MODIFIED CYCLODEXTRIN COATED MAGNETITE NANOPARTICLES FOR TARGETED DELIVERY OF HYDROPHOBIC DRUGS

Abstract

The invention discloses a composition comprising surface modified iron oxide nanoparticles with citric acid modified cyclodextrin with a hydrodynamic diameter of less than 10 nm and a hydrophobic molecule.

The composition finds use in targeted delivery of a hydrophobic drug and as contrast agent in imaging applications.

Claims

1. Beta-cyclodextrin-citrate coated magnetic nanoparticles of size 3 to 10 nm.

2. A process for the preparation of Beta-cyclodextrin-citrate coated magnetic nanoparticles comprising the steps of: i. dissolving β-cyclodextrin and citric acid in a ratio ranging between 3:1 to 5:2 in water followed by stirring the mixture at a temperature in a range of 70-80° C. for period in the range of 3-4 hrs to obtain a transparent solution; ii. treating the transparent solution as obtained in step (i) with alcohol, followed by washing and drying at a temperature in the range of 60-70° C. for a period in the range of 24-26 hrs to obtain cyclodextrin-citrate complex; iii. dissolving cyclodextrin-citrate complex as obtained in step (ii) in water to obtain a cyclodextrin-citrate solution; iv. mixing ferric chloride hexahydrate and ferrous chloride tetrahydrate molar ratio ranging from 2:1 to 5:3 in water and ammonium hydroxide solution followed by stirring and washing to obtain magnetic nanoparticle; v. redispersing the magnetic nanoparticles as obtained in step (iv) followed by adding the Cyclodextrin-citrate solution as obtained in step (iii) with stirring for period in the range of 4-5 hours at temperature in the range of 80-90° C. to obtain a stable dispersion of beta-cyclodextrin coated magnetite nanoparticles; vi. dialyzing the beta-cyclodextrin coated magnetite nanoparticles dispersion obtained in step (v) against water for a period in range of 3-4 days and at a temperature in the range of 60-70° C. to obtain coated solid nanoparticles; vii. dispersing the coated nanoparticles obtained in step (vi) in water at the physiological pH to obtain Beta-cyclodextrin-citrate coated magnetite nanoparticles;

3. The Beta-cyclodextrin-citrate coated magnetic nanoparticles as claimed in claim 1, wherein the coated nanoparticles are useful for targeted drug delivery.

4. The Beta-cyclodextrin-citrate coated magnetic nanoparticles as claimed in claim 1, wherein said coated nanoparticles are loaded with hydrophobic drugs.

5. The process for preparation of hydrophobic drugs loaded beta-cyclodextrin-citrate coated magnetic nanoparticles comprising the steps of: a. dissolving curcumin in solvent followed by adding beta-cyclodextrin-citrate coated magnetite nanoparticles as obtained in claim 6 and stirring gently for 6-8 hrs to obtain a mixture; b. stirring the mixture as obtained in step (a) for a period in the range of 8-12 hrs followed by centrifuging at 4000-5000 rpm for a period in the range of 5-8 minutes to followed by drying to obtain Curcumin loaded beta-cyclodextrin-citrate coated magnetite nanoparticles.

6. The process as claimed in step (a) wherein the solvent used is selected from acetone, cyclohexane, DMSO, etc.

7. The hydrophobic drugs loaded beta-cyclodextrin-citrate coated magnetic nanoparticles as claimed in claim 4 wherein the hydrophobic drugs are selected from curcumin, doxorubicin, taxol, methotrexate, vincritine and such like.

8. The hydrophobic drug loaded beta-cyclodextrin-citrate coated magnetic nanoparticles as claimed in claim 4, wherein the drug loaded is curcumin.

9. The hydrophobic drug loaded beta-cyclodextrin-citrate coated magnetic nanoparticles as claimed in claim 4, wherein the coated nanoparticle is useful as contrast enhancement agents in MRI Scanning

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1 depict IR spectra of citric acid (CIT), citric acid-cyclodextrin complex (CD-CIT), β-cyclodextrin (CD), citric acid (CIT) and the CD-CIT coated magnetite nanoparticles (CDmf).

[0047] FIG. 2 depict (a) TEM image of CDmf with the inset showing a single particle of size 5 nm, and (b) the log-normal size distribution from DLS measurement showing a mean particle size of 7.7 nm.

[0048] FIG. 3 depicts TGA curves of the CD-CIT complex, CDmf and curcumin loaded CDmf (CDmf20). The inset shows the corresponding differential thermograms (DTG).

