A PREPARATION METHOD FOR A DNA ORIGAMI BASED CARRIER

SYSTEM

Field of The Invention

The present invention relates to a new way to prepare multifunctional DNA origami based drug nano- carrier and -delivery vehicle.

Background of the Invention

Adenine-thymine and guanine-cytosine Watson-Crick hydrogen bonding allows the preparation of artificial 2D and 3D DNA based nanostructures. ¹ Using DNA and artificial oligonucleotides, molecular structures with different sizes and shapes can be constructed. Since the first report for the construction of DNA origami structure by Paul Rothemund, the preparation of many 2D and 3D structures using simple base pairing (or DNA hybridization) has been demonstrated. ² The first 2D DNA structure was designed by Winfree et al. using DNA tiles ³ . In 1999, the first discrete 3D structures were reported by Seeman ⁴ .

The DNA tiles are rectangular shaped DNA structures that contain multiple antiparallel DNA crossovers. The first developed DNA tiles were consisted of two DNA double helices with two crossovers ⁴ , subsequently structures contain three double helices with four crossovers were developed. ⁵ The DNA tiles are designed to include short and unpaired single stranded DNA strands, which allow the binding of another tile. Through these free regions, called sticky ends, the DNA tiles can be combined to create larger DNA structures ⁶ or modified with different molecules for several applications.

Although the use of DNA brings several advantages such as biocompatibility and biodegradability, it also has some disadvantages such as inefficient cellular uptake. Cellular uptake is an important parameter that should be considered for use of DNA constructs in drug delivery applications. Therefore, a molecular moiety that may utilize this function can be introduced into the DNA construct. The use of the viral vectors, peptides, and liposomes was reported as carrier structures to increase the cellular uptake efficiency. ⁷ Carbohydrates are a group of biomacromolecules that may serve for this goal as well. Sugar conjugates were reported to be as effective as commercial transfection agents. The transfection efficiencies of the lactosylated and a-glucosylated polylysine structures were found high as commercial transfection agents, lipofectamin and PEL ⁸

Doxorubicin is typical anthracycline type drug molecule and it is commonly used as chemotherapeutic agents for the cancer treatments, more specifically for breast cancer treatment. ⁹ It mainly induces apoptotic cell death in cancer cells. ¹⁰ Beside cancer cells, doxorubicin also affects other cells and shows side effects on healthy cells. ¹¹ To reduce side effects of doxorubicin, the novel vehicles have always been investigated.

The first DNA based system investigated for doxorubicin delivery was an aptamer conjugated DNA icosa, which was used for killing the cancer cells. ¹² Triangular- and tubular- shaped DNA origami structures were used as carriers of doxorubicin. The effect of doxorubicin intercalated triangular- and tubular- shaped DNA origami structures was investigated on regular human breast adenocarcinoma cancer cells, MCF7, and doxorubicin resistant subline and overcome to doxorubicin resistance. ¹³ The two 18-helix bundle nanotubes were evaluated as delivery systems for doxorubicin. ¹⁴

Summary of the Invention

The objective of the present invention is to prepare DNA origami based nano- carrier system, which can be used as drug delivery and targeting vehicle. A targeting agent such as a RGD peptide, folic acid, or a sugar molecule can be bound through the sticky ends of the DNA origami purposely designed. Figure 10 shows the proposed structure and binding points of the oligonucleotide modified agents including an RGD peptide, folic acid, sugar molecule and a drug molecule. A multifunctional DNA based nanocarrier system can be constructed by maintaining the basic origami tile structure and attaching the mentioned molecules to the olgonucleotides complementary to the sticky ends of the DNA origami structure.

A further objective of the present invention is to improve a cellular uptake of the DNA origami based carrier with modification using sugar (carbohydrate) molecules, lactose.

Detailed Description of the Invention

A preparation method for the modified DNA origami based carrier developed to fulfill the objective of the present invention is illustrated in the accompanying figures, wherein

Figure 1 is the agarose gel electrophoresis image of DNA tile formation obtained. Figure 2 is the AFM image of DNA tiles on a representative area on mica surface. Figure 3 is the dynamic light scattering analysis results of the DNA origami carrier.

