present invention can offer a wider scope of magnetic resonance directed imaging in the clinic than the agents known to date.
  • the concentration of the liposomes in the magnetic resonance contrast agents of the invention is 1-50 mg/mL, more preferably 1-30 mg/mL, but the invention is not limited to these ranges.
  • a pharmaceutically acceptable carrier for use in the preparation of the magnetic resonance contrast agents is an aqueous carrier such as a HEPES.
  • a method of magnetic resonance imaging of an organ or organ structure in a mammal comprising the steps of:
  • the method is used for enhancing a magnetic resonance image of a tumour in a mammal.
  • a concentration of liposomes in the magnetic resonance contrast agent of 1-50 mg/mL, more preferably 1-30 mg/mL, but the invention is not limited to this range.
  • Liposome A is depicted in FIG. 8 .
  • Liposome A is a novel MRI active liposome with tumour imaging ability, as we shall demonstrate below.
  • the Liposome A formulation consists of Gd.DOTA.DSA/DSPC/Cholesterol/DSPE_PEG2000: 30/33/30/7 mol %.
  • a 1 mol % DOPE-Rhodamine is also added to the formulation and 32 mol % of DSPC is used.
  • Liposome A was developed to observe signal enhancement of tumour tissue in vivo by MRI.
  • the structures of the lipids comprising this liposome system are shown in FIG. 9 .
  • MRI signal enhancement is achieved by incorporating the paramagnetic lipid Gd.DOTA.DSA into the liposome formulation.
  • the size distribution of the particles was measured as per FIG. 11 .
  • the Liposome A particle sizes in various PBS dilutions are shown in the bottom graph while those of the control particle are shown in the top graph.
  • the Liposome A formulation consists of Gd.DOTA.DSA/DSPC/Cholesterol/DSPE_PEG2000:30/33/30/7 mol % and the control nanoparticle is DOTA.DSA/DSPC/Cholesterol/DSPE_PEG2000:30/33/30/7 mol %.
  • NB The peaks at 1000 nm+ are removed post filtration through 0.2 ⁇ m filters (data not shown).
  • Liposome A and the control particle were extremely stable, and were sized below 100 nm at various dilutions in PBS.
  • the particle also had a very low polydispersity index, indicating a uniform and homogenous sample.
  • Liposome A The measured sizes for Liposome A are smaller than previously published DOPC liposomes, and the polydispersity index (PdI) is also much lower than those measured for the same formulation containing DOPC (see Table 2). This indicates that the new DSPC formulation offers a smaller size distribution, which is more favourable for liver clearance of the liposomes and gradual accumulation within tumour tissue, and also a lower polydispersity index confirms a more homogenous and uniform liposome sample.
  • PdI polydispersity index
  • Liposome A and the control nanoparticle of the same composition but without Gd chelated in the DOTA macrocycle was assessed using the MTT and LDH toxicity assays.
  • the liposomes were formulated in buffer [20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], pH 6.8, 150 mM NaCl) at a total concentration of 25 mg mL ⁇ 1 .
  • MTT assay measures the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability (balance between proliferation and cell death). This assay involves the reduction of tetrazolium salts by mitochondrial dehydrogenase enzymes.
  • the yellow tetrazolium MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) is converted to the purple product Formazan, by metabolically active cells, through the action of dehydrogenase enzymes.
  • the resulting intracellular purple formazan can be solubilised and quantified spectrophotometrically. In this manner, the viability of cells in the presence of the added gadolinium liposomes can be measured and quantitated.
  • the MTT viability assay on kidney LLC PK1 cells revealed a good level of cell viability, after the addition of the liposomes, and viability was shown to fall only at the higher dose and incubation periods.
  • the toxicity of Liposome A is lower than the control nanoparticle, this effect is perhaps due to the carboxylic acids of the DOTA head group, which in the Liposome A formulation, are chelated to Gd 3+ , and therefore become neutral and relatively inert within the cellular environment.