[0049] FIG. 4 depict Magnetization curves of the uncoated and different coated iron oxide nanoparticles, measured at room temperature.

[0050] FIG. 5 depict ZFC and FC magnetization curves of (a) uncoated magnetite nanoparticles (Unmf), (b) CIT coated nanoparticles (CITmf), (c) CD-CIT coated nanoparticles (CDmf), and (d) CUR loaded CDmf nanoparticles(CDmf20), measured in a field of 50 Oe. The insets of (c) and (d) show the enlarged FC curves at low temperatures.

[0051] FIG. 6 depict graph showing the amount of CUR loaded per mg of the sample (CDmf10, CDmf20 and CDmf30) compared with CUR inclusion complex of bare β-CD (CD20) and CD-CIT (CD-CIT20). The numbers in percentage represent the loading efficiency.

[0052] FIG. 7 depicts UV-visible spectra of CD-CIT coated (CDmf) and curcumin loaded (CDmf20) nanoparticles dispersed in water. Spectra of CDmf20 dispersed in DMSO as well as in buffer solutions (pH of 7.4 and 5.5) are also shown which show the distinct peak of curcumin.

[0053] FIG. 8 depicts the drug (curcumin) release profile of (a) CDmf and (b) CURmf at pH7.4 and 5.5. The inset of (a) shows the zero order fitting curves of the release profile.

[0054] FIG. 9 depicts the reciprocals of (a) spin lattice (T.sub.1) and (b) spin-spin (T.sub.2) relaxation times plotted against concentration.

DETAILED DESCRIPTION OF THE INVENTION

[0055] The present invention provides a composition that can be used for more efficient loading/encapsulation of a hydrophobic molecule.

[0056] The composition comprises surface modified iron oxide nano particles, wherein the modification is done using an ester of an acid and a biocompatible entity. The acid used is citric acid and the biocompatible entity is β-cyclodextrin (CD).

[0057] The composition comprising the surface modification of iron oxide nanoparticles with the ester of citric acid and β-cyclodextrin and the mean size of 5 nm and the hydrodynamic size as obtained is 7.7 nm. The composition increases the encapsulation efficiency of curcumin into the cyclodextrin cavity and the drug can be targeted to the infected site by an external magnetic field. The CD-citrate coated nanoparticles were treated with curcumin at different weight ratios.

[0058] The presence of magnetic core in the composition is beneficial for using it as a contrast enhancement agent in magnetic resonance imaging (MRI). The synthesized nanoparticles can be a used as a multifunctional probe that can be used in targeted drug delivery, magnetic hyperthermia and contrast enhancement agent in MRI.

[0059] The present invention discloses the process for the synthesis of the composition comprising CD-citrate coated nanoparticles encapsulated with curcumin.

[0060] The coated, curcumin loaded, nanoparticles are water dispersible for delivery of curcumin at the cancerous sites. The as-synthesized nanoparticle which forms a stable fluid in water can be effectively used for targeting and delivery of hydrophobic drug to the affected site.

[0061] In an aspect, the individual components of the composition comprising super paramagnetic iron oxide nanoparticles, β-cyclodextrin, citric acid and curcumin are known to be biocompatible and non-toxic for biomedical applications. The composition is applicable to other hydrophobic entities and anti cancer drugs selected from, but not limited to cisplatin, doxorubicin, taxol, methotrexate, vincritine and such like.

EXAMPLES

[0062] Following examples are given by way of illustration and therefore should not be construed to limit the scope of the invention

Example 1

[0063] Materials: Ferric chloride hexahydrate (≧98%), ferrous chloride tetrahydrate (99%), citric acid monohydrate, curcumin and β-cyclodextrin were purchased from Sigma Aldrich. Ammonium hydroxide (25%), dimethyl sulphoxide (DMSO), nitric acid and 2-propanol were procured from Merck. All the chemicals were used without further purification and double distilled water was used throughout this work.

[0064] Preparation of CD-CIT complex: 3 g of β-cyclodextrin and 1 g of citric acid was dissolved in 10 ml of water and the mixture was stirred at 80° C. for three hours. The transparent solution obtained was treated with 2-propanol, which gave a white precipitate. The product was washed thoroughly to remove unreacted components and further dried at 60° C. for 24 hours to get the white CD-CIT complex. The formation of the product was confirmed by FT-IR.