Figure 4 is the lactose modification of 5'-NH2-01igonucleotide (A) and agarose gel electrophoresis image of lactose modification reaction sample and pure oligonucleotide (B).

Figure 5 is the cytotoxicity comparison of the DNA tile structure with/without lactose modification on MDA-MB- 231 cells.

Figure 6 is the cytotoxicity of the doxorubicin loaded DNA origami carrier with/without lactose modification.

Figure 7 is the intracellular uptake of DNA origami based carrier with/without lactose modification.

Figure 8 is the flow cytometry graphs of BT-474, HeLa and MDA-MB -231 cancer cells incubated with free doxorubicin and DNA origami based carrier with/without lactose modification.

Figure 9 is the confocal microscopy images of the cancer cells incubated with free doxorubicin,doxorubicin loaded DNA tile and lactose modified DNA origami based carrier.

Figure 10 is the schemiatc representation of proposed DNA origami structure with binding points of modified oligonucleotides. A preparation method for the modified DNA origami based carrier developed to fulfill the objective of the invention comprises the steps of

- preparing DNA origami based carrier,

mixing complementary oligonucleotides in hybridization buffer, incubating in a water bath at 95 °C,

- slowly cooling from to 95 °C room temperature,

characterizing hybridized DNA origami carrier,

- synthesizing lactose modified oligonucleotide,

- mixing lactose, 5'-aminohexyloligonucleotide and sodium

cyanoborohydride in borate buffer (pH 8, 0.1 M),

- stirring the mixture at room temprature for 12 hours

centrifuging the mixture to remove unreacted chemicals,

hybridizing lactose modified oligonucleotide to the DNA origami carrier mixing DNA origami structure and lactosylated oligonucleotide in hybridization buffer at room temprature for 18 hours.

- intercalating the doxorubicin into DNA origami based carrier

mixing DNA origami based carrier and doxorubicin in hybridization buffer,

stirring the mixture at room temperature for 24 hours,

centrifuging the mixture to collect the DNA origami -doxorubicin pellet

In the inventive method, first DNA origami structures, tile shaped, was prepared incubating nine complementary oligonucleotides in hybridization buffer solution applying slowly cooling method. The oligonucleotides complementary to the stick ends of the DNA origami (Figure 10) are modified with lactose molecule. The newly prepared DNA origami carrier was hybridized from the sticky ends to the complementary oligonucleotide-lactose adjuncts. The chemotherapy drug, doxorubicin, was intercalated to the modified DNA origami structure stirring overnight at room temperature.

Figure 1 shows the agarose gel electrophoresis image DNA origami formation. After the addition of each oligonucleotide and heating/cooling step, sample was taken from the hybridization solution and loaded into the gel to monitor the size change. On the agarose gel electrophoresis image of the hybridization samples, the line 1 shows the hybridization mixture containing only one oligonucleotide (oligonucleotide 1). The line 2 belongs to the hybridization mixture containing two oligonucleotides (oligonucleotide 1 and oligonucleotide 2). The line 3 represents the hybridization mixture containing three oligonucleotides (oligonucleotide 1, oligonucleotide 2 and oligonucleotide 3) and the finally line 9 is the sample of hybridization mixture of nine oligonucleotides, which are necessary to form complete DNA tile structure. M represents the DNA ladder. When the oligonucleotides forming DNA tile are added sequentially into the hybridization buffer and the molecular weights of the DNA constructs increase, the bands (from line 1 to line 9) in the agarose gel migrates slower. The increase of the molecular weights of the DNA constructs as observed from the bands on the agarose gel electrophoresis image confirms the hybridization of oligonucleotides step by step to form DNA tile structure.

Figure 2 shows the atomic force microscopy (AFM) image of DNA origami structure. The DNA tile samples taken from the hybridization media were dropped on mica surface for deposition and subsequently washed with deionized water. When the dropped DNA samples on mica were dried, images acquired at non- contact mode. Considering the distance between the base pairs in the DNA as 0.34 nm and the number of the base pairs in the DNA tile structure from one end to another, which is 40, the theoretical length of an assembled DNA tile can be estimated as 40 nm. A representative AFM image obtained from the DNA tile containing dried droplet area is shown on Figure 2. On the right hand side of the image, the graph shows the image of one structure on the surface as marked with the red arrows. The marked structure is measured as 64 nm, which is larger than the theoretical value of 40 nm. However, as seen on the image, there are smaller and larger structures than the theoretical value. Considering the fact that the formation of DNA based structures is not always 100 percent efficient, this deviation can be acceptable. In conclusion, the analysis of the AFM image supports the DNA origami formation as combined with the gel electrophoresis results.

