DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Formal Matters
Applicant’s response in the reply filed on 23 September 2024 are acknowledged and have been fully considered. Claims 1-24 are pending. Claims 1-6 are under consideration in the instant office action. Claims 7-24 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Information Disclosure Statement
The information disclosure statements (IDSs) submitted on 28 January 2025 are noted and the submissions are in compliance with the provisions of 37 CFR 1.97. Accordingly, the examiner has considered the references. Signed copies are attached herein.
Election/Restriction
REQUIREMENT FOR UNITY OF INVENTION
As provided in 37 CFR 1.475(a), a national stage application shall relate to one invention only or to a group of inventions so linked as to form a single general inventive concept (“requirement of unity of invention”). Where a group of inventions is claimed in a national stage application, the requirement of unity of invention shall be fulfilled only when there is a technical relationship among those inventions involving one or more of the same or corresponding special technical features. The expression “special technical features” shall mean those technical features that define a contribution which each of the claimed inventions, considered as a whole, makes over the prior art.
The determination whether a group of inventions is so linked as to form a single general inventive concept shall be made without regard to whether the inventions are claimed in separate claims or as alternatives within a single claim. See 37 CFR 1.475(e).
When Claims Are Directed to Multiple Categories of Inventions:
As provided in 37 CFR 1.475 (b), a national stage application containing claims to different categories of invention will be considered to have unity of invention if the claims are drawn only to one of the following combinations of categories:
(1) A product and a process specially adapted for the manufacture of said product; or
(2) A product and a process of use of said product; or
(3) A product, a process specially adapted for the manufacture of the said product, and a use of the said product; or
(4) A process and an apparatus or means specifically designed for carrying out the said process; or
(5) A product, a process specially adapted for the manufacture of the said product, and an apparatus or means specifically designed for carrying out the said process.
Otherwise, unity of invention might not be present. See 37 CFR 1.475 (c).
Restriction is required under 35 U.S.C. 121 and 372.
This application contains the following inventions or groups of inventions which are not so linked as to form a single general inventive concept under PCT Rule 13.1.
In accordance with 37 CFR 1.499, applicant is required, in reply to this action, to elect a single invention to which the claims must be restricted.
Group I, claim(s) 1-6, drawn to a magnetoelectric nanoparticle composition.
Group II, claim(s) 7-10, drawn to a system for targeted and controlled drug release.
Group III, claim(s) 11-19, drawn to a method of treating a subject having cancer or at risk of developing cancer.
Group IV, claim(s) 20-24, drawn to a method of making a magnetic nanoparticle composition.
The groups of inventions listed above do not relate to a single general inventive concept under PCT Rule 13.1 because, under PCT Rule 13.2, they lack the same or corresponding special technical features for the following reasons:
Groups I-IV lack unity of invention because even though the inventions of these groups require the technical feature of a magnetoelectric nanoparticle composition, this technical feature is not a special technical feature as it does not make a contribution over the prior art in view of Betal et al. (US2018/0297858, IDS reference) and Balg et al. (US 2010/0150848, IDS reference). Betal et al. teach certain embodiments are directed to Magneto-elasto-electroporation (MEEP), which is a phenomenon where nanopores open in a cell membrane due to interaction with core shell magnetoelectric nanoparticles under the influence of ac magnetic field. Embodiments of the invention use a core-shell magnetoelectric nanoparticle (CSMEN) comprising a magnetostricitve core and a ferroelectric shell to achieve MEEP across cell membranes. The core of the CSMEN is encapsulated by piezoelectric shell. The encapsulated core is capable of producing a photoacoustic emission and/or a magnetoelastic emission under influence of alternating current (AC) magnetic field. The core of the CSMEN will experience strain in the form of expansion and contraction in presence of an AC magnetic field. The strain on the CSMEN core will generate a magnetoelastic wave that is absorbed by the shell as pressure wave. The absorbing of the pressure wave changes the surface potential due to the shell's piezoelectric property. The continuous change of surface potential of CSMENs under influence of AC magnetic field results in a transmembrane voltage change across a lipid membrane when CSMENs are positioned nanometers from lipid membrane. This transmembrane voltage result in opening of nanopores on cell membrane. The CSMENs will penetrate the lipid membrane through these electrically opened nanopores due to the magnetic moment of CSMENs towards magnets. In certain aspects the CSMENs can be exposed to an AC magnetic field for a period of time sufficient for CSMENs to penetrate and pass through multiple lipid membranes, e.g., from one cell to another. In certain aspects the frequency and amplitude of the AC magnetic field can be optimized for various lipid membrane compositions, i.e., for different cell or tissue types (paragraph 0005). In certain aspects the core comprises cobalt ferrite CoFe2O4. The core can be substituted with transition metal (M), e.g. Co1-xMxFe2O4 where x<0.1 g/ml. The core can be used to form a biocompatible and non-cytotoxic (as tested with MTS assay) nanoparticle. In certain aspects the core is a single crystalline CoFe2O4 core (paragraph 0006). Silica coating on previously synthesized particles was achieved via sol-gel method. As prepared BaTiO3 coated CFO nanoparticles (10 mg) were suspended in ethanol. The pH of the suspension was adjusted to 10 using 0.1M NaOH to stabilize the particle and to catalyze the sol gel reaction. Under magnetic stirring, 250 μl of Tetraethylorthosilicate (TEOS) was then added to the suspension and allowed to react for 2 hour at 50° C. The hydrolysis and condensation of TEOS forms the silica coating on the surface of the particles. The reaction mixture was then dried overnight to achieve the powder form of the particles (see paragraph 0083). FITC was first conjugated to APTES. Typically, FITC (2 mg) was dissolved in 0.1M APTES in ethanol. The solution was stirred in dark for 24 hour. FITC-APTES (5 ml) solution was then added to silica coated particles (10 mg) and was stirred vigorously for 1 hour. The solution was then incubated for 24 hour at 40° C. The resulting solution was washed repeatedly by ethanol to remove unconjugated FITC (paragraph 0087). The experimental results also indicate that cell membrane's elasticity is influenced by the voltage change at nanometer distance by the particles due to externally applied AC Magnetic field. TEM imaging, DLS measurement and AFM imaging have confirmed the size of CSMEN as ˜78.8 nm with a coating of 19-20 nm of the piezoelectric layer on magnetostricitve cobalt ferrite nanoparticles. PFM measurement has confirmed the single crystalline state of barium titanate shell. Acoustic measurement reveals the opto-acoustic and magneto-acoustic property