Example 2

[0065] Preparation of Surface Functionalized Magnetite Nanoparticles:

[0066] Magnetite nanoparticles were prepared by the reverse co-precipitation method. A mixed solution of 2 mmol of FeCl.sub.3.6H.sub.2O and 1 mmol of FeCl.sub.2.4H.sub.2O in water was added to 100 ml of 19% ammonium hydroxide solution under argon atmosphere. The mixture was stirred well for complete formation and growth of magnetite nanoparticles. The nanoparticles were washed with distilled water to remove excess base. Then the pH was brought down to 7 by washing with water and the resultant nanoparticles were re-dispersed in 100 ml distilled water. 2 g of the CD-CIT complex dissolved in water was added drop-wise to the dispersion and stirred for 4 hours at 80° C. The stable dispersion obtained was then dialyzed against water for three days to remove excess CD-CIT complex. The dispersion was then dried at 70° C. to get solid nanoparticles. The coated nanoparticles were well dispersible in water and at the physiological pH to form a nanofluid. The sample was labeled as CDmf. Citric acid coated magnetite nanoparticles were also synthesized following the same procedure for comparison. The nanoparticles coated with citric acid also formed stable dispersion in aqueous media and was labeled as CITmf. Uncoated nanoparticles were also prepared under the same reaction conditions and labeled as Unmf.

[0067] Magnetite nanoparticles directly coated with curcumin was synthesized by the procedure reported earlier. A mixture of ferric chloride hexahydrate and ferrous chloride tetrahydrate, taken in the molar ratio of 2:1, was added to ammonia solution to form magnetite nanoparticles. After stirring for 30 minutes, dilute nitric acid was added to bring down the pH to ˜8-9. Curcumin solution at the same pH was added and the dispersion was stirred for the effective coating of curcumin to magnetite nanoparticles. The final dispersion was dialyzed against water to remove unreacted excess curcumin and ammonia. The dispersion was dried to get a powder which forms stable dispersion in dimethyl sulfoxide. The curcumin encapsulated sample was labeled as CURmf.

Example 3

[0068] Preparation of CUR Inclusion Complex:

[0069] 20 mg of the CD-CIT coated sample (CDmf) was dispersed in 30 ml water in a 50 ml vial. To this dispersion, varying amounts of curcumin (10 mg, 20 mg and 30 mg), dissolved in 1 ml acetone, were added while stirring gently. The mixture was stirred for 6 hours to evaporate acetone. The dispersion was then stirred overnight and centrifuged at 5000 rpm for 5 minutes. The supernatant liquid which contains highly water dispersed inclusion complex was dried and stored at 5° C. for further use. The resultant inclusion complexes were labeled as CDmf10, CDmf20 and CDmf30. Inclusion complexes were also prepared using CD alone and the CD-CIT conjugate using 20 mg curcumin and 20 mg of the compound. They were designated as CD20 and CD-CIT20, respectively.

Example 4

[0070] Curcumin Loading Studies:

[0071] 1 mg of the solid curcumin inclusion complex was dispersed in 10 ml dimethyl sulfoxide (DMSO) to extract the curcumin to the solvent. This dispersion was shaken on a vortex shaker for 24 hours at room temperature. The vial containing the dispersion was covered with an aluminium foil to prevent exposure to light. The dispersion was then centrifuged at 10000 rpm to remove the curcumin-free CD-CIT coated sample and the clear yellow supernatant solution of curcumin in DMSO was collected and used for estimation. The amount of curcumin released was estimated from the absorbance measured at 425 nm using a standard graph of absorbance of curcumin dissolved in DMSO.

[0072] The curcumin entrapment efficiency (EE) is calculated using the formula:

[00001]$\mathrm{EE}\left(\%\right)=\frac{\mathrm{mass}.Math..Math.\mathrm{of}.Math..Math.\mathrm{curcumin}.Math..Math.\mathrm{trapped}}{\mathrm{mass}.Math..Math.\mathrm{of}.Math..Math.\mathrm{curcumin}.Math..Math.\mathrm{used}}\times 100$

[0073] In Vitro Release:

[0074] The release of curcumin from the CD-CIT coated sample was done at pH 7.4 and pH 5.5, by the dialysis bag method. The CDmf20 sample which showed maximum curcumin loading was dispersed in the phosphate buffer (pH=7.4) at a concentration of 1 mg/ml, sonicated to form a stable dispersion and was transferred to a dialysis bag. The dialysis bag tied at both ends was immersed in 50 ml buffer solution and stirred gently. 2 ml of the buffer was withdrawn at particular intervals and replenished with the same amount of fresh buffer. The absorbance was measured at 425 nm, the λ.sub.max for curcumin. The amount of curcumin released was then plotted against time. Release rate of curcumin was also determined using acetate buffer (pH=5.5) using the same procedure.

