Patent Description:
Metal compounds and complexes are widely used for treating and detecting disease and they are playing an increasingly important role in the emerging field of nanomedicine. Gold nanoparticles (AuNP) in particular offer a number of attractive features for visualization, detection, and treatment of disease. They exhibit a range of surface chemistries for drug or biomaterial modification and, when internalized by the cell, they appear to have minimally toxic effects. For example, DNA-capped AuNP has been used as intracellular gene regulation agents for the control of protein expression in cells, and platinum drugs conjugated to AuNP show considerable promise as chemotherotherapeutic agents. In addition to drug attachment, the NP core itself could be used in treatment strategies and one emerging approach is photothermal therapy, in which the particle is heated to cause damage to the cell.

Most clinically used anticancer drugs have relatively narrow therapeutic windows indicating that the distribution of the drug between normal and diseased tissue is small. For example, the anthracycline antibiotic doxorubicin (DOX, or adriamycin) is a clinically approved chemotherapy agent that binds to DNA via sequence specific intercalation. The binding mechanism involves intercalation of the aglycone portion of the drug at a high affinity site, e.g., <NUM>'-CG/CG, with the daunosamine sugar lying in the minor groove, occluding an additional DNA base pair adjacent to the intercalation site. When bound to DNA, the drug inhibits the enzyme topoisomerase II and the action of DNA polymerase causing cell death. DOX intercalation stabilizes the double-helix, which has been shown to result in an increase in thermal denaturation (e.g. melting) temperature of duplex DNA.

<NPL> discloses the binding of a first oligonucleotide to gold nanoparticles (AuNP), and the binding of a fluorophore to a second, oligonucleotide which is complementary to the first one. The fluorophore-containing oligonucleotide is released at a certain temperature. <NPL> discloses AuNP which are linked to a single-stranded DNA. The AuNP are used to administer a complementary antisense-DNA which is released from the AuNP by light. <CIT> and <NPL> describe the binding of double-stranded DNA to AuNP. A fluorescent moiety is bound to the first strand, and a quencher is bound to the second strand. The moieties are separated by a DNAse which results in fluorescence. <CIT> discloses nanoparticles which are coated with organized self-assembled monolayers (SAMs) and present nucleic acid binding entities, such as complementary nucleic acids. The nanoparticles are used for loading a carrier vehicle with RNAi. <CIT> discloses the attachment of single-stranded DNA to an AuNP. Complementary DNA, e.g. antisense therapeutic, is bound to a dye as label. The label is released by using external electromagnetic energy. <NPL> discloses the binding of single-stranded DNA to AuNP. Dye-tagged complementary DNAs of different lengths are complexed with these AuNP. <CIT> describes the use of double-stranded oligonucleotides for linking a doxorubicin molecule to iron oxide nanoparticles.

It is therefore a principal object and advantage of the present invention to provide a system and method for delivering doxorubicin.

