WO2014141287A1 - The art, method, manner, process and system of multifunctional nanobiomaterial for molecular imaging and drug- delivery - Google Patents
The art, method, manner, process and system of multifunctional nanobiomaterial for molecular imaging and drug- delivery Download PDFInfo
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- WO2014141287A1 WO2014141287A1 PCT/IN2013/000142 IN2013000142W WO2014141287A1 WO 2014141287 A1 WO2014141287 A1 WO 2014141287A1 IN 2013000142 W IN2013000142 W IN 2013000142W WO 2014141287 A1 WO2014141287 A1 WO 2014141287A1
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- multifunctional
- imaging
- nanocontrast
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- agent
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/6458—Fluorescence microscopy
Definitions
- TITLE OF THE INVENTION "The Art, Method, Manner, Process and System of Multifunctional Nanobiomaterial for molecular imaging and drug- delivery
- the Invention relates to the art, method and manner and process of Multifunctional Nanobiomaterial for molecular imaging and drug- delivery
- the present invention relates to a nanosized material that can provide contrast enhancement for multiple molecular imaging methods and also deliver therapeutic nucleic acids or chemodrugs at a specific site of disease such as cancer.
- the present invention relates to nanosized synthetic calcium apatite based materials showing contrast imaging for visible to near-infrared fluorescence, magnetic resonance imaging and X-ray imaging together with targeted delivery of nucleic acid drugs (D A or RNA) to specific cancer types.
- D A or RNA nucleic acid drugs
- This invention relates to the preparation of a nanobiomaterial based on doped 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 magnetic resonance imaging (MRI), X-ray computed tomography (CT) and near-infrared (NIR) fluorescence is an emerging area of research and development in oncology [1,2].
- Combinatorial imaging can provide simultaneous high definition anatomical, physiological and functional information about the disease like cancer leading to early stage diagnostics, image guided drug delivery and therapeutics, and efficacy analysis of the treatment [3,4].
- separate 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 opticalJA orjescenceLtechniques-can- ⁇
- MRI or CT 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.
- MRI requires paramagnetic, super paramagnetic contrast agents
- computed tomography (CT) requires X-ray absorbing contrast agent
- in vivo fluorescence imaging requires near- infrared fluorescent contrast agents.
- 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 and fluorescent molecules
- proteins Green fluorescent protein or Red fluorescent proteins
- QDs quantum dots
- Luminescent quantum dots, superparamagnetic iron oxide nanoparticles and Au nanocrystals were studied for optical, magnetic 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 toxicity associated with_the_
- 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 NIR fluorescence, MRI, X-ray, ultrasound or scintillation imaging, all at the same time.
- the inventors disclose a method of preparation of bionanomaterial based on calcium apatite doped with multiple impurities for deriving different physical properties suitable for simultaneous multi-modal contrast imaging.
- Calcium apatite preferably calcium hydroxyapatite (HAp)
- HAp calcium hydroxyapatite
- Biomimetic and synthetic HAp is extensively studied and used in clinical practice as implant material for in vivo bone tissue re-generation.
- the present invention discloses, the preparation of an improved calcium apatite doped with multiple impurities and impurity properties such as near- infrared fluorescence light together with X-ray absorption, magnetism and preferably scintillation emission.
- 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 photos 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 haemoglobin and water absorb minimally in this spectral window (680-900nm) so as to allow photons to penetrate for several centimetres deep inside the tissue. This allows mapping of molecular events in intact tissues using near- infrared fluorescence mediated molecular tomography (FMT) [8], 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.
- fluorescent probes can also absorb X-ray or gamma-ray radiation and emit NIR light
- FMT can be combined directly with X-ray imaging or radiation therapy. This allows doctors to visualize regions of disease, like tumor, where a particular molecular event takes place more compared to other regions. For example, differentiation between necrotic region and apoptotic regions or expression of a biomarker in cancer region compared to neighbouring tissues. This allows doctors to plan the treatment in advance, using non-invasive visualization techniques like X-ray imaging or MRI.
- stoichiometric hydroxyapatite Caio(OH)2(P04) 6
- an ammonium phosphate solution to a solution of calcium and ammonium hydroxide mixture.
- Doped apatite particles may also be prepared by replacing calcium ions with fluorescent or paramagnetic metal ions.
- By replacing the OH" with simple anions, including F ⁇ , C1-, Br-, I ⁇ , or 1/2[C03 2 ] other apatite derivatives can also be prepared.
- a novel biocompatible nanoparticle that can provide, simultaneous contrast enhancement for multiple molecular imaging modalities such as MRI, X-ray, visible and near Infrared (NIR) fluorescence, and preferably scintillation tomography together with targeted drug or gene delivery is disclosed.
- the particle is based on calcium apatite, preferably nanosized calcium hydroxyapatite, termed hereafter as nHAp, doped with more than one impurity and impurity clusters of amorphous phase located at interstitial regions of apatite microstructure.
- This multifunctional nano-composite particle can enable combined molecular imaging using MRI, CT, optical near-infrared fluorescent imaging and scintillation tomography.
- Another aspect of this invention relates to a method of making nanomedicine formulations based on the said nHAp particles capable of delivering the chemodrugs or nucleic acid drugs such as DNA, RNA, small interfering RNA (siRNA) specifically to a targeted disease like cancer.
- these nanomedicines may preferably deliver embedded with the help of an external control / trigger that can be activated by some unique physical properties of said particles.
- nanoparticle refers to primary inventive nanoparticles formed by biomineral, hydroxy apatite, measuring size about 1-100 nm, preferably 5-50nm, most preferably around 20-30 nm in size, showing " multifunctional' properties such as visible and NIR fluorescence, paramagnetism, X-ray absorption and X-ray, Gamma ray or alpha, beta or neutron excited fluorescence, called scintillation, simultaneously or separately as the case of interest may be.
- therapeutics refers to chemical drugs and nucleic acid drugs such as gene therapy materials DNA or RNA or small interfering RNA (siRNA) or antibodies, peptides, proteins, etc that have a therapeutic effect against a disease condition like cancer, inflammatory disease or autoimmune disease.
- siRNA small interfering 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 cancer / tumor type tissues.
- Targeting ligand include antibodies, peptides, aptamers, vitamins like folic acid, sugar molecules like mannose ligands
- nanomedicine refers to a composite construct based on tlie said nHAp nanoparticle which is connected or alternatively loaded with any of tlie above said therapeutics which ivill be delivered to the desired site using tlie said targeting ligands.
- the above said “nanomedicine” construct is formed by either : i) directly connecting tlie therapeutics and targeting ligands to the surface of said nHAp particles or alternatively, ii) the said nanoparticle will be taken as a core with anotlter biodegradable polymer containing the therapeutics as shell with targeting ligands connected to its surface, or Hi) a biodegradable polymeric nanoparticle which embed one or more said nHAp particles together with tlie therapeutic agents and connected with tlie targeting ligands on its surface
- the said nanomedicine construct preferably will have a size of 50-200nm, more preferably 80-150nm and most preferably ⁇ 100-120nm.
- the nanomedicine may be produced in tlieform of dry powders or liquid dispersions. In the present invention, the application as pharmaceutical suspension is envisioned.
- 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 in it (doped nanoparticles)
- SBF simulated body fluid
- tlie dopant ions include ions or molecular clusters that can provide visible/NIR fluorescence or magnetic contrast by paramagnetic, superparamagnetic, ferromagnetic or ferrimagnetic properties or X-ray contrast by X-ray absorption or scintillation.
- dopant ions are preferably situated at TC ro rffite ⁇ la ⁇ ttice positions including siibTtifufional7 ⁇ interstiTial positions or in combination or as ⁇ atomic clusters' of atoms or molecules of different phase formed by a few tens of molecules at particular location or locations within the crystal lattice of said nanoparticle.
- the material for making the biodegradable polymeric shell or bigger nanoparticle that can embed the said nanoparticles may include polymerizable monomers preferably connected with said therapeutics having 1 or preferably more tlian 1, 2, 3, 4 or 5 different components connected through intermolecular bonding (electrostatic, hydrogen, Van der Waals, coordinating or covalent), so as to form a network of interconnected precursors containing therapeutics, that self-assemble around the said nanoparticle to form the multifunctional nanomedicine.
- Fig 1 Schematic Flow chart for the synthesis of multifunctional nanobiomaterial
- Fig.2 a. Differential light scattering based particle size distribution of nHAp, b) Transmission Electron Microscope image of nHAp
- Fig. 7a Magnetization curves of undoped and doped nHAP, b) MRI imaging showing bright -contr-ast-provided-by-doped-nHAp—
- Fig. 8a Schematic diagram of X-ray contrast measurement set-up, b) X-ray contrast of undoped and doped nHAp with varied doping concentrations, c) X-ray opacity plot
- Fig. 9 Cytotoxicity study of nHAp showing no toxicity by the nanoparticles in both normal cells and cancer cells
- Fig. 10 Flow cytometry data showing no production of reactive oxygen species by nHAp when incubated with cell lines
- FIG. 11 Fluorescence microscopic images showing specific attachment of nHAp on to cancer cells ( c, d) while leaving normal cells (a,b) untouched
- Fig. 12 Schematic diagram of nHAp based siRNA delivery vehicle targeted to cancer cells
- Fig. 13 Fourier transform infrared spectrum of unconjugated, polymer conjugated (PEI) and folic acid (FA) conjugated nHAp Detailed Description:
- the method of preparation of said nanoparticle and nanomedicine formulation containing the same 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 ), potassium (K 3 PO 4 , K 2 HP0 4/ KH 2 PO 4 ) lithium (Li 3 P0 4/ Li 2 HP0 4/ ⁇ 2 ⁇ 4), ammonium ((NH 4 ) 3 P0 / (NH ) 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 ions (Part D) in the nanoparticle that gives near-infrared fluorescence is europium (III) and europium phosphate clusters.
