WO2014141288A1 - Art, procédé, manière, processus et systèmes d'un nano-biominéral pour imagerie de contraste multimodale et administration de médicament - Google Patents

Art, procédé, manière, processus et systèmes d'un nano-biominéral pour imagerie de contraste multimodale et administration de médicament Download PDF

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WO2014141288A1
WO2014141288A1 PCT/IN2013/000143 IN2013000143W WO2014141288A1 WO 2014141288 A1 WO2014141288 A1 WO 2014141288A1 IN 2013000143 W IN2013000143 W IN 2013000143W WO 2014141288 A1 WO2014141288 A1 WO 2014141288A1
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imaging
nanocontrast
multifunctional
calcium
nanoparticles
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PCT/IN2013/000143
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English (en)
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Manzoor Koyakutty
Anusha ASHOKAN
Shantikumar V. Nair
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Amrita Vishwa Vidyapeetham University
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Priority to PCT/IN2013/000143 priority Critical patent/WO2014141288A1/fr
Publication of WO2014141288A1 publication Critical patent/WO2014141288A1/fr

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
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    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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    • A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
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    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
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Definitions

  • the present invention relates to a nano-sized material that can provide contrast enhancement for multiple imaging methods and also deliver therapeutic molecules such as nucleic acids or chemo drugs to diseased sites such as cancer.
  • the present invention relates to nano-sized synthetic calcium phosphates and calcium apatite nanomaterials showing simultaneous contrast for at least any two of the medical imaging modalities including radio-, raman-, near-infrared fluorescence-, magnetic resonance- and X ray- imaging.
  • This invention relates to the preparation of a nanomaterial based on doped calcium phosphate or calcium apatite contrast agent for multimodal contrast imaging, drug-delivery and therapy applications.
  • Multimodal molecular imaging of disease using a combination of techniques such as single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), positron emission tomography (PET), X-ray computed tomography (CT) and near- infrared (NIR) fluorescence, raman spectroscopy is an emerging area of research [1,2].
  • SPECT single photon emission computed tomography
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • CT X-ray computed tomography
  • NIR near- infrared fluorescence
  • imaging systems and contrast agents are used independently to derive different anatomical and functional information such as location, size, angiogenesis, metastasis etc. It will be highly useful, if low-cost, but high resolution imaging modalities like optical (fluorescence) techniques can be combined with SPECT, MRI, CT Or raman spectroscopy to derive molecular information about the expression level of specific disease marker or biological process that influence the therapeutic outcome.
  • imaging modalities like optical (fluorescence) techniques can be combined with SPECT, MRI, CT Or raman spectroscopy to derive molecular information about the expression level of specific disease marker or biological process that influence the therapeutic outcome.
  • suitable contrast agents that can show different physical properties compatible with basic principles of imaging technique.
  • different molecular imaging techniques requires separate contrast agents because their working principles are distinctly different.
  • SPECT requires gamma radiation emitting nuclides
  • MRI requires paramagnetic
  • CT computed tomography
  • raman imaging requires raman active metals or dyes
  • in vivo fluorescence imaging requires near-infrared fluorescent contrast agents.
  • radionuclides such as Tc9 m is used for scintigraphy and 3D SPECT imaging
  • paramagnetic gadolinium complexes such as Gd-carboxylates or superparamagnetic iron oxide particles are used as MRI contrast agents
  • iodine or colloidal barium sulphate contrast agents are used in CT
  • Au Au
  • proteins Green fluorescent protein or Red fluorescent proteins
  • QDs quantum dots
  • contrast agents differ significantly.
  • An ideal solution is to provide a single contrast agent that can show all important contrast functionalities at the same time.
  • Luminescent quantum dots, superparamagnetic iron oxide nanoparticles, Ag nanoparticles and Au nanocrystals were studied for optical, magnetic, raman and photo- acoustic/two- photon imaging applications, respectively.
  • bi-functional nanosystems combining two separate agents such as luminescent QDs and magnetic iron oxide or fluorescent dyes and magnetic iron oxide were also reported for combinatorial (MRI and optical) imaging applications.