  • HepG2 cellular viability was minimally affected as a result of the addition of Liposome A or the control nanoparticle ( FIG. 13 ). However, Liposome A toxicity was lower than the control nanoparticle, where cell viability reductions were observed at the higher dose and longer incubation periods (Top graph, FIG. 13 ).
  • the LDH assay is a non-radioactive colourimetric cytotoxicity assay that quantitatively measures LDH, which is a stable cytosolic enzyme that is released upon cell lysis during cell death.
  • LDH is a stable cytosolic enzyme that is released upon cell lysis during cell death.
  • the amount of LDH released in the cell media is measured with a 30-minute coupled enzymatic assay, which results in the conversion of a tetrazolium salt (INT) into a red formazan product.
  • INT tetrazolium salt
  • the amount of colour formed is proportional to the number of lysed and therefore dead cells.
  • the results are then normalised against controls such as the LDH released from cells with no compounds added to them.
  • FIG. 14 presents the NCL LDH assay results on kidney LLC PK1 cells for the control nanoparticle and Liposome A.
  • Cytotoxicity of the control liposomes is more variable for the control nanoparticles, and it appears that these particles are more toxic to HepG2 liver cells ( FIG. 15 , top) than Liposome A. HepG2 toxicity and therefore the liver toxicity of Liposome A appear quite low at all concentrations and incubation periods. These data confirm an overall low toxicity for Liposome A.
  • mice Mouse tumours of human cancer are a good model for preliminary investigations of imaging agents and their effectiveness as tumour signal enhancers.
  • the human ovarian cancer cell line IGROV-1 was used to induce tumours in Balb/c nude mice.
  • cells were injected under the right flanks of 6-8 week old female mice, and after two weeks the mice had grown large enough tumours suitable for imaging.
  • Liposome A particles were prepared in HEPES buffer and injected through the tail vein of tumour bearing mice, a method that ensures rapid entry of the liposomes into the blood circulation.
  • Prior to injection baseline MRI scans were obtained on a 4.7 T magnet in order to identify the tumour and measure baseline signal intensity values.
  • Post liposome injection the mice were then imaged at 2 h, 16 h and 24 h post injection. T 1 -weighted images for each time point were obtained and the percent signal intensity enhancement as a result of the accumulation of the liposomes within the tumour tissue was calculated from tumour signal intensity values generated from the tumour tissue (see FIG. 16 ).
  • FIG. 16 presents MR images of tumour bearing mice, the tumour appears dark prior to injection of Liposome A, and becomes more enhanced post-administration of the liposomes. This effect is persistent up to the 24 h end point of the experiment.
  • the tumour signal enhancement of Liposome A is further confirmed by FIG. 17 , where at different TR time points the tumour signal intensity is seen to rise consistently over time, confirming the gradual accumulation of Liposome A in the tumour due to the EPR effect.
  • tumour signal intensity increase in FIG. 18 , we can see that the tumour signal intensity increases over 24 h, and a 72% signal increase is achieved up to the 24 h end point of the experiment.
  • This data is very impressive and demonstrates the utility of Liposome A according to the present invention as a “passively” targeted tumour imaging agent.
  • mice were sacrificed and their tumours excised.
  • the tumours were frozen, fixed and subjected to cryo-sectioning, where 7 m sections were cut and the slides analysed for their fluorescence using microscopy.
  • the inclusion of the red fluorescent lipid DOPE-Rhodamine in the Liposome A formulation allowed for the bimodal assessment of liposome localisation within the tumour tissue.