of cobalt ferrite nanoparticles and absorption of acoustic wave by the BaTiO3 coating/shell. Fluorescence microscopy, confocal microscopy and transwell experiments recorded the penetration of particle inside the HEP2 when subjected to an external AC magnetic field (paragraph 0089). Certain embodiments are directed to nanoparticle compositions and conjugates to facilitate delivery of molecules into a biological system such as cells. The nanoparticles described herein can be directly or indirectly coupled a moiety to be delivered or localized to a cell. The moiety/nanoparticle complex is referred herein as a nanoparticle conjugate or conjugate. The moiety can be permanently coupled to the nanoparticles or reversibly coupled, e.g., the moiety is released from the conjugate at some time after the conjugate is transported across a lipid membrane. The conjugates can impart therapeutic activity by transferring therapeutic compounds across cellular membranes. Certain aspects are directed to nanoparticle agents for the delivery of molecules, including but not limited to small molecules, lipids, nucleosides, nucleotides, nucleic acids, negatively charged polymers and other polymers, for example proteins, peptides, carbohydrates, or polyamines (paragraph 0045). Betal et al. do not teach wherein the silica is fused silica. Baig et al. teach fused silica is a high-purity amorphous silicon dioxide. It is sometimes referred to as fused quartz, vitreous silica, silica glass, or quartz glass. Fused silica is a type of glass, which, typical of glasses, lacks long-range order in its atomic structure. But the optical and thermal properties of fused silica are unique from those of other glasses, as fused silica typically has more strength, thermal stability, and ultraviolet transparency. For these reasons, fused silica is known to be used in situations such as semiconductor fabrication and laboratory equipment (paragraph 0051). For example, dentifrices incorporating fused silica have superior stability and bioavailability (paragraph 0058). It would have been prima facie obvious to one of ordinary skill in the art to choose fused silica by routine experimentation because it is less reactive (see Baig Para [0058] it is believed that the fused silica, with its low BET specific surface area, low porosity, and low number of surface hydroxyl groups, is less reactive than precipitated silica. Consequently, the fused silica may adsorb less of other components leading to better availability for these other components. For example, dentifrices incorporating fused silica have superior stability and bioavailability). Additionally, claim 1 would have been obvious based on the teachings of the references used below under the 35 USC 103 rejection as well.
During a telephone conversation with Bernard A. Brown II on 27 May 2026 a provisional election was made without traverse to prosecute Invention I (claims 1-6). Affirmation of this election must be made by applicant in replying to this Office action. Claims 7-24 are withdrawn from further consideration by the examiner, 37 CFR 1.142(b), as being drawn to a non-elected invention.
Applicant is reminded that upon the cancelation of claims to a non-elected invention, the inventorship must be corrected in compliance with 37 CFR 1.48(a) if one or more of the currently named inventors is no longer an inventor of at least one claim remaining in the application. A request to correct inventorship under 37 CFR 1.48(a) must be accompanied by an application data sheet in accordance with 37 CFR 1.76 that identifies each inventor by his or her legal name and by the processing fee required under 37 CFR 1.17(i).
The examiner has required restriction between product or apparatus claims and process claims. Where applicant elects claims directed to the product/apparatus, and all product/apparatus claims are subsequently found allowable, withdrawn process claims that include all the limitations of the allowable product/apparatus claims should be considered for rejoinder. All claims directed to a nonelected process invention must include all the limitations of an allowable product/apparatus claim for that process invention to be rejoined.
In the event of rejoinder, the requirement for restriction between the product/apparatus claims and the rejoined process claims will be withdrawn, and the rejoined process claims will be fully examined for patentability in accordance with 37 CFR 1.104. Thus, to be allowable, the rejoined claims must meet all criteria for patentability including the requirements of 35 U.S.C. 101, 102, 103 and 112. Until all claims to the elected product/apparatus are found allowable, an otherwise proper restriction requirement between product/apparatus claims and process claims may be maintained. Withdrawn process claims that are not commensurate in scope with an allowable product/apparatus claim will not be rejoined. See MPEP § 821.04. Additionally, in order for rejoinder to occur, applicant is advised that the process claims should be amended during prosecution to require the limitations of the product/apparatus claims. Failure to do so may result in no rejoinder. Further, note that the prohibition against double patenting rejections of 35 U.S.C. 121 does not apply where the restriction requirement is withdrawn by the examiner before the patent issues. See MPEP § 804.01.
Objection to the title
The title of the invention is not descriptive. A new title is required that is clearly indicative of the invention to which the claims are directed. The title of the instant application is “MAGNETIC NANOPARTICLES AND METHODS OF DRUG RELEASE”. The title should be brief but technically accurate and descriptive and should contain fewer than 500 characters. The title does not reflect the main inventive concept of Applicant’s invention and the major components of the ‘MAGNETIC NANOPARTICLES AND METHODS OF DRUG RELEASE”. The title is generic and can be applicable to any “MAGNETIC NANOPARTICLES AND METHODS OF DRUG RELEASE”. The examiner advises Applicant to consider including major components of the composition in the title to precisely reflect the inventive concept. Inasmuch as the words "new," "improved," "improvement of," and "improvement in" are not considered as part of the title of an invention, these words should not be included at the beginning of the title of the invention and will be deleted when the Office enters the title into the Office’s computer records, and when any patent issues. Similarly, the articles "a," "an," and "the" should not be included as the first words of the title of the invention and will be deleted when the Office enters the title into the Office’s computer records, and when any patent issues.
Claim Objections
Claims 2-6 are objected to because of the following informalities: Independent claim 1 in the preamble recites “A magnetoelectric nanoparticle composition”. Claims 2-6 which depends from claim 1 directly or indirectly recite in their preambles and in some cases in the recitations of the claims “The composition”. For consistency and clarity reasons claims 2-6 should recite in the preamble “The magnetoelectric composition”. Appropriate correction is required.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1 and 4-5 are rejected under 35 U.S.C. 103 as being unpatentable over Sikima et al. (US2009/0269284) in view of Morks (J OURNAL OF THE MECHANICAL BEHAVIOR OF BIOMEDICAL MATERIALS 1, 105–111, 2008).
Applicants’ claims
Applicants claim a magnetoelectric nanoparticle composition comprising the components as recited. Dependent claims thereof recite additional features.