  • one of the major concerns about nanoparticle based contrast imaging is the efficient incorporation of multiple impurities and toxicity associated with each component within the nanomaterial.
  • luminescent semiconductor nanoparticles were found to be toxic to biological systems due to their heavy- metal composition (cadmium, selenium, tellurium, lead). Further, there exist no biocompatible single nanoparticle that can provide contrast enhancement for multiple imaging options such as SPECT, raman imaging, NI fluorescence, MRI and X-ray imaging.
  • HAp and calcium phosphates are extensively studied and used in clinical practice as implant material for in vivo bone tissue re-generation.
  • the present invention discloses, the preparation of improved calcium apatite and calcium phosphates (tricalcium phosphate, dicalcium phosphate, monocalcium phosphate) nanoparticles doped and labeled with multiple impurities that confer properties such as radioactivity, raman scattering, near-infrared fluorescence, X-ray absorption and super/para-magnetism.
  • the preparation of undoped hydroxyapatite and calcium phosphate nanoparticles is disclosed in several prior arts.
  • stoichiometric hydroxyapatite, Cai 0 (OH) 2 (P04)6 and calcium phosphate nanoparticles are prepared by adding ammonium phosphate or phosphoric acid and ammonium hydroxide mixture solution to a solution of Ca 2 ⁇ precursor.
  • Doped apatite/calcium phosphate nanoparticles is prepared by replacing calcium ions with fluorescent or paramagnetic metal ions.
  • Organic dye doped calcium phosphates can also be prepared by the incorporation of the dye within calcium phosphate matrix during its synthesis, wherein the dye can confer properties such NIR fluorescence or raman scattering.
  • Near infrared fluorescence based imaging is a recent advancement in biomedical research. Generally, in vivo fluorescence imaging using visible light is limited by low penetration of light photons into living tissue due to high absorption and scattering properties of tissue components. However, near- infrared region of the spectrum offers certain advantages because the hemoglobin and water absorb minimally in this spectral window (68o-90onm) so as to allow photons to penetrate for several centimeters deep inside the tissue. This allows mapping of molecular events in intact tissues using near-infrared fluorescence mediated molecular tomography (FMT), a new technique that can three- dimensionally image molecular events like gene expression by resolving fluorescence activation in deep tissues.
  • FMT near-infrared fluorescence mediated molecular tomography
  • an essential component of successful FMT imaging is the development of biocompatible NIR emitting fluorescent probes that can be targeted to the tissue.
  • various paramagnetic and superparamagnetic nanocontrast agents are being developed that has superior magnetic properties that their macro size counterparts.
  • Combining such nanocontrast agents with other imaging modalities such as NIR imaging provides added advantage of combining imaging of anatomic and functional characteristics of the diseased condition.
  • Research in combinatorial imaging approaches are exploring the advantages of combining anatomic imaging modalities such as MRI, 2D/3D CT imaging together with imaging modalities such as NIR, raman and radioactive imaging that give details on molecular characteristics of the pathological condition. It is therefore imperative to develop a biocompatible multimodal contrast agent that can combine the above mentioned modalities in combination to aid better and early stage disease diagnosis and treatment.
  • a novel biocompatible nanoparticle that can provide contrast enhancement for multiple molecular imaging modalities such as radioactive imaging, raman imaging, MRI, X-ray and/or near infrared (NIR) fluorescence together with targeted drug or gene delivery is disclosed.
  • the particle is based on nanoparticles of calcium phosphate (nCP) and calcium hydroxyapatite (nHAp), doped and/or conjugated with more than one impurity ion/molecule.
  • This multifunctional nano-composite particle enable combined molecular imaging using single photon emission computed tomography (SPECT), raman imaging, MRI, CT and/or optical near-infrared fluorescent imaging.
  • Another aspect of this invention relates to a method of making nanomedicine formulations based on the said nCP and nHAp particles, capable of delivering chemodrugs, small molecule inhibitors or nucleic acid drugs such as DNA, RNA, small interfering RNA (siRNA), micro RNA (miRNA) specifically to a targeted disease such as cancer.