  • tumour tissue has microvessels with large fenestrations and as such the liposomes are able to extravasate into the tumour. These extravasated liposomes are not cleared due to an impaired lymphatic drainage system and may accumulate within the tumour extracellular fluid over time.
  • Liposome A is a novel liposome nanoparticle formulation that is capable of effective tumour imaging by MRI.
  • the incorporation of DSPC, a fully saturated phospholipid for use in the Gd.DOTA.DSA liposomes of the present invention gives excellent results.
  • the results demonstrate clearly that Liposome A has low liver toxicity and a very high MRI signal enhancement activity. This is believed to be due to the optimal size of Liposome A, a typical Gd.DOTA.DSA liposome of the present invention, as it is small enough to be accumulated in the tumour due to the EPR effect and this smaller size also prevents it being accumulated in the liver in particular due to the reduction of Kupffer cell uptake.
  • Liposome B a further tumour targeted MRI active liposome referred to hereafter as Liposome B.
  • Liposome B is a novel tumour targeted liposome nanoparticle for MRI.
  • Liposome B see the depiction of Liposome B in FIG. 20 .
  • the particles were formulated to ensure a size distribution of approximately 100 nm with a low polydispersity index.
  • IGROV-1 cells were used to induce tumours in nude Balb/c mice and the folate-targeted liposomes were injected intravenously. Rapid accumulation of the folate-targeted liposomes within the tumour tissue compared to non-targeted liposomes was observed.
  • Liposome B The formulation for Liposome B is similar to Liposome A, with the exception that the molar percentage of the DSPE-PEG2000 stealth lipid is reduced by 1.5 mol % in order to incorporate the targeting amphiphile: DSPE-PEG-2000(Folate) [distearoylphosphatidylethanolamine-polyethylene glycol (2000)-folate] (see FIG. 21 for particle composition).
  • the human nasopharyngeal KB carcinoma cell line is considered to have the highest level of FR expression, yet the number of cases for this cancer are low in comparison to ovarian cancer which has the highest frequency (>90% of cases).
  • the ⁇ -FR isoform which is a glycosyl phosphatidylinositol (GPI)-anchored membrane protein is highly expressed in ovarian carcinoma.
  • the ⁇ -FR isoform has also been shown to have specific biomarker value, aiding in the identification of metastatic tumour site origin. 29 Therefore, we were interested in using this receptor in order to test the efficacy of folate targeted bimodal liposomes for the imaging of ovarian tumours using MRI.
  • FR targeted bimodal fluorescent and paramagnetic liposomes were formulated and compared to non-targeted liposomes by both MRI and fluorescence microscopy.
  • a human ovarian carcinoma cell line expresses a sufficient level of the folate receptor
  • FACS analysis of four different cell lines was carried out.
  • ⁇ -FR ⁇ -folate receptor
  • OVCAR-3 and HeLa cervical cancer
  • flow cytometry experiments were carried out.
  • SKBR-3 breast cancer cell line
  • Immunostaining was carried out with a monoclonal antibody (MAb Mov18/ZEL) specific for the ⁇ -FR, and post incubation with this antibody, a secondary FITC labelled antibody (Goat anti-body IgG, FITC conjugated) was allowed to incubate with the cells. Post staining, the cells were fixed and analyzed by fluorescence microscopy. From the FACS ⁇ -FR expression analysis (see FIG. 22 ), where all cell lines were cultured under the same standardized conditions using folate free cell culture medium, it was shown that the IGROV-1 cell line exhibited a distinctly higher level of ⁇ -FR expression. From these typical FACS data the ⁇ -FR expression was measured to be in the order: IGROV-1>>OVCAR-3>HeLa>SKBR-3 (three days post-seeding).
  • Liposome B targeted liposomes were prepared for specific cell receptor binding and uptake into IGROV-1 tumour cancer cells.
  • Table 3 shows a series of liposomes with varying folate amphiphile formulated for incubation with IGROV-1 cells.
  • FIG. 23 presents the obtained data. From this data we can see that the liposome formulation with the highest uptake into IGROV-1 cells is the one containing 1.5 mol % of DSPE-PEG-2000(Folate).
