A dual-mode drug NANO-carrier bioconjugate combination

Abstract

The present invention relates to an anti-cancer drug-loaded aprotinin nano-bioconjugate which is synthesized by using a microfluidic synthesis method in a single step using aprotinin, that is a multifunctional (moonlighting) protein acting as a nano-drug carrier for any compound / drug comprising anti-cancer properties.

Description

[0001] A DUAL-MODE DRUG NANO-CARRIER BIOCONJUGATE COMBINATION

[0002] Technical Field

[0003] The present invention relates to an anti-cancer drug-loaded aprotinin nanobioconjugate which is synthesized by using a microfluidic synthesis method in a single step using aprotinin, that is a multifunctional (moonlighting) protein acting as a nano-drug carrier for any compound / drug comprising anti-cancer properties.

[0004] Background of the Invention

[0005] Chemotherapeutic drugs generally used in cancer treatment are cytotoxic, i.e. they kill cancer cells as well as healthy cells in the living system. Therefore, there is a need for targeted drugs that can deliver cytotoxic drugs to cancer cells without harming the healthy cells of the body.

[0006] The International patent document no. W02007009229, an application included in the state of the art, discloses conjugates comprising aprotinin. The invention subject to the said international patent document discloses conjugates comprising carriers selected from the group consisting of aprotinin, a biologically active aprotinin fragment, Angiopep-1, Angiopep-2 and biologically active analogs, derivatives or fragments thereof and a label or drug. The use of these conjugates in the treatment and diagnosis of cancer is described.

[0007] Summary of the Invention

[0008] An object of the present invention is to enable the efficiency and sensitivity of any anti-cancer agent alone that is used as an anti-cancer drug in cancer treatment and is conjugated with a carrier to be enhanced by the anti-cancer drug-loaded aprotinin nano-bioconjugate combination.

[0009] Another object of the present invention is to obtain a nano-bioconjugate combination which allows to increase drug uptake from cancerous cells compared to healthy cells, thus helping to protect healthy body cells.

[0010] A further object of the present invention is to enable a nano-bioconjugate combination to be obtained in a single step by using a microfluidic co-flow mixing platform without the need for strong chemicals that can disrupt the structure of the aprotinin protein.

[0011] Detailed Description of the Invention

[0012] “A Dual-Mode Drug Nano-Carrier Bioconjugate Combination” realized to fulfil the objectives of the present invention is shown in the figures attached, in which:

[0013] Figure 1 is an image of the microfluidic co-flow mixing platform on which the inventive nano-bioconjugate combination is synthesized.

[0014] Figure 2 is a characterization of the synthesized Apr-Dox nano-bioconjugate showing: (A) Isothermal titration calorimetric analysis for the binding affinity of aprotinin with doxorubicin molecules. (B) Zeta potential of aprotinin alone and after conjugation with doxorubicin molecules. (C) DLS analysis of aprotinin alone and after conjugation with doxorubicin. (D) Transmission electron microscopy (TEM) images of the synthesized bioconjugate, i- Scale 50nm ii- Scale 20nm iii- Size of the average graph obtained from TEM images.

[0015] Figure 3 A) UV- Visible spectrophotometric analysis of Apr-Dox nanobioconjugate. (B) Circular Dichroism (CD) spectroscopic analysis of aprotinin alone and after conjugation with Dox.

[0016] Figure 4 (A) Loading of Dox into aprotinin conjugate and (B) % encapsulation efficiency of aprotinin at different drug / protein ratios for the preparation of Apr- Dox nano-bioconjugate. The Dox concentration ranges from O.lmM to 1.5mM while the aprotinin concentration in all solutions is O.lmM. (C) In vitro release analysis of Apr-Dox nano-bioconjugate in PBS and glycine buffer (pH 4.8, 6.5 and 7.3) (D) Structure change of aprotinin for Dox release in acidic conditions by CD spectroscopy.

[0017] Figure 5 Cellular uptake analysis of Dox and Apr-Dox nano-bioconjugate alone by flow cytometry at concentrations of 2pM and 4pM in (A) MCF-7 breast cancer cells (B) H-1299 small cell lung carcinoma (C) C4-2 prostate cancer and (D) Human umbilical vein endothelial cells (HUVECs).

[0018] Figure 6 Hemolysis test of Apr-Dox nano-bioconjugate. (A) Images of centrifuged RBC solutions after incubation with different concentrations of nano-bioconjugate. (B) Hemolysis percentage of Apr-Dox nano-bioconjugate at different concentrations. Trition X-100 and deionized water were used as positive controls while PBS was used as negative control.

[0019] Figure 7 is the results of in vitro cytotoxic analysis after treatment of (A) HUVECs (healthy cells) (B) C4-2 (prostate cancer) (C) MCF-7 (breast cancer) (D) H-1299 (lung cancer) with Apr-Dox nano-bioconjugate, Dox alone, aprotinin alone and PBS as control group.

[0020] Figure 8 is the in vitro cytotoxic analysis after treatment of HUVECs, MCF-7, C4- 2 and H-1299 with Apr-Dox nano-bioconjugate, Dox alone, aprotinin alone and PBS as control.