  • nucleic acid drugs such as DNA, RNA, small interfering RNA (siRNA), micro RNA (miRNA) specifically to a targeted disease such as cancer.
  • nanoparticle refers to primary inventive nanoparticles formed by calcium phopshate or hydroxyapatite, measuring size about 1-500 nm, preferably l-ioonm, most preferably around 1- 50 nm in size, showing "multifunctional' properties such as radioactivity, raman scattering, NIR fluorescence, magnetism and X-ray absorption in different combinations with at least two properties together.
  • therapeutics refers to chemodrugs, nucleic acids such as DNA or RNA [small interfering RNA (siRNA) or micro RNA (miRNA)], bisphosphonates, antibodies, peptides, proteins, etc that have a therapeutic effect against a disease condition such as cancer, inflammatory disease or autoimmune disease.
  • nucleic acids such as DNA or RNA [small interfering RNA (siRNA) or micro RNA (miRNA)]
  • siRNA small interfering RNA
  • miRNA micro RNA
  • targeting ligand refers to biomolecules that can specifically identify and target another molecule like an antigen or receptor on the surface of cell-membrane of diseased cells such as that of cancer / tumor.
  • Targeting ligand include antibodies, peptides, aptamers, vitamins like folic acid, sugar molecules like mannose ligands, carbohydrates.
  • nanomedicine refers to a composite construct based on the said calcium phosphate or HAp nanoparticle which is connected or alternatively loaded with any of the above said therapeutics which will be delivered to the desired site using the said targeting ligands.
  • nanomedicine construct is formed by either : i) directly connecting the therapeutics and targeting ligands to the surface of said nanoparticles or alternatively, ii) the said nanoparticle will be taken as a core with another biodegradable polymer containing the therapeutics as shell with targeting ligands connected to its surface, or Hi) a biodegradable polymeric nanoparticle which embed nCP or nHAp together with the therapeutic agents and connected with the targeting ligands on its surface
  • the said nanomedicine construct preferably will have a size of 50- 20onm, more preferably 8o-i50nm and most preferably ⁇ ioo-i2onm.
  • the nanomedicine may be produced in the form of dry powders or liquid dispersions.
  • the nanoparticle may also be synthesized via colloidal synthesis or self- assembly of ions in saturated solutions like simulated body fluid (SBF) and may take the form of colloidal crystals with dopant ions or molecules in it.
  • SBF simulated body fluid
  • the term "doped” as used herein, refers to incorporation of small amount of (about 0.001-30 mole % or up to 30 weight %) another substance (inorganic elements, ions or organic molecules or ratioactive isotopes) within the said nanoparticles.
  • the dopant molecules include, elements, ions, dyes or molecular clusters that can provide radioactivity, raman scattering, NIR fluorescence, magnetic contrast by paramagnetic, superparamagnetic, ferromagnetic or ferrimagnetic properties or X-ray absorption.
  • the dopant ions may substitute either Ca or P ions, or may be present as molecular clusters within the interstitial space of nHAp or nCP lattice.
  • labelled refers to attaching the impurity ions, molecules or radioactive isotopes on the surface of the said nanoparticle with the help of a ligand such as bisphosphonates or functional groups such as carboxyl on the surface of the nanoparticle. This may involve electrostatic interactions , covalent-, ionic-, coordinate, or hydrogen bonding or hydrophobic, lipophilic or hydrophilic interactions
  • Figure 1 Schematic showing the method of preparation of multimodal and therapeutic nCP and nHAp
  • Figure 2 (a) XRD of MF-nHAp showing hexagonal HAp crystal structure (b) SEM image of MF-nHAp showing particle size ⁇ 100 nm. (c) DLS data of MF- nHAp giving a size distribution of 100 ⁇ 20nm (d) Digital photograph of MF- nHAp and nHAp solutions ( ⁇ 20 mg/mL) indicating the greenish body color due to ICG doping .