  • Liposome B is a novel formulation that incorporates an optimized ratio of the targeting ligands, established using the same cell line from which tumours were grown for in vivo MR imaging experiments.
  • FIG. 24 presents data on the size characterization of Liposome B particles.
  • the particles have an average size of approximately 100 nm, with the filtered particles having an excellent polydispersity index.
  • MTT assays on LLC PK1 kidney cells were performed on Liposome B liposomes and cell viability was not affected to a great degree at the majority of doses and incubation times (see FIG. 25 ). The higher dose and incubation period did lead to a reduction in cellular viability, indicating the optimal dose range to be between 0.001 and 0.5 mg/mL.
  • the LDH assay data are presented in FIG. 26 , the toxicity effects of Liposome B here appear to become much more pronounced at the 48 h incubation period.
  • the relaxivity of Liposome B liposomes was measured by formulating liposomes with varying concentrations of the Gd.DOTA.DSA lipid to obtain 5 formulations with atomic Gd concentrations within the range 1.972 to 0.2466 mM.
  • the relaxivities of Liposome B and folate targeted liposomes containing DOPC lipid are shown in Table 4.
  • the MRI active Gd lipid Gd.DOTA.DSA and its concentration is the same in both formulations, the r 1 and r 2 relaxivities obtained at 4.7 T are comparable.
  • Liposome B particles (total liposome concentration; 15 mg mL ⁇ 1 ) were prepared in HEPES buffer (20 mM, NaCl, 135 mM, pH 6.5) and injected through the tail vein of IGROV-1 tumour bearing mice. Prior to injection, baseline MRI scans were obtained on a 4.7 T magnet in order to identify the tumour and measure T i baseline values. The mice were then imaged at 2 h, 16 h and 24 h intervals post injection. Percent signal enhancement as a result of the accumulation of the Liposome B particles within the tumour tissue was calculated from signal intensities generated from the tumours.
  • FIG. 27 presents the MR images of tumours at pre-injection, 2, 16 and 24 h post administration of Liposome B. The tumour images reveal a bright rim of enhanced signal around the tumour area at the 24 h time point, showing the great effectiveness of the folate receptor targeted paramagnetic Liposome B according to the present invention.
  • the measured tumour signal intensity values show that within just 2 h post i.v. injection the active and specific targeting effect of the folate liposomes is apparent where the tumour signal is enhanced by 20%.
  • the signal enhancement is then continually increased up to the 16 h imaging time point, where a 62% tumour signal enhancement is achieved. This substantial enhancement is observed despite injection of Liposome B particles which contain half the amount of Folate targeting ligand as compared to previous DOPC-3 mol % DSPE-PEG2000 (Folate) containing liposomes.
  • Liposome B is an optimal MRI active liposomal nanoparticle which within the ⁇ M dose range can enhance tumour tissue substantially, clear after the signal enhancement saturation point, and demonstrates advantages over current clinically available small molecular weight MRI contrast agents.
  • FIG. 29 presents fluorescence microscopy images of sectioned tumours 24 h post Liposome B injection. The presence of intense red fluorescence from these sectioned tumour slices is indicative of the accumulation of the targeted B Liposomes in the tumour tissue.
  • Phosphatidylethanolamine-lissamine rhodamine B DOPE-Rhodamine
  • Cholesterol Cholesterol
  • distearoylphosphocholine DSPC
  • 1,2-Distearoyl-sn-Glycero-3-Phospocholine-N-Methoxy(Polyethylene glycol)-2000 DSPE-PEG2000
  • All other chemicals were of analytical grade or the best grade available and purchased from Sigma-Aldrich (UK) or Macrocyclics (USA).
  • Gd.DOTA.DSA was synthesised as follows.
  • IR Infrared
  • IR-620 Infrared-620 infra-red spectrophotometer
  • absorption's are recorded in wavenumbers (vmax in cm ⁇ 1 ).