In accordance with the foregoing objects and advantages, the present invention comprises a system and method for loading the front line anticancer drug, doxorubicin (DOX) onto DNA-capped gold nanoparticles whose duplex DNA has been designed for specific DOX intercalation. Drug binding was confirmed by monitoring the increase in DNA melting temperature, the shift in the plasmon resonance maximum, and the increase in the NP hydrodynamic radius as a function of [DOX]/[DNA] ratio. Specifically, the present invention provides a drug delivery molecule, comprising: (a) a gold nanoparticle; (b) a first nucleic acid molecule covalently linked to said gold nanoparticle, wherein said first nucleic acid molecule comprises three doxorubicin binding sites of the sequence (<NUM>'-TCG-<NUM>'); and (c) a second, complementary nucleic acid molecule hybridized to said first nucleic acid molecule to form a double-stranded nucleic acid molecule. In a preferred embodiment, the second nucleic acid molecule comprises the sequence of SEQ ID NO:<NUM>. In another preferred embodiment, the first nucleic acid molecule comprises the sequence of SEQ ID NO: <NUM>. In another preferred embodiment, the drug delivery molecule further comprises a plurality of doxorubicin molecules bound to said plurality of binding sites. In another preferred embodiment, the first nucleic acid molecule is longer than said second, complementary nucleic acid molecule. In another preferred embodiment, the gold nanoparticle has a diameter of about <NUM> nanometers. In another aspect, the invention relates to a method of forming a drug delivery molecule, said method comprising the steps of: (a) covalently linking a first nucleic acid molecule to a gold nanoparticle, wherein said first nucleic acid molecule comprises three doxorubicin binding sites of the sequence (<NUM>'-TCG-<NUM>'); and (b) hybridizing a second, complementary nucleic acid molecule to said first nucleic acid molecule to form a double-stranded nucleic acid molecule. In a preferred embodiment, the second nucleic acid molecule comprises the sequence of SEQ ID NO:<NUM>. In another preferred embodiment, the first nucleic acid molecule comprises the sequence of SEQ ID NO:<NUM>. In another preferred embodiment, the step of covalently linking a first nucleic acid molecule to a gold nanoparticle further comprises covalently linking a plurality of said first nucleic acid molecules to said gold nanoparticle. In another preferred embodiment, the step of hybridizing a second, complementary nucleic acid molecule to said first nucleic acid molecule to form a double-stranded nucleic acid molecule further comprises hybridizing a plurality of said second, complementary nucleic acid molecules to said plurality of said first nucleic acid molecules to form a plurality of said binding sites. In another preferred embodiment, a plurality of doxorubicin molecules are bound to said plurality of binding sites. In another preferred embodiment, each of said plurality of first nucleic acid molecules is longer than each of said second nucleic acid molecules. In another aspect, the invention relates to a method of forming a drug loaded nanoparticle, said method comprising: (a) providing at least one gold nanoparticle covalently linked to a plurality of first nucleic acid molecules, each of which is hybridized with a plurality of second, complementary nucleic acid molecules to form a plurality of double-stranded nucleic acid molecule each having a binding site with a high affinity for a doxorubicin molecule, wherein said first nucleic acid molecule comprises three doxorubicin binding sites of the sequence (<NUM>'-TCG-<NUM>'); and (b) binding a plurality of said doxorubicin molecules to said binding sites to form a doxorubicin loaded nanoparticle. In a preferred embodiment, the second nucleic acid molecule comprises the sequence of SEQ ID NO:<NUM>. In another preferred embodiment, the first nucleic acid molecule comprises the sequence of SEQ ID NO:<NUM>. In another preferred embodiment, the gold nanoparticle has a diameter of about <NUM> nanometers.

The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which:.

Referring now to the drawings, wherein like reference numerals refer to like parts throughout, there is seen in <FIG> a system according to the presention invention that uses the sequence specificity of DOX and engineer two DNA-capped AuNPs, one capable of binding multiple copies of DOX with high affinity and a second having reduced binding for drug. The particles were prepared by covalently attaching multiple copies of a <NUM>'-thiol-modified <NUM>-mer oligonucleotide to the surface of <NUM> ± <NUM> AuNP followed by hybridization of the complementary <NUM>-mer, 1b or 2b, to yield 1ab/2ab-AuNP. The 1ab duplex was designed to include three identical high affinity DOX intercalation sites having the sequence, <NUM>'-TCG. Binding sites were separated by a <NUM>-nucleotide spacer, allowing each 1ab to simultaneously bind three DOX molecules. The duplex 2ab was designed to have length and melting characteristics similar to 1ab, but to lack high affinity DOX intercalation sites. In order to quantify the number of 1ab or 2ab at each AuNP (e.g. dsDNA), a fluorescently tagged 1b was employed, 1b-CY3. Fluorescence spectroscopy was then utilized to determine the quantity of 1b-CY3 hybridized at the 1a-AuNP. Using this approach an average number of <NUM> ± <NUM>1ab/AuNP was measured. From this value and the spectrophotometrically determined concentration of AuNP, the value of, [DOX]/[1ab] = r, was determined for the various experiments. Considering that three binding sites exist per 1ab, one can expect ∼<NUM> high affinity DOX sites per AuNP.

