NANOPARTICLES

Technical Field of the Invention

The invention relates to nanotechnology-based gene and drug carrier systems.

State of the Art of the Invention (Prior Art)

Chemotherapy is used as the main treatment modality for cancer. However, combination treatment strategies of concurrent gene therapy and chemotherapy have been studied for reasons such as drug resistance. Carrier systems are needed since gene therapy agents cannot enter cancer cells per se. Mostly liposomes and cationic polymers are used as gene carrier systems, however these structures have toxic effects and these structures are often not suitable for simultaneous delivery of gene therapy agent and chemotherapy drug.

In the state of the art, there are drugs formulated with liposome, polymer or peptide -based carrier systems. The company Alnylam received a FDA approval for the first siRNAliposome carrier system in 2018 (Onpattro). This treatment is for hereditary transthyretin- mediated amyloidosis (hATTR), which is a rare hereditary disease. In this technique, for gene transport, liposome structures are used which are known to have toxicity problems.

Secondly, Dicerna Pharmaceuticals developed Nedosiran for Primary Hyperoxaluria, which is a rare disease associated with liver metabolism in children. In this technique, for gene transport, liposome structures, which are known to have toxicity problems are used.

With Inclisiran, a gene therapy system for cholesterol, developed by Alnylam and Novartis Pharmaceuticals together, it has been moved in to a more common disease from rare diseases. In this technique, liposome structures, which are known to have toxicity problems are used for gene transport. There are gold nanoparticles, surface of which is coated with siRNAs specific to the Bcl-2 gene against brain tumors in the state of the art. In this technique, chemotherapy and gene therapy agents are not loaded into the carrier system simultaneously.

In the patent document No. US 20120244230A1 chemotherapy drug Paclitaxel is delivered to cancer cells by using nucleic acid coated gold nanoparticles. In this technique, the chemotherapy agent is chemically modified and bound to nucleic acids.

In brief, the problems with the prior arts can be listed as follows: Chemotherapy and gene therapy drugs cannot be loaded into the same carrier at the same time, the need for chemical modification on the drug molecules and carrier material in techniques where chemotherapy and gene therapy drugs are loaded into the same carrier; chemical modifications being inconvenient in terms of efficiency, cost and difficulty; and liposome and polymeric -based nanocarrier systems being toxic.

Brief Description and Objects of the Invention

Within the scope of the invention, during the loading of the chemotherapy drug into the gold nanoparticle -based carrier system, chemical modification / chemical (primary) bonding or layer-by-layer coating with structures such as loaded polymers used in the prior arts are not performed; instead anthracycline-type chemotherapy drug such as doxorubicin, aclarubicin, daunorubicin, epirubicin, mitoxantrone, valrubicin is loaded into the structure with intercalation by inserting between the base pairs of siRNA, miRNA and DNA/RNA antisense oligonucleotides analogous. Intercalation is the reversible insertion of anthracycline-type chemotherapy drugs between the bases of nucleic acids. Thus, drug loading can be carried out easily and efficiently without changing the chemical structure of the drug/ without the need for chemical modification.

In the known cases where anthracycline-type chemotherapy drugs such as doxorubicin are used in the prior art, the drug is bound to the structure with chemical modifications, and instead of this method; which causes a change in the structure of the chemotherapy drug, is costly and technically difficult; the aforementioned problems have been overcome by intercalating directly onto the gene therapy agent with the invention. Furthermore, while the release of the chemotherapy drug chemically bound to the carrier following delivery into cancer cells is difficult, its release from the carrier system is facilitated by the loading with intercalation used in the invention.

The advantages of the invention are listed below:

• Ease of synthesis,

• Synthesis on large scales (upscale),

• Lower costs,

• High activity against chemotherapy resistance due to lower excretion of the drug from the cell compared to the direct drug administration since the release of the chemotherapy drug from the carrier is sustained,

• Capability to develop products in the field of personalized treatment by performing concurrent gene therapy and chemotherapy.