- other elements such as neodymium, ytterbium, erbium, manganese and their corresponding phosphate clusters_can-be_used
- the dopant ion pairs (Part D) in the nanoparticle that gives simultaneous near-infrared fluorescence and magnetism is combination of europium or europium phosphates clusters with magnetic ions such as chromium(III), manganese(II), iron(II), iron (III), praseodymium (III), neodymium (III), samarium(III), ytterbium(III), gadolinium(III), terbium(III), dysprosium(III), holmium(III), erbium(III), or combinations thereof
- magnetic ions such as chromium(III), manganese(II), iron(II), iron (III), praseodymium (III), neodymium (III), samarium(III), ytterbium(III), gadolinium(III), terbium(III), dysprosium(III), holmium(III), erbium(III), or combinations thereof
- the dopant ions (Part D)that provide X-ray contrast together with NIR emission and magnetism are: a combination of europium or europium phosphates clusters with the above said magnetic ions and iodine or barium, bismuth, strontium tungsten, tantalum, hafnium, lanthanum, molybdenum, niobium, zirconium and combinations thereof
- the dopant ions (Part D) in the nanoparticle that provide X-ray, Gamma ray or neutron, alpha or beta scintillation property together with NIR emission, magnetism and X-ray contrast properties are: a combination of europium or europium phosphates clusters with the above said magnetic and X-ray contrast ions and cerium, lutetium, sodium, potassium and combinations thereof.
- the nanoparticle with dopant ions (Part D) or their combinations were formed by reacting precursor Part-A, B, C and D at a temperature range of 50-150°C and pH range of 4 -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 pH of the reaction is varied using a hydroxide salts of either sodium, potassium, lithium, ammonium or calcium or combinations thereof, added during or prior to the reaction between Part A, B, C and D.
- the nanoparticles doped with preferred ions can alternatively be formed by self-assembly of precursor Part-A and Part - B in simulated body fluid containing the suitable dopant ions mentioned above
- the formed nanoparticles are grown to a preferred size scale, between 1-lOOnm by aging of the reactants at time scales of any range between 0-12 Hrs, preferably, 0-6 Hrs and most preferably 2-4 hrs at a temperature range of 30-150°C, most preferably at 90°C.
- the nanoparticles formed by the above described method is highly monodispersed with > 90% of the particles showing an average size of ⁇ 30nm with no aggregation. This is evident from the particle size analysis by dynamic light scattering method shown in Fig. 2a and transmission electron micrograph (TEM) shown in Fig 2b.
- TEM transmission electron micrograph
- the said nanoparticles show major phase of pure hydroxyapatite as given in X-ray diffraction patterns of undoped and Eu 3+ -Gd 3+ doped samples Fig.3 All the patterns can be indexed to the hexagonal Caio(P0 4 )6(OH)2 in P63m space group (JCPDS No. 09-0432). All major crystal planes can be seen in the spectrum. Lattice constant values for all the three samples were calculated and compared with that of standard microcrystalline HAp.
- c 0.6882 nm
- c 0.6874 nm
- c 0.6876 nm.
- this invention leads to an important aspect of doping hydroxyapatite nanoparticles with molecular clusters of dopant ions within the interatomic lattice spaces within calcium apatite microstructure, leading to new properties such as near-infrared fluorescence, which is not known in the case of individual atoms doped calcium apatite of prior art.
- the high resolution transmission electron micrographs Fig.
- NIR near-infrared fluorescence
- the said nanoparticle can be used for molecular imaging based NIR fluorescence for biomedical applications.
- This is displayed in phantom experiments as shown in Fig. 6 where the circular pellets formed from the said nanoparticles are imaged by detecting both red light (Fig 6a) and NIR light, 700nm (Fig 6b), emitted by the nanoparticles. While undoped nanoparticles (1, 2) show only blue emission, doped nanoparticles (3-8) show red and NIR emission and its signal strength increases with doping concentration with sample 8 showing maximum contrast .
- the said NIR emitting nanoparticles show magnetism together with NIR fluorescence when co-doping of Eu ions with paramagnetic ions, Gadolinium as an example taken here.
- Fig. 7a 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 Europium and Gadolinium doped samples show paramagnetic property, which increases with the concentration of dopant ions, gadolinium in this case.
- This property makes it possible to use the said nanoparticle as a contrast agent for magnetic resonance imaging, as demonstrated in Fig. 7b wherein the said nanoparticles taken in low (0.05mg/ml) to high concentration (1.5mg/ml) and imaged under a clinical MRI system show excellent Tl weighted contrast properties, which increases with increase in concentration.
- the nanoparticles show X-ray contrast properties (radio opacity) together with NIR and magnetic resonance imaging.
- Fig. 8 where the X-ray contrast imaging of powder nanoparticle samples of different dopant concentrations are displayed. Undoped and doped samples were taken in eppendroff tube, half-filled, 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).
- Fig.8a shows the schematic of the optics construction of the X-ray imaging system.
- Incident X-ray passes through the sample and then through a phosphor plate where it is converted to visible light that is quantified by the detector.
- Fig. 8b shows the X-ray contrast images of the undoped and doped nHAp. Samples clearly shows dark contrast for the portion filled with said nanoparticles of HAp.
- the X-Ray density of the material which is a measure of the X-ray absorbed or attenuated, was calculated and plotted in Fig. 8c.
- the results discloses that, while undoped nanoparticle of HAp attenuated only -50% of incident X-ray, Eu-Gd doped samples attenuated - 80% of incident X-ray energy, clearly suggesting that the said nanoparticles can provide very good X-ray contrast together with both magnetic and NIR fluorescence contrast imaging.
- the said nanoparticles are highly bio-compatible (non- toxic) and cause no adverse effects on the cell viability. This is tested as shown in Fig. 9 where the inventors tested the cytotoxicity of said nanoparticles in three different cell lines; KB, A549 and L929 cells using MTT assay. The nanoparticle was found to show no toxicity to all the three cell lines even up to relatively high dose of 500 g/ ml for 48Hrs of incubation.
- the said nanoparticle can serve as a contrast agent for a number of molecular imaging techniques at the same time.
- the paramagnetic property or radio- opacity of the nanoparticle can be used to image a disease like tumor mass, its location, size, etc using imaging modalities like MRI and CT
- the near-infrared fluorescence emission from the same nanoparticle can be used to study the microscopic cellular level expression of a particular biomarker in the same rumor, for example over expression of a folate receptor in oral cancer lesions or her2-nue gene in breast cancer cell or drug- resistant protein in rumor by connecting the said nanoparticle with corresponding complimentary ligands or antibodies targeted to the said receptor molecule.
- the said nanoparticle can help doctors to improve the surgical procedure of removing cancer with the help of combined X-ray and fluorescence imaging.
- one the major challenge faced by oncology surgeons around the world is that, after detecting and removing to larger solid tumor with the help of CT or RI, they face difficulty in identifying the localised microscopic spread of cancer near to the solid tumor, using naked eye.
- a contrast agent that can also give visible or near infrared emission that can mark the tumor edges such that, after removing the solid tumor, doctors can easily visualize and remove the localized microscopic spread areas by detecting fluorescence emission from said nanoparticles.
- 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 -sur-g-ical-procedure ⁇ 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 MR or CT contrast for solid tumor.
- the said nanoparticle can, together with providing MR or CT contrast to solid tumor, can detect and illuminate individual cancer cells that normally circulate through the blood during metastatic spread of the disease.
- the cancer-targeted nanoparticles can attach specifically to the cell membrane of the targeted cancer cells while leaving the normal cells untouched.
- the nanoparticle targeted cells can be detected using high resolution and blood penetrating red or near-infrared fluorescence from the nanoparticles. This is demonstrated in Fig. 11 where the inventors tested specific targeting capability of folate conjugated nanoparticle to detect individual cancer cells without affecting the normal cells.
- Normal lung fibroblast cell line L929 and folate receptor positive (FR+) KB cell lines where used for the study.
- FIG. 11a and Fig lib shows microscopic images of L929 cells treated with folic acid conjugated nanoparticles of HAp.
- the red emitting nHAp can be seen randomly distributed all around the cells without any specific interaction with the cell membrane even after 4 and 24 Hrs of incubation.
- the nature of interaction changed quite dramatically when the said nanoparticles are treated with FR+ve KB cells (Fig. 11c and Fig lid). Large concentration of the particles was found specifically attached to the cell membrane as early as 1-2 Hr of incubation. This clearly suggests that, the said nanoparticle can specifically detect individual cancer cells.
- the doped nanoparticles are ca ⁇ xonrc ⁇ m _ natarerthe-sam
- nanomedicine constructs can be targeted using monoclonal antibodies (mAbs), peptides (pep) or similar ligands
- the said doped nanoparticles can also be coated with a biodegradable polymers containing anticancer drugs or nucleic acid drugs and using the magnetic property of the nanoparticle, the said nanomedicine construct can be delivered to a specific site of cancer with the help of an applied magnetic field near to tumor (magnetic drug delivery)
- the biodegradable polymers can be formed by polymers such as poly-lactic acid (PLA), poly (lactic-co-gly colic acid) (PLGA), polyvinylpyrrolidon (PVP), polyvinyl alcohol (PVA), polyethyleneimine (PEI), polyethelene glycol (PEG), chitosan, carboxymethyl chitosan, cyclodextrin, thermosensitive polymers such as Poly(N-isopropylacrylamide) and its derivative or proteins such as bovine or human serum albumin.
- the said doped 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 offhe nanoparticle
- the present invention provides a method of delivery of "drug using an external trigger. This is preferably done as follows: After assessing the concentration of the drug at the diseased site by any of the molecular imaging method, the magnetically active nanoparticle having a shell of chemodrugs embedded in a thermosensitive polymer will be subjected to inductive heating under an externally applied magnetic field such that drug will be released from the polymeric shell in a controlled fashion.
- the inventive nanoparticle provide a unique opportunity for image guided drug delivery, drug dose estimation and controlled, externally stimulated drug release or therapy.
- 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 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 imaging modalities like MRI, CT or 2D X-ray, which gives micro/ macroscopic anatomical information.
- the inventive step provide a nanomedicine formulation, containing the said nanoparticle as a main component together with therapeutic molecules that can treat disease in a targeted (specific) manner by making use of one of the functional properties of nanoparticle such as magnetism (magnetic drug -df]-iv&r-y-)-Qr— . by— molecular_recep,tQr_ligand targeting.
- the drugs incorporated within the nanomedicine constructs can be released, in a controlled fashion, by making use of the magnetically inductive heating property of the said nanoparticle.
- One of the major aspect of synthesis of doped hydroxyapatite, in the present invention, is the incorporation of multiple impurities into the calcium apatite crystal lattice by a single step process, while maintaining the particle size ⁇ 50nm without the use of any additional surfactants.