The 1ab-AuNP (with [AuNP] = <NUM>) were incubated with DOX, at r = <NUM> to <NUM> for <NUM> at RT in PBS (<NUM> Phosphate buffer, <NUM> NaCl, pH = <NUM>), and separated from unbound drug by centrifugation. To probe the uptake of DOX via intercalation to 1ab-AuNP, we investigated the thermal denaturation of the 1ab strands by measuring increase in extinction at <NUM> using UV-visible spectroscopy (UV-vis). The melting of duplex DNA at NP interfaces is known to be influenced by a number of cooperative effects resulting from the confined DNA environment at the nano-interface, namely; increased local DNA concentration and ionic strengths. These effects are known to result in an increase in melting cooperativity, as well as an increase in Tm.

<FIG> shows the observed melting profiles as a function of r. At r = <NUM>, the 1ab-AuNP exhibits cooperative melting associated with duplex 1ab with Tm = <NUM>. However, upon addition of drug, the Tm increases from <NUM> to <NUM> in the range, r = <NUM> to ~<NUM> (Fig. 1a). This is consistent with drug binding to DNA on 1ab-AuNP thereby increasing Tm. An interesting aspect of the melting profiles shown in <FIG> is the low Tm feature between <NUM>-<NUM> when r >-<NUM>. This reversible melting feature appears to be the result of increased drug loading on the DNA and/or particle when all high affinity DOX sites on 1ab are saturated (r~<NUM>). This feature strongly correlates with the surface plasmon resonance band, λSPR, of the AuNP (<FIG>), which undergoes a λSPR red shift from <NUM> to <NUM> at r><NUM>. A red shift in λSPR is indicative of a change in the dielectric medium immediately surrounding the AuNP, and/or interactions between AuNP as the result of assembly or clustering. Such clustering is likely the result of the increasingly hydrophobic nature of 1ab-AuNP at high r, which can approach ~<NUM> if all DOX present is bound to AuNP.

Drug binding to 1ab-AuNP was also probed via dynamic light scattering (DLS). DLS measures the hydrodynamic diameter, Dh, of the 1ab-AuNP, which is influenced by both the diameter of AuNP as well as the thickness of the DNA shell. Since drug initially binds at each site on 1ab by intercalation, we can expect an increase in the length of 1ab by ~<NUM> (<NUM> x <NUM>. 6Å), which would correlated with a net increase of ~<NUM> in Dh. <FIG> shows a Dh increase with r of about ~<NUM> at r~<NUM>, and a net increase of ~<NUM> at r><NUM>. This observation is consistent with drug intercalation at r < ~<NUM> followed by less specific interaction at r>~<NUM>.

In order to better understand the interaction between DOX and 1ab-AuNP, parallel experiments were carried out with 2ab-AuNP which contains DNA sequences that more weakly bind drug. At r = <NUM>, the Tm value for 2ab-AuNP is <NUM> (<FIG>) and the increase in Tm with r is notably less than with 1ab-AuNP, <FIG>. Interestingly, for 2ab-AuNP there is no change in either λSPR (<FIG>) or Dh (<FIG>) with r which collectively suggest that DOX binding to this gold bound duplex is weaker than for 1ab and that the binding mechanism may be non-intercalative in nature.

If 1ab-AuNP is to potentially serve as a carrier for delivering high payloads of drug to a tumor site, it is important to show that DOX can be released from the loaded particle to a receptor target DNA. That this is possible was demonstrated by loading a dialysis membrane with DOX-1ab-AuNP (r = <NUM>), exposing the membrane to a solution containing a high concentration of calf thymus DNA (CT-DNA) and after <NUM>, measuring the concentration of DOX transferred from particle-DNA to the target CT-DNA outside the membrane (see supporting information). Spectrophotometric analysis showed that ~<NUM>% of the DOX originally bound to 1ab-AuNP was transferred to CT-DNA, demonstrating that DOX can be released from particle-DNA and captured by a receptor DNA.

These results show that 1ab-AuNP binds DOX at high affinity sites (<NUM>'-TCG), and once these sites are saturated (r~<NUM>) drug continues to weakly bind to other regions of the DNA and/or AuNP, increasing AuNP hydrophobicity. Moreover, the particle-bound drug can be transferred to a receptor DNA, raising the possibility that drug delivered by the particle to a cell could be available for interaction with genomic DNA. Aside from simple diffusion of bound drug away from particle DNA, it may be possible to initiate drug release by photothermal melting, apatmer recognition, and/or degradation of the drug-DNA complex by nucleases. These studies are part of our ongoing work to explore the potential of AuNPs as new delivery vehicles for clinically approved anticancer drugs.