[0075] As seen in the FIG. 3, the amount of CUR released from β-CD cavity is less at pH 5.5 initially compared to pH 7.4. In the case of CDmf there is an initial burst release whereas the curcumin directly coated to magnetite nanoparticles shows a pulsatile release at the initial stage itself.

[0076] Characterization:

[0077] Phase purity of the iron oxide nanoparticles was determined by powder X-ray diffraction (XRD) using a PANalytical X'PERT PRO model X-ray diffractometer, in the 20 range of 10 to 80 degrees, using Cu Kα radiation. TEM analysis was performed on a FEI, TECNAI G2 TF30 instrument. Samples were prepared by placing a drop of dilute dispersion on a carbon coated 200 mesh copper grid and imaged at an accelerating voltage of 300 kV. Zeta potential and hydrodynamic particle size were measured using the dynamic light scattering (DLS) technique using a Brookhaven instruments 90Plus Particle Size Analyzer equipped with a 632.8 nm laser. Infrared spectra were recorded on a Tensor 27 Bruker FT-IR spectrometer, using KBr pellets, in the frequency range of 4000-400 cm.sup.−1. Thermogravimetric analysis (TGA) of the synthesized samples, in air, was performed on a Perkin-Elmer TGA7 analyzer.

[0078] Magnetic measurements were carried out on a Quantum Design MPMS 7TSQUID-VSM. Zero field cooled (ZFC) and field cooled (FC) magnetization measurements were carried out in an applied field of 5 mT (50 Oe) and magnetization versus field measurements, at room temperature, were carried out from −3 T to +3 T. UV-Visible spectra were recorded using a Cary 5000 UV-Vis-NIR spectrophotometer and the measurements were carried out in a Quartz cell of 10 mm path length. The absorbance measurements for the study of curcumin release were also done on the same instrument. Fluorescence measurements were performed using a Photon Technology International fluorescence QM40 spectrophotometer with a Quartz cell of 10 mm path length. The T.sub.1 and T.sub.2 relaxation studies were done on a Bruker AV400 NMR spectrometer at a magnetic field of 9.4 Tesla and 400 MHz frequency.

[0079] The IR spectra of CD, citric acid, CD-CIT complex and CDmf20 are shown in FIG. 1. The spectra of the CD-CIT complex resemble the spectra of CD. The major bands at 3350 cm.sup.−1, 2925 cm.sup.−1, 1158 cm.sup.−1 and 1029 cm.sup.−1 of CD correspond to the stretching vibrations of —OH, —CH.sub.2, —C—C and bending vibration of —OH groups, respectively. The band at 1645 cm.sup.−1 corresponds to the H—O—H deformation band of water present in the cavity of CD. The band at 1750 cm.sup.−1 in the spectra of citric acid is due to the vibration of the C═O group of the carboxylic acid and this band is shifted to 1730 cm.sup.−1 in CD-CIT due to the formation of ester. The band at 1730 cm.sup.−1, which is due to the C═O stretching of the ester group, is a clear indication for the formation of the CD-CIT conjugate. The intensity of this band of CD-CIT is reduced in CDmf, after coating on the magnetite nanoparticles, indicating that the CD-CIT conjugate binds to the nanoparticle via the C═O group of the citric acid. The bands in the IR spectra of the coated nanoparticles resemble that of the CD-CIT conjugate indicating the bonding of the conjugate to nanoparticles surface. The band at 1645 cm.sup.−1 corresponding to the vibrations of water in the CD cavity is found to be retained in the coated nanoparticles also, indicating that the iron oxide nanoparticles are not occupied inside the cavity and binds to the CD-CIT complex without disturbing the cavity.

[0080] The average crystallite size of the CDmf nanoparticles is calculated as 5 nm from the XRD pattern using the Scherrer equation. The TEM image in FIG. 2(a) shows isolated particles with average particle size of 5 nm, comparable to the crystallite size. Average particle size of 7.7 nm, with a polydispersity of 0.261, is obtained from DLS measurements as shown in FIG. 2(b).