- Different sequences of reaction are possible in this method to achieve successful doping. These sequences are presented in different examples as follows:
- nHAp hydroxyapatite nanoparticles
- dopant ions including 6% Eu 3+ (Europium) and 4% Gd 3+ (Gadolinium) with respect to total Ca 2+ (Calcium) content
- this method can be followed for more than two dopant ions and to any higher or lower concentrations preferably between 0-20%.
- Aqueous solution containing Calcium chloride (CaCl 2 ), lOOmL, 0.5M, 98%, Sigma, USA (Part A) is mixed with 6ml of 0.5M, Europium chloride, EuCl 3 and 4 mL of 0.5M Gadolinium nitrate, Gd(NO 3 ) 3 and stirred well for 30min and then the temperature is raised to ⁇ 90°C.
- Part B Another two solutions containing phosphate group (Part B), ortho- phosphoric acid, H 3 (PO) 4 , (llOmL, 0.3M, 98%, Qualigens, India) and hydroxyl group, 100ml Ammonium hydroxide, ,NH 4 OH, Part C (0.1M, 25% NH 3 , Qualigens, India) are prepared separately and taken in two different burettes. Both these solutions are added drop wise to the warm precursor mixture of Part A+D with continuous stirring over a period of 1 hour. The rate of addition of NH 4 OH solution is adjusted to maintain the pH of the reaction medium at ⁇ 7.4 throughout the reaction. After completion of precipitation, the mixture is heated at 100° C for another 2 hours while stirring.
- the mixture is then left overnight followed by separation of the precipitate by centrifugation at 3000 rpm for lOminutes and washing with hot water for 6-8 times. It is then dried in hot air oven at 60° C for 24 hours and powdered using mortar and pestle.
- dopant ions Part D
- Part A dopant ions
- Part B is reacting in presence of Part C.
- Aqueous solution of a different calcium precursor Ca(OH) 2 lOOmL, 0.5M, 98%, Sigma, USA (Part A) is heated to temperature ⁇ 90°C.
- the mixture is then left overnight followed by separation of the precipitate by centrifugation at 3000 rpm for lOminutes and washing with hot water for 6-8 times. It is then dried in hot air oven at 60° C for 24 hours and powdered using mortar and pestle.
- Example 3 Production of fluorescent magnetic and X-ray absorbing Hydroxyapatite nanoparticles doped with more than two cationic and anionic dopant ions using, Scheme -
- total 04 dopant ions, three cationic and one anionic, are incorporated into Hydroxyapatite nanoparticles.
- Ca(OH) 2 lOOmL, 0.5M, 98%, Sigma, USA (Part A) was heated to temperature ⁇ 90°C.
- a representative anionic dopant ions 4 ml, 0.5M iodine (Part D)(4% wrt calcium), which can enhance X-ray contrast, is mixed with Part B, ortho-phosphoric acid, H 3 (PO)4, (llOmL, 0.3M, 98%, Qualigens, India) and taken in a burette, where as three other cationic dopant ions, 6ml of 0.5M EuCh, 4ml of 0.5M Gd(N0 3 ) 3 and 4ml, 0.5M BaC , all 99.9%, Sigma, USA were taken in another burette. Both orthophosphoric acid and cationic dopant ions are then added separately, drop wise to hot CaCl 2 solution while maintaining the pH of reaction at 7.4 using lOOml NH 4 OH (0.1M, 25% NH , Qualigens, India).
- Example 4 Preparation of nHAp-siRNA 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)) arid sonicated for 10 minutes to get a fine dispersion.
- PBS phosphate buffer saline
- the nHAp-siRNA conjugates were further treated with O.Olmg/ml BSA (Bovine Serum Albumin) and O.lmg/ml EDC (1- ethyl-3-(3-dimethylaminopropyl) carbodiimide) for 30min 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 1- ethyl-3-(3-dimethylaminopropyl) carbodiimide
- Example 5 Preparation of nHAp-DNA nanomedicines capable of image guided delivery
- this example provides preparation of a representative nanomedicine formed by loading the said multifunctional hydroxyapatite 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 colour 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 tumour growth.
- Part B Another two solutions containing phosphate group (Part B), ortho-phosphoric acid, H 3 (PO) 4 , (llOmL, 0.3M, 98%, Qualigens, India) and 100ml NH 4 OH (0.1 M, 25% NH 3 , Qualigens, India) are prepared separately and taken in two different burettes. Both these solutions are added drop wise to the warm precursor mixture of Part A+D with continuous stirring over a period of 1 hour. The rate of addition of ammonium hydroxide solution is adjusted to maintain the pH of the reaction medium at ⁇ 7.4 throughout the reaction.
- the mixture is kept stirring for 2 hours and kept overnight followed by separation of the precipitate by centrifugation at 3000 rpm for lOminutes and washing with ice cold water for 6-8 times.
- the DNA embedded within the doped calcium phosphate matrix is protected from enzymatic degradation using a protective coat of BSA, by treating the nHAp-DNA conjugates with O.Olmg of BSA and 0.1 mg of EDC in 5ml PBS for 30min at 37°C, followed by washing with water.
- Example 6 Preparation of nHAp-polymer nanomedicine containing chemo drugs capable of image guided delivery
- a representative nanomedicine formed by the said multifunctional hydroxyapatite nanoparticles embedded within polymeric nanoparticle containing chemical drugs, for example a small molecule inhibitor, Rapamycin is presented.
- Img/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
- ⁇ ⁇ Rapamycine in lmg/ml PLGA poly(lactic-co- glycolic acid)
- ⁇ 500 ⁇ of H 2 O is added to the HAp-Rapamycine-PLGA mixture to precipitate the polymeric nanoparticles that contain HAp and Rapamycin embedded in it.
- This nanomedicine formulation can be imaged using multiple imaging modalities or delivered to a specific site using magnetic properties of embedded HAp nanoparticles or connecting a targeting ligand.
- other biodegradable polymers such as PEI, PLA, PCL, PVA, PPV etc also can be used for embedding the nHAp and chemical drug.
- Example 7 Preparation of nHAp-polymer core-shell nanomedicine containing chemo drugs capable of delivering the drug by external trigger by inductive heating
- PNIPAm Poly(A -isopropylacrylamide)
- MB Am ⁇ , ⁇ '-methylene-bis-acrylamide
- CBAm ⁇ , ⁇ '-cystamine-bis- acrylamide
- PNIPAm Since PNIPAm expels its liquid contents at a temperature near to that of the human body, PNIPAm can be used to deliver drug embedded in it by heating using an external trigger.
- nHAp-PNIPAm-Rapamycine nanomedicine formulation wherein the inductive heating property of magnetic nHAp under an externally applied magnetic field can heat up the PNIPAm polymer to change its phase and release the embedded drug. This process also can be monitored by molecular imaging contrast provided by the embedded nHAp.
- nHAp-PNIPAm-Rapamycine nanomedicines 5mg bare nHAp nanoparticles prepared as said in any of the above examples 1-3, is re-dispersed in 5ml water and sonicated for 10 minutes to get a fine suspension, in another solution, ⁇ ⁇ Rapamycine dissolved in ⁇ 3 ml water-DMSO (1:1) solution containing 0.4 gm PNIPAm (3.4 mmol) and 0.06 gm MBAm (0.4 mmol) is formed and the same is added to the nanoparticle suspension and mixed well by sonication for 30 min.
- the temperature of this reaction mixture is elevated to 75°C and after ⁇ 30 min, O.Olgm of pottassium persulfate in 4ml water is injected in to the polymer-nHAp-Rapamycine mixture to initiate the polymerization reaction.
- the reaction is then allowed to continue for 2 Hrs and the precipitate is removed by centrifugation and washing with distilled water several times.
- the nanomedicines thus formed will have nHAp nanoparticles and Rapamycine embedded in thermosenstive PNIPAm nanoparticles of size ⁇ 10-20% bigger than nHAp.
- This nanoconjugate is capable of releasing the embedded drug Rapamycin, when subjected to magnetically inductive heating of embedded magnetic nHAp.
- Amine reactive succinimidyl ester activated FA is prepared by reacting FA (5mL, 0.1 M, Sigma Aldrich, USA) with EDC (lmL, 1M, Sigma Aldrich, USA) at pH 5.5 in MES (99%,Sigma Aldrich,USA) buffer for 15 minutes, in dark, at room temperature.
- Sulfo-NHS N-hydroxysuccinimide
- MES MES (2-(N- morpholine) ethanesulfonic acid) buffer (pH 5.5) is added to EDC-FA and reacted for 5 Hrs in the similar condition.
Abstract
The present invention relates to a nano-sized material that can provide contrast enhancement for multiple molecular imaging mthods and also deliver therapeutic nucleic acids or chemodrugs at a specific site of disease such as cancer. In particular, the present invention relates to a nanosized synthetic calcium apatite based materials showing contrast imaging for visible to near-infrared fluroscence, magnetic resonance imaging and x-ray imaging together with targeted delivery of nucleic acid drugs (DNA or RNA) to specific cancer types.
Description
1. TITLE OF THE INVENTION: "The Art, Method, Manner, Process and System of Multifunctional Nanobiomaterial for molecular imaging and drug- delivery
2.
2. APPLICANT(S)
(a) Name : AMRITA VISHWAVIDYAPEETHAM UNIVERSITY represented by its Director, Amrita Centre of Nano Sciences, Dr. Shantikumar Nair
(b) Nationality : Indian.
(c) Address : "Elamakkara P.O., Cochin 682 026, Kerala
-3TPREA-MBrE-TO-THE DESCRIPTION~
COMPLETE SPECIFICATION
The following specification describes the invention
The Invention relates to the art, method and manner and process of Multifunctional Nanobiomaterial for molecular imaging and drug- delivery
FIELD OF THE INVENTION
The present invention relates to a nanosized material that can provide contrast enhancement for multiple molecular imaging methods and also deliver therapeutic nucleic acids or chemodrugs at a specific site of disease such as cancer. In particular, the present invention relates to nanosized synthetic calcium apatite based materials showing contrast imaging for visible to near-infrared fluorescence, magnetic resonance imaging and X-ray imaging together with targeted delivery of nucleic acid drugs (D A or RNA) to specific cancer types.