Determination of the Scope and Content of the Prior Art
(MPEP 2141.01)
Sikima et al. teach in some embodiments, the present invention provides a magnetic resonance contrast agent composition comprising one or more T1 contrast agent portions, one or more T2 contrast agent portions, and one or more linker regions. In some embodiments, the T2 contrast agent portion of the present invention comprises a superparamagnetic nanoparticle. (paragraph 0008). FIG. 2 shows analysis of nanoparticles: a) TEM image of CoFe2O4 nanoparticles, b) the XRD spectrum of CoFe2O4 nanoparticles, c) TEM image of silica coated cobalt ferrite nanoparticles revealing the dark center and homogeneous gray silica shell, d) X-ray phoyoelectron spectra of Nis and S 2s peaks indicating the monolayer adsorption of APTEM and MPTMS (see paragraph 0015). FIG. 5 shows TEM images of cobalt ferrite cores (left), SiOx-coated cobalt ferrite core-shell nanoparticles (middle), and Gd(III)-DTPA-modified nanoparticles (right) (paragraph 0018). In an exemplary embodiment, the present invention provides for the design, synthesis, and characterization of an enzyme cleavable T1-T2 agent for the detection of MMP-7 enzyme activity. The T1 agent is a Gd(III)-DTPA chelate and the T2 agent is a SPM silica-coated cobalt ferrite core-shell particle with surface amine groups. The linker is a peptide backbone containing an MMP-7-specific cleavage site and a PEG spacer to provide extra room for the enzyme to interact with the peptide substrate as well as increase stability in aqueous conditions. More generally, this type of design provides applications for the diagnosis and monitoring of disease progression by real-time tracking of a variety of enzymes that are responsible for a multitude of diseases (paragraph 0044). After enzymatic cleavage of the peptide, it is contemplated that the relaxation properties of the agent changes. In some embodiments, a PEG spacer was inserted between the peptide and the nanoparticle to allow ample space for the enzyme to interact with the peptide substrate. A (PEG)3 spacer was chosen for initial studies due to cost and simplicity, however, many different lengths of this PEG spacer or other spacers are commercially available and equally amenable for use with these embodiments of the present invention. The exemplary amino acid sequence used is based on consensus sequences for MMP-7. Diethylenetriamine pentaacetic acid (DIPA) was chosen as the Gd(III) chelator due to cost and availability, however, other Gd(III) chelators may be used. Embodiments of the present invention include, but are not limited to, varying the peptide length and composition as well as by varying the length of the PEG spacer between the peptide and the nanoparticle. For example, in some embodiments, longer spacers are used. For example, in some embodiments, a Gd(III)-DTPA-modified MMP-7 peptide linker contains and longer spacer, (PEG)6 (paragraph 0045). In some embodiments, the present invention provides a nanoparticle with a diameter of approximately 5-50 nm (e.g. 5 nm . . . 10 nm . . . 20 nm . . . 30 nm . . . 40 nm . . . 50 nm) In some embodiments, the present invention provides a nanoparticle with a diameter of greater than 50 nm. In some embodiments, the nanoparticle of the present invention comprises core comprised of for example ferrite (MFe2O4, M=Fe, Co, Mn, Ni), iron, cobalt, or other. In some embodiments the nanoparticle is coated with a biocompatible surfactant that permits further modification, prevents aggregation, and/or renders the nanoparticle useful in a biological setting, although the present invention is not limited to any particular mechanism of action and an understanding of the mechanism of action is not necessary to practice the present invention. Such biocompatible coatings include, but are not limited to, silica, PEG, and dextran. In some embodiments, the surface of the nanoparticle is functionalized to allow further modification, although the present invention is not limited to any particular mechanism of action and an understanding of the mechanism of action is not necessary to practice the present invention. In some embodiments the surface modification of the nanoparticles includes thiol-modification (paragraph 0051). A modified inverse micro-emulsion based sol-gel approach was used to fabricate stable and well-dispersed CoFe2O4@SiO2 nanoparticles with improved control over shell thickness and core diameters. The CoFe2O4 core particles were synthesized according using the method of Sun et al. with slight modifications (Sun et al. J. Am. Chem. Soc. 2004, 126, 273-279, herein incorporated by reference in its entirety). A quantity of 40 mL of benzyl ether solution containing 2 mmol concentrated iron and cobalt acetylacetonate (Fe(acac)3 and Co(acac)) was reduced by 10 mM hexadecanediol under N, blanket and heating at 275° C. to yield stable cobalt ferrite (CoFe2O4) nanoparticles in non-aqueous conditions. The solution was heated in presence of 6 mmol lauric acid and lauryl amine, which play the key role of surface stabilizers for the nanoparticles. The mixture turns brown after mixing at 100° C. and holding the mixture at this temperature for 30 min eliminates the water and other organic moistures. The solution turns dark brown indicating that nanoparticles are nucleated at 200° C.; however, holding the reaction mixture at this temperature for an hour homogenizes the nanoparticle growth during this time. By turning the temperature reaction around the boiling point of solvent (265-285° C.), it is observed that the rate of growth of nanoparticles is considerably enhanced and, consequently, all nanoparticle syntheses were carried out at this temperature The iron oxide nanoparticle solution was subjected to magnetic separation by ethanol precipitation, and the resulting aggregate was washed with copious amounts of ethanol and acetone to remove any uncoordinated stabilizer molecules. The aggregate was then dispersed in hexane for further studies (paragraph 0114). The cobalt ferrite nanoparticles were coated with SiO2 by base-catalyzed silica formation from tetraethylorthosilicate (TEOS) in a water-in-oil microemulsion (Lee et al. J. Phys. Chem. B 2006, 110, 11160-11166. and Deng et al. Colloids Surf, A 2005, 262, 87-93., herein incorporated by reference in their entireties) with slight modifications. IGEPAL CO-520 (1 mL) was mixed with 20 ml of anhydrous cyclohexane and stirred for 10 minutes. Cobalt ferrite nanoparticles were dispersed in cyclohexane at a concentration of 1 mg/mL and then poured slowly into the cyclohexane/Igepal solution. The amount of nanoparticle was adjusted so as to achieve desired silica shell thickness of approximately 10 nm. Then 120 μl of 30% NH4OH aqueous solution was added drop-wise and stirred for 15 minutes, followed by the addition of 190 μl of tetraethylorthosilicate. Depending on the desired silica shell thickness, the amount of TEOS can be varied. The mixture was stirred for 48 h before adding ethanol to precipitate the particles. The precipitate with ethanol was collected by centrifugation at 10,000 rpm and particles were washed by redispersing in ethanol. The CoFe2O4@SiO2 nanoparticles were washed using this procedure at least three times to remove excess surfactant The final