  • Flash column chromatography was performed using Merck 0.040 to 0.063 mm, 230 to 400 mesh silica gel. Microscopy experiments were conducted on a Nickon Eclipse E600 microscope. FACS analysis was conducted on a Becton Dickinson FACSCalibur machine. All MRI experiments were conducted on a 4.7 T Magnex magnet (Oxford, UK) Varian Unity Inova console (Palo Alto, Calif., USA).
  • Scheme 1 presents the synthetic route undertaken to produce the only in-house synthesised component of the liposomal nanoparticles put forward: Gd.DOTA.DSA lipid 4. This lipid is produced with ⁇ 98% purity as assessed by analytical HPLC.
  • DOTA-NHS-ester 100 mg, 0.120 mmol
  • bis(steroylamide) 80.17 mg 0.139 mmol
  • Triethylamine 66.90 l, 0.480 mmol
  • the solvents were removed in vacuo and the crude mixture was purified by flash column chromatography (eluted with (CH 2 Cl 2 :MeOH:NH3 34.5:9:1):CH 2 Cl 2 1:9 ⁇ 9:1, v/v) to yield a white solid.
  • FTIR vmax (nujol)/cm ⁇ 1 3750.56, 2726.56, 1889.87, 1793.63, 1681.21, 1534.22.
  • HRMS (FAB+) calculated for 54H104N6O8 m/z 964.7916, found 987.7833 (M+Na) + .
  • FTIR vmax (nujol)/cm ⁇ 1 3750.23, 2234.78, 1991.59, 1889.89, 1793.44, 1681.90.77.
  • MS (ESI+) calculated for C54H101GdN6O8 m/z 1119.67, found 1120.10 (M+H) + .
  • Boc-glycine (310 mg, 1.77 mmol) and dioctadecylamine (923.96 mg, 1.77 mmol) were dissolved in dry chloroform (30 ml).
  • HBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) (804.12 mg, 2.12 mmol) and DMAP (4-dimethylanninopyridine) (648.72 mg, 5.31 mmol) were added to the solution and the reaction was stirred at room temperature under N 2 for 12 h. The solvents were removed in vacuo. The mixture was dissolved in CH 2 Cl 2 (50 mL) and extracted with H 2 O (3 ⁇ 50 mL).
  • NMR spectroscopy was not suitable as a characterisation tool due to the extreme peak broadening caused by the paramagnetic gadolinium metal.
  • All gadolinium lipids were analysed by electrospray mass spectrometry (ESI-MS), HPLC and the xylenol orange assay was used to test for the presence of any free Gd 3+ in the product samples.
  • the xylenol orange assay is a colourimetric test whereby a colour change from orange to purple is indicative of Gd 3+ complexation to the xylenol orange dye. This causes a bathochromic shift from 440 nm to 573 nm.
  • T 1 analysis Gd.DOTA.DSA 4, Gd.DTPA.BSA, and controls of the metal free compound, and Magnevist (Schering AG, Germany) were added to water to give a final concentration of 0.5 mM.
  • the solutions 200 ⁇ L were placed in eppendorf tubes and T 1 relaxation values measured on a 4.7 T Varian MR scanner at ambient temperature.
  • gadolinium liposome formulations were prepared in order to obtain five different gadolinium concentrations between 0.20 to 0.66 M in 200 ⁇ L of distilled water and the molar relaxivity r 1 (mM ⁇ 1 s ⁇ 1 ) determined.
  • lipids were stored as stock solutions in anhydrous organic solvents (CHCl 3 , MeOH or a mixture of both), at ⁇ 20° C. under argon. Appropriate volumes of each lipid stock were placed in a round bottom flask containing chloroform and stirred to ensure thorough mixing of the lipids. The solvent was slowly removed in vacuo to ensure production of an even lipid film. The film was re-hydrated with buffer (HEPES, NaCl, 150 mM, pH 6.8) at a defined volume (20 mL per 500 mg liposome). The resulting solution was sonicated for 60 min (at 30° C.). The pH of the liposomal suspension was checked by pH Boy (Camlab Ltd., Over, Cambridgeshire, UK). For each preparation, the size and polydispersity of liposomes was measured by photon correlation spectroscopy (PCS).
  • PCS photon correlation spectroscopy
  • IGROV-1 cells (5 ⁇ 10 6 /0.1 mL PBS) were implanted into the flanks of 6-8 weeks old Balb/c nude mice for generation of subcutaneous tumours. After ⁇ 2 weeks (estimated tumour weights 40-50 mg) the mice were anaesthetized with an isoflurane/O 2 mix and placed into a quadrature 1 H volume coil and positioned into the magnet.
  • HEPES 200 ⁇ L liposome solution
  • the animals were sacrificed and the tumours, livers and kidneys were excised, frozen in liquid nitrogen, embedded in OCT (VWR) embedding fluid and either 10 or 7 m thick sections cut, mounted on slides and studied for fluorescence microscopy.
  • VWR OCT