  • Figure 3 (A) Graph showing integrated fluorescence emission intensity at 800 nm with increase in MF-nHAp concentration. Inset: Fluorescence spectra of MF- nHAp showing excitation and emission peaks at 765 nm and 800 nm, respectively.
  • Figure 4 (a) Radioactive emission of 20mg/mL of MF-nCP samples conjugated to Tc-99m of varying concentrations (b) Fluorescence images of MF-nCP samples conjugated to varying concentrations of ICG (c) Graph showing the variation in radioactive emission of MF-nCP samples conjugated to varying concentrations of Tc-99m (d) Graph showing the fluorescence intensity variation of MF-nCP with varying doping % of ICG
  • FIG. 5 (A) Hemolysis data of nHAp and D-nHAp treated whole blood showing non-hemolytic action of the tested samples (B) Digital photograph of the whole blood treated with PBS, D-nHAp and triton showing no leakage of hemoglobin from PBS/D-nHAp treated samples compared to triton treated sample.
  • Figure 6 Scanning electron micrograph of RBC treated with (A) PBS (B) D-nHAp. Enlarged image (C))showing uniform distribution of D- nHAp particles although out the RBC without affecting its morphology (D) EDX spectrum of the D-nHAp that are distributed on the RBC.
  • Figure 7 Cytotoxicity data of D-nHAp treatment on peripheral blood derived mononuclear cells after 24 hours incubation showing no toxicity up to a concentration of 500 ug/mL.
  • Figure 8 Radioactive emission images (a-d) and fluorescence images (e-h) of MF-nCP sample imaged at varying depths in pork tissue.
  • the bottle containing the sample placed in pork tissue was rotated and images taken at varying angles. Depth at which the sample was imaged from, in tissue, is denoted as d and the angle at which the bottle is rotated is denoted as ⁇ .
  • Figure 9 In vivo multimodal imaging of MF-nCP after tail vein injection into mice.
  • Figure (a) shows the fluorescence image
  • (b) shows radioactive emission image
  • (c) shows radioactive emission image overlaid over X-ray images.
  • the method of preparation of said nanoparticle and nanomedicine formulation consists the steps of:
  • the precursor compound, part A is formed from a material selected from the group consisting of sulphate, phosphate, hydroxide, chloride, bromide, iodide, fluoride, nitrate, carbonate or oxide salts of calcium.
  • another precursor compound, Part B is formed from water soluble or miscible salt of phosphates including sodium (Na 3 P0 4 , Na 2 HP0 4 , NaH 2 P0 4 ), potassium (K 3 P0 4 , KsHPC-4, KH 2 P0 4 ) lithium (Li 3 P0 4 , Li 2 HP0 4 , LiH 2 P0 4 ), ammonium ((NH 4 ) 3 P0 4 , (NH 4 ) 2 HP0 4 , NH 4 H 2 P0 4 ) or phosphoric acid
  • the precursor compound containing hydroxyl anions (Part C) is formed by hydroxide salts of sodium, potassium, lithium, ammonium or calcium .
  • the dopant molecules (Part D) in the nanoparticle that gives raman scattering is formed by cyanine dyes (Cy3, Cy5, ICG), rhodamine (rhodamine 6G, tetramethyl rhodamine, rhodamine no), aniline blue, crystal violet, malachite green, basic fuchsin, variamine blue RT salt, triaminopyrimidine sulphate or porphyrins.
  • cyanine dyes Cy3, Cy5, ICG
  • rhodamine rhodamine 6G, tetramethyl rhodamine, rhodamine no
  • aniline blue crystal violet
  • malachite green malachite green
  • basic fuchsin basic fuchsin
  • variamine blue RT salt triaminopyrimidine sulphate or porphyrins.
  • the dopant molecules (Part D) that gives near-infrared fluorescence to the nanoparticle is NIR emitting dyes such as Indocyanine green (ICG).
  • the dopant ions (Part D) in the nanoparticle that gives magnetic contrast includes ions such as chromium(III), manganese(II), iron(II), iron (III), praseodymium (III), neodymium (III), samarium(III), ytterbium(III), gadolinium(lll), terbium(III), dysprosium(III), holmium(III), erbium(III).