[0081] TGA curve of CDmf is compared with that of CD and the CD-CIT conjugate in FIG. 3. The total weight loss for CDmf is about 60% and the weight loss path resembles that of bare CD-CIT conjugate, except for a shift in the third weight loss to higher temperatures. Dehydration of CDmf and CD-CIT caused a total mass loss of 8.2% and 8.5% (first weight loss below 100° C.), and this corresponds to loss of 6.3 and 6.1 water molecules, respectively, from the cavity of CD. The dehydration of CDmf20 results in a mass loss of 6.1%, indicating the removal of 4.5 water molecules from the CD cavity.

[0082] The M vs H curves of the iron oxide samples measured at room temperature, before and after surface modifications, are shown in FIG. 4.

[0083] The zero field cooled (ZFC) and field cooled (FC) magnetization curves of the uncoated and the different coated nanoparticles are compared in FIG. 5. The superparamagnetic blocking temperature (T.sub.B), corresponding to the temperature at which a maximum is observed in the zero field cooled (ZFC) magnetization curve, for the uncoated (Unmf) and citric acid coated (CITmf) samples are obtained as 110 K and 40 K, respectively.

[0084] CDmf and the inclusion complex CDmf20 show almost comparable values of T.sub.B as 20 K. The FC curve of Cdmf shows a saturating trend at very low temperatures (inset of FIG. 5(c)) whereas this trend is not observed for CDmf20 (inset of FIG. 5(d)).

[0085] The curcumin inclusion complexes, CDmf10, CDmf20 and CDmf30, are analyzed for their curcumin loading capacity (FIG. 6). The encapsulation efficiency was found to be higher in the case of 1:1 weight ratio of the sample and curcumin (CDmf20) is used (20 g each). The encapsulation efficiency of the coated nanoparticles is also compared with that of bare CD as well as the CD-CIT complex (FIG. 6). Higher efficiency is observed for CD-CIT (4.1%) compared to CD (1.5%). The solubility of bare β-cyclodextrin in water is found to be ˜18mg/ml, whereas the solubility of CD-CIT complex is obtained as ˜60 mg/ml. PLGA nanoparticles show a maximum curcumin loading of about 5-10%.

[0086] The zeta potential of the different formulations is measured by dispersing them in water. The zeta potential of CDmf is measured as −19.2 mV. CITmf also gave stable water dispersion with a zeta potential of −21.8 mV. The zeta potential for CDmf10, CDmf20 and CDmf30 are obtained as −33.2, −30.3 and −35.8 mV, respectively, indicating the high stability of the dispersions.

[0087] The UV-visible spectra also do not show any sharp peak at 425 nm which is the characteristic absorption maximum of curcumin. However, the inclusion complex once treated with dimethyl sulfoxide (DMSO) gives the characteristic peak of curcumin, as shown in FIG. 7, where the UV spectra of CDmf dispersed in different media are compared.

[0088] The release profile of CUR from CDmf20 sample was analyzed at the physiological pH 7.4 and that of the diseased cells pH 5.5. As shown in FIG. 8, the amount of CUR released from the CD cavity is very low at pH 5.5, initially, compared to that released at the physiological pH 7.4.

[0089] The release profile of CURmf follows the zero order kinetics at both the investigated pH values from the initial time itself whereas the CDmf shows a burst release of CUR followed by constant release. The amount of CUR released from the CDmf sample at a particular time is larger than that compared to CURmf.

[0090] The relaxivity of cyclodextrin coated magnetite nanoparticles is measured on an NMR spectrophotometer at a magnetic field of 9.4 T and frequency of 400 MHz. The CDmf sample was dispersed in water at different concentrations and the spin-lattice relaxation time T.sub.1 and spin-spin relaxation time T.sub.2 are measured. The reciprocals of the relaxation times are plotted against concentration (FIG. 9) to obtain the corresponding relaxivity values r.sub.1 and r.sub.2 which describe the ability to shorten the relaxation times per millimole of the concentration of contrast agent. The relaxivity values, r.sub.1 and r.sub.2, calculated from the slopes of the plots are 0.0082 mM.sup.−1s.sup.−1 and 6.875 mM.sup.−1s.sup.−1, respectively. The r.sub.2/r.sub.1 ratio is obtained as 838. The r.sub.2 and r.sub.1 values are calculated by considering the particle diameter as 5 nm as obtained from TEM, which will have approximately 880 magnetic iron ions. The r.sub.2/r.sub.1 ratio is larger than the minimum threshold (=2) value required to be used as an effective contrast agent.

Advantages of the Invention

[0091] Higher loading of drug [0092] Smaller hydrodynamic diameter of complex facilitating better lodging of hydrophobic drug