product was stored as a toluene dispersion for further surface modification with 3-aminopropyl triethoxysilane (APTES) or 3-mercaptopropyl trimethoxysilane (MPTMS) (the examiner notes that this entails salinization). The surface modification was carried out at room temperature after injecting 100 μl of APTES or MPTMS in 1 mg of CoFe2O4@SiO2 toluene-nanoparticle dispersion. The mixture was stirred rigorously for 2-4 days. The precipitated mixture was rinsed copiously with absolute alcohol and later dispersed directly in water or DMSO (paragraph 0115). The core-shell nanoparticles were characterized by transmission electron microscopy (TEM), energy-dispersive spectroscopy, elemental mapping, and surface characterization techniques such as FTIR and x-ray photoelectron spectroscopy (XPS). The TEM analysis of the bare particles (SEE FIG. 2 a) reveals the uniformity in shape and size (7 nm, Std deviation ≦10%) XRD spectrum (SEE FIG. 2 b) confirmed the cobalt ferrite phase formation and Debye-Scherer equation was used to calculate the particle size and found to be in good agreement with TEM results. The particle core size can be tuned from 7-20 nm by varying the surfactant concentration and seed-mediated growth process TEM of the initial silica-coated particles (SEE FIG. 2 c) suggests that the 7 nm core is uniformly isolated in an individual shell (10 nm thickness). This silica shell can be tuned from 10 to 50 nm by varying the TEOS concentration (paragraph 0116). The presence of anchoring sites on the nanoparticles for attachment of T1 agents was shown by FTIR and XPS FTIR spectrum of core-shell particles (CoFe2O4@SiO2) clearly shows the presence of hydroxyl groups on the silica shell by the peak at 940 cm−1. These surface functional groups are treated with APTES or MPTMS in dry toluene to convert them to surface amine or thiol groups for further attachment of T1 agent. The presence of amine or thiol monolayer was confirmed by XPS(N 1s peak BE-400 eV and S 2s peak at 2285 eV) (SEE FIG. 2 d) (paragraph 0117). Thiol-modified silica-coated cobalt ferrite nanoparticles (CoFeSiOx—SH) were prepared in three subsequent stages: 1) cobalt ferrite core synthesis, 2) silica shell formation, and 3) surface thiol functionalization; although the present invention is not limited to any single synthesis strategy. Thiol functionalization was achieved through surface modification with (3-mercaptopropyl)trimethoxysilane (MPTMS). The final nanoparticles were characterized using TEM, XRD, FTIR, and XPS (paragraph 0123). This approach resulted in controlled core diameter and silica shell thickness with core-shell nanoparticles highly dispersed in aqueous solution. TEM analysis of the CoFeSiOx—SH nanoparticles revealed the uniformity in shape and size of the core (7 nm, std. dev.≦10%) and SiOx coating of approximately 10 nm (SEE FIG. 5). XRD confirmed cobalt ferrite phase formation and the particle sizes calculated by the Debye-Scherer equation were found to be in good agreement with TEM results. FTIR and XPS of the core-shell nanoparticles verified surface modification of the cobalt ferrite core with silica and MPTMS. The hydrodynamic size of the nanoparticles was determined to be 55±9 nm (paragraph 0124). The morphological features of the silica-coated cobalt ferrite nanoparticles did not alter upon conjugation of Gd(III)-DTPA-SS-pyridyl as observed by TEM (SEE FIG. 5). EDS analysis showed the presence of Gd, Fe, Co, and S, which supports the successful attachment of Gd(III)-DTPA to the nanoparticle surface and illustrates the integrity of the nanoparticle. The hydrodynamic size (62±20 nm) revealed that upon conjugation of Gd(III)-DTPA-SS-pyridyl the hydrodynamic size increased only slightly indicating the CoFeSiOx—Gd(III) nanoparticles are not aggregated in solution (paragraph 0126). During development of embodiments of the present invention, a modified inverse micro-emulsion based sol-gel approach was used to fabricate stable and well-dispersed magnetic CoFe2O4@SiO2 nanoparticles with improved control over shell thickness and core diameters. The CoFe2O4 core particles were synthesized according using the method of Sun et al. with slight modifications (Sun, et al., J Am Chem Soc 2004, 126:273-279, herein incorporated by reference in its entirety). A quantity of 40 mL of benzyl ether solution containing 2 mmol concentrated iron and cobalt acetyl acetonate (Fe(acac)3 and Co(acac)3) was reduced by 10 mM hexadecanediol under N2 blanket and heating at 275° C. to yield stable cobalt ferrite (CoFe2O4) nanoparticles in non-aqueous conditions. The solution was heated in presence of 6 mmol lauric acid and lauryl amine which play the key role of surface stabilizers for the nanoparticles. The mixture turns brown after mixing at 100° C. and holding the mixture at this temperature for 30 min eliminates the water and other organic moistures. The solution turns dark brown indicating that nanoparticles are nucleated at 200° C.; however, holding the reaction mixture at this temperature for an hour homogenizes the nanoparticle growth during this time. By turning the temperature reaction around the boiling point of solvent (265-285° C.), it is observed that the rate of growth of nanoparticles is considerably enhanced and, consequently, all nanoparticle syntheses were carried out at this temperature. The iron oxide nanoparticle solution was subjected to magnetic separation by ethanol precipitation, and the resulting aggregate was washed with copious amounts of ethanol and acetone to remove any uncoordinated stabilizer molecules. The aggregate was then dispersed in hexane for further studies (paragraph 0156). The cobalt ferrite nanoparticles were coated with SiO2 by base-catalyzed silica formation from tetraethylorthosilicate in a water-in-oil microemulsion, using the methods of Lee et al. and Deng et al. with slight modifications. Igepal CO-520 (1 mL) was mixed with 20 ml of anhydrous cyclohexane and stirred for 10 minutes. Cobalt ferrite nanoparticles were dispersed in cyclohexane at a concentration of 1 mg/mL and then poured slowly into the cyclohexane/Igepal solution. The amount of nanoparticle was adjusted so as to achieve desired silica shell thickness of approximately 10 nm. Then 120 μl of 30% NH4OH aqueous solution was added dropwise and stirred for 15 minutes, followed by the addition of 190 μl of tetraethylorthosilicate (TEOS). Depending on the desired silica shell thickness, the amount of TEOS can be varied. The mixture was stirred for 48 h before adding ethanol to precipitate the particles. The precipitate with ethanol was collected by centrifugation at 10,000 rpm and particles were washed by redispersing in ethanol. The CoFe2O4@SiO2 nanoparticles were washed using this procedure at least three times to remove excess surfactant. The final product was stored as a toluene dispersion for further surface modification with 3-aminopropyl triethoxysilane (APTES). The surface modification was carried out at room temperature after injecting 100 μl of APTES in 1 mg of CoFe2O4@SiO2 toluene-nanoparticle dispersion. The mixture was stirred rigorously for 2-4 days. The precipitated mixture was rinsed copiously with absolute alcohol and later dispersed directly in water or DMSO (see paragraph 0157). The core-shell nanoparticles