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Radiology & Medical Imaging (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Magnetic Resonance Imaging Apparatus (AREA)
US13/511,126 2009-11-20 2010-11-19 Novel Liposome Nanoparticles for Tumor Magnetic Resonance Imaging Abandoned US20130129636A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB0920304.3 2009-11-20
GBGB0920304.3A GB0920304D0 (en) 2009-11-20 2009-11-20 Novel liposome nanoparticles for tumour magnetic resonance imaging
PCT/GB2010/051925 WO2011061541A1 (en) 2009-11-20 2010-11-19 Liposome nanoparticles for tumor magnetic resonance imaging

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2010/051925 A-371-Of-International WO2011061541A1 (en) 2009-11-20 2010-11-19 Liposome nanoparticles for tumor magnetic resonance imaging

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/463,964 Continuation US11369697B2 (en) 2009-11-20 2017-03-20 Liposome nanoparticles for tumor magnetic resonance imaging

Publications (1)

Publication Number Publication Date
US20130129636A1 true US20130129636A1 (en) 2013-05-23

Family

ID=41565567

Family Applications (2)

Application Number Title Priority Date Filing Date
US13/511,126 Abandoned US20130129636A1 (en) 2009-11-20 2010-11-19 Novel Liposome Nanoparticles for Tumor Magnetic Resonance Imaging
US15/463,964 Active US11369697B2 (en) 2009-11-20 2017-03-20 Liposome nanoparticles for tumor magnetic resonance imaging

Family Applications After (1)

Application Number Title Priority Date Filing Date
US15/463,964 Active US11369697B2 (en) 2009-11-20 2017-03-20 Liposome nanoparticles for tumor magnetic resonance imaging

Country Status (9)

Country Link
US (2) US20130129636A1 (zh)
EP (1) EP2501415B1 (zh)
JP (2) JP5883797B2 (zh)
CN (1) CN102711840B (zh)
DK (1) DK2501415T3 (zh)
ES (1) ES2538255T3 (zh)
GB (1) GB0920304D0 (zh)
HK (1) HK1170437A1 (zh)
WO (1) WO2011061541A1 (zh)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140050670A1 (en) * 2010-12-07 2014-02-20 The University Of Western Australia Multifunctional nanoparticles
US20140255470A1 (en) * 2013-01-22 2014-09-11 The Charles Stark Draper Laboratory, Inc. Multivalent liposome formulations
WO2014152211A1 (en) 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
WO2015034928A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Chimeric polynucleotides
WO2015034925A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Circular polynucleotides
WO2015051214A1 (en) 2013-10-03 2015-04-09 Moderna Therapeutics, Inc. Polynucleotides encoding low density lipoprotein receptor
WO2016014846A1 (en) 2014-07-23 2016-01-28 Moderna Therapeutics, Inc. Modified polynucleotides for the production of intrabodies
WO2017112943A1 (en) 2015-12-23 2017-06-29 Modernatx, Inc. Methods of using ox40 ligand encoding polynucleotides
WO2017120612A1 (en) 2016-01-10 2017-07-13 Modernatx, Inc. Therapeutic mrnas encoding anti ctla-4 antibodies
WO2020112822A1 (en) * 2018-11-28 2020-06-04 Kansas State University Research Foundation Nanocarrier systems for imaging and delivery of active agents
EP4011451A1 (en) 2015-10-22 2022-06-15 ModernaTX, Inc. Metapneumovirus mrna vaccines
EP4159741A1 (en) 2014-07-16 2023-04-05 ModernaTX, Inc. Method for producing a chimeric polynucleotide encoding a polypeptide having a triazole-containing internucleotide linkage
WO2023161350A1 (en) 2022-02-24 2023-08-31 Io Biotech Aps Nucleotide delivery of cancer therapy
US11786468B2 (en) * 2015-06-08 2023-10-17 King's College London Nanoparticles
US11964948B2 (en) 2022-06-07 2024-04-23 Actinium Pharmaceuticals, Inc. Bifunctional chelators and conjugates

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0920304D0 (en) * 2009-11-20 2010-01-06 Medical Res Council Novel liposome nanoparticles for tumour magnetic resonance imaging
EP2777715A1 (en) * 2013-03-14 2014-09-17 Centre National de la Recherche Scientifique (CNRS) Particles comprising luminescent lanthanide complexes
JP6623479B2 (ja) * 2015-02-08 2019-12-25 トンリ・バイオメディカル・カンパニー・リミテッド 脂質ナノ粒子及びその使用
GB201509935D0 (en) * 2015-06-08 2015-07-22 Globalacorn Ltd And King S College London Precision therapeutics
AU2016349577B2 (en) 2015-11-06 2023-10-26 Wisconsin Alumni Research Foundation Long-lived gadolinium based tumor targeted imaging and therapy agents
US11052162B2 (en) * 2016-09-16 2021-07-06 Texas Children's Hospital Liposomal gadolinium (GD) contrast agent “NMRX” for T1-MRI
CN108904818B (zh) * 2018-09-03 2022-02-18 国家纳米科学中心 Ttd核杂化纳米颗粒、其制备方法及应用
CN111840581B (zh) * 2020-08-05 2022-12-09 牡丹江医学院 一种用于诊断脑梗死的核磁共振造影剂及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4766046A (en) * 1985-09-27 1988-08-23 Liposome Technology, Inc. Stabilized liposome/amphotericin composition and method
US20070292494A1 (en) * 2004-03-19 2007-12-20 Let There Be Hope Medical Research Institute Carbohydrate-Derivatized Liposomes for Targeting Cellular Carbohydrate Recognition Domains of Ctl/Ctld Lectins, and Intracellular Delivery of Therapeutically Active Compounds