  • the dopant ions (Part D) in the nanoparticle that gives X-ray contrast includes ions such as iodine or barium, bismuth, strontium tungsten, tantalum, hafnium, lanthanum, molybdenum, niobium, zirconium.
  • the doped nanoparticle is conjugated with a radioactive element that includes Sm-153, Tc- 99m, 12 3I, 18F, l 3 » I, '"In, l88 Re, l66 Ho, ⁇ ⁇ 0 r 82 Rb through ligands such as bisphOsphonates.
  • a radioactive element that includes Sm-153, Tc- 99m, 12 3I, 18F, l 3 » I, '"In, l88 Re, l66 Ho, ⁇ ⁇ 0 r 82 Rb through ligands such as bisphOsphonates.
  • the nanoparticle with dopant ions / molecules were formed by reacting precursor Part-A, B, C and D at a temperature range of 25-i50°C and pH range of 5 -12.
  • the dopant ions may be mixed preferably with either Part-A or Part B or added separately during the reaction between Part A, B and C.
  • the formed nanoparticles are grown to a preferred size scale, between 1-200 nm by aging of the reactants at time scales of any range between 0-24 Hrs, preferably, 0-6 Hrs and most preferably 1-4 hrs at a temperature range of 5-i50°C.
  • Figure 2b and 2c shows the SEM and DLS images of ICG and Gd3 + co-doped multifunctional hydroxyapatite nanoparticles (MF-nHAp). 90% of the particles synthesized were ⁇ 100 ⁇ 25 nm in size as seen in DLS and SEM data.
  • the XRD data of MF-nHAp gave characteristic peaks of hexagonal HAp crystal lattice as shown in Figure 2a.
  • inset of Figure 3A shows the fluorescence excitation and emission spectra of ICG and Gd3 + co-doped MF-nHAp, peaking at 760 nm and 830 nm proving the capability of the material for non invasive in vivo NIR imaging.
  • the total weight percentage is varied in the range of 0.01 - 1%.
  • the total concentration of ICG in the precursor solution is maintained less than 2xio _ s M to avoid agglomeration of dye molecules.
  • the said nanoparticle can be used for molecular imaging based NIR fluorescence for biomedical applications. This is displayed in phantom experiments as shown in Figure 3B that shows increase in fluorescence with increase in MF-nHAp concentration.
  • the nanoparticles show X-ray contrast properties (radio opacity) together with NIR imaging.
  • X-ray contrast imaging of powder nanoparticle samples of different dopant concentrations are displayed.
  • Undoped and doped samples were made into pellets and imaged using 12.87 KeV X-ray energy (0.4mm filter) for an exposure time of 30 seconds in a digital X-ray imaging station (Kodak in vivo multispectral imaging system, Carestream, USA).
  • the X-Ray density of the material which is a measure of the X-ray absorbed or attenuated, was calculated and plotted in Figure 3C.
  • the said NIR emitting nanoparticles show paramagnetism together with NIR fluorescence and X-ray contrast when co-doping ICG with paramagnetic Gd ions.
  • Figure 3E refers to magnetic studies carried out by keeping the sample under a varying magnetic field, in an instrument called vibrating sample magnetometer. When undoped sample show diamagnetic property the ICG and Gadolinium doped samples show paramagnetic property, which increases with the concentration of dopant ions, gadolinium in this case.
  • FIG. 4a shows the radioactive emission from MF- nCP samples. As seen in the figure tagging of Tc-99m increases with increase in Tc-99m concentration till a saturation value of 250 ⁇ ' (Figure 4c). Figure 4b shows increase in fluorescence with increase in % doping of ICG till a saturation value of 0.16 mole% ( Figure 4d).
  • FIG. 5A shows the hemocompatibility of the doped nHAp (D-nHAp) where D-nHAp and D- nHAp cause hemolytic effects upto a concentration of as shown in Figure 5A .
  • Figure 5B shows the blood samples treated with D-nHAp and triton where hemolysis is clearly visible in triton treated samples and D-nHAp treated sample is comparable to PBS treated blood.