were characterized by transmission electron microscopy (TEM), energy-dispersive spectroscopy, elemental mapping, and surface characterization techniques such as FTIR and x-ray photoelectron spectroscopy (XPS). The TEM analysis of the bare particles reveals the uniformity in shape and size (7 nm, Std. deviation ≦10%). XRD spectrum confirmed the cobalt ferrite phase formation and Debye-Scherer equation was used to calculate the particle size and found to be in good agreement with TEM results. The particle core size can be tuned from 7-20 nm by varying the surfactant concentration and seed-mediated growth process. TEM of the initial silica-coated particles suggests that the 7 nm core is uniformly isolated in an individual shell (10 nm thickness). This silica shell can be tuned from 10 to 50 nm by varying the TEOS concentration (see pragarph 0158). During development of embodiments of the present invention, the synthesis of silica-coated cobalt ferrite core-shell nanoparticles (CoFe2O4@SiO2) modified at the surface with 3-aminopropyltriethoxysilane (APTES). Standard peptide coupling techniques were used to covalently attach the C-terminus of the Gd(III)-DTPA-APLALWA-PEGn-A peptide (n=3-6) to the amine groups on the surface of the APTES-modified nanoparticle (SEE FIG. 10). Briefly, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC, 4.2 μmol) and N-hydroxysulfosuccinimide (sulfo-NHS, 4.2 μmol) were dissolved in 100 μL DMSO and 50 μL 100 mM MES pH 6.2. Gd(III)-DTPA-APLALWA-PEGn-A (0.8 μmol) was dissolved in 100 μL DMSO and added to the solution of EDC and sulfo-NHS (the examiner notes that this are coupling agents similar to the succinic acid anhydride recited in claim 1). The reaction was incubated at room temperature for 15 minutes and subsequently added to a solution of APTES-modified CoFe2O4@SiO2 nanoparticles (0.7 μmol Fe) in DMSO. The reaction mixture was incubated at room temperature with end-over-end rotation for 24 h. Excess reagents were purified by extensive dialysis against a 5% DMSO in water mixture followed by size filtration using Millipore Amicon Ultra-4 100,000 MWCO centrifugal filter devices. ICP-MS was used to quantify the Co, Fe, and Gd content of the agents (paragraph 0160). Various reaction conditions have been investigated in order to optimize the conjugation yield. Aqueous and organic solvents were tested and it was found that higher conjugation was achieved in polar aprotic solvents, namely DMSO. The coupling agents (EDC and sulfo-NHS) were added in 6-fold excess and preliminary data indicates that the addition of more equivalents of the coupling agents (up to 12) does not have as large of an effect on the yield as the solvent (see paragraph 0161). In some embodiments, an additional functional portion is a biomolecule, such as for example, a ligand, antibody, peptide, polypeptide, protein, nucleic acid, polysaccharide, carbohydrate, lipid, glycoprotein, phospholipid, sterol, hormone, disaccharide, amino acid, nucleotide, phosphate, monsacharide, etc. In some embodiments, a biomolecule functional portion serves to localize the present invention in a specific cell type, for example, blastomere, embryonic stem cell, erythrocyte, fibroblast, hepatocyte, myoblast, myotube, neuron, oocyte, osteoblast, osteoclast, T-Cell, zygote, prokaryotic cell, a specific bacteria, plant cells, fungal cells, etc. In some embodiments, a biomolecule functional portion serves to localize the present invention in a specific cellular region, for example cytoplasm, nucleus, intracellular space, golgi complex, endoplasmic reticulum, mitochondria, chloroplasts, etc. In some embodiments, a biomolecule functional portion serves to localize the present invention in a specific tissue, for example, epithelial, connective, muscle, neural, etc. In some embodiments, a biomolecule functional portion serves to localize the present invention in specific diseased cells, for example, cancer cells, virally infected cells, etc. In some embodiments, a biomolecule functional portion serves to interact with native biomolecules in a subject, sample, tissue, or cell, such as for example, cell surface markers, antibodies, receptor proteins, nucleic acid, specific classes of proteins, etc., (paragraph 0086). In some embodiments, an additional functional portion is a biomolecule which serves as a targeting moiety. By “targeting moiety” herein is meant a functional group which serves to target or direct the complex to a particular location, cell type, diseased tissue, or association. In general, the targeting moiety is directed against a target molecule. As will be appreciated by those in the art, the MRI contrast agents of the invention may be injected intravenously; thus targeting moieties may be those that allow concentration of the agents in a particular localization. In some embodiments, the agent is partitioned to the location in a non-1:1 ration. Thus, for example, antibodies, cell surface receptor ligands and hormones, lipids, sugars and dextrans, alcohols, bile acids, fatty acids, amino acids, peptides and nucleic acids may all be attached to localize or target the contrast agent to a particular site (paragraph 0087). In some embodiments, the targeting moiety is a peptide. For example, chemotactic peptides have been used to image tissue injury and inflammation, particularly by bacterial infection (see paragraph 0089).
Ascertainment of the Difference Between Scope of the Prior Art and the Claims
(MPEP 2141.02)
Sikima et al. do not specifically teach fused silica. This deficiency is cured by the teachings of Morks.
Morks teaches fabrication and characterization of plasma-sprayed HA/SiO2 coatings for biomedical application (see title). Fused silica powder has been mixed with hydroxyapatite (HA) powder and plasma sprayed by using gas tunnel-type plasma jet. The influence of silica content (10 wt% and 20 wt%) on the microstructure and mechanical properties of HA–silica coatings was investigated. For investigating the microstructure and mechanical properties of HA–silica coatings, SUS 304 stainless steel was used as substrate material. The spraying was carried out on roughened substrate in an atmospheric chamber. Scanning electron microscope micrographs of cross sectioned HA/SiO2 coatings showed that the sprayed HA coatings with 10 and 20 wt% SiO2 have dense structure with low porosity compared to the pure HA coatings. On the other hand, as the amount of silica was increased the coatings became denser, harder and exhibited high abrasive wear resistance. The presence of silica significantly improved the adhesive strength of HA/SiO2 coatings mainly due to the increase in bonding strength of the coating at the interface (see abstract). The surfaces of metallic implants are usually coated with bioactive material to improve the implant integration by promoting the growth of bony tissue (apatite) on the surface of porous biomaterial (see introduction). The adhesive strength was significantly improved by incorporating silica particles in HA coatings. On the other hand, the adhesion increased with increasing the amount of silica. The presence of silica improved the adhesive strength at the interface as well as the bonding strength among the individual splats of HA (see adhesion properties, page 110).