[0021] Figure 9 3D in vitro cytotoxic analysis after treatment with Apr-Dox nanobioconjugate (A) Schematics of spheroid production for drug treatment studies (B) Brightfield images of individual spheroids after drug treatment on day 5 for C4-2 and graphical description of the size change. (C) H-1299 (D) Stained images of spheroids after treatment with drug in C4-2 (E) H-1299.

[0022] The inventive chemotherapeutic nano-bioconjugate combination which targets especially lung, breast, prostate and colon cancer cells and all other cancer cells in the treatment of cancer comprises anti-cancer drug which is any small molecule cytotoxic anti-cancer agent in the form of doxorubicin, paclitaxel, etoposide, taxol, vinblastine or any pharmaceutical compound with anti-cancer properties or derivatives thereof, and multifunctional aprotinin which has the ability to bind to at least one anti-cancer compound and acts as a nano-carrier protein for small molecule cytotoxic drugs and exhibits anti-cancer properties.

[0023] Aprotinin included in the inventive nano-bioconjugate combination has the ability to carry small drug particles and acts as a nano-carrier for anti-cancer drug (preferably doxorubicin). Aprotinin is used for increasing the uptake of anti-cancer drugs by cancer cells by inhibiting the proteolytic degradation of any therapeutically active substance in the extracellular matrix (ECM), making it specifically available for uptake by cancer cells and decreasing the uptake of anticancer drugs by healthy cells. Therefore, it protects the healthy cells of the body from the cytotoxic effect of the drug. Aprotinin is used as a nano-carrier for anticancer drugs in cancer treatment and is used to enable the chemotherapeutic drug dose used in cancer treatment to be 10 to 100 times less than the drug dose administered without the nano-carrier. The structure of aprotinin enables the anticancer (chemotherapeutic) drug it carries therein to be released and to target the cancer cell by being opened at acidic pH, i.e. in an acidic tumour environment. Aprotinin exhibits a synergistic effect in anti-cancer therapy.

[0024] The anti-cancer drug included in the inventive nano-bioconjugate combination is loaded onto the nano-carrier aprotinin protein. The anti-cancer drug is taken up by cancer cells by the opening of the aprotinin structure in the acidic pH environment around the cancer cell and used in the treatment of these cells. The anti-cancer drug is doxorubicin, paclitaxel, etoposide, taxol, vinblastine and any anti-cancer agent or any compound having anti-cancer properties or derivatives thereof. In the following parts of the invention, doxorubicin, which is an exemplary name for an anti-cancer drug, is used, and this use expresses the concept of anti-cancer drug and does not include any limitation. The inventive nano-bioconjugate combination is synthesized by using a microfluidic co-flow mixing platform with two 'Y' shaped inlet and one outlet microchannels. During the one-step synthesis with the platform, aprotinin is first dissolved in PBS (phosphate buffered salt). Doxorubicin is then dissolved in water. The prepared aprotinin and doxorubicin solutions are separately carried to the platform inlet by means of syringe pumps and fed into the inlet microchannels separately. Aprotinin and doxorubicin solutions that are fed from separate inlet microchannels are combined as they move on the platform and the product is obtained from the outlet microchannel by synthesizing Apr-Dox (Aprotinin- Doxorubicin) nano-bioconjugate. Then, dialysis (dialysis membrane-MWCO IKDa) is performed in order to remove the excess / unbound drug from the Apr-Dox nano-bioconjugate solution. In one embodiment of the invention, Img-lOOOmg aprotinin is dissolved in PBS (phosphate buffered salt) and O.OOlmM-lOOmM doxorubicin is dissolved in water. The prepared aprotinin and doxorubicin solutions are fed into the inlet microchannels at a flow rate of 10-10000 ul / hour.

[0025] The inventive nano-bioconjugate combination is a chemotherapeutic combination targeting cancer cells. The nano-bioconjugate combination acts as a targeting agent and facilitates the release of doxorubicin in the acidic tumour microenvironment by enabling doxorubicin drug to be released only at acidic pH (acidic tumour environment). The nano-bioconjugate combination enables increased efficiency and sensitivity of doxorubicin molecules in cancer treatment compared to doxorubicin alone. The nano-bioconjugate combination specifically increases drug uptake from cancer cells compared to healthy cells by means of aprotinin, thus protecting healthy body cells. The nano-bioconjugate combination has a pH- sensitive property and the structure of aprotinin allows the drug to be encapsulated by being opened at acidic pH (tumour acidic microenvironment).

[0026] Within these basic concepts; it is possible to develop various embodiments of the inventive “A Dual-Mode Drug Nano-Carrier Bioconjugate Combination (1)”; the invention cannot be limited to examples disclosed herein and it is essentially according to claims.

Claims

CLAIMS1. A chemotherapeutic nano-bioconjugate combination which targets especially lung, breast, prostate and colon cancer cells and all other cancer cells in the treatment of cancer; characterized in that it comprises anti-cancer drug which is any small molecule cytotoxic anti-cancer agent in the form of doxorubicin, paclitaxel, etoposide, taxol, vinblastine or any pharmaceutical compound with anticancer properties or derivatives thereof, and multifunctional aprotinin which has the ability to bind to at least one anti-cancer compound and acts as a nano-carrier protein for small molecule cytotoxic drugs and