  • Figure 6 shows SEM image of RBC treated with D-nHAp ( Figure 6B) that shows no membrane damage compared to PBS treated RBC ( Figure 6A).
  • Figure 6D shows EDAX data of D-nHAp distributed over RBC.
  • the said nanoparticles are highly bio-compatible to primary human cells, for eg; peripheral blood mononuclear cells, as shown in Figure 7 .
  • Phantom imaging using Tc tagged and ICG doped MF-nCP was done in pork tissue samples to check the efficiency of contrast when imaged in tissue samples.
  • Figure 8 shows that both NIR fluorescence and radioactivity is imageable at varying depths up to 2.5 cm.
  • Later imaging in mice was carried out using the same MF-nCP nanoparticles that proved the ability of the material for multimodal imaging under in vivo conditions.
  • Figure 9 shows the fluorescent and radioactive imaging of mice after tail vein injection of the MF-nCP sample. All these imaging experiment were carried out in an in vivo imaging station (Kodak in vivo multispectral imaging system, Carestream, USA).
  • the high resolution and blood penetration capability of near-infrared light can help doctors, during a surgery, to identify angiogenic blood vessels in the vicinity of a solid tumor, for its effective removal during a surgical procedure.
  • Angiogenesis is a process of formation of larger concentration of new blood vessels around a growing tumor and the removal of such blood vessels is critical in stopping recurrence of tumor.
  • the said nanoparticles conjugated with antibody targeted against angiogenic growth factors may illuminate the cancer related blood vessels together with providing contrast for the tumor.
  • the doped nanoparticles are cationic in nature, the same can be loaded with nucleic acid drugs such as DNA or RNA or its small fractions like siRNA, for therapeutic gene delivery applications.
  • nucleic acid drugs such as DNA or RNA or its small fractions like siRNA, for therapeutic gene delivery applications.
  • mAbs monoclonal antibodies
  • pep peptides
  • similar ligands ligands
  • the said nanoparticles can also be Coated with a biodegradable polymers containing anticancer drugs or nucleic acid drugs.
  • the biodegradable polymers can be formed by polymers such as poly-lactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyvinylpyrrolidon (PVP), polyvinyl alcohol (PVA), polyethyleneimine (PEI), polyethelene glycol (PEG), chitosan, carboxymethyl chitosan, cyclodextrin, thermosensitive polymers such as Poly(JV- isopropylacrylamide) and its derivative or proteins such as bovine or human serum albumin.
  • PLA poly-lactic acid
  • PLGA poly(lactic-co-glycolic acid)
  • PVP polyvinylpyrrolidon
  • PVA polyvinyl alcohol
  • PEI polyethyleneimine
  • PEG polyethelene glycol
  • chitosan carboxymethyl chitosan
  • cyclodextrin thermosensitive polymers
  • thermosensitive polymers such as Poly(JV- isopropylacrylamide) and its derivative or proteins such as
  • the said nanoparticle or drug loaded nanomedicine construct is connected, at the surface with cancer targeting ligands including antibodies, peptides or small molecular ligands such as folic acid or aptamers
  • the present invention provides an injectable composition or composition for oral administration comprising the said nanomedicine according to the present invention as described above, together with a pharmaceutically acceptable medium.
  • the present invention provides a method of image guided delivery of the drug, estimation of drug concentration at diseased site with respect to other regions, estimation of pharmacokinetics and pharmacodynamics, treatment planning and estimation of treatment efficacy after preferred treatment, all the above using any of the said contrast imaging property of the nanoparticle
  • essentially the present invention provides a method of simultaneously detecting and treating the disease like cancer with the help of more than one and upto 05 different molecular imaging techniques using a single nanoparticle system.
  • the inventors have discovered primarily a nanoparticle that provides novel concept for combining a number of important molecular imaging techniques such as radioactivity, raman scattering, near-infrared fluorescence imaging, which can register both spatial and temporal functional properties of a disease at microscopic level such as expression level of a cancer biomarker, with other properties such as super/para-magnetism and X-ray absorbance which gives micro/ macroscopic anatomical information.