Finding of Prima Facie Obviousness Rational and Motivation
(MPEP 2142-2143)
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the instant invention to modify the teachings of Sikima et al. by utilizing fused silica as the coating agent because Morks for instance teach fabrication and characterization of plasma-sprayed HA/SiO2 coatings for biomedical application (see title). Fused silica powder has been mixed with hydroxyapatite (HA) powder and plasma sprayed by using gas tunnel-type plasma jet. The influence of silica content (10 wt% and 20 wt%) on the microstructure and mechanical properties of HA–silica coatings was investigated. For investigating the microstructure and mechanical properties of HA–silica coatings, SUS 304 stainless steel was used as substrate material. The spraying was carried out on roughened substrate in an atmospheric chamber. Scanning electron microscope micrographs of cross sectioned HA/SiO2 coatings showed that the sprayed HA coatings with 10 and 20 wt% SiO2 have dense structure with low porosity compared to the pure HA coatings. On the other hand, as the amount of silica was increased the coatings became denser, harder and exhibited high abrasive wear resistance. One of ordinary skill in the art would have been motivated to do so because Morks teach valuable properties of fused silica as a coating agent in biomaterial applications. Morks teaches that e.g., the presence of silica significantly improved the adhesive strength of HA/SiO2 coatings mainly due to the increase in bonding strength of the coating at the interface (see abstract). The surfaces of metallic implants are usually coated with bioactive material to improve the implant integration by promoting the growth of bony tissue (apatite) on the surface of porous biomaterial (see introduction). The adhesive strength was significantly improved by incorporating silica particles in HA coatings. On the other hand, the adhesion increased with increasing the amount of silica. The presence of silica improved the adhesive strength at the interface as well as the bonding strength among the individual splats of HA (see adhesion properties, page 110). The selection of a known material based on its suitability for its intended use supported a prima facie obviousness determination in Sinclair & Carroll Co. v. Interchemical Corp., 325 U.S. 327, 65 USPQ 297 (1945) (Claims to a printing ink comprising a solvent having the vapor pressure characteristics of butyl carbitol so that the ink would not dry at room temperature but would dry quickly upon heating were held invalid over a reference teaching a printing ink made with a different solvent that was nonvolatile at room temperature but highly volatile when heated in view of an article which taught the desired boiling point and vapor pressure characteristics of a solvent for printing inks and a catalog teaching the boiling point and vapor pressure characteristics of butyl carbitol.). Furthermore, in the case where the claimed particle size "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). Furthermore, differences in concentration or any measurable parameters will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233,235 (CCPA 1955). One of ordinary skill in the art would have had a reasonable chance of success in combining the teachings of Sikima et al. and Morks because both references are drawn to biomaterial substrates coated with silica for biomedical applications.
In light of the forgoing discussion, the Examiner concludes that the subject matter defined by the instant claims would have been obvious within the meaning of 35 USC 103. Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art before the effective filing date of the instant invention, as evidenced by the references, especially in the absence of evidence to the contrary.
Claim(s) 3 is/are rejected under 35 U.S.C. 103 as being unpatentable over Sikima et al. (US 2009/0269284) in view of Morks (J OURNAL OF THE MECHANICAL BEHAVIOR OF BIOMEDICAL MATERIALS 1, 105–111, 2008) as applied to claims 1 and 4-5 above, and further in view of Nasiri et al. (Journal of the Chinese Chemical Society 65(2), 231-242, 2018, abstract only provided).
Applicants’ claims
Applicants claim a magnetoelectric nanoparticle composition comprising the components as recited. Claim 3 recites the composition of claim 1, further comprising a polyethylene glycol (PEG)-linked folate or folic acid molecule covalently conjugated to the fused silica shell.
Determination of the Scope and Content of the Prior Art
(MPEP 2141.01)
The teachings of Sikima et al. and Morks are described above in detail and are incorporated herein by reference.
Ascertainment of the Difference Between Scope of the Prior Art and the Claims
(MPEP 2141.02)
Sikima et al. and Morks do not specifically teach wherein the composition further comprising a polyethylene glycol (PEG)-linked folate or folic acid molecule covalently conjugated to the fused silica shell. It should be noticed that Sikima teach attaching PEG linkers to the silica coating surface to be able to couple active agents and targeting moieties. This deficiency is cured by the teachings of Nasir et al.
Nasir et al. teach a stable and biocompatible targeting complex CFNs@PEG-FA is developed. The initially synthesized cobalt ferrite nanoparticles (CFNs) were treated with poly(ethylene glycol) (PEG) in order to improve biocompatibility of the CFNs. Citric acid (CA) was used as the coupling agent, which made PEG to bond with the CFNs. CFNs@PEG were conjugated with folic acid (FA) to synthesize CFNs@PEG-FA, which was capable of targeting the FA receptor positive (FAR+) cancer cells. Synthesized nanoparticles were physically and chemically analyzed using EDX, FT-IR, XRD, TGA, FESEM, TEM, DLS, and VSM. The biocompatibility of CFNs@PEG-FA was assessed in vitro on HSF 1184 (human skin fibroblast cells) and HeLa (human cervical cancer cell, FAR+) using MTT assay and AO/EB staining florescence method. High level of CFNs@PEG-FA binding to HeLa was confirmed through quantitative and qualitative in vitro targeting studies. Results show that CFNs@PEG-FA can be a potential biomaterial for use in biomedical trials, especially magnetic hyperthermia. The findings through this in vitro study are to be compared in future with those of in vivo studies.