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050112065A1 (en) 2003-07-09 2005-05-26 Drummond Daryl C. Remote detection of substance delivery to cells
CN1960707B (zh) 2004-06-03 2012-11-14 博莱科瑞士股份有限公司 用于治疗和/或诊断用途的脂质集合体
JP4715133B2 (ja) * 2004-08-26 2011-07-06 コニカミノルタエムジー株式会社 抗腫瘍リポソーム製剤およびその製造方法
CA2578921A1 (en) 2004-08-31 2006-03-09 Astellas Pharma Inc. Intracellular drug delivery improving liposome
CN101496906B (zh) * 2009-02-19 2011-11-09 浙江大学 含磁共振对比剂的脂质纳米粒及其制备方法和应用
GB0920304D0 (en) * 2009-11-20 2010-01-06 Medical Res Council Novel liposome nanoparticles for tumour magnetic resonance imaging

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4766046A (en) * 1985-09-27 1988-08-23 Liposome Technology, Inc. Stabilized liposome/amphotericin composition and method
US20070292494A1 (en) * 2004-03-19 2007-12-20 Let There Be Hope Medical Research Institute Carbohydrate-Derivatized Liposomes for Targeting Cellular Carbohydrate Recognition Domains of Ctl/Ctld Lectins, and Intracellular Delivery of Therapeutically Active Compounds

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Charrois et al. Drug release rate influences the pharmacokinetics, biodistribution, therapeutic activity, and toxicity of pegylated liposomal doxorubicin formulations in murine breast cancer. 2004 Biochim. Biophys. Acta. 1663: 167-177. *
Kamaly et al. Folate receptor targeted bimodal liposomes for tumor magnetic resonance imaging. 2009 Bioconjug. Chem. 20: 648-655. *
Mulder et al. A liposomal system for contrast-enhanced magnetic resonance imaging of molecular targets. 2004 Bioconjug. Chem. 15: 799-806. *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140050670A1 (en) * 2010-12-07 2014-02-20 The University Of Western Australia Multifunctional nanoparticles
US20140255470A1 (en) * 2013-01-22 2014-09-11 The Charles Stark Draper Laboratory, Inc. Multivalent liposome formulations
US9408890B2 (en) * 2013-01-22 2016-08-09 The Charles Stark Draper Laboratory, Inc. Multivalent liposome formulations
WO2014152211A1 (en) 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
WO2015034928A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Chimeric polynucleotides
WO2015034925A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Circular polynucleotides
WO2015051214A1 (en) 2013-10-03 2015-04-09 Moderna Therapeutics, Inc. Polynucleotides encoding low density lipoprotein receptor
EP4159741A1 (en) 2014-07-16 2023-04-05 ModernaTX, Inc. Method for producing a chimeric polynucleotide encoding a polypeptide having a triazole-containing internucleotide linkage
WO2016014846A1 (en) 2014-07-23 2016-01-28 Moderna Therapeutics, Inc. Modified polynucleotides for the production of intrabodies
US11786468B2 (en) * 2015-06-08 2023-10-17 King's College London Nanoparticles
EP4011451A1 (en) 2015-10-22 2022-06-15 ModernaTX, Inc. Metapneumovirus mrna vaccines
EP4349404A2 (en) 2015-10-22 2024-04-10 ModernaTX, Inc. Respiratory virus vaccines
EP4349405A2 (en) 2015-10-22 2024-04-10 ModernaTX, Inc. Respiratory virus vaccines
EP4039699A1 (en) 2015-12-23 2022-08-10 ModernaTX, Inc. Methods of using ox40 ligand encoding polynucleotides
WO2017112943A1 (en) 2015-12-23 2017-06-29 Modernatx, Inc. Methods of using ox40 ligand encoding polynucleotides
WO2017120612A1 (en) 2016-01-10 2017-07-13 Modernatx, Inc. Therapeutic mrnas encoding anti ctla-4 antibodies
WO2020112822A1 (en) * 2018-11-28 2020-06-04 Kansas State University Research Foundation Nanocarrier systems for imaging and delivery of active agents
WO2023161350A1 (en) 2022-02-24 2023-08-31 Io Biotech Aps Nucleotide delivery of cancer therapy
US11964948B2 (en) 2022-06-07 2024-04-23 Actinium Pharmaceuticals, Inc. Bifunctional chelators and conjugates
US11975081B2 (en) 2022-06-07 2024-05-07 Actinium Pharmaceuticals, Inc. Bifunctional chelators and conjugates