  • the inventive step provide a nanomedicine formulation, containing the said nanoparticle as a mairi component together with therapeutic molecules such as chemo drugs or nucleic acids that can treat disease in a targeted manner by conjugation of a ligand specific to a receptor in the diseased tissue /cell.
  • Example 1 Multifunctional contrast agent based on calcium phosphate nanoparticles with 2 dopants for combined NIR and radioactive imaging.
  • the pH during the entire process was maintained at ⁇ 7.4 by the addition of 0.1M NH 4 OH solution.
  • the precipitate was left stirring for another 30 minutes, centrifuged and washed at least 2 times.
  • the entire process was carried out room temperature at ⁇ 25°C. - 3omg/mL of the washed product was treated with lmL of 250 ⁇ Tc-MDP (Tc- 99m conjugated to methylene diphosphate). The mixture is vortexed for 2 minutes and incubated for another 20 minutes.
  • the conjugate is washed at least twice and resuspended in PBS or distilled water for further applications.
  • ICG can be replaced with any NIR emitting dye or raman active dye molecules for the synthesis of other NIR emitting or raman active calcium phosphate nanoparticles.
  • Example 2 Multifunctional contrast agent based on calcium phosphate nanoparticles with more than 2 dopants for combined raman. magnetic. X-ray and radioactive imaging.
  • the entire process was carried out room temperature at ⁇ 25 C. ⁇ 3omg/mL of the washed product was treated with lmL of 250 ⁇ Tc-MDP(Tc-99m conjugated to methylene diphosphate). The mixture was vortexed for 2 minutes and incubated for another 20 minutes. The conjugate was washed at least twice and resuspended in PBS or distilled water for further applications.
  • Aniline blue can be replaced by any other raman active or NIR emitting dye for the synthesis of other raman active or NIR emitting calcium phosphate nanoparticles.
  • Gadolinium can be replaced by any other impurity ion to give magnetic or X-ray contrast properties.
  • Tc-99m can be replaced with any other radioactive element to synthesize other radioactive calcium phosphate nanoparticles.
  • Example 3 Multifunctional contrast agent based on hydroxyapatite nanoparticles with more than 2 dopants for combined raman. magnetic. X-rav and radioactive imaging,
  • Aniline blue can be replaced by any other raman active or NIR emitting dye for the synthesis of other raman active or NIR emitting hydroxyapatite nanoparticles.
  • Gadolinium can be replaced by any other impurity ion to give magnetic or X-ray contrast properties.
  • Tc-99m can be replaced with any other radioactive element to synthesize other radioactive hydroxyapatite nanoparticles.
  • Example 4 Preparation of siRNA incorporated calcium phosphate nanoparticles based nanomedicines capable of image guided delivery.
  • siRNA therapeutic small interfering RNA
  • Antisense strand 3 1 -dTdTCGGCGAGCAACCUUGAGGU— 5 1
  • lmg/ml bare nanoparticles prepared as said in any of the above examples 1-3 is prepared in phosphate buffer saline (PBS, Sigma, USA)) and sonicated for 10 minutes to get a fine dispersion.
  • PBS phosphate buffer saline
  • the nCP-siRNA conjugates were further treated with o.oimg/ml BSA (Bovine Serum Albumin) and o.img/ml EDC (i-ethyl-3-(3- dimethylaminopropyl) carbodiimi.de) for 3omin at 37°C. After 30min, the nanoconjugates are removed by centrifugation and re-suspended in PBS. This forms a protective shell of albumin protein that can be derivatized using targeting ligands.
  • BSA Bovine Serum Albumin
  • EDC i-ethyl-3-(3- dimethylaminopropyl) carbodiimi.de
  • this example provides preparation of a representative nanomedicine formed by loading the said multifunctional calcium phosphate nanoparticles with a representative gene.
  • a gene preferably a marker gene that can be readily detected by simple laboratory tools
  • An appropriate marker gene selected for this example is beta.- galactosidase ( ⁇ -gal) since its expression can be readily detected by addition of X- gal, a substrate which yields a blue color when the active enzyme is present.