BACKGROUND OF THE INVENTION
This invention relates to the preparation of a nanobiomaterial based on doped 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 magnetic resonance imaging (MRI), X-ray computed tomography (CT) and near-infrared (NIR) fluorescence is an emerging area of research and development in oncology [1,2]. Combinatorial imaging can provide simultaneous high definition anatomical, physiological and functional information about the disease like cancer leading to early stage diagnostics, image guided drug delivery and therapeutics, and efficacy analysis of the treatment [3,4]. At present, separate 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 opticalJA orjescenceLtechniques-can-^
MRI or CT to derive molecular information about the expression level of specific disease marker or biological process that influence the therapeutic outcome. However, one of the major challenges in multi-modal molecular imaging is the requirement of suitable contrast agents that can show different physical properties compatible with basic principles of imaging technique. At present, different molecular imaging techniques requires separate contrast agents because their working principles are distinctly different. For example, MRI requires paramagnetic, super paramagnetic contrast agents, computed tomography (CT) requires X-ray absorbing contrast agent and in vivo fluorescence imaging requires near- infrared fluorescent contrast agents. Accordingly, 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 and fluorescent molecules, proteins (Green fluorescent protein or Red fluorescent proteins) or quantum dots (QDs) are used for optical imaging [5-7]. For combining all these different imaging techniques to obtain both functional as well as anatomical information, separate contrast agents need to be injected in to the human body.
Further, these contrast agents should reach to the disease site at the same time, at sufficient concentrations. This invite formidable challenges because the pharmacokinetics of all the above mentioned 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.
With the emergence of nanotechnology, a number of new nanocontrast agents were proposed for molecular imaging applications. Luminescent quantum dots, superparamagnetic iron oxide nanoparticles and Au nanocrystals were studied for optical, magnetic and photo- acoustic/ two-photon imaging applications, respectively. In addition, 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. However, one of the major concerns about nanoparticle based contrast imaging is the toxicity associated with_the_
— _____ ■ __-—__ ■ — ■ #
nanomaterials. For example, luminescent semiconductor nanoparticles (quantum dots) 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 NIR fluorescence, MRI, X-ray, ultrasound or scintillation imaging, all at the same time.
In the present invention, the inventors disclose a method of preparation of bionanomaterial based on calcium apatite doped with multiple impurities for deriving different physical properties suitable for simultaneous multi-modal contrast imaging. Calcium apatite, preferably calcium hydroxyapatite (HAp), have a general formula Caio(P04)6(OH)2. It is a well-known biomineral constituent of human bone and teeth. Biomimetic and synthetic HAp is extensively studied and used in clinical practice as implant material for in vivo bone tissue re-generation.
Owing to the high stability and flexibility of the apatite structure, atomic substitutions mainly with functional elements, in place of calcium or hydroxyl ion, can be introduced into the crystal lattice in order to obtain properties such as fluorescence, magnetism, radio-opacity, etc. In the prior art, the preparation of calcium apatite particles doped with either paramagnetic or X-ray absorbing impurities or ultrasound contrast were reported [US patent no. 5342609, 5,690,908, 5468465, 5344640] as listed in the reference. There are also publications related to visible light (from blue 400nm to red 620nm) emission from calcium apatite crystals doped with different types of impurities.
However, there exist no prior art on the preparation of doped calcium apatite nanocrystals with medically significant ^near-infrared fluorescence' (~700nmV together with magnetism. X-ray contrast and scintillation properties suitable for medical imaging.
Accordingly, the present invention discloses, the preparation of an improved calcium apatite doped with multiple impurities and impurity properties such as near- infrared fluorescence light together with X-ray absorption, magnetism and preferably scintillation emission.
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 photos 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 haemoglobin and water absorb minimally in this spectral window (680-900nm) so as to allow photons to penetrate for several centimetres deep inside the tissue. This allows mapping of molecular events in intact tissues using near- infrared fluorescence mediated molecular tomography (FMT) [8], a new technique that can three-dimensionally image molecular events like gene expression by resolving fluorescence activation in deep tissues. Obviously, an essential component of successful FMT imaging is the development of biocompatible NIR emitting fluorescent probes that can be targeted to the tissue.
In addition, if such fluorescent probes can also absorb X-ray or gamma-ray radiation and emit NIR light, FMT can be combined directly with X-ray imaging or radiation therapy. This allows doctors to visualize regions of disease, like tumor, where a particular molecular event takes place more compared to other regions. For example, differentiation between necrotic region and apoptotic regions or expression of a biomarker in cancer region compared to neighbouring tissues. This allows doctors to plan the treatment in advance, using non-invasive visualization techniques like X-ray imaging or MRI.
In general, the preparation of undoped apatite particles is disclosed in several prior arts. For example, stoichiometric hydroxyapatite, Caio(OH)2(P04)6, is prepared by adding an ammonium phosphate solution to a solution of calcium and ammonium hydroxide mixture. Doped apatite particles may also be prepared by replacing calcium ions with fluorescent or paramagnetic metal ions. By replacing the OH" with simple anions, including F~, C1-, Br-, I~, or 1/2[C032 ] other apatite derivatives can also be prepared.
Various techniques for controlling the particle size for calcium apatite are also disclosed in the prior arts. For example, slower addition of reactants, faster stirring, higher reaction temperatures, and lower concentrations generally result in smaller particles. In addition, sonication during precipitation, turbulent flow or impingement mixers, homogenization, and pH modification are also used to control particle size. Other means, such as computer controlled auto-burettes, peristaltic pumps, and syringes, may be used to control the release of precipitating ions to produce smaller particles.
Although, the above said methods allow one to prepare either magnetic or radio- opaque calcium apatite for its individual use as contrast agents for MRI or X ray, there exist no prior art describing preparation of apatite crystals having a number of important contrast properties such as near-infrared fluorescence, magnetism, X-ray absorption and scintillation in a single system.
There exists a need to prepare such a contrast agent based on non-toxic materials like calcium apatite. This is achieved, in the present invention, by doping apatite with multiple impurities and impurity clusters, while maintaining the particle size <100nm. In the prior art, while the doping of apatite with individual atoms, that replaces either calcium or hydroxyl ions were disclosed, here we disclose, an improved novel method of doping of calcium apatite with impurity clusters of a different phase, sometimes amorphous, at localised regions within the microstructure of apatite. These clusters show its own new properties of practical significance such as near-infrared fluorescence. In addition, we also disclose, a method of utilising such new materials for producing nanomedical formulation for its use in human health care, more specifically image guided drug delivery and therapy.
SUMMARY OF THE INVENTION
In the present invention, a novel biocompatible nanoparticle that can provide, simultaneous contrast enhancement for multiple molecular imaging modalities such as MRI, X-ray, visible and near Infrared (NIR) fluorescence, and preferably scintillation tomography together with targeted drug or gene delivery is disclosed. Specifically, the particle is based on calcium apatite, preferably nanosized calcium hydroxyapatite, termed hereafter as nHAp, doped with more than one impurity and impurity clusters of amorphous phase located at interstitial regions of apatite microstructure. This multifunctional nano-composite particle can enable combined molecular imaging using MRI, CT, optical near-infrared fluorescent imaging and scintillation tomography. Another aspect of this invention relates to a method of making nanomedicine formulations based on the said nHAp particles capable of delivering the chemodrugs or nucleic acid drugs such as DNA, RNA, small interfering RNA (siRNA) specifically to a targeted disease like cancer. In addition, these nanomedicines may preferably deliver embedded with the help of an external control / trigger that can be activated by some unique physical properties of said particles.
DETAILED DESCRIPTION OF THE INVENTION
Definitions : Tlie term "nanoparticle" as used herein refers to primary inventive nanoparticles formed by biomineral, hydroxy apatite, measuring size about 1-100 nm, preferably 5-50nm, most preferably around 20-30 nm in size, showing "multifunctional' properties such as visible and NIR fluorescence, paramagnetism, X-ray absorption and X-ray, Gamma ray or alpha, beta or neutron excited fluorescence, called scintillation, simultaneously or separately as the case of interest may be.
Tlie term " therapeutics" as used herein refers to chemical drugs and nucleic acid drugs such as gene therapy materials DNA or RNA or small interfering RNA (siRNA) or antibodies, peptides, proteins, etc that have a therapeutic effect against a disease condition like cancer, inflammatory disease or autoimmune disease.
The term "targeting ligand" as used herein refers to biomolecules that can specifically 'identify and target another molecule like an antigen or receptor on the surface of cell-membrane of cancer / tumor type tissues. Targeting ligand include antibodies, peptides, aptamers, vitamins like folic acid, sugar molecules like mannose ligands
The term "nanomedicine' as used herein refers to a composite construct based on tlie said nHAp nanoparticle which is connected or alternatively loaded with any of tlie above said therapeutics which ivill be delivered to the desired site using tlie said targeting ligands.
The above said "nanomedicine" construct is formed by either : i) directly connecting tlie therapeutics and targeting ligands to the surface of said nHAp particles or alternatively, ii) the said nanoparticle will be taken as a core with anotlter biodegradable polymer containing the therapeutics as shell with targeting ligands connected to its surface, or Hi) a biodegradable polymeric nanoparticle which embed one or more said nHAp particles together with tlie therapeutic agents and connected with tlie targeting ligands on its surface
The said nanomedicine construct preferably will have a size of 50-200nm, more preferably 80-150nm and most preferably ~100-120nm. The nanomedicine may be produced in tlieform of dry powders or liquid dispersions. In the present invention, the application as pharmaceutical suspension is envisioned.
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 in it (doped nanoparticles)
The term "doped" as used herein, refers to incorporation of small amount of (about 0.001- 15mol%) another substance (ions) ivithin the said nanoparticles. In the present case, tlie dopant ions include ions or molecular clusters that can provide visible/NIR fluorescence or magnetic contrast by paramagnetic, superparamagnetic, ferromagnetic or ferrimagnetic properties or X-ray contrast by X-ray absorption or scintillation. These dopant ions are preferably situated at TC ro rffite~la~ttice positions including siibTtifufional7~interstiTial positions or in combination or as ^atomic clusters' of atoms or molecules of different phase formed by a few tens of molecules at particular location or locations within the crystal lattice of said nanoparticle.