Finding of Prima Facie Obviousness Rational and Motivation
(MPEP 2142-2143)
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the instant invention to modify the teachings of Sikima et al. and Morks by incorporating a polyethylene glycol (PEG)-linked folate or folic acid molecule covalently conjugated to the fused silica shell because Nasir et al. teach a stable and biocompatible targeting complex CFNs@PEG-FA is developed. The initially synthesized cobalt ferrite nanoparticles (CFNs) were treated with poly(ethylene glycol) (PEG) in order to improve biocompatibility of the CFNs. Citric acid (CA) was used as the coupling agent, which made PEG to bond with the CFNs. CFNs@PEG were conjugated with folic acid (FA) to synthesize CFNs@PEG-FA, which was capable of targeting the FA receptor positive (FAR+) cancer cells. Synthesized nanoparticles were physically and chemically analyzed using EDX, FT-IR, XRD, TGA, FESEM, TEM, DLS, and VSM. The biocompatibility of CFNs@PEG-FA was assessed in vitro on HSF 1184 (human skin fibroblast cells) and HeLa (human cervical cancer cell, FAR+) using MTT assay and AO/EB staining florescence method. High level of CFNs@PEG-FA binding to HeLa was confirmed through quantitative and qualitative in vitro targeting studies. One of ordinary skill in the art would have been motivated to do so because Nasir et al. teach that results show that CFNs@PEG-FA can be a potential biomaterial for use in biomedical trials, especially magnetic hyperthermia. The findings through this in vitro study are to be compared in future with those of in vivo studies. One of ordinary skill in the art would have had a reasonable chance of success in combining the teachings of Sikima et al., Morks, and Nasir et al. because all of the references are drawn to the delivery of biomaterials for biomedical applications.
In light of the forgoing discussion, the Examiner concludes that the subject matter defined by the instant claims would have been obvious within the meaning of 35 USC 103. Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art before the effective filing date of the instant invention, as evidenced by the references, especially in the absence of evidence to the contrary.
Claim(s) 2 and 6 is/are rejected under 35 U.S.C. 103 as being unpatentable over Sikima et al. (US 2009/0269284) in view of Morks (J OURNAL OF THE MECHANICAL BEHAVIOR OF BIOMEDICAL MATERIALS 1, 105–111, 2008) as applied to claims 1 and 4-5 above, and further in view of Mohapatra et al. (J. Mater. Chem., 2011, 21, 9185-9193).
Applicants’ claims
Applicants claim a magnetoelectric nanoparticle composition comprising the components as recited. Claim 2 recites the composition of claim 1, wherein the one or more therapeutic agents comprise one or more chemotherapeutic drugs comprising doxorubicin or diphyllin. Claim 6 recites the composition of claim 1, wherein the one or more therapeutic agents are covalently conjugated to the fused silica shell through silanization with a succinic acid anhydride moiety on the fused silica shell.
Determination of the Scope and Content of the Prior Art
(MPEP 2141.01)
The teachings of Sikima et al. and Morks are described above in detail and are incorporated herein by reference.
Ascertainment of the Difference Between Scope of the Prior Art and the Claims
(MPEP 2141.02)
Sikima et al. and Morks do not specifically teach wherein the one or more therapeutic agents are covalently conjugated to the fused silica shell through silanization with a succinic acid anhydride moiety on the fused silica shell and the therapeutic agent is doxorubicin. These deficiencies are cured by the teachings of Mohapatra et al.
Mohapatra et al. teach a multifunctional folic acid decorated superparamagnetic mesoporous CoFe2O4 based nanocarrier with the particle size of 35–40 nm was synthesized by a simple method. The particles show excellent aqueous dispersion stability in physiological pH without any deterioration in hydrodynamic size and zetapotential. The cytotoxicity and internalization efficiency of these nanocarriers have been evaluated on folate receptor overexpressed HeLa cells (see page 9185). Amine functionalized superparamagnetic CoFe2O4 nano particles were prepared by thermal decomposition of CoCl2.6H2O and FeCl3 in ethylene glycol in presence of sodium acetate and ethanol amine. Briefly, anhydrous FeCl3 (0.683 g, 4.2 mmol) and CoCl2.6H2O (0.576 g, 2.1 mmol) were taken in 30 mL ethylene glycol and 0.5 gm of sodium acetate was added to it. The reddish black color solution thus obtained was stirred for 30 min at 80 C followed by addition of 15 mL of ethanol amine. The entire solution was allowed to reflux for 6h during which fine black colloidal particles appeared in the reaction mixture. Then it was cooled down to room temperature. The CoFe2O4 particles were recovered using a magnetic separator (DynaMag2, Invitrogen) washed with millipore water (5 5 mL) and dried in hot air oven at 80 ºC for 2h (see Preparation of CoFe2O4-NH2 nanoparticles page 9186). 100 mg of amine functionalized CoFe2O4 nanoparticles were dispersed in 5mL of water. To a stirred solution of EDC (18 mg, 0.09 mmol)in5mLofH2O,NHS(41mg,0.35mmol)wasadded. To the resulting solution, alkaline solution of folic acid (13 mg, 0.031 mmol) in 5 mL of H2O was added followed by addition of 100 mg amine functionalized CoFe2O4 nanoparticles dispersed in 5mLH2O.Theentiresolutionwasstirredinthe darkfor12hand the particles were separated magnetically, washed thoroughly with (3 5mL) millipore H2O and dispersed in 5 mL H2O (CoFe2O4-FA). To this dispersion RITC (100 mg in 1mL DMSO/ H2O) was added. The resulting suspension was sonicated for 1 h in the dark. Particles were recovered by magnetic separation and washed thoroughly with millipore water (CoFe2O4-FA-RITC) (see Synthesis of folate decorated fluorescent magnetic carriers page 9186). Fluorescent molecule RITC and antitumor agents such as methotrexate and doxorubicin were successfully attached to the surface amine groups following simple organic coupling reactions thus endowing the nanoparticle with therapeutic and optical properties. These drug loaded nanoagents exhibit elevated cytotoxicity and induce apoptosis in HeLa cells. To load DOX, the amine functionalized magnetic nanoparticles (CoFe2O4-FA) were converted to acid terminated particles through ring opening reaction with succinic anhydride in the presence of DMAP. For this purpose, 10 ml aqueous dispersion (0.1 mg mL1) of nanoparticles were washed thoroughly with millipore water and then dispersed in 5 mL of methanol. In a 100 mL round bottom flask, succinic anhydride (0.05g, 0.5 mmol) and DMAP (0.06g, 0.5 mmol) were taken in 15mL methanol followed by addition of CoFe2O4nanoparticles. The total mixture was refluxed for 24 h at 80 C (CoFe2O4-FA COOH). The particles were recovered, washed thoroughly with methanol (5 5 mL), dried at 80 C. 5 mL of aqueous solution containing NHS (41 mg, 0.358 mMol) and EDC (18 mg, 0.0938 mmol) was taken in a 50 ml of round bottom flask and was kept in the dark for 2 h. After that the aqueous solution of DOX (74 mg in 12 mL water, molar ratio FA:DOXz1:5) was added to the above solution followed by addition of CoFe2O4 FA-COOH nanoparticles. The resulting suspension was stirred at room temperature for overnight and the particles (CoFe2O4 FA-DOX) were isolated magnetically. In order to study release behavior, 5 ml suspension of CoFe2O4-FA-DOX particles (0.1 mg mL1) was suspended in a solution containing 0.1mg ml 1 protease from bovine pancreas and 5 ml of PBS (0.1 mg ml 1). The solution pH was adjusted to 3, 5 and 7.4 using 0.1 M HCl and NaOH. Subsequent to incubation for 5, 10, 20, 25h, at 37 C, the nanoparticles were isolated magnetically and the recovered DOX in the supernatant was quantified spectro photometrically in UV spectrophotometer (lmax ¼ 255 nm).