Description of the Drawings

Figure 1: Schematic representation of loading the gene therapy agent and chemotherapy drug into the gold nanoparticle.

Figure 2: A: UV/Vis spectroscopy and B: Size analysis of nanomaterials prepared by dynamic light scattering. C: toxicities of the gold nanoparticles (AuNPs) alone and gene therapy agent-loaded nanoparticles (siRNA-AuNPs) and D: nanoparticles (Drug-siRNA- AuNPs) loaded both with chemotherapy drug and gene therapy agent, and free drug on cancer cells.

Figure 3: A: Qualitative analysis of intracellular uptake of chemotherapy drug by flow cytometry. B: Graphical representation of the percentage of cells that have uptaken the chemotherapy drug.

Figure 4: A: Analysis of cells entering the death pathway by Annexin- V-FITC staining, B: Early and late apoptosis ratios in cancer cells, C: Cell cycle phase distributions, D: Survival and proliferation ratios of cancer cells in the presence of 0.05 pM and 0.1 p M chemotherapy drug after dual therapy.

Figure 5: Light microscope images (xlO magnification) showing the changes occurred in the morphology of cancer cells after the treatments applied.

Figure 6: A: Representation of a decrease in the ability of cancer cells to proliferate by colonizing and survive after the treatment, B: % normalized colony numbers formed and C: % normalized absorbance values obtained after staining.

Detailed Description of the Invention

The drug carrying system of the invention contains gold nanoparticles surface of which is coated with SH (thiol) group containing gene therapy agents intercalants containing anthracycline-type chemotherapy drug.

The gene therapy agent is polynucleic acid or antisense oligonucleotide selected from deoxyribonucleic acid and ribonucleic acid.

Deoxyribonucleic acid must be complementer to the messenger RNA of Bcl-2 or a cancer- associated gene.

Antisense oligonucleotide can be phosphorodiamidate (morpholino), Phosphorothioate (SDNA), peptide nucleic acid (PNA), Lock nucleic acid (LNA), tricyclo-DNA (tcDNA), phosphoroamidite (2’-0me) or 2‘-O-(2-Methoxyethyl)-oligoribo-nucleotides (2’-M0E).

The drug carrying system of the invention contains gold nanoparticles in the range of 1-300 nanometers (nm), small interference RNAs (siRNA) or micro RNAs (miRNA) providing gene therapy, or antisense oligonucleotides such as morpholino, SDNA, plasmid DNA (pDNA), and an anthracycline-type chemotherapy drug such as doxorubicin, aclarubicin, daunorubicin, epirubicin, mitoxantrone, valrubicin. The nanocarrier system prepared within the scope of the invention has advantages such as ease of synthesis, loading of the chemotherapy drug into the carrier without changing its chemical structure, being biocompatible and capability to be synthesized on industrial scales. It has been shown that the prepared carrier system is biocompatible as a result of toxicity tests and suppress the targeted gene effectively. The carrier system has effectively delivered the drug molecules into the cancer cells and induced controlled cell death at a high rate and suppressed their proliferation. It has been shown that the nanocarrier system developed within the scope of the invention can be used effectively for the combination therapy strategy of gene and chemo-therapy in treating cancer and in personalized treatment methods.