In the nanomedicine construct, the material for making the biodegradable polymeric shell or bigger nanoparticle that can embed the said nanoparticles may include polymerizable monomers preferably connected with said therapeutics having 1 or preferably more tlian 1, 2, 3, 4 or 5 different components connected through intermolecular bonding (electrostatic, hydrogen, Van der Waals, coordinating or covalent), so as to form a network of interconnected precursors containing therapeutics, that self-assemble around the said nanoparticle to form the multifunctional nanomedicine.
Figure Captions:
Fig 1. Schematic Flow chart for the synthesis of multifunctional nanobiomaterial
Fig.2 a. Differential light scattering based particle size distribution of nHAp, b) Transmission Electron Microscope image of nHAp
Fig. 3 X-ray Diffraction pattern of undoped, Eu doped and Eu-Gd doped nHAp
Fig.4 High resolution TEM showing dopant ions related lattice defects in nHAp
Fig.5 Fluorescence emission spectrum of nHAP doped with different concentration of Eu
Fig 6. Visible and NIR fluorescence imaging of undoped ( sa.ple 1 & 2) and doped nHAp ( sample 3-8)
Fig. 7a. Magnetization curves of undoped and doped nHAP, b) MRI imaging showing bright -contr-ast-provided-by-doped-nHAp—
Fig. 8a. Schematic diagram of X-ray contrast measurement set-up, b) X-ray contrast of undoped and doped nHAp with varied doping concentrations, c) X-ray opacity plot
Fig. 9. Cytotoxicity study of nHAp showing no toxicity by the nanoparticles in both normal cells and cancer cells
Fig. 10. Flow cytometry data showing no production of reactive oxygen species by nHAp when incubated with cell lines
Fig. 11. Fluorescence microscopic images showing specific attachment of nHAp on to cancer cells ( c, d) while leaving normal cells (a,b) untouched
Fig. 12. Schematic diagram of nHAp based siRNA delivery vehicle targeted to cancer cells
Fig. 13. Fourier transform infrared spectrum of unconjugated, polymer conjugated (PEI) and folic acid (FA) conjugated nHAp
Detailed Description:
Referring the flowchart as shown in Fig.l, the method of preparation of said nanoparticle and nanomedicine formulation containing the same, consists the steps of:
• Providing the nanoparticle precursor solutions containing Ca2+ (Part A), PO4- (Part B) and OH- (Part C) ions
• Providing the precursor solutions containing multiple dopant ions, Part D, that provide visible and near infrared fluorescence, magnetism, X-ray absorption and scintillation properties
• Forming the impurity doped nanoparticle by reacting the Part A, B C and D by drop wise mixing each reactants at appropriate sequences, rates, pH and temperature conditions
• Aging the colloidal precipitate at 100-120°C for 1-4 Hrs for crystallization of particles to desired size
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• Coupling the said nanoparticles with anticancer chemodrugs or nucleic acid drugs or making the nanomedicine formulation containing said nanoparticle in biodegradable polymeric shell containing the drugs, or biodegradable polymer containing both said nanoparticles and drugs
• Connecting the said nanoparticles or associated nanomedicines with a suitable targeting ligands
In a preferred embodiment of said method of making nanoparticles, 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.
In another preferred embodiment of said method of making nanoparticles, another precursor compound, Part B, is formed from water soluble or miscible salt of phosphates including sodium (Na3P04, Na2HP04/ NaH2P0 ), potassium (K3PO4, K2HP04/ KH2PO4) lithium (Li3P04/ Li2HP04/ ΠΗ2ΡΟ4), ammonium ((NH4)3P0 / (NH )2HP04/ NH4H2P04) or phosphoric acid
In another preferred embodiment of said method, the precursor compound containing hydroxyl anions (Part C) is formed by hydroxide salts of sodium, potassium, lithium, ammonium or calcium
In another preferred embodiment of said method, the dopant ions (Part D) in the nanoparticle that gives near-infrared fluorescence is europium (III) and europium phosphate clusters. In addition, preferably, other elements such as neodymium, ytterbium, erbium, manganese and their corresponding phosphate clusters_can-be_used
In yet another preferred embodiment of said method, the dopant ion pairs (Part D) in the nanoparticle that gives simultaneous near-infrared fluorescence and magnetism is combination of europium or europium phosphates clusters with magnetic ions such as chromium(III), manganese(II), iron(II), iron (III), praseodymium (III), neodymium (III), samarium(III), ytterbium(III), gadolinium(III), terbium(III), dysprosium(III), holmium(III), erbium(III), or combinations thereof
In yet another preferred embodiment of said method, the dopant ions (Part D)that provide X-ray contrast together with NIR emission and magnetism are: a combination of europium or europium phosphates clusters with the above said magnetic ions and iodine or barium, bismuth, strontium tungsten, tantalum, hafnium, lanthanum, molybdenum, niobium, zirconium and combinations thereof
In yet another preferred embodiment of said method, the dopant ions (Part D) in the nanoparticle that provide X-ray, Gamma ray or neutron, alpha or beta scintillation property together with NIR emission, magnetism and X-ray contrast properties are: a combination of europium or europium phosphates clusters with the above said magnetic and X-ray contrast ions and cerium, lutetium, sodium, potassium and combinations thereof.
In yet another preferred embodiment of said method, the nanoparticle with dopant ions (Part D) or their combinations were formed by reacting precursor Part-A, B, C and D at a temperature range of 50-150°C and pH range of 4 -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.
In yet another preferred embodiment of said method, the pH of the reaction is varied using a hydroxide salts of either sodium, potassium, lithium, ammonium or calcium or combinations thereof, added during or prior to the reaction between Part A, B, C and D.
In yet another embodiment of said method, the nanoparticles doped with preferred ions can alternatively be formed by self-assembly of precursor Part-A and Part - B in simulated body fluid containing the suitable dopant ions mentioned above
In yet another preferred embodiment of said method, the formed nanoparticles are grown to a preferred size scale, between 1-lOOnm by aging of the reactants at time scales of any range between 0-12 Hrs, preferably, 0-6 Hrs and most preferably 2-4 hrs at a temperature range of 30-150°C, most preferably at 90°C.
Accordingly, in one of the novel aspect of the present invention, the nanoparticles formed by the above described method is highly monodispersed with > 90% of the particles showing an average size of ~ 30nm with no aggregation. This is evident from the particle size analysis by dynamic light scattering method shown in Fig. 2a and transmission electron micrograph (TEM) shown in Fig 2b. This controlled particle size distribution and well dispersed, rounded particle morphology is achieved without the help of any additional surface capping agent or surface treatment as used in prior arts. Thus, the inventors present an improved method of making doped calcium apatite nanoparticles.
In another aspect of the invention, the said nanoparticles show major phase of pure hydroxyapatite as given in X-ray diffraction patterns of undoped and Eu3+-Gd3+ doped samples Fig.3 All the patterns can be indexed to the hexagonal Caio(P04)6(OH)2 in P63m space group (JCPDS No. 09-0432). All major crystal planes can be seen in the spectrum. Lattice constant values for all the three samples were calculated and compared with that of standard microcrystalline HAp. The calculated values of undoped nHAp is = b = 0.9412 nm, c = 0.6882 nm, nHAp:Eu3+ is a = b = 0.9427 nm, c = 0.6874 nm and nHAp:Eu3+-Gd3+ is a = b = 0.9429 nm, c = 0.6876 nm. The values of undoped nHAp is in good agreement with the standard data of a = b = 0.9418 nm, c = 0.6884 nm (space group P63/m), whereas both the doped samples showed deviations, suggesting lattice distortions caused by the incorporation of dopant ions.
In relation to the above aspect of doping associated lattice distortions, this invention leads to an important aspect of doping hydroxyapatite nanoparticles with molecular clusters of dopant ions within the interatomic lattice spaces within calcium apatite microstructure, leading to new properties such as near-infrared fluorescence, which is not known in the case of individual atoms doped calcium apatite of prior art. For example, in one of the preferred embodiment of europium doped nanoparticles of HAp, the high resolution transmission electron micrographs (Fig. 4) shows that along with the ordered lattice of HAp, indicated by parallel lines in the figure 4, dark spots or amorphous nature (marked in circle) indicating inter atomic precipitation of dopant clusters of sub- nanometer size ~ 0.3-0.6nm which is formed by molecular clusters of dopant ions.
In relation to the above aspect of doping the hydroxyapatite nanoparticle with molecular clusters of dopant ions, this invention disclose new properties such as near infrared fluorescence at ~ 700nm, which is not observed in prior arts related to doped calcium apatite nanoparticles. Fig. 5 shows fluorescence emission spectra of europium doped nanoparticles doped with varying concentration of Eu3+. Upto -1.3% of Eu3+, the sample showed typical fluorescence characteristics of individual europium ions substituted at Ca (II) lattice positions. But above 1.3 %, the emission band at 700nm started increasing and become dominant at ~ 6% of Eu3+ doping. This emission is not related to individual europium ions doped at Ca site, as reported by many other publications. However this emission is a characteristic emission caused by 5Do→ 7¥i transitions in europium phosphate molecules. Observation of this characteristic NIR emission together with the appearance of molecular cluster like defects in the microstructure of heavily doped HAp (TEM images, Fig.4) indicate that at > 1.3% Eu 3+, europium phosphate phases are getting stabilized in the HAp crystal lattice leading to new advantageous properties such as near-infrared fluorescence (NIR).
In relation to the above said novel aspect of NIR fluorescence, the said nanoparticle can be used for molecular imaging based NIR fluorescence for biomedical applications. This is displayed in phantom experiments as shown in Fig. 6 where the circular pellets formed from the said nanoparticles are imaged by detecting both red light (Fig 6a) and NIR light, 700nm (Fig 6b), emitted by the nanoparticles. While undoped nanoparticles (1, 2) show only blue emission, doped nanoparticles (3-8) show red and NIR emission and its signal strength increases with doping concentration with sample 8 showing maximum contrast .
In another aspect of the present invention, the said NIR emitting nanoparticles show magnetism together with NIR fluorescence when co-doping of Eu ions with paramagnetic ions, Gadolinium as an example taken here. Fig. 7a 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 Europium and Gadolinium doped samples show paramagnetic property, which increases with the concentration of dopant ions, gadolinium in this case. This property makes it possible to use the said nanoparticle as a contrast agent for magnetic resonance imaging, as demonstrated in Fig. 7b wherein the said nanoparticles taken in low (0.05mg/ml) to high concentration (1.5mg/ml) and imaged under a clinical MRI system show excellent Tl weighted contrast properties, which increases with increase in concentration.
In yet another aspect of this present invention, the nanoparticles show X-ray contrast properties (radio opacity) together with NIR and magnetic resonance imaging. This is demonstrated in Fig. 8 where the X-ray contrast imaging of powder nanoparticle samples of different dopant concentrations are displayed. Undoped and doped samples were taken in eppendroff tube, half-filled, 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). Fig.8a shows the schematic of the optics construction of the X-ray imaging system. Incident X-ray passes through the sample and then through a phosphor plate where it is converted to visible light that is quantified by the detector. Fig. 8b shows the X-ray contrast images of the undoped and doped nHAp. Samples clearly shows dark contrast for the portion filled with said nanoparticles of HAp. The X-Ray density of the material, which is a measure of the X-ray absorbed or attenuated, was calculated and plotted in Fig. 8c. The results discloses that, while undoped nanoparticle of HAp attenuated only -50% of incident X-ray, Eu-Gd doped samples attenuated - 80% of incident X-ray energy, clearly suggesting that the said nanoparticles can provide very good X-ray contrast together with both magnetic and NIR fluorescence contrast imaging.
In another important aspect of the present inventions, the said nanoparticles are highly bio-compatible (non- toxic) and cause no adverse effects on the cell viability. This is tested as shown in Fig. 9 where the inventors tested the cytotoxicity of said nanoparticles in three different cell lines; KB, A549 and L929 cells using MTT assay. The nanoparticle was found to show no toxicity to all the three cell lines even up to relatively high dose of 500 g/ ml for 48Hrs of incubation.
In addition to cell-viability, inventors also tested reactive oxygen stress caused by the nanoparticles. This was investigated by flow cytometry using intracellular ROS chemical sensor, Dichlorofluorescin diacetate (DCFH-DA). FACS data (Fig. 10) suggests that, after 12 Hrs of incubation, the said nanoparticles treated with FR+ve KB cells show practically no additional ROS levels (P2 scatter) compared to the untreated cells (negative control). These studies clearly suggest that the FA conjugated, lanthanide doped
functions and hence suitable for in vivo applications.
As described above, another important aspect of the present invention is, the said nanoparticle can serve as a contrast agent for a number of molecular imaging techniques at the same time. This makes it possible to combine different imaging techniques for the better understanding of disease. For example, while the paramagnetic property or radio- opacity of the nanoparticle can be used to image a disease like tumor mass, its location, size, etc using imaging modalities like MRI and CT, the near-infrared fluorescence emission from the same nanoparticle can be used to study the microscopic cellular level expression of a particular biomarker in the same rumor, for example over expression of a folate receptor in oral cancer lesions or her2-nue gene in breast cancer cell or drug- resistant protein in rumor by connecting the said nanoparticle with corresponding complimentary ligands or antibodies targeted to the said receptor molecule.
In another aspect of this invention, the said nanoparticle can help doctors to improve the surgical procedure of removing cancer with the help of combined X-ray and fluorescence imaging. For example; one the major challenge faced by oncology surgeons around the world is that, after detecting and removing to larger solid tumor with the help of CT or RI, they face difficulty in identifying the localised microscopic spread of cancer near to the solid tumor, using naked eye. It is therefore advantages to use a contrast agent that can also give visible or near infrared emission that can mark the tumor edges such that, after removing the solid tumor, doctors can easily visualize and remove the localized microscopic spread areas by detecting fluorescence emission from said nanoparticles.
In yet another novel aspect of the present invention, 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 -sur-g-ical-procedure^ 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 MR or CT contrast for solid tumor.
In yet another novel aspect of the present invention, the said nanoparticle can, together with providing MR or CT contrast to solid tumor, can detect and illuminate individual cancer cells that normally circulate through the blood during metastatic spread of the disease. This is possible because, the cancer-targeted nanoparticles can attach specifically to the cell membrane of the targeted cancer cells while leaving the normal cells untouched. The nanoparticle targeted cells can be detected using high resolution and blood penetrating red or near-infrared fluorescence from the nanoparticles.
This is demonstrated in Fig. 11 where the inventors tested specific targeting capability of folate conjugated nanoparticle to detect individual cancer cells without affecting the normal cells. Normal lung fibroblast cell line L929 and folate receptor positive (FR+) KB cell lines where used for the study. Fig. 11a and Fig lib shows microscopic images of L929 cells treated with folic acid conjugated nanoparticles of HAp. The red emitting nHAp can be seen randomly distributed all around the cells without any specific interaction with the cell membrane even after 4 and 24 Hrs of incubation. However, the nature of interaction changed quite dramatically when the said nanoparticles are treated with FR+ve KB cells (Fig. 11c and Fig lid). Large concentration of the particles was found specifically attached to the cell membrane as early as 1-2 Hr of incubation. This clearly suggests that, the said nanoparticle can specifically detect individual cancer cells.
In another important aspect of the present invention, since the doped nanoparticles are ca^xonrc~m_natarerthe-sam
as DNA or RNA or its small fractions like siRNA, for therapeutic gene delivery applications, as depicted in Fig. 12. These nanomedicine constructs can be targeted using monoclonal antibodies (mAbs), peptides (pep) or similar ligands
In another aspect of the present invention, the said doped nanoparticles can also be coated with a biodegradable polymers containing anticancer drugs or nucleic acid drugs and using the magnetic property of the nanoparticle, the said nanomedicine construct can be delivered to a specific site of cancer with the help of an applied magnetic field near to tumor (magnetic drug delivery)
The biodegradable polymers, as said in above novel aspect, can be formed by polymers such as poly-lactic acid (PLA), poly (lactic-co-gly colic acid) (PLGA), polyvinylpyrrolidon (PVP), polyvinyl alcohol (PVA), polyethyleneimine (PEI), polyethelene glycol (PEG), chitosan, carboxymethyl chitosan, cyclodextrin, thermosensitive polymers such as Poly(N-isopropylacrylamide) and its derivative or proteins such as bovine or human serum albumin.
In another aspect of the present invention the said doped 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
In another aspect, 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.
In another aspect, 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 offhe nanoparticle
In yet another aspect, the present invention provides a method of delivery of "drug using an external trigger. This is preferably done as follows: After assessing the concentration of the drug at the diseased site by any of the molecular imaging method, the magnetically active nanoparticle having a shell of chemodrugs embedded in a thermosensitive polymer will be subjected to inductive heating under an externally applied magnetic field such that drug will be released from the polymeric shell in a controlled fashion. Thus, the inventive nanoparticle provide a unique opportunity for image guided drug delivery, drug dose estimation and controlled, externally stimulated drug release or therapy.
In another aspect, 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 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 imaging modalities like MRI, CT or 2D X-ray, which gives micro/ macroscopic anatomical information. In addition, the inventive step provide a nanomedicine formulation, containing the said nanoparticle as a main component together with therapeutic molecules that can treat disease in a targeted (specific) manner by making use of one of the functional properties of nanoparticle such as magnetism (magnetic drug -df]-iv&r-y-)-Qr— . by— molecular_recep,tQr_ligand targeting. In addition, optionally, the drugs incorporated within the nanomedicine constructs can be released, in a controlled fashion, by making use of the magnetically inductive heating property of the said nanoparticle.
Method of preparation of Hydroxyapatite nanoparticles
One of the major aspect of synthesis of doped hydroxyapatite, in the present invention, is the incorporation of multiple impurities into the calcium apatite crystal lattice by a single step process, while maintaining the particle size < 50nm without the use of any additional surfactants. Different sequences of reaction are possible in this method to achieve successful doping. These sequences are presented in different examples as follows:
Example 1. Production of fluorescent magnetic and X-ray absorbing Hydroxyapatite nanoparticles with two dopant ions: Scheme -1
A procedure of preparing hydroxyapatite nanoparticles (nHAp) with total 10% dopant ions including 6% Eu3+ (Europium) and 4% Gd3+ (Gadolinium) with respect to total Ca2+ (Calcium) content is presented. However, this method can be followed for more than two dopant ions and to any higher or lower concentrations preferably between 0-20%._ Aqueous solution containing Calcium chloride (CaCl2), lOOmL, 0.5M, 98%, Sigma, USA (Part A) is mixed with 6ml of 0.5M, Europium chloride, EuCl3 and 4 mL of 0.5M Gadolinium nitrate, Gd(NO3)3 and stirred well for 30min and then the temperature is raised to ~90°C. Another two solutions containing phosphate group (Part B), ortho- phosphoric acid, H3(PO)4, (llOmL, 0.3M, 98%, Qualigens, India) and hydroxyl group, 100ml Ammonium hydroxide, ,NH4OH, Part C (0.1M, 25% NH3, Qualigens, India) are prepared separately and taken in two different burettes. Both these solutions are added drop wise to the warm precursor mixture of Part A+D with continuous stirring over a period of 1 hour. The rate of addition of NH4OH solution is adjusted to maintain the pH of the reaction medium at ~ 7.4 throughout the reaction. After completion of precipitation, the mixture is heated at 100° C for another 2 hours while stirring. The mixture is then left overnight followed by separation of the precipitate by centrifugation at 3000 rpm for lOminutes and washing with hot water for 6-8 times. It is then dried in hot air oven at 60° C for 24 hours and powdered using mortar and pestle.
Tc
Example 2. Production of fluorescent, magnetic and X-ray absorbing Hydroxyapatite nanoparticles with two dopant ions, Scheme -2
In this alternative scheme, dopant ions, Part D, is added separately while Part A and Part B are reacting in presence of Part C. Aqueous solution of a different calcium precursor Ca(OH)2 lOOmL, 0.5M, 98%, Sigma, USA (Part A) is heated to temperature ~90°C. Other three solutions containing phosphate group ortho-phosphoric acid, H3(PO)4, (Part B), (llOmL, 0.3M, 98%, Qualigens, India), a mixture of 6ml, 0.5M EuCl3 and 4ml, 0.5M Gd(NO3)3, both 99.9%, Sigma, USA (Part D) are prepared separately and both the solutions are taken in two different burettes and added separately drop wise to the warm precursor solution of Calcium hydroxide with continuous stirring over a period of 1 hour. In this scheme, ammonium hydroxide solution is added intermittently to maintain the pH of the reaction medium at ~ 7.4 throughout the reaction. After completion of precipitation, the mixture was heated at 100° C for another 2 hours while stirring. The mixture is then left overnight followed by separation of the precipitate by centrifugation at 3000 rpm for lOminutes and washing with hot water for 6-8 times. It is then dried in hot air oven at 60° C for 24 hours and powdered using mortar and pestle.
Example 3. Production of fluorescent magnetic and X-ray absorbing Hydroxyapatite nanoparticles doped with more than two cationic and anionic dopant ions using, Scheme -
3
In this example, total 04 dopant ions, three cationic and one anionic, are incorporated into Hydroxyapatite nanoparticles. First, Ca(OH)2 lOOmL, 0.5M, 98%, Sigma, USA (Part A) was heated to temperature ~ 90°C.
A representative anionic dopant ions, 4 ml, 0.5M iodine (Part D)(4% wrt calcium), which can enhance X-ray contrast, is mixed with Part B, ortho-phosphoric acid, H3(PO)4, (llOmL, 0.3M, 98%, Qualigens, India) and taken in a burette, where as three other cationic dopant ions, 6ml of 0.5M EuCh, 4ml of 0.5M Gd(N03)3 and 4ml, 0.5M BaC , all 99.9%, Sigma, USA were taken in another burette. Both orthophosphoric acid and cationic dopant ions are then added separately, drop wise to hot CaCl2 solution while maintaining the pH of reaction at 7.4 using lOOml NH4OH (0.1M, 25% NH , Qualigens, India).
After completion of precipitation, the mixture is heated at 100° C for another 2 hours while stirring. The mixture is then left overnight, followed by separation of the precipitate by centrifugation at 3000 rpm for lOminutes and washing with hot water for 6-8 times. It is then dried in hot air oven at 60° C for 24 hours and powdered using mortar and pestle. In all the above schemes, more cationic or anionic dopant ions or alternative source of hydroxyl ions can also be used- Example 4: Preparation of nHAp-siRNA nanomedicines capable of image guided delivery.
In this example, preparation of a representative nanomedicine formulation formed by loading the said multifunctional hydroxyapatite nanoparticles (MF-nHAp) with small length nucleic acid drugs, for example therapeutic small interfering RNA (siRNA) is presented. The sequence of siRNA selected for this example is the one targeted against 315T mutations in the kinase domain (KD) of BCR-ABL that is prevalent mechanism of acquired drug resistance in patients with chronic myeloid leukaemia (CML).
Sequence details are:
Sense strand: 51 -GCCGCUCGUUGGAACUCCAdTdT— 31
Antisense strand: 31 -dTdTCGGCGAGCAACCUUGAGGU—51
Length: 21 base pair
For the loading reaction, lmg/ml bare nanoparticles prepared as said in any of the above examples 1-3 , is prepared in phosphate buffer saline (PBS, Sigma, USA)) arid sonicated for 10 minutes to get a fine dispersion. To this, ~ Ο.ΙμΜ siRNA
(Sense strand: 51 -GCCGCUCGUUGGAACUCCAdTdT-- 31 Antisense strand: 31 -dTdTCGGCGAGCAACCUUGAGGU—51 )
solution prepared in PBS is mixed and reacted for 3 Hrs at 37°C in a water bath, while stirring ultrasonically. The sequence of the siRNA is only representative and any other sequence of therapeutic importance shall be used. After 03 hrs, the nanoparticle-siRNA conjugates were removed from the medium by centrifugation and unbound siRNA from the supernatant is discarded. To protect the siRNA bound to the surface of nanoparticles from enzymatic degradation during the transfection, the nHAp-siRNA conjugates were further treated with O.Olmg/ml BSA (Bovine Serum Albumin) and O.lmg/ml EDC (1- ethyl-3-(3-dimethylaminopropyl) carbodiimide) for 30min 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.
Example 5: Preparation of nHAp-DNA nanomedicines capable of image guided delivery
In an alternative method, this example provides preparation of a representative nanomedicine formed by loading the said multifunctional hydroxyapatite nanoparticles with a representative gene. Here we show encapsulating a gene, preferably a marker gene that can be readily detected by simple laboratory tools, into the nanoparticle during the synthesis of nanoparticles.
An appropriate marker gene selected for this example is beta.-galactosidase (β-gal) since its expression can be readily detected by addition of X-gal, a substrate which yields a blue colour when the active enzyme is present. However, this example is not limited to the said marker gene, but any gene intended for a desired function such as inhibition of tumour growth. For the encapsulation of linear and super coiled β-gal coding DNA into HAp, lmg/ml DNA is dissolved in PBS and mixed with lOOmL CaCl2, 0.5M, 98%, Sigma, USA (Part A) and stirred for ~ 30min at 37°C. To this, 4ml each of 0.5M dopant ion precursors (Part D) EuCl3 and Gd(N03)3 are added and stirred well for 30min and then raised the temperature to ~50°C. Another two solutions containing phosphate group (Part B), ortho-phosphoric acid, H3(PO)4, (llOmL, 0.3M, 98%, Qualigens, India) and 100ml NH4OH (0.1 M, 25% NH3, Qualigens, India) are prepared separately and taken in two different burettes. Both these solutions are added drop wise to the warm precursor mixture of Part A+D with continuous stirring over a period of 1 hour. 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 2 hours and kept overnight followed by separation of the precipitate by centrifugation at 3000 rpm for lOminutes and washing with ice cold water for 6-8 times. The DNA embedded within the doped calcium phosphate matrix is protected from enzymatic degradation using a protective coat of BSA, by treating the nHAp-DNA conjugates with O.Olmg of BSA and 0.1 mg of EDC in 5ml PBS for 30min at 37°C, followed by washing with water.
Example 6: Preparation of nHAp-polymer nanomedicine containing chemo drugs capable of image guided delivery
In this example, preparation of a representative nanomedicine formed by the said multifunctional hydroxyapatite nanoparticles embedded within polymeric nanoparticle containing chemical drugs, for example a small molecule inhibitor, Rapamycin, is presented. Img/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. To this solution, ~ ΙμΜ Rapamycine in lmg/ml PLGA (poly(lactic-co- glycolic acid)) , dissolved in DMSO is added and reacted for ~ 2 Hrs with continuous stirring. After 02 hrs, ~ 500μί of H2O is added to the HAp-Rapamycine-PLGA mixture to precipitate the polymeric nanoparticles that contain HAp and Rapamycin embedded in it. This nanomedicine formulation can be imaged using multiple imaging modalities or delivered to a specific site using magnetic properties of embedded HAp nanoparticles or connecting a targeting ligand. Alternative to PLGA, other biodegradable polymers such as PEI, PLA, PCL, PVA, PPV etc also can be used for embedding the nHAp and chemical drug.
Example 7: Preparation of nHAp-polymer core-shell nanomedicine containing chemo drugs capable of delivering the drug by external trigger by inductive heating
In this example, formulation of multifunctional hydroxyapatite containing chemical drug used against cancer, example Rapamycine, incorporated in a temperature responsive polymer for drug delivery triggered by external magnetic field by inductive heating is presented. Poly(A -isopropylacrylamide) (PNIPAm) forms a three-dimensional hydrogel when cross linked with Ν,Ν'-methylene-bis-acrylamide (MB Am) or Ν,Ν'-cystamine-bis- acrylamide (CBAm).
When heated in water above 37°C, it undergoes reversible phase transition from a swollen hydrated state to a shrunken dehydrated state, losing about 90% of its mass. Since PNIPAm expels its liquid contents at a temperature near to that of the human body, PNIPAm can be used to deliver drug embedded in it by heating using an external trigger. Here we show an example of making nHAp-PNIPAm-Rapamycine nanomedicine formulation wherein the inductive heating property of magnetic nHAp under an externally applied magnetic field can heat up the PNIPAm polymer to change its phase and release the embedded drug. This process also can be monitored by molecular imaging contrast provided by the embedded nHAp.
For the preparation nHAp-PNIPAm-Rapamycine nanomedicines, 5mg bare nHAp nanoparticles prepared as said in any of the above examples 1-3, is re-dispersed in 5ml water and sonicated for 10 minutes to get a fine suspension, in another solution, ~ ΙμΜ Rapamycine dissolved in ~ 3 ml water-DMSO (1:1) solution containing 0.4 gm PNIPAm (3.4 mmol) and 0.06 gm MBAm (0.4 mmol) is formed and the same is added to the nanoparticle suspension and mixed well by sonication for 30 min. The temperature of this reaction mixture is elevated to 75°C and after ~ 30 min, O.Olgm of pottassium persulfate in 4ml water is injected in to the polymer-nHAp-Rapamycine mixture to initiate the polymerization reaction. The reaction is then allowed to continue for 2 Hrs and the precipitate is removed by centrifugation and washing with distilled water several times. The nanomedicines thus formed will have nHAp nanoparticles and Rapamycine embedded in thermosenstive PNIPAm nanoparticles of size ~ 10-20% bigger than nHAp. This nanoconjugate is capable of releasing the embedded drug Rapamycin, when subjected to magnetically inductive heating of embedded magnetic nHAp.
Example 8: Bioconjugation of nanoparticles with cancer targeting ligands
In this example, bio-conjugation of nHAp nanoparticles with a promising cancer targeting ligand folic acid (FA) is presented. First 0.001 % Polyetheleimine, PEI (MW 25 kDa, Sigma Aldrich, USA ) having a large number of amine groups was reacted with ~lmg of nHAp overnight (-12 Hrs). The sample is centrifuged and washed to remove unconjugated polymer and resuspended in PBS (pH 7.4) to obtain surface aminized nHAp. Amine reactive succinimidyl ester activated FA is prepared by reacting FA (5mL, 0.1 M, Sigma Aldrich, USA) with EDC (lmL, 1M, Sigma Aldrich, USA) at pH 5.5 in MES (99%,Sigma Aldrich,USA) buffer for 15 minutes, in dark, at room temperature. Sulfo-NHS (N-hydroxysuccinimide) (2.5mL, 1M, Sigma Aldrich, USA) dissolved in MES (2-(N- morpholine) ethanesulfonic acid) buffer (pH 5.5) is added to EDC-FA and reacted for 5 Hrs in the similar condition. FA-NHS ester thus formed is reacted with surface aminized MF-nHAp at pH 7.4 in PBS for 2 Hrs in dark condition resulting in the formation of the FA-PEI-nHAp through amide linkage that is washed several rimes and re-dispersed in fresh PBS for cell studies. The successful bioconjugation reaction leading to presence of FA on nHAp surface can be seen in FIG. 13.
Claims
1. A doped nano-biomaterial showing multi-functional properties such as X-ray absorption, optical fluorescence (visible and near-infrared), para- or superpara- magnetism, and optionally scintillation emission, making it suitable for application as a contrast agent for medical or molecular imaging technique that uses, in combination or separately, the X-ray absorption, reflection, transmission, scintillation, optical fluorescence, para- or superpara-magnetism for the imaging of biological cells, tissue, organs, biological processes, any part of the human body, for the examination of health condition Including the detection, diagnosis and treatment efficacy analysis of disease.
2. The multifunctional nanocontrast agent as in claim 1, is capable of absorbing X-ray radiation that provide contrast difference with the background, optionally, convert the absorbed X-ray into light radiation which can be detected using optical detectors, emit fluorescence emission in the visible and near-infrared region, show paramagnetism or
-sijp&r-paramagne.tism,_all the above at the same time, making it possible to apply multiple medical imaging techniques such as X-ray computed tomography, magnetic resonance imaging, fluorescence imaging, near infrared optical tomography, scintillation tomography imaging in combination or independently for examining the human health condition.
3. The multifunctional nanocontrast agent as in claiml, is nanosized hydroxyapatite ((Ca+X)io(PC>4)6 (OH+Y)2 where X represents the doped rare earth elements/ transition metals and Y represents the halogen ions.
4. The dopant ions according to claim 3, is a combination of impurities includes Gd3+-Eu3+, Eu3+-Gd3+-Tb +, Gd3+-Yb3+-Er3+, Mn2+-Yb3+-Er3+, Mn +-Eu3+, Mn2+-Eu3+-Halogen, Mn2+- Tb3+, Mn2+-Tb3+-Halogen, Gd3+-Eu3+-Halogen, Gd3+-Eu3+-Mn2+-Halogen, Gd3+-Tb3+- Halogen, Gd3+-Tb3+-Mn2+-Halogen, Gd3+-Tb +-F", Fe2+-Eu3+-Cl", Fe3+- Eu3+-Cl", Fe2+-Tb3+-Cl-, Fe3+- Tb3+-Cl- . The above ions may also form their phosphate clusters located at the interstitial lattice
5. The multifunctional nanocontrast agent as in claim 1, the dopant ions are substituting calcium or hydroxy 1 ion site depending on the charge neutrality /compensation or interstitially placed at the apatite lattice as individual atoms or cluster of atoms.
6. The multifunctional nanocontrast agent as in claim 4, each dopant ion concentration varies from 0-20 atomic percentage (at %) compared to the concentration of calcium, phosphate or hydroxyl ions in the lattice.
7. The multifunctional nanocontrast agent as in claim 6 have spherical, elongated or diamond like shape.
8. The multifunctional nanocontrast agent as in claim 6, the spherical shape particles has size range of 1-50 or 50-1 OOnm which can be used for delivery through blood circulation and imaging of all organs in human body where the blood circulation can reaches and
-d-iseases-sueh-as-tu-mo r—
9. The multifunctional nanocontrast agent as in claim 6, the diamond shaped or elomgated particles has length of 10-300nm and width l-250nm and can be used for oral delivery and imaging of gastrointestinal (GI) track.
10. The multifunctional nanocontrast agent as in claim 1, the level of X-ray absorption by the material can be increased or decreased in the range of 0-90 % of the total X-ray irradiated using -the concentration of dopant ions and processing conditions.
11. The multifunctional nanocontrast agent as claim in 1, the range of fluorescence emission varies from UV-visible to near-infrared which is tunable by the selection of dopant ions, concentration and processing. The material preferably emits near infrared light which has higher tissue penetration suitable for better contrast during optical imaging in the background of auto fluorescence from the tissue.
12. The multifunctional nanocontrast agent as in claim 1, the pararamagnetic or superparamagentic properties depend on the choice of dopant ions. Paramagnetic ion doped contrast agents are suitable for Tl weighted MR imaging (bright images) and superparamagnetic ion doped contrast agents are suitable for T2 weighted MR imaging (dark images).
13. The multifunctional nanocontrast agent as in claim 1 is non-toxic to human cells, tissue or organs.
14. The multifunctional nanocontrast agent as in claim 1 can be preferably delivered specifically (active targeting) or non-specifically (passive targeting) to disease sites such as cancer, inflammatory disease, cardiovascular disease, brain damage or intra-cellular or intra-nuclear regions using surface conjugated molecular targeting ligands such as peptides, antibodies, aptamer, folic acid, proteins, polysaccharide chains.
_15._The multifunctional nanocontrast agent as in claim 1, can be conjugated or loaded with drug molecules against diseases such as cancer, cardiovascular disease, brain damage, inflammatory disease, organ malfunction or failure.
16. The multifunctional nanocontrast agent as in claim 15, where the therapeutic drugs that can be conjugated or loaded (embedded) with the contrast agent include but limited to chemodrugs, anticancer gene therapy agents, RNA fragments (siRNA, mRNA), photosensitive drugs, small molecule inhibitors, antibiotics.
17. The multifunctional nanocontrast agent as in claim 15, can be used for medical/ molecular imaging based quantification of in vivo pharmacokinetics, pharmacodynamics, accumulation of drug at the diseased site, drug concentration, bio- distribution within the organ or tissue, off-target effects, pre- and post- treatment analysis etc using MRI, X-Ray tomography and NIR fluorescence, in combination or independently.
18. The multifunctional nanocontrast agent as in claim 15, wherein, the property of magnetism can be utilised to guide the embedded drug to a particular location using externally implanted tiny magnets at the location or applied magnetic field (Magnetic drug delivery).
19. The multifunctional nanocontrast agent as in claim 15, the X-ray absorption or scintillation can be used to trigger the drug release.
20. The multifunctional nano-contrast agent as in claim 1 act as contrast agent at least for the following combined molecular imaging modes: a) 2D X-ray imaging with MRI, b) 2D X-ray imaging with visible and near infrared fluorescence imaging c) 3D X-ray computed tomography with MRI, d) 3D X-ray computed tomography with visible or near-infrared fluorescence imaging e) MRI with visible or near-infrared fluorescence imaging f) 2D X- ray imaging with MRI and visible or near-infrared fluorescence imaging, g) 3D X-ray computed tomography with MRI and visible or near-infrared fluorescence imaging
21. A method of making the multifunctional nanocontrast agent based on doped hydroxyapatite as in claim 1, consist the steps of (a) preparing an aqueous or Simulated Body Fluid (SBF) based solution containing calcium (Ca2+) ions as Part-A (b) preparing an aqueous or SBF based solution containing phosphate PO43" ions as Part-B c) preparing an aqueous or SBF based solution containing hydroxyl, OH", ions as Part-C d) preparing an aqueous or SBF based solution containing dopant ions separately or in combination as Part-D e) reacting Part-A, Part-B, and Part-C and Part-D in suitable sequence at a pH range and temperature range for a stipulated period of time in open air on a hot plate, e) Continuously stirring the reaction medium to precipitate inventive particles while maintaining the pH at a particular range by adding Part B intermittently f) aging the precipitated particles for a stipulated period at a stipulated temperature range g) separating the precipitated particles from the growth medium by centrifugation h) washing the precipitated particles with hot water followed by cold water and i) redispersing the precipitate in water or phosphate buffer saline for further application.
22. A method of making multifunctional nanocontrast as in claim 21, the reaction of Part A to D are done in following possible sequences, a) Part B and Part D are added drop wise separately or combined to Part A while Part C is mixed with Part A or added at predetermined intervals b) Part A is reacted with Part D first, Part B is added drop wise separately, Part C is added at pre-determined intervals c) Part B, Part C and Part D added to Part A separately or combined d) Part A is added to a mixture of Part B ,Part C and Part D e) Part A and Part C are added separately or combined to mixture of Part B and Part D f) Part A, Part C and Part D are added to Part B g) Part B is added to mixture of Part A , Part C and Part D.
23. A method of multifunctional nanocontrast as in claim 21, the part A is formed from water soluble, miscible or dispersible hydroxide, chloride, bromide, iodide, fluoride, nitrate, sulphate, carbonate or oxide salts of calcium.
24. A method multifunctional nanocontrast as in claim 21, the part B is formed from water soluble or miscible salt of phosphate including sodium (Na3P04, Na2HP04, NaH2P04), potassium (K3PO4, Κ2ΗΡ04, KH2PO4) lithium (L13PO4, Li2HP04, ΠΗ2ΡΟ4), ammonium ( (NH4)3P04, (NH4)2HP04, NH4H2P04) and phosphoric acid (H3PO4).
25. A method of making multifunctional nanocontrast as in claim 21, Part C is formed by sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonium hydroxides, calcium hydroxide or all possible water soluble hydroxides.
26. A method of making multifunctional nanocontrast as in claim 21, the Part D is formed by water soluble or miscible chloride, bromide, iodide, fluoride, nitrate, sulphate, carbonate or oxide salts of gadolinium, europium, terbium, ytterbium, erbium, manganese and iron (Fe2+ and Fe3+ salts) and halogen ions.
27. A method of making multifunctional nanocontrast as in claim 21, all reactions are carried out within a pH range of 5-12
28. A method of making multifunctional nanocontrast as in claim 21, SBF based reaction is carried out at temperature range of 25-40°C and aqueous reaction at a temperature range of 70-120°C.
29. A method of making multifunctional nanocontrast as in claim 21, the aging of the precipitated nanoparticles is carried out for a period of 2-24 Hrs at temperature range of 70-120°C.
30. A method of making multifunctional nanocontrast as in claim 21, the precipitated particles are washed with water 01 to 06 times and separated the particles from the medium after each washing step by centrifugation at a speed varying from 100 to 12000 rpm.
31. A method of making multifunctional nanocontrast as in claim 21, the precipitated particles are redispersed in water or phosphate buffer saline for its further use
32. A method of making multifunctional nanocontrast as in claim 21, the water or PBS dispersed particles are preferentially conjugated at its surface with cancer targeting ligands including folic acid, antibodies, peptides, aptamers or polysaccharides.
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