All materials, unless otherwise specified, were purchased from Sigma Aldrich. All oligonucleotides, including 1a/2a precursors, 1b/2b, and 1b-CY3 (and excluding CT-DNA) were purchased from Integrated DNA Technologies.

The gold nanoparticles (AuNP) with average diameters of <NUM> ± <NUM> AuNPs were synthesized by standard citrate reduction method (<FIG>). Next, the AuNP were functionalized with ssDNA using methods for high DNA coverage (<FIG>). Briefly, the 1a and 2a, compounds of <FIG>, were purchased as disulfides, and first reduced using dithiothrietol, to produce 1a/2a (containing a <NUM>'-terminal thiol), which were then purified using a Sephadex G-<NUM> column. Next, the AuNP were incubated with 1a or 2a at 300x molar ratio ([1a]/[AuNP]), and then subjected to the salt aging process (S1, <NUM>). The [1a] and [2a] stock concentrations were determined using UV-visible spectroscopy (UV-vis), based on extinction coefficients, ε<NUM> = <NUM>,<NUM> and <NUM>,<NUM>-<NUM>cm-<NUM>, respectively. The [AuNP] was similarly determined based on ε<NUM> = <NUM> ×<NUM><NUM> M-<NUM>cm-<NUM>.

The 1a-AuNP or 2a-AuNP were then purified via centrifugation. The average DNA loading on each AuNP (~<NUM> ± <NUM>) was estimated based on measurement of DNA uptake, as measured during purification. The number of 1ab molecules per AuNP was later confirmed using fluorescence spectroscopy (<NUM>), see below.

In a typical hybridization experiment, 1a- or 2a-AuNP was combined with <NUM> molar excess of the respective partial complement 1b, or 2b, forming 1ab or 2ab dsDNA functionalized AuNP (1ab-AuNP, 2ab-AuNP). To promote full hybridization, the solution was heated to <NUM>, and allowed to cool to room temperature for <NUM>. The 1ab- or 2ab-AuNP were then purified of free 1b or 2b via centrifugation. All final 1ab- or 2ab-AuNP was resuspended in PBS (<NUM> NaCl, <NUM> Phosphate buffer, pH=<NUM>). Washing/resuspension was repeated at least three times.

Fluorescence spectroscopy using a dye-modified 1b ssDNA was used to determine the average loading of 1ab on AuNP. Briefly, samples of 1a-AuNP with 1b-CY3 (1b modified by covalent attachment of the fluorescent dye, CY3) as described above were used, except that 1a-AuNP was combined with <NUM> molar excess of 1b-CY3. Following hybridization, each solution was centrifuged, removing the 1ab-Cy3-AuNP, and the supernatant containing excess 1b-CY3 was removed and compared to a concentration calibration curve prepared for 1b-CY3. Each experiment was performed in triplicate. Using this method (<NUM>), an average number of <NUM> ± <NUM>1ab dsDNA was calculated at each AuNP. Using this value, it was possible to calculate the appropriate number of DOX drugs to add to a known concentration of AuNP, as described next.

We next utilized the 1ab- or 2ab-AuNP prepared above, with dsDNA-functionalization, for drug (DOX) loading. For each DOX binding and melting experiment, 1ab- or 2ab-AuNP were incubated for <NUM> with DOX at specific ratios, r = [DOX]/[1ab/2ab]. In this study, we explored r = <NUM> - <NUM>, based on the number of 1ab per AuNP, as well as the number of binding sites at each 1ab. Next, each DOX-1ab-AuNP was analyzed via thermal denaturation melting experiments using temperature controlled UV-vis at Abs = <NUM> and a heating rate of <NUM>/min from <NUM> to <NUM>. Each melting temperature was taken as the maximum of a peak-fitted first derivative plot of its corresponding melting curve (using PeakFit® Peak Separation and Analysis Software, V. The [DOX] was calculated for DOX stock solutions based on ε<NUM> = <NUM>,<NUM>-<NUM>cm-<NUM>(<FIG>).