Figure 3 shows the hydrodynamic sizes of the DNA origami, doxorubicin loaded DNA origami, and lactose modified doxorubicin loaded DNA origami structures obtained with dynamic light scattering analysis. For DLS measurements, DNA tiles are dispersed in water. In water, DNA tiles are solvated by water molecules. This hydration layer causes grater diameter than theoretical size of the structure. Hydrodynamic size of DNA tiles was determined as approximately 50 nm, which is in good agreement with AFM results. The averaged size of lactose modified DNA tiles and doxorubicin loaded DNA tile structures was measured as approximately 60 nm and 78 nm, respectively.

Figure 4 shows the lactose modification of complementary 5'-NH ₂ - oligonucleotide (A) and agarose gel electrophoresis image of lactose modification reaction sample and pure oligonucleotide. The lactose modified oligonucleotides were synthesized with reductive amination as described in Maruyama et al.. ²⁹ , Lactose molecule was directly ligated to the 5' and 3' amino modified oligonucleotides in borate buffer (pH 8, 0.1 M) using same procedure. Samples taken from the reaction mixtures were carried out in 2 % agarose gel. On the agarose gel electrophoresis image, M represents the DNA ladder; the line 1 represents the pure oligonucleotide (without modification), line 2 belongs to the lactose-modified oligonucleotide. With the attachment of lactose molecule to the oligonucleotide structure, a slower migration was observed a result of increased molecular weight the conjugate.

Figure 5 shows the cytotoxicity of the DNA origami carrier. The cytotoxicity assessments of the DNA origami structure was carried out on MDA-MB-231 cell line at a concentration range of 50-400 nM. According to the cell viability results of the experiment, the DNA-tiles are non-toxic to the cells.

Figure 6 shows the cytotoxicity of free doxorubicin, doxorubicin loaded DNA origami carrier and the lactose modified-doxorubicin loaded DNA origami carrier, a slight improvement with lactose modified DNA tiles was observed on cell viability and incorporation of doxorubicin into the DNA origami with/without lactose modification does not significantly reduce the cellular cytotoxicity. This indicated that the effect of doxorubicin remains intact.

Figure 7 shows the intracellular uptake of doxorubicin content, which determined quantitatively by using fluorescence spectroscopy. According to the Figure7, when the fluorescence intensities are compared, the free doxorubicin and doxorubicin loaded DNA origami based carrier with/without lactose modification, the lactose modified DNA origami based carrier showed the highest intensity. Results suggest that more doxorubicin taken into the cells, when the cancer cells were treated with lactose modified DNA origami based carrier.

Figure 8 shows the flow cytometry results of the cancer cells incubated with free doxorubicin, doxorubicin loaded DNA origami based carrier with/without lactose modification. According to the flow cytometry results, lactose modification increases the number of cells internalized the DNA origami based carrier indicating faster entrance of the lactose modified DNA tile structures into the cells. It was observed that the lactose-modified doxorubicin loaded DNA origami based carriers are internalized by the each cancer cells more than those of the unmodified DNA origami based carrier.

Figure 9 shows the localization of free doxorubicin and doxorubicin loaded-DNA origami based carrier with/without lactose modification monitored with confocal microscopy. According to the images, the fluorescence intensity from the cells exposed to the lactose modified and doxorubicin loaded DNA origami based carrier was higher (brighter) under the same experimental conditions indicating the faster uptake of the lactose modified DNA origami based carrier.

Figure 10 shows the proposed structure and binding points of the oligonucleotide modified agents. A multifunctional DNA based nanocarrier system can be constructed by maintaining the basic origami tile structure and attaching the mentioned molecules to the olgonucleotides complementary to the sticky ends of the DNA origami structure. DNA origami (Figure 10) are modified with lactose molecule. The newly prepared DNA origami carrier was hybridized from the sticky ends to the complementary oligonucleotide-lactose adjuncts.