  • ⁇ -gal beta.- galactosidase
  • this example is not limited to the said marker gene, but any gene intended for a desired function such as inhibition of tumor growth.
  • Part B Another two solutions containing phosphate group (Part B), ammonium dihydrogen phosphate, (5 mL, 0.3M, 98%, Qualigens, India) and 1 ml NH 4 OH (0.1M, 25% NH 3 , Qualigens, India) are prepared. Both these solutions are added drop wise to the precursor mixture of Part A+D with continuous stirring over a period of 15 minutes. The rate of addition of ammonium hydroxide solution is adjusted to maintain the pH of the reaction medium at ⁇ 7.4 throughout the reaction. After completion of precipitation, the mixture is kept stirring for 1 hour followed by separation of the precipitate by centrifugation at 3000 rpm for lominutes and washing with ice cold water for 3-5 times.
  • the DNA embedded within the doped calcium phosphate matrix is optionally protected from enzymatic degradation using a protective coat of BSA, by treating the nCP-DNA conjugates with o.oimg of BSA and o.img of EDC in 5ml PBS for 30mm at 37°C, followed by washing with water.
  • Example 6 Preparation of nCP-polymer nanomedicine containing chemo drugs capable of image guided delivery
  • a representative nanomedicine formed by the said multifunctional calcium phosphate nanoparticles embedded within polymeric nanoparticle containing chemical drugs, for example a small molecule inhibitor, Sorafenib is presented, l mg/ml bare nanoparticles prepared as said in any of the above examples 1-3 is prepared in DMSO (dimethyl sulfoxide) medium and sonicated for 10 minutes to get a fine dispersion.
  • DMSO dimethyl sulfoxide
  • ⁇ ⁇ Sorafenib in lmg/ml PLGA poly(lactic-co-glycolic acid)
  • DMSO dimethyl sulfoxide
  • ⁇ 500 ⁇ , of H 2 0 is added to the CP-Sorafenib-PLGA mixture to precipitate the polymeric nanoparticles that contain CP and Sorafenib embedded in it.
  • This nanomedicine formulation can be used to deliver drug actively or passively to tumor sites together with imaging using multiple imaging modalities.
  • PLGA other biodegradable polymers such as PEI, PLA, PCL, PVA, PPV etc also can be used for embedding the nCP and chemical drug.
  • Example 7 Bisphosphonate tagged nCP for imaging and therapeutic applications
  • synthesis of bisphosphonate drug, alendronate, tagged Tc99m labeled doped calcium phosphate nanoparticles for imaging and therapy of diseases such as bone metastasis or other bone metabolic disorders, osteoporosis etc is presented.
  • the precipitate was left stirring for another 30 minutes, centrifuged and washed at least 2 times. The entire process was carried out room temperature at ⁇ 25°C. ⁇ 30mg/mL of the washed product was treated with lmL of 250 ⁇ Tc99m-alendronate (Tc-99m conjugated to alendronate drug). The mixture is vortexed for 2 minutes and incubated for another 20 minutes. The conjugate is washed at least twice and resuspended in PBS or distilled water for further applications.

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Abstract

La présente invention concerne un matériau nanométrique qui peut produire une accentuation de contraste pour de multiples procédés d'imagerie et également délivrer des molécules thérapeutiques telles que des acides nucléiques ou des médicaments chimiothérapeutiques à des sites pathologiques tels qu'un cancer. En particulier, la présente invention concerne des phosphates de calcium synthétique nanométriques et des nanomatériaux d'apatite calcique présentant un contraste simultané pour au moins deux quelconques des modalités d'imagerie médicale comprenant l'imagerie radio, raman, de fluorescence dans l'infrarouge proche, de résonance magnétique et radiographique.
PCT/IN2013/000143 2013-03-12 2013-03-12 Art, procédé, manière, processus et systèmes d'un nano-biominéral pour imagerie de contraste multimodale et administration de médicament WO2014141288A1 (fr)

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