Preparation of the drug carrier system consists of the following steps: i. After reduction of gold salt with synthetic reducing agents such as sodium citrate, surfactants, dimethylformamide, ascorbic acid, cetyltrimethylammonium chloride or reducing agents of vegetable/biological origin, gold nanoparticles are synthesized and are characterized by UV spectroscopy and dynamic light scattering. Figures 2A and 2B show an increase in the size of the structure after loading the gene therapy agent and chemotherapy drug. ii. The surface of gold nanoparticles is coated with siRNA, miRNA or antisense oligonucleotides, which are SH (thiol)-modified gene therapy agents that suppress the Bcl-2 gene. At this point, 30-150 gene- silencing oligonucleotides per nanoparticle on average are bound by using salt ageing method. The Bcl-2 gene is a gene that prevents cancer cells from apoptosis, and its suppression triggers controlled death in cancer cells. iii. Unbound siRNAs are removed by centrifugation at 13.000 rpm for 20 minutes. iv. The chemotherapy drug doxorubicin is added to siRNA-coated gold nanoparticles and mixed for 1-5 hours at room temperature. siRNA nucleic acids have binding spots for doxorubicin drug, and with this binding method called intercalation, the drug molecule is loaded into the carrier system by inserting between the base pairs. The schematic representation of the prepared carrier system is given in Figure 1. v. The unbound drug is removed by centrifugation of the siRNA and doxorubicin-loaded structure at 13.000 rpm for 20 minutes. The prepared multi-functional carrier system provides ease of synthesis and the capability to be synthesized in large volumes. It has been proved that the nanocarrier is not toxic, as shown in Figure 2C. Besides, it is shown in Figure 2D that, it causes death in cancer cells when the chemotherapy agent is loaded. The nanocarrier system also delivers a gene therapy agent to cancer cells, as well as chemotherapy, and it has been shown in Figure 2E that a specifically targeted gene in cancer cells reduces the protein level by using the Western impregnation method. In addition, the carrier system has been used to effectively deliver the chemotherapy drug to cancer cells. The drug uptake efficiency is shown in Figure 3.

Simultaneous Bcl-2 gene therapy and doxorubicin chemotherapy in metastatic breast cancer cells were provided with prepared multi-functional carrier system, and it has been shown that a higher rate of cancer cell death occurred when compared to chemotherapy alone. It is shown in Figure 4A, 4B and 4C that cancer cells enter the controlled death pathway at a higher ratio, compared to free drug, with concurrent gene therapy and chemotherapy carried out using nanocarrier system in cancer cells analyzed by flow cytometry, while it is shown in Figure 4D that cancer cell proliferation is suppressed at a higher rate.

Cancer cells have the characteristics of uncontrolled proliferation, escaping the death pathway, and proliferating by colonizing from a single cell without being dependent on other cells. It has been shown that after gene therapy and chemotherapy carried out with the carrier system prepared within the scope of the invention, there are deteriorations in the morphological characteristics of the cells (Figure 5) and there is a decrease greater than 90% in their ability to colonize from a single cell (Figure 6).

Delivery of chemotherapy drugs into cells by using carrier systems is more efficient when compared to treatment with free drugs. Besides, gene therapy agents such as siRNA, miRNA, and antisense oligos cannot enter cells per se and must be delivered with carrier systems. Delivery of both treatment agents simultaneously provides a greater advantage.

It is shown that both gene therapy agents and chemotherapy drugs can be delivered simultaneously with gold nanoparticles with the invention. The novel and critical feature of the invention is the binding of chemotherapy drug to the structure by direct interaction method. Direct loading of the chemotherapy drug into gene therapy agents provides ease of synthesis and does not require the chemical structure changes occurred in the structure of the drug due to the chemical binding used in other methods. The drug is loaded into the carrier without deteriorating the function and structure of the drug. The direct binding of the chemotherapy drug to the gene therapy agents ensures that both therapeutic agents are taken into the cell together.

The surface of gold nanoparticles can be coated with immune-stimulating CpG sequences instead of gene therapy agents. CpG sequences are rich in cytosine and guanine, and since they are similar to nucleic acids of bacterial origin, they can serve as a stimulant in the immune system and increase the immune response against cancer. It can be used for concurrent immune therapy and chemotherapy strategy by using CpG sequences instead of gene therapy agents. Furthermore, by adding polyethylene glycol (PEG) derivatives to the carrier system, multi-functional carrier can be made more biocompatible in biological systems. The PEG structure is used for increasing the efficiency of the carrier system by increasing the compatibility with the biological system in circulation due to its highly hydrophilic (water-loving) nature. Furthermore, targeting agents such as sugar, peptide, amino acid, lipid derivatives, small molecule, antibodies can be conjugated to the carrier system both through PEG block and directly on the particle against a number of diseases.