Finding of Prima Facie Obviousness Rational and Motivation
(MPEP 2142-2143)
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the instant invention to modify the teachings of Sikima et al. and Morks by incorporating wherein the one or more therapeutic agents are covalently conjugated to the fused silica shell through silanization with a succinic acid anhydride moiety on the fused silica shell and the therapeutic agent is doxorubicin because first Sikima et al. the silanization processes as described above wile preparing the silica coating with the formation of available functional groups for further coupling with therapeutic agents on the surface of the silica coating. Furthermore, Mohapatra et al. teach a multifunctional folic acid decorated superparamagnetic mesoporous CoFe2O4 based nanocarrier with the particle size of 35–40 nm was synthesized by a simple method. The particles show excellent aqueous dispersion stability in physiological pH without any deterioration in hydrodynamic size and zetapotential. The cytotoxicity and internalization efficiency of these nanocarriers have been evaluated on folate receptor overexpressed HeLa cells (see page 9185). One of ordinary skill in the art would have been motivated to link the active doxorubicin via a succinic anhydride linker because Mohapatra et al. teach that amine functionalized superparamagnetic CoFe2O4 nano particles were prepared by thermal decomposition of CoCl2.6H2O and FeCl3 in ethylene glycol in presence of sodium acetate and ethanol amine. Briefly, anhydrous FeCl3 (0.683 g, 4.2 mmol) and CoCl2.6H2O (0.576 g, 2.1 mmol) were taken in 30 mL ethylene glycol and 0.5 gm of sodium acetate was added to it. The reddish black color solution thus obtained was stirred for 30 min at 80 C followed by addition of 15 mL of ethanol amine. The entire solution was allowed to reflux for 6h during which fine black colloidal particles appeared in the reaction mixture. Then it was cooled down to room temperature. The CoFe2O4 particles were recovered using a magnetic separator (DynaMag2, Invitrogen) washed with millipore water (5 5 mL) and dried in hot air oven at 80 ºC for 2h (see Preparation of CoFe2O4-NH2 nanoparticles page 9186). 100 mg of amine functionalized CoFe2O4 nanoparticles were dispersed in 5mL of water. To a stirred solution of EDC (18 mg, 0.09 mmol)in5mLofH2O,NHS(41mg,0.35mmol)wasadded. To the resulting solution, alkaline solution of folic acid (13 mg, 0.031 mmol) in 5 mL of H2O was added followed by addition of 100 mg amine functionalized CoFe2O4 nanoparticles dispersed in 5mLH2O.Theentiresolutionwasstirredinthe darkfor12hand the particles were separated magnetically, washed thoroughly with (3 5mL) millipore H2O and dispersed in 5 mL H2O (CoFe2O4-FA). To this dispersion RITC (100 mg in 1mL DMSO/ H2O) was added. The resulting suspension was sonicated for 1 h in the dark. Particles were recovered by magnetic separation and washed thoroughly with millipore water (CoFe2O4-FA-RITC) (see Synthesis of folate decorated fluorescent magnetic carriers page 9186). Fluorescent molecule RITC and antitumor agents such as methotrexate and doxorubicin were successfully attached to the surface amine groups following simple organic coupling reactions thus endowing the nanoparticle with therapeutic and optical properties. These drug loaded nanoagents exhibit elevated cytotoxicity and induce apoptosis in HeLa cells. To load DOX, the amine functionalized magnetic nanoparticles (CoFe2O4-FA) were converted to acid terminated particles through ring opening reaction with succinic anhydride in the presence of DMAP. For this purpose, 10 ml aqueous dispersion (0.1 mg mL1) of nanoparticles were washed thoroughly with millipore water and then dispersed in 5 mL of methanol. In a 100 mL round bottom flask, succinic anhydride (0.05g, 0.5 mmol) and DMAP (0.06g, 0.5 mmol) were taken in 15mL methanol followed by addition of CoFe2O4nanoparticles. The total mixture was refluxed for 24 h at 80 C (CoFe2O4-FA COOH). The particles were recovered, washed thoroughly with methanol (5 5 mL), dried at 80 C. 5 mL of aqueous solution containing NHS (41 mg, 0.358 mMol) and EDC (18 mg, 0.0938 mmol) was taken in a 50 ml of round bottom flask and was kept in the dark for 2 h. After that the aqueous solution of DOX (74 mg in 12 mL water, molar ratio FA:DOXz1:5) was added to the above solution followed by addition of CoFe2O4 FA-COOH nanoparticles. The resulting suspension was stirred at room temperature for overnight and the particles (CoFe2O4 FA-DOX) were isolated magnetically. In order to study release behavior, 5 ml suspension of CoFe2O4-FA-DOX particles (0.1 mg mL1) was suspended in a solution containing 0.1mg ml 1 protease from bovine pancreas and 5 ml of PBS (0.1 mg ml 1). The solution pH was adjusted to 3, 5 and 7.4 using 0.1 M HCl and NaOH. Subsequent to incubation for 5, 10, 20, 25h, at 37 C, the nanoparticles were isolated magnetically and the recovered DOX in the supernatant was quantified spectro photometrically in UV spectrophotometer (lmax ¼ 255 nm). One of ordinary skill in the art would have had a reasonable chance of success in combining the teachings of Sikima et al., Morks, and Mohapatra et al. because all of the references are drawn to the delivery of biomaterials for biomedical applications.
In light of the forgoing discussion, the Examiner concludes that the subject matter defined by the instant claims would have been obvious within the meaning of 35 USC 103. Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art before the effective filing date of the instant invention, as evidenced by the references, especially in the absence of evidence to the contrary.
Conclusion
No claims are allowed.
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/TIGABU KASSA/Primary Examiner, Art Unit 1619
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