The monitor the release of DOX bound to the 1ab-AuNP, we employed a transfer dialysis experiment. Briefly, a concentrated solution ([AuNP] = <NUM>) of DOX-1ab-AuNP (r = <NUM>) was placed inside a dialysis membrane (Spectra/Por Biotech regenerated cellulose dialysis membrane, MWCO = 15kD), and the loaded membrane was placed in a <NUM> x <NUM>-<NUM> M solution of calf thymus DNA (CT-DNA) in PBS (<NUM> phosphate buffer, <NUM> NaCl, pH = <NUM>) and stirred for <NUM>. After this time, a UV-vis spectral analysis of the solution containing CT-DNA showed the characteristic spectrum of DOX bound to DNA. Using, ε<NUM> = <NUM>,<NUM>-<NUM>cm-<NUM>, (<FIG>), the [DOX] in the solution containing CT-DNA was calculated and the percentage of particle-bound drug transferred to the CT-DNA outside the membrane was determined to be <NUM>%. Two controls, which only deviated from the experimental conditions by the contents of the dialysis membrane, were carried out. A control containing only 1ab-AuNP in the dialysis membrane verified that no AuNP passed through the membrane, and a second control containing only DOX in the dialysis membrane verified that DOX passes through the membrane and binds CT-DNA to produce the spectrum noted in the drug transfer experiment.

UV-visible Absorption (UV-vis): The UV-vis measurements were collected on a Varian Cary <NUM> Bio UV-vis spectrophotometer between <NUM>-<NUM>. The instrument is equipped with an <NUM>-cell automated holder with high precision Peltier heating controller.

Dynamic Light Scattering (DLS): Dynamic Light Scattering (DLS) measurements were collected using a Malvern Zetasizer ZS instrument equipped with a <NUM> laser source, and a backscattering detector at <NUM>°.

The PL emission and excitation measurements were collected on a Fluoromax-<NUM> photon counting spectrofluorometer (Horiba Jobin Yvon). The instrument is equipped with a 150W xenon white light excitation source and computer controlled monochromator. The detector is a R928P high sensitivity photon counting detector that is calibrated to emission wavelength. All PL emission and excitation spectra were collected using both wavelength correction of source intensity and detector sensitivity.

Transmission Electron Microscopy (TEM): TEM measurements were performed on either a FEI T12 Twin TEM operated at <NUM> kV with a LaB6 filament and Gatan Orius dual-scan CCD camera (Cornell Center for Materials Research), or a JEOL 2000EX instrument operated at <NUM> kV with a tungsten filament (SUNY-ESF, N. Brown Center for Ultrastructure Studies). Particle size was analyzed manually by modeling each qdot as a sphere, with statistical analsysis performed using Imaged software on populations of at least <NUM> counts.

If 1ab-AuNP is to potentially serve as a carrier for delivering high payloads of drug to a tumor site, it is important that DOX can be released from the loaded particle to a receptor target DNA. In addition to DNA melting, we also investigated DOX release to receptor DNA in solution using a dialysis membrane loaded with DOX-<NUM> ab-AuNP (at rd = <NUM>), and exposed to a solution containing a high concentration of duplex calf thymus DNA (CT-DNA). The dialysis membrane itself serves only to separate the AuNP from DOX for UV-vis analysis. The DOX release, and uptake by CT-DNA was then measured by UV-vis (<NUM>) in which we measured the release of DOX from the 1ab-AuNP. In this test, -<NUM>% of DOX was released after only <NUM>, demonstrating a significant drug release in a timely manner to a receptor DNA.

Cytotoxicity experiments were performed using neuroblastoma (SK-N-SH) cells under standard conditions in an incubator, using Eagle's minimum essential media (MEM) containing <NUM>% fetal bovine serum (FBS), <NUM>µg/mL streptomycin, and <NUM> IU/mL penicillin. Solutions containing free DOX and 1ab-AuNP loaded with DOX at a saturated loading ratio (<NUM> DOX/1ab), were prepared at [DOX] = <NUM>, <NUM>, <NUM>, and <NUM>, for <NUM> exposure, <NUM> recovery, in media. Cells were plated in a <NUM>-well microplate, and, following incubation, media was removed, and media containing specified concentrations of drug were added. Following exposure time, media containing drug was removed, and cells were washed with fresh media. Following recovery time, cell viability was determined using the CCK-<NUM> assay. To evaluate the degree of cytotoxicity attributable to the DNA-AuNP in the absence of drug, a control experiment was performed using 1ab-AuNP at an effective [DOX] = <NUM>, <NUM>, <NUM>, and <NUM>, for a <NUM> exposure, and <NUM> recovery time.

In these preliminary cytotoxicity experiments, the DOX -1ab/2ab-AuNP systems produced similar inhibition in comparison to DOX alone. The 1ab-AuNP control was observed to not produce inhibition at any concentrations explored. Interestingly, the DOX-1ab-AuNP was noted to produce a greater percent inhibition at the lowest two [DOX] = <NUM> and <NUM>, in comparison to DOX alone, demonstrating the potential of the DOX-1ab-AuNP systems to effectively kill cells at low concentration. The internalization of a single DOX 1ab/2ab-AuNP is perhaps extremely likely to kill a cell when internalized, whereas DOX internalization may result in only a slightly increased likelihood of cell death, and it may take many internalized DOX molecules to kill a cell. Upcoming cytotoxicity experiments will likely clarify the inhibition differences between DOX-1ab/2ab-AuNP and DOX alone.

Collectively, these results demonstrate that DNA-capped nanoparticles can be designed to bind multiple copies of an intercalating drug. The 1ab-AuNP is clearly shown to bind DOX at high affinity sites, which indicated by Tm and Dh elevation. The particle-bound DOX was shown to be released and transferred under mild conditions to a receptor DNA. Preliminary cytotoxicity studies demonstrated similar inhibition of neuroblastoma cells by the DOX-1ab/2ab-AuNP systems as by the drugs alone and slightly elevated inhibition at low concentration. Fluorescence-based kinetic drug transfer experiments are in progress, and are intended to determine the rate and extent of drug release. Other upcoming work includes varying conditions of cytotoxicity studies.

Most clinically used anticancer drugs have relatively narrow therapeutic windows indicating that the distribution of the drug between normal and diseased tissue is small. The present invention provides for multiple copies of certain clinically used anticancer drugs to be attached to particle bound DNA to yield a new type of drug delivery device. When the device is equipped with a vector capable of targeting a cancer cell, it will be possible to direct high copy numbers of drug to tumors. As the gold nanoparticles with attached DNA can be internalized by the cell, the present invention is capable of delivering a high payload of drug specifically to cancer cells, thereby effectively enhancing the therapeutic window of the antitumor agent. An additional attractive feature of the present invention is that since existing clinically used anticancer drugs can be used, FDA approval of the drug which is incorporated into the device, is not required. Implementation of the present invention may require determining the cytotoxicity of the invention toward various cancer cell lines, incorporation of a cellular targeting vector, equipping the device with other DNA binding drugs, and exploring methods of triggering drug release such as degradation of DNA by nucleases and photothermal heating.

The nanoparticle delivery system of the present invention may be adapted to deliver other drugs by tailoring the DNA encoding to accommodate additional chemotherapy drugs, such as actinomycin D (ActD), epirubicin (EPI), idarubicin (IDA), and bleomycin (BLM). The present invention can also incorporate folic acid (FA) and RGB targeting vectors and PEG groups to the dsDNA functionality. A list of the ssDNA sequences having binding affinity for these drugs are shown in Table <NUM> below. We recently began a collaboration with the drug company Transo-Pharm GmbH, which has provided samples of DOX, EPI and IDA (see letters). Transo-Pharm became interested in our work from our recent publication (<NUM>), and is currently evaluating our preliminary patent.

Claim 1:
A drug delivery molecule, comprising:
(a) a gold nanoparticle;
(b) a first nucleic acid molecule covalently linked to said gold nanoparticle, wherein said first nucleic acid molecule comprises three doxorubicin binding sites of the sequence (<NUM>'-TCG-<NUM>'); and
(c) a second, complementary nucleic acid molecule hybridized to said first nucleic acid molecule to form a double-stranded nucleic acid molecule.