Also Published As

Publication number Publication date
US20170348440A1 (en) 2017-12-07
WO2011061541A1 (en) 2011-05-26
JP2015143262A (ja) 2015-08-06
ES2538255T3 (es) 2015-06-18
DK2501415T3 (en) 2015-06-15
HK1170437A1 (zh) 2013-03-01
JP2013511503A (ja) 2013-04-04
EP2501415A1 (en) 2012-09-26
CN102711840B (zh) 2014-06-18
EP2501415B1 (en) 2015-03-18
JP5883797B2 (ja) 2016-03-15
CN102711840A (zh) 2012-10-03
US11369697B2 (en) 2022-06-28
GB0920304D0 (en) 2010-01-06

Similar Documents

Publication Publication Date Title
US11369697B2 (en) Liposome nanoparticles for tumor magnetic resonance imaging
Kamaly et al. Bimodal paramagnetic and fluorescent liposomes for cellular and tumor magnetic resonance imaging
Pan et al. Manganese-based MRI contrast agents: past, present, and future
Zhou et al. Peptide targeted tripod macrocyclic Gd (III) chelates for cancer molecular MRI
Hak et al. A high relaxivity Gd (III) DOTA-DSPE-based liposomal contrast agent for magnetic resonance imaging
EP3283493B1 (en) Texaphyrin-phospholipid conjugates and methods of preparing same
AU2004264857B2 (en) Remote detection of substance delivery to cells
Corbin et al. Low-density lipoprotein nanoparticles as magnetic resonance imaging contrast agents
JP4057646B2 (ja) リポソーム剤
US10039843B2 (en) Paramagnetic solid lipid nanoparticles (pSLNs) containing metal amphiphilic complexes for MRI
Li et al. AS1411 aptamer-modified theranostic liposomes co-encapsulating manganese oxide nano-contrast agent and paclitaxel for MRI and therapy of cancer
Kamaly et al. A novel bimodal lipidic contrast agent for cellular labelling and tumour MRI
Liu et al. Hierarchical theranostic nanomedicine: MRI contrast agents as a physical vehicle anchor for high drug loading and triggered on-demand delivery
Fang et al. Gd-DTPA-dialkylamine derivatives: Synthesis and self-assembled behaviors for T1-enhanced magnetic resonance imaging and drug carriers
Vabbilisetty Functional Anchoring Lipids for Drug Delivery Carrier Fabrication and Cell Surface Re-Engineering Applications
Bryson The Design and Study of Lanthanide-Chelating Macromolecular Diagnostic and Delivery Agents

Legal Events

Date Code Title Description
AS Assignment

Owner name: IMPERIAL INNOVATIONS LIMITED, UNITED KINGDOM

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:THANOU, MAYA;MILLER, ANDREW DAVID;REEL/FRAME:039928/0169

Effective date: 20121205

Owner name: MEDICAL RESEARCH COUNCIL, UNITED KINGDOM

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KAMALY, NAZILA;KALBER, TAMMY LOUISE;KENNY, GAVIN DAVID;AND OTHERS;SIGNING DATES FROM 20121121 TO 20121123;REEL/FRAME:039931/0176

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION