KR20140096821A - Gd-PEI Nanogel and Contrast Agent or Pharmaceutical Composition Comprising the Same - Google Patents

Abstract

The present invention relates to a Gd-PEI nanogel and a contrast agent or a pharmaceutical composition comprising the same. The Gd-PEI nanogel has an amorphous shape and a flexible nature, and thus passes through an RES system. The Gd-PEI nanogel expresses minimum cytotoxin and enables excellent targeting of in-vivo tumor, thereby ensuring high biomedical potential power as the contrast agent and a drug carrier.

Description

Gd-PEI Nanogel, and a contrast agent or pharmaceutical composition containing the same.

The present invention relates to Gd-PEI nanogels and to contrast agents or pharmaceutical compositions comprising them.

Hybridization of organic and inorganic components has been a subject of much research since it allows complementary and synergistic integration at the interface. ¹ as a typical metal hybrid material is formed by a balance between a metal ion and organic ligands are the organic chelating agent. Metal-organic frameworks (MOFs) are one of the most exciting discoveries with a wide range of potential applications, due to their high porosity and variety of structures. ^In biomedical imaging and pharmaceutical applications, nanoscale metal-organic frameworks (nano MOFs) have received much attention in recent years as promising nanoproducts. The primary biomedical application of ^4-8 nano-MOFs has shown high potential as a drug delivery vehicle, but their use in in vivo, particularly with respect to tumor targeting has not been studied.

Nanomaterials circulating in the blood have the potential to passively target tumors, due to increased permeability and retention (EPR) effects, which accumulate in tumor tissues with vascular and lymphatic drainage defects Depending on the propensity to be retained. ^9-11 However, systemically administered rigid / crystalline hybrid cores (e.g., nano MOFs and / or other hybrid nanomaterials) are excreted by the network endothelial system (RES), despite the anti-fouling surface protective layer , Which shortens their circulation time and thus limits tumor accumulation. ^{8, 12, 13 A} commonly known method that can minimize RES filtering is to reduce the size of the robust core to below 10 nm. ^14-16 However, in some cases, the size reduction represents a side effect on the size-dependent activation function of the core (eg, drag loading capacity, EPR effect, or transverse magnetization relaxation), thereby limiting the use of nanomaterials . ^{4-6, 9-11, 17, 18} A ductile material with structural deformation capability may pass through the RES perforations without necessarily accompanied by a reduction in size, as observed in the liposome and polymer nanoparticles It is known to be able to. ^{19 Although} some MOFs are crystalline, they have been reported to be somewhat flexible, responding to external stimuli. Despite ^7,20-25 However, such flexibility and nano-MOFs of crystalline if the in vivo administration were observed to be rapidly excluded by the RES organ. ^17,18

On the other hand, gadolinium belongs to the rare earth element of element number 64 and there are seven isotopes in the natural state. Gadolinium has strong magnetic properties and is superior to other elements in its ability to absorb neutrons and is used as an MRI contrast agent. In addition, the use of neutron absorptive capacity as a therapeutic agent for neutron cancer has also been attempted. However, as with most contrast media, gadolinium contrast agents also have disadvantages such as vomiting, diarrhea and dizziness after administration, and they cause toxicity in the body. In order to eliminate the side effects of gadolinium and to improve the vascular retention time of gadolinium with a short vascular retention time, research is needed.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent No. 10-1080499

The present inventors have made efforts to develop a biocompatible gadolinium contrast agent or a drug delivery system capable of tightly chelating gadolinium and effectively passing the RES system and achieving systemic tumor targeting without toxicity or side effects and as a result gadolinium has been impregnated into the polyethyleneimine network The present invention has been accomplished by preparing an amorphous and flexible gadolinium-PEI nano-gel including a core structure of the present invention.

It is therefore an object of the present invention to provide a gadolinium-PEI hybrid nanogel.

It is another object of the present invention to provide a contrast agent composition comprising a Gd-PEI nanogel.

It is another object of the present invention to provide a pharmaceutical composition for treating cancer or tumor comprising Gd-PEI nanogel.

It is still another object of the present invention to provide a method for producing a gadolinium-PEI hybridized nanogel.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

One aspect of the present invention is to provide a Gd-PEI nanogel comprising spherical cores impregnated with gadolinium (Gd) in the network structure of PEI (polyethyleneimine).

In order to compensate for the rapid dissipation of a crystalline nanohybrid having a rigid structure such as the previously known iron oxide nanoparticles (SPION) by the RES system, the present inventors have found that a novel " soft "substitute having an amorphous and flexible hybrid core I invented a concept.

The Gd-PEI nanogel of the present invention is characterized by being flexible and amorphous. This amorphous, flexible network network architecture has the advantage of being able to pass through the RES without reducing its size due to the advantage of elastic straining forces, allowing increased blood circulation time and in vivo tumor targeting.

The Gd-PEI nanogels of the present invention also provide another advantage in that the network structure of PEI tightly kills gadolinium to minimize toxic effects of free gadolinium ions.

In one embodiment, the Gd-PEI nanogel of the present invention is characterized in that it does not include diethylene triamine pentaacetic acid (DTPA) and iron oxide. The Gd-PEI nanogel of the present invention is based on a flexible PEI network network structure, and gadolinium ions are tightly chelated therein, and other polymers (for example, DTPA) conventionally used in gadolinium contrast agents Structure, and has a completely different structure from the structure in which PEI is conjugated or additionally introduced. Of course, the Gd-PEI nanogels of the present invention can also be conjugated or further introduced with other functional materials to PEI, which is a polymer forming the basic structure. However, since the basic structure is a flexible amorphous PEI network structure .

In one embodiment, the total diameter of the Gd-PEI nanogel is 100 to 500 nm, preferably 100 to 200 nm.

In another embodiment, the core of the Gd-PEI nanogel has a diameter of 10 to 100 nm, preferably 40 to 80 nm.

In another embodiment, the Gd-PEI nanogels of the present invention may incorporate additional chemical or biofunctional materials using functional groups present in PEI, such as polyethylene glycols (PEGs) or The fluorescent indicator or the phosphor can be bonded.

Suitable phosphors include, for example, fluorescein, phycoerythrin, rhodamine, lissamine, Cy3, Cy5 (Pharmacia), chromophore, chemiluminescent moiety, magnetic particles, radioactive isotopes S35), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), but are not limited thereto.

In one preferred embodiment, the PEI used in the Gd-PEI nanogel of the present invention is characterized by being a branched PEI.

Another aspect of the present invention is to provide a contrast agent composition comprising the Gd-PEI nanogel described above and a pharmaceutically acceptable carrier.

Such pharmaceutically acceptable carriers are those conventionally used in the field of manufacture and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The contrast agent composition of the present invention is preferably parenterally administered. In the case of parenteral administration, intravenous infusion, intramuscular injection, intra-articular injection, intra-synovial injection, intrathecal injection, intrahepatic injection, intralesional injection, Intracranial injection or the like. A suitable dose of the contrast agent composition of the present invention may be variously prescribed by such factors as the formulation method, the administration method, the age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient .

In one embodiment, the contrast agent composition of the present invention is characterized by being for Magnetic Resonance Imaging (MRI).

In another embodiment, the contrast agent composition of the present invention is characterized by being for fluorescent imaging.

In a preferred embodiment, the contrast agent composition of the present invention is characterized by being a dual-modality contrast agent composition capable of simultaneously performing MRI imaging and fluorescent imaging.

The method of obtaining the fluorescent image and the MRI image using the contrast agent of the present invention can be carried out according to the conventional method. For example, these methods are described in U.S. Patent Nos. 6,151,377, 6,072,177, 5,900,636, 5,608,221, 5,532,489, 5,272,343 and 5,103,098, 6,151,377, 6,144,202, 6,128,522, 6,127,825, 6,121,775, 6,119,032, 6,115,446, 6,111,410 and 602,891, the disclosures of which are incorporated herein by reference.

Since the contrast agent composition of the present invention can be used simultaneously as a contrast agent for fluorescence imaging and MRI imaging, MRI and fluorescence images can be obtained from the contrast agent reaching the tissue to be obtained. This feature makes it possible to take full advantage of both MRI and fluorescence imaging, and ultimately, images with high sensitivity and high temporal resolution of fluorescence imaging and high spatial resolution of MRI can be obtained at the same time.

The dual-system contrast agent of the present invention is very useful for imaging the internal region of the human body. The imaging process is accomplished by administering a diagnostically effective amount of contrast agent to a human and then scanning the human body using fluorescence imaging and MRI imaging to obtain a visible image of the internal region (tissue) of the body.

In particular, the dual-mode contrast agents of the present invention are useful for cancer imaging.

Another aspect of the present invention is to provide a pharmaceutical composition for treating cancer or tumor comprising the Gd-PEI nanogel described above and a pharmaceutically acceptable carrier.

The composition of the present invention may contain a drug such as a chemical substance or biomolecules exhibiting bioactivity, in order to further impart functions other than imaging (for example, cancer treatment), wherein the drug is PEI Polyethyleneimine). ≪ / RTI >

The bioactive chemical material includes various functional monomolecules, polymers, inorganic supports, and the like.

Examples of such monomers include, but are not limited to, anticancer drugs, antibiotics, vitamins, drugs including folic acid, fatty acids, steroids, hormones, purines, pyrimidines, monosaccharides and disaccharides.

Examples of the biomolecules include proteins, peptides, DNA, RNA, antigens, hapten, avidin, streptavidin, neutravidin, protein A, protein G, a cytotoxic agent such as lectin, selectin, a hormone, an interleukin, an interferon, a growth factor, a tumor necrosis factor, an endotoxin, a lymphotoxin, an urokinase, a streptokinase, a tissue plasminogen activator, a hydrolase, But are not limited to, enzymatically active enzymes such as isomerization enzymes and synthetic enzymes, enzyme enzymes and enzyme inhibitors, and the like.

Another aspect of the present invention is a method of preparing a Gd-PEI nanogel comprising the steps of: mixing a gadolinium (Gd) salt and PEI (polyethyleneimine) in an aqueous solution to form a Gd- PEI nanogel, And a method for producing the PEI nanogel.

Because PEI is a flexible water-soluble polymer with very high density of ethylenediamine units (strong metal-chelating ligands), when mixed in gadolinium ions and aqueous solutions, irregular cross-linking of the flexible polymer chain results in the formation of gadolinium ions in the interpolymeric Impregnated, causing fast and irregular gelation.

In one embodiment, the separation of the Gd-PEI nanogel can be performed by emulsion braking, centrifugation, washing, resuspension in water, but is not limited thereto.

The Gd-PEI nanogels of the present invention exhibit amorphous and flexible nature, exhibit minimal cytotoxicity, and are capable of superior in vivo tumor targeting, thus having a high biomedical potential as contrast agents and drug delivery agents.

Figure 1. (a) Schematic of network structure in GdNGs. (b) Gd-coordinated PEI gel formed in water. (c) Scheme of preparation of colloidal GdNGs by inverse microemulsion technique. (D) cryogenic TEM image of GdNGs and (e) EDX spectrum. The L and M shell ionization of Gd is indicated by the red arrow. The blue arrow indicates emission from the copper grid.
Figure 2. (a) AFM topographic image of GdNGs immobilized on submerged glass substrates. Arrows (i) and (ii) represent nanogel and epoxy silane coated glass, respectively. (b) An indentation curve obtained underwater at the position indicated by arrows (i) and (ii) in (a). The solid line represents the theoretical pit which calculates the Young's modulus value as indicated. (c) Color and NIR fluorescence (NIRF) images showing filtration of SPION and GdNGs through filters with different pore sizes as indicated. (d) Relative absorbance (Abs) and fluorescence (FL) of the filter in (c). (e) Cytotoxicity of the sample for 6 hours of the evaluation performed by the MTT analysis, cells SCC7 GdNGs, Gd-DTPA and GdCl3 (0.1 mM Gd ^{3 +)} for. The control group is a cell to which no sample is added. Error bars represent standard deviations for independent experiments (n = 10). (f) the glass Gd ^{3 +} released from GdNGs incubated in PBS (pH 7.4, containing 10% FBS) at 37 ℃. Error bars represent standard deviations for independent experiments (n = 3).
Figure 3. (a) GdNGs vivo image of the NIRF ^{(200 L, 3.8 mM Gd 3} +) tail vein injection to before and after SCC7 tumor model mouse. And represents the imaging time after injection. The white dot indicates the tumor location. (b) Biodistribution of GdNGs identified from NIRF signals from resected organs and tumors before (control) and after injection. Error bars represent the standard deviation of independent experiments ( n = 3).
Figure 4. (a) Gd ^{3 +} concentration (1: 0 mM, 2: 0.0625 mM, 3: 0.125 mM, 4: 0.25 mM, 5: 0.5 mM, 6: 1 mM, 7: 1.5 mM, 8: 3 mM ), R ₂ map image (b) R ₁ and R ₂ of the curve GdNGs according to the action. The slope provides the indicated r ₁ and r ₂ . wt MR images - (c) T ₂ in the tail vein before injection and 2 hours after SCC7 tumor including mice GdNGs (200 L, 3.8 mM Gd 3 +). Tumor tissues were marked with dotted lines.
Figure 5. Absorption and emission spectra of GdNGs dispersed in water.
Figure 6. Number of GdNGs dispersed in water Average hydrodynamic size (measured by DLS).
Figure 7. (a) Small powder x-ray diffraction (left), diffraction (insertion) and modeling (NanoFit, Bruker, Germany) results of GdNGs powder (right). (b) broad powder x-ray diffraction (left) and diffraction diagram of GdNGs powder.
Figure 8. (a) Number-averaged hydrodynamic size (measured in DLS) of SPION dispersed in water. (b) TEM images of SPION and GdNG and SPION mixtures.
Figure 9. Near infrared fluorescence (NIRF) image of SCC7 cells treated with GdNGs.
10. NIRF image of excised organs extracted from SCC7 tumor bearing mice. Mice were sacrificed before and after intravenous administration of GdNGs (control) and 1 day / 1 week (control group, n = 1, 1 il / week sample is n = 3).
Figure 11. (a) Gd-DTPA, and (b) another Gd ^{3 +} concentrations (0 mM ~ 3 mM) R 1 maps of a phantom containing a GdNGs. wt MR images - (c) T ₁ of GdNGs (3.8 mM Gd ^{3 +} of 200 L) to the tail vein injection before and 2 hours after injection SCC7 tumor include mouse.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not to be construed as limiting the scope of the present invention. It will be self-evident.

Example

<Experimental Method>

Experimental tools

The morphology of the nanogels was observed using a transmission electron microscope (TEM, CM200 electronmicroscope, Philips, Netherlands) and a cryo-transmission electron microscope (Cryo TEM, Tecnai G2 F20 Cryo, FEI, Netherlands). The surface topography and force curves of the nanogels fixed on the glass substrate were obtained using a liquid atomic microscope (NTEGRA, NT-MDT, Russia) equipped with a cantilever (NT-MDT). Absorbance and fluorescence spectra were obtained using a UV-visible spectrophotometer (8453, Agilent, USA) and a fluorescence spectrometer (F-7000, Hitachi, Japan). The concentration of Gd ^{+ 3} is ICP was measured by using the mass spectrometer (ICP-MS, Elan 6100DRC Plus , Perkin Elmer, USA). The hydrodynamic size and zeta potential were measured using Zetasizer Nano ZS (Malvern, UK). The power X-ray diffraction pattern was acquired on a GADDS (general area detector diffraction system, Bruker AXS, Germany). NIR fluorescence of SCC7 cells was imaged using a Nuance FX multispectral imaging system (Cambridge Research & Instrumentation, Inc., USA). NIR fluorescence images of 96-well plates and cut-off organ morphology of SCC7 tumor-bearing mice were prepared using a 12-bit CCD camera (Image Station 4000MM, manufactured by Dow Corning, Inc.) equipped with a 150-W quartz halogen illuminator (PL900, Dolan- Kodak, USA) and an excitation / emission filter set for Cy5.5 (Omega optical, USA). In vivo NIR fluorescence images of SCC7 tumor bearing mice were obtained using an exploratory optics system (ART, Canada) located in mice on animal plates heated at 37 ^° C. Magnetic resonance images were also obtained from a 7T MRI system (70/20 BioSpec, Bruker BioSpin GmbH, Germany).

GdNGs Manufacturing

In reverse micelles of Tween 80, minutes to prepare a branched PEI (bPEI, Mw 1.8k, Polysciences Inc., USA) and GdNGs situ by the colloid-impregnated between Gd ^{+ 3.} Specifically, for uniform dispersion of GdNGs, 20 mL of 20 wt% aq. Solution was added to 1 mL of 20 w% tween in cyclohexane under vigorous stirring conditions. GdCl ₃ .H ₂ O ₂ (Sigma-Aldrich) 20 L, 20 wt% aq. bPEI, 30 L, aq. Cy5.5-vinyl sulfone (10 mg mL- ¹ , BioActs Co. Ltd., Korea) 10 L and aq. 15 L of PEG-NHS (Mw 5k, 10 mg mL ^-1 , Sunbio Inc., Korea) was continuously added. After gently shaking at room temperature for 12 hours, the emulsion mixture was poured into ethanol. The precipitated GdNGs were purified by centrifugation (10000 rpm, 1 h), careful removal of supernatant and resuspension in ethanol under probe sonication three times. After the last centrifugation, the purified GdNGs precipitate was resuspended in Milli-Q water (1 mL) using a probe sonication and used for further experiments.

Nano Indentation  Experiment

An epoxy silane-coated glass plate Cut into 5 mm x 5 mm, and placed in a well of a 96-well plate. 100 L of a GdNGs suspension was added to the wells and kept at ambient temperature for 24 hours to fix GdNGs on the surface of the glass. Prior to AFM measurement, the sample-immobilized glass was washed three times with Milli-Q water and immersed in a liquid AFM cell filled with water. NT-MDT AFM imaging was performed at room temperature with an NSG20 cantilever (NT-MDT, force constant = 48 N / m) in semi-contact mode to obtain surface topography information. After dealing with a single GdNG from the surface topography, the force curves were measured by raising the liquid cell against the cantilever, recording the deflection distance of the cantilever in GdNG cells, and for comparison, near the epoxy silane-coated glass surface. Young's modulus was calculated according to the Hertz model by applying the following equation to the obtained force curve:

d = [(2 / π) [E / (1- v) 2] (d - z) 2 tan (α)] / k

Where d is the deflection distance, E is the Young's modulus, v is the Poisson ratio of the sample (estimated 0.5 for rubber), z is the piezo motion (height of sample in Figure 2 (b) Half of the indenting cone opening angle (11 ^o for the NSG20 cantilever), and k represents the force constant (48 N / m for the NSG20 cantilever).

GdNGs Cytotoxicity

SCC7 cells were cultured in 96-well plates and cultured for 6 hours with GdNGs, Gd-DTPA and GdCl ₃ .H ₂ O ₂ (0.1 mM Gd ^{3 + for} all samples). After washing with fresh medium, cell viability was assessed by MTT assay.

Intracellular introduction and fluorescence imaging in vitro

Murine SCC7 (squamous carcinoma cell) cells were cultured in RPMI1640 medium containing 10% fetal calf serum (FBS). The tested cells were passed on a 35 mm glass cover slip dish and left for 2 days (1 x ¹⁰⁶ cells per dish). The cells were then washed with DPBS and incubated with GdNGs suspension (200 L, 1 mM Gd ³⁺ ) in 10% FBS medium for 3 hours. Cells were then washed twice with DPBS and incubated directly with LEICA DMI 3000B equipped with a Nuance FX multispectral imaging system (Cambridge Research & Instrumentation, Inc., USA) set at 620-660 nm excitation and 100 ms exposure time Respectively.

Animal imaging experiment

All animal experiments were approved by the Korea Institute of Science and Technology animal protection and use ethics committee, and all mice were operated according to the regulations of the committee. Five - week - old BALB / c nude mice (Orient, Korea) were subcutaneously injected with 1 × 10 ⁷ SCC7 cell suspension in RPMI1640 cell culture medium to make a tumor xenograft model. After two to three weeks after inoculation, the GdNGs (200 mL, 3.8 mM Gd 3 +) was injected into mice via the tail vein. For fluorescence imaging, in vitro visualization was performed using a 7 T MRI system 70/20 BioSpec (Bruker BioSpin GmbH, Germany) for MR imaging at 37 ^° C using the Optics explorer system (Advanced Research Technologies Inc., Canada) Imaging experiments were performed. In order to evaluate the tissue distribution of GdNGs, major organs and tumors were excised from the mice at predetermined time points. Fluorescent images of excised organs and tumors were obtained using a 12-CCD camera (Image Station 4000MM, Kodak, USA). Fluorescence intensity was quantified from tissue images by the region of interest (ROI) function of the analytical workstation software (Advanced Research Technologies Inc., Canada).

<Experimental Results>

GdNGs  Structure verification

Considering that the crystalline nano-MOFs are rapidly excluded by the RES organs when administered in vivo, the present inventors have found that in order to obtain a high degree of deformability, an amorphous / flexible metal-organic hybrid platform, Structure of the nanocomposite structure. In order to take advantage of the flexibility of organic components, branched poly (ethyleneimine) (PEI, MW = 1.8 kD) was adopted as a multidentate polymeric ligand, which is chelated in metal ions and aqueous solutions , Because of the irregular cross-linking of the flexible polymer chain, it was expected to cause rapid and irregular gelation. The resulting amorphous network structure will form a "soft" nanogel similar to a rubber that can pass through the RES because of its elastic deformability, if it is confined within the nanoscopic space.

The inventors first confirmed that the complex between Gd ^{3 +} and PEI actually took place by the bulk complex in solution. As expected, the mixing of GdCl ₃ and PEI aqueous solution caused immediate formation of a frozen phase gel at room temperature (Fig. 1b), which is very efficient in cross-linking the Gd-impregnated PEI, To form a network structure that is robust enough to hold. To prepare nanoscale GdNGs, gelation was performed in a water-in-oil microemulsion (Tween 80 / cyclohexane / water) system (FIG. The nanogels formed in the microemulsion were further functionalized by coupling polyethylene glycol (PEGs) and NIR staining labels (Cy5.5) to the extra free PEI amine groups for surface coating and dual-mode imaging. The anti-fouling coating by pegylation was to prevent opsonization of plasma proteins that could occur on the cation-charged surface of PEI nanogels. Finally, GdNGs were separated by emulsion blanching in ethanol, centrifugation, washing and resuspension in water to produce an aqueous suspension with NIR absorption and fluorescence (FIG. 5).

By ICP-MS (inductively coupled plasma- mass spectrometry), was Gd ^{+ 3} concentration is measured with a 3.84 mM, which indicates that the Gd ^{+ 3} ion complex with PEI as a 37% yield of the emulsion. Cryo-transmission electron microscopy and DLS (dynamic light scattering) studies have shown that the nanogels thus obtained have a spherical hybrid core impregnated with Gd having an average size of 65 4 nm (Fig. 1d), and the hydrodynamic size of the whole 159 ± 62 nm (FIG. 6).

The energy dispersive X-ray (EDX) spectrum of GdNGs obtained under TEM observation showed L and M shell peaks characteristic of Gd, where no Cl was detected, indicating complete removal of GdCl ₃ ). In the powder X-ray diffraction (PXRD) pattern, only one large diffraction peak was observed in the small angular region (2? = 2.6 占 corresponding to a diameter of 56 nm that fits into a spherical nano- (Fig. 7A). Particularly, it should be noted that no crystalline structure was observed over a wide range of angles, confirming that the Gd-impregnated core was a randomly cross-linked amorphous gel as expected (FIG. 7b).

GdNGs Form of Strain

The elastic deformation force of GdNGs was evaluated in water by atomic force microscope using NSG-20 cantilever (48 N / m). For AFM indentation measurements, GdNGs were pre-immobilized on epoxy-coated glass surfaces through surface amine bonds and placed in water. 2a-b show the surface topography scanned in the semi-contact mode and the nanoindentation results analyzed according to the Hertz model ^26. FIG. From the curve pits in the initial force indentation range, the apparent Young's modulus of GdNGs was measured at 3.0 MPa, which is much less than that of the epoxy silane-coated substrate (9.2 MPa) without GdNGs. This value is in the modular range of the inflated gelatin film ²⁶ , which confirms that GdNGs are mechanically soft and flexible. Indeed, GdNGs with an average size of 158 nm easily passed through the membrane filter regardless of the pore size of the membrane as small as 0.2 m (Fig. 2c-d). In the steep contrast, the hard / non-deforming iron oxide nanoparticles (SPION), despite their much smaller core and overall size (9 and 54 nm, respectively, 8) Lt; / RTI &gt; This apparent flexibility, which is dependent on flexibility, strongly supports that the flexible GdNGs have a morphotropic force that allows them to pass through a filtration system such as RES ²⁷ .

GdNGs Cytotoxicity of

In addition to the RES blockade, the potential toxicity resulting from metal ions is another issue to consider in nanohybrid applications for biomedical applications. Gd ^{3 +} ions because the disturbance within the relevant Ca ^{2 +} cell signal has a high toxicity, and therefore are known to require strong chelation of the ligand such as DTPA (diethylenetriamine pentaacetate) in order to avoid the free Gd ³⁺ emission. ^28. In this regard, PEI is because "ethylenediamine" rich multi-projection property (polydentate) have a property, the use of PEI as a ligand is advantageous in tight chelation. In addition, cationic branched PEI is known to provide not only biocompatibility but also cell permeability. ²⁹ Indeed, when tested under NIR fluorescence microscopy, PEI-based GdNGs with a positive zeta potential (38 mV) were observed in the cytoplasm of a strong signal from the Cy5.5 labeling, and into SCC7 (squamous cell carcinoma) cells (Fig. 9). In order to evaluate that the benefit of PEI chelation, the inventors have found that by performing the MTT assay has the SCC7 cells treated for 6 hours with GdNGs, Gd-DTPA and GdCl ₃ (both 0.1 mM Gd ^{3 +} included), the cell viability (Fig. 2E). Under the given conditions, where the toxicity of liberated Gd ^{3 +} (GdCl ₃ ) is severe, the effect on ligand-chelated Gd-DTPA (a commercially available MRI contrast agent), although reduced in cell survival, still has a notable effect. However, GdNGs with PEI chelation showed much improved results in cell viability without noticeable toxicity, suggesting that the toxic Gd ^{3 +} ions are strongly caught in the PEI network. In order to prove this, the inventors have observed a 10% fetal bovine serum (FBS) 37 ^o C PBS of the temporarily released from the culture from GdNGs (phosphate-buffered saline, pH 7.4 ) Gd 3 + , which includes a. Seated go Nano gel to separate the sample centrifuged each time point, the supernatant fraction of the free ion of the entire Gd ^{3 +} content was calculated according to analysis by ICP-MS. Figure 2f shows the Gd ^{3 +} profiles obtained in the supernatant, where the presence of initial Gd ^{3 +} (ca. 1.2%) is due to incomplete centrifugation or the initial rapid release of weakly bound ions. It should be noted here that no additional increase in the supernatant fraction was observed during the experimental period (2 weeks), because PEI-induced polyprotein chelation minimizes the release of toxic Gd ^{3 +} ions under physiological conditions It is confirmed that it is robust enough to do.

GdNGs In vivo blood circulation, tumor Targeting  And excretion through the kidneys

With the advantages of the elastic modulus and minimal cytotoxicity described above, the present inventors have tested the possible utility of GdNGs as a RES-pass nano platform for in vivo tumor targeting and dual-mode imaging. For this purpose, GdNGs were administered systemically into SCC7 tumor-bearing mice via tail vein infusion and their pharmacokinetic behavior was observed by NIR fluorescence imaging. For our interest, GdNGs exhibited excellent blood circulation and tumor accumulation, as shown in Figure 3a, despite its large core and hydrodynamic size (65 and 158 nm, respectively). Immediately after injection, a remarkable image contrast in the whole body signal was found to strongly increase in the tumor. Tumor signals increased further up to 12 hours after injection, indicating tumor targeting by EPR effects. FIG. 3B shows the distribution of mean fluorescence intensity obtained from the x-vivo images (FIG. 10) of excised tumors and organs at various time points. It should be noted that the initial signal was much higher in the tumor than in the RES organs, such as the liver and spleen, which are expected to rapidly capture robust nanomaterials. In addition, while other organs progressively darkened, the renal signal remained elevated throughout the entire time, indicating renal excretion of GdNGs. These results clearly demonstrate that GdNGs are capable of being modified in the blood, and thus capable of passing through the RES filter despite its large size. Further experiments with GdNGs (prolonged blood circulation, tumor targeting by EPR effect and body cleansing through elongation) demonstrate the design strategy of the present invention for the structural strain of metal-organic nanohybrid.

GdNGs As a contrast medium

From the Gd ^{3 +} -rich structure of GdNGs, the inventors have contemplated and tested their use as contrast agents for magnetic resonance imaging (MRI). MRI is an excellent non-invasive tool for medical image diagnosis that measures the density and relaxivity of water hydrogen atoms between normal and diseased tissues. To speed up the vertical axis of the water proton relaxation T ₁ - a paramagnetic Gd ^{+ 3} complex to facilitate weight MRI contrast is generally used. In the case of nano-gadolinium, relaxation times on the longitudinal axis ( T ₁ ) and the transverse axis ( T ₂ ) are known to depend on the number or size of Gd ³⁺ ions. ^{30,31 3 +} as a multi -Gd complex of nano-size, 37 ^o C 7 T and the vertical axis in the MR field (r ₁₎ and a horizontal axis (r ₂₎ of the magnetic GdNGs was determined by measuring the relaxation ability. _{R 1 (= 1 / T 1} ) and _{R 2 (= 1 / T 2} ) The map image is Gd ^{+ 3} concentration showed that the water proton relaxation obviously increased with the increased (Fig. 4a and Fig. 11). R ₁ and R ₂ From the amount dependence of the plot, r ₁ and r ₂ of GdNGs were estimated to be 2.1 and 82.6 mM ^-1 s ^-1 , respectively (FIG. 4b). R ₁ and r ₂ value for Gd-DTPA (chelating of mono -Gd ^{+ 3)} are under the same experimental conditions, 3.6 and 5.5 mM ^-1 s ^{- 1} was determined to be. Compared to Gd-DTPA, the reduced r ₁ of GdNGs results from large nanogel core sizes with small surface-to-volume ratios, which interfere with direct Gd ^{3 +} -water contact, a prerequisite to affect longitudinal axis relaxation. On the other hand, the transverse ion increased significantly at a high r ₂ / r ₁ ratio of ~ 40, due to the high density of Gd ^{3 +} ions in the large size core. The horizontal axis relaxation also obtained ^{_{(r 2 = 82.6 mM -1 s}} -1) is to fall within the scope of the superparamagnetic iron oxide nanoparticles, and ³² which The use of GdNGs as a T ₂ MRI contrast agent is presented.

In vivo MRI performance of GdNGs was assessed by tumor imaging in the 7 T MR field. For NIR fluorescence imaging, the same amount of GdNGs was intravenously administered to SCC7 tumor-bearing mice, corresponding to 77 molar Gd / kg, which was slightly lower than the standard dose of Gd-DTPA (100 molar Gd / kg). ³³ No noticeable contrast enhancement was observed in the spin-echo T ₁ -weight image under these experimental conditions (FIG. 11). However, in a T ₂ -weighted image with a refocusing echo time (40 ms, RARE factor: 4) using rapid acquisition, within 2 hours after injection, in certain regions of the tumor, at high spatial resolution Significant signal darkening was clearly observed while visualizing the tumor internal tissues (Figure 4c). With enhanced tumor visualization with strong NIR fluorescence (FIG. 3A), the negative contrast enhancement suggests that GdNGs have potential as dual-mode ( T ₂ MRI and optical) contrast agents that enable systemic tumor targeting.

GdNGs of As a drug delivery agent  Usage

GdNGs was systemically administered to SCC7 tumor-bearing mice and tested under NIR fluorescence microscopy. As a result, a strong signal from the Cy5.5 label was observed in the cytoplasm, confirming that it was highly efficiently introduced into SCC7 (squamous cell carcinoma) cells (Fig. 9). In addition, a comparison of cell viability by MTT assay showed that GdNGs with PEI chelation showed excellent cell survival without toxicity by free Gd ^{3 +} ion. In addition, GdNGs not only have an elastic deformation force that allows them to pass through a filtration system such as the RES (Fig. 2 cd), but also forms a network structure that is robust enough to hold an excess of water inside 1a). These results support the drug loading and delivery of GdNGs, which can efficiently transfer drugs and the like to tumor tissues by impregnating drugs or other biomolecules into the crosslinked nanogel cores.

<Reference Literature>

1. Taylor-Pashow, KML; Rocca, JD; Huxford, RC; Lin, W. Hybrid Nanomaterials for Biomedical Applications. Chem . Commun . 2010 , 46 , 5832-5849.

2. Stock, N .; Biswas, S. Synthesis of Metal-Organic Frameworks (MOFs): Routes to Various MOF Topologies, Morphologies, and Composites. Chem . Rev. 2012 , 112 , 933-969.

3. Zacher, D .; Schmid, R .; C. Woll, Fischer, RA Surface Chemistry of Metal-Organic Frameworks at the Liquid-Solid Interface. Angew . Chem . Int . Ed. 2011 , 50 , 176-199.

4. Rocca, JD; Liu, D .; Lin, W. Nanoscale Metal-Organic Frameworks for Biomedical Imaging and Drug Delivery. Acc . Chem . Res . 2011 , 44 , 957-968.

5. Keskin, S .; Kizilel, S. Biomedical Applications of Metal Organic Frameworks. Ind . Eng . Chem . Res . 2011 , 50 , 1799-1812.

6. McKinlay, AC; Morris, RE; Horcajada, P .; Ferey, G .; Gref, R .; Couvreur, P .; Serre, C. BioMOFs: Metal-Organic Frameworks for Biological and Medical Applications. Angew . Chem . Int . Ed . 2010 , 49 , 6260-6266.

7. Horcajada, P .; Serre, C .; Maurin, G .; Ramsey, NA; Balas, F .; Vallet-Regi M .; Sebban, M .; Taulelle, F .; Ferey, G. Flexible Porous Metal-Organic Frameworks for a Controlled Drug Delivery. J. Am . Chem . Soc . 2008 , 130 , 6774-6780.

8. Horcajada, P .; Chalati, T .; Serre, C .; Gillet, B .; Sebrie, C.; Baati, T .; Eubank, JF; Heurtaux, D .; Clayette, P .; Kreuz, C. et al . Porous Metal-Organic-Framework Nanoscale Carriers as a Potential Platform for Drug Delivery and Imaging. Nat . Mater . 2010 , 9 , 172-178.

9. Matsumura, Y .; Maeda, H. A New Concept for Macromolecular Therapeutics in Cancer Chemotherapy: Mechanism of Tumoritropic Accumulation of Proteins and Antitumor Agent Smancs. Cancer Res . 1986 , 6 , 6397-6392.

10. Brigger, I .; Dubernet, C .; Couvreur, P. &lt; / RTI &gt; Nanoparticles in Cancer Therapy and Diagnosis. Adv . Drug Deliver . Rev. 2002 , 54 , 631-651.

11. Ferrari, M. Cancer Nanotechnology: Opportunities and Challenges. Nat. Rev. Cancer 2005, 5, 161-171.

12. Berry, CC; Curtis, ASG Functionalization of Magnetic Nanoparticles for Applications in Biomedicine. J. Phys . D: Appl . Phys . 2003 , 36 , R198-R206.

13. Neuberger, T .; Schopf, B .; Hofmann, H .; Hofmann, M .; von Rechenberg, B. Superparamagnetic Nanoparticles for Biomedical Applications: Possibilities and Limitations of a New Drug Delivery System. J. Magn . Magn . Mater. 2005 , 293 , 483-496.

14. Åtkerman, ME; Chan, WCW; Laakkonen, P .; Bhatia, SN; Ruslahti, E. Nanocrystal Targeting In Vivo . Proc . Natl . Acad . Sci . USA 2002 , 99 , 12617-12621.

15. Choi, HS; Liu, W .; Misra, P .; Tanaka, E .; Zimmer, J. P; Ipe, BI; Bawendi, MG; Frangioni, JV Renal Clearance of Quantum Dots. Nat . Biotechnol. 2007 , 25 , 1165-1170.

16. Xie, J .; Liu, G .; Eden, HS; Ai, H .; Chen, X. Surface-Engineered Magnetic Nanoparticle Platforms for Cancer Imaging and Therapy. Acc. Chem . Res . 2011 , 44 , 883-892.

17. Jun, Y .; Lee, J.-H .; Cheon, J. Chemical Design of Nanoparticle Probes for High-Performance Magnetic Resonance Imaging. Angew . Chem . Int . Ed . 2008 , 47 , 5122-5135.

18. Jun, YW; Seo, JW; Cheon, J. Nanoscaling Laws of Magnetic Nanoparticles and Their Applicabilities in Biomedical Sciences. Acc . Chem . Res. 2008 , 41 , 179-189.

19. Wang, M .; Thanou, M. Targeting Nanoparticles to Cancer. Pharmacol. Res . 2010 , 62 , 90-99.

20. Serre, C .; Mellot-Draznieks, C .; Surble S .; Audebrand, N .; Filinchuk, Y .; Ferey, G. Role of Solvent-Host Interactions That Lead to Very Large Swelling of Hybrid Frameworks. Science 2007 , 315 , 1828-1831.

21. Jones, JTA; Holden, D .; Mitra, T .; Hasell, T .; Adams, DJ; Jelfs, KE; Trewin, A .; Willock, DJ; Day, GM; Bacsa, J. et al . On-Off Porosity Switching in a Molecular Organic Solid. Angew . Chem . Int . Ed . 2011 , 50 , 749-753.

22. Coudert, F.-X .; Jeffroy, M .; Fuchs, AH; Boutin, A .; Mellot-Draznieks, C. Thermodynamics of Guest-Induced Structural Transitions in Hybrid Organic-Inorganic Frameworks. J. Am . Chem . Soc . 2008 , 130 , 14294-14302.

23. Ma, S .; Sun, D .; Wang, X.-S .; Zhou, H.-C. A Mesh-Adjustable Molecular Sieve for General Use in Gas Separation. Angew . Chem . Int . Ed . 2007 , 46 , 2458-2462.

24. Moggach, SA; Bennett, TD; Cheetham, AKA The Effect of Pressure on ZIF-8: Increasing Pore Size with Pressure and Formation of a High-Pressure Phase 1.47 GPa. Angew . Chem . Int . Ed . 2009 , 48 , 7087-7089.

25. Yang, C .; Wang, X .; Omary, M. Crystallographic Observation of Dynamic Gas Adsorption Sites and Thermal Expansion in a Breathable Fluorous Metal-Organic Framework. Angew . Chem . Int . Ed . 2009 , 48 , 2500-2505.

26. Domke, J .; Radmacher, M. Measuring the Elastic Properties of Thin Polymer Films with the Automotive Force Microscope. Langmuir 1998 , 14 , 3320-3325.

27. Kim, K .; Kim, JH; Park, H .; Kim, Y.-S .; Park, K .; Nam, H.; Lee, S .; Park, JH; Park, R.-W .; Kim, I.-S. meat al . Tumor-Homing Multifunctional Nanoparticles for Cancer Theragnosis: Simultaneous Diagnosis, Drug Delivery, and Therapeutic Monitoring. J. Control . Release . 2010 , 146 , 219-227.

28. Caravan, P .; Ellison, JJ; McMurry, TJ; Lauffer, RB Gadolinium (III) Chelates as MRI Contrast Agents: Structure, Dynamics, and Applications. Chem . Rev. 1999, 99, 2293-2352.

29. Boussif, O .; Lezoualc'h, F .; Zanta, MA; Mergny, MD; Scherman, D .; Demeneix, B .; Behr, J.-P. A Versatile Vector for Gene and Oligonucleotide Transfer into Cells in Culture and In Vivo : polyethylenimine. Proc. Natl . Acad . Sci . USA 1995 , 92 , 7297-7301.

30. Taylor, KML; Jin, A .; Lin, W. Surfactant-Assisted Synthesis of Nanoscale Gadolinium Metal-Organic Frameworks for Potential Multimodal Imaging. Angew . Chem . Int . Ed . 2008 , 47 , 7722-7725.

31. Park, JY; Baek, MJ; Choi, ES; Woo, S .; Kim, JH; Kim, TJ; Jung, JC; Chae, KS; Chang, Y .; Lee, GH Paramagnetic Ultrasmall Gadolinium Oxide Nanoparticles as Advanced T ₁ MRI Contrast Agent: Optimal Particle Diameter, and In Vivo T ₁ MR Images. ACS Nano 2009 , 3 , 3663-3669.

32. Weinstein, JS; Varallyay, CG; Dosa, E .; Gahramanov, S.; Hamilton, B .; Rooney, WD; Muldoon, LL; Neuwelt, EA Superparamagnetic Iron Oxide Nanoparticles: Diagnostic Magnetic Resonance Imaging and Potential Therapeutic Applications in Neurooncology and Central Nervous System Inflammatory Pathologies, a Review. J. Cereb . Blood Flow Metab . 2010 , 30 , 15-35.

33. Weinmann, H. -J .; Brasch, RC; Press, W.-R .; Wesbey, GE Characteristics of Gadolinium-DTPA Complex: a Potential NMR Contrast Agent. Am. J. Roentgenol . 1984 , 142 , 619-624.

Claims (13)

A Gd-PEI nanogel comprising spherical cores impregnated with gadolinium (Gd) in the network of PEI (polyethyleneimine).
The Gd-PEI nanogel according to claim 1, wherein the Gd-PEI nanogel does not comprise DTPA (diethylene triamine pentaacetic acid) and iron oxide.
The Gd-PEI nanogel of claim 1, wherein the Gd-PEI nanogel is flexible and amorphous.
The Gd-PEI nanogel according to claim 1, wherein the diameter of the Gd-PEI nanogel is 100 to 500 nm.
The Gd-PEI nanogel according to claim 1, wherein the core has a diameter of 10 to 100 nm.
The Gd-PEI nanogel according to claim 1, wherein a phosphor is bonded to the amine group of the PEI.
The Gd-PEI nanogel according to claim 1, wherein the PEI is a branched PEI.
A Gd-PEI nanogel according to any one of claims 1 to 7; And a pharmaceutically acceptable carrier.
The contrast agent composition of claim 8, wherein the contrast agent composition is for MRI (Magnetic Resonance Imaging).
9. The contrast agent composition of claim 8, wherein the contrast agent composition is for Fluorescence Imaging.
9. The contrast agent composition of claim 8, wherein the contrast agent is a dual-modality contrast agent capable of simultaneous MRI imaging and fluorescent imaging.
A Gd-PEI nanogel according to any one of claims 1 to 7; A pharmaceutical composition for the treatment of cancer or tumor comprising a pharmaceutically acceptable carrier.
Mixing a gadolinium (Gd) salt and PEI (polyethyleneimine) in an aqueous solution to form a Gd-PEI nanogel; And
Separating the formed Gd-PEI nanogel
Wherein the Gd-PEI nanoparticles are prepared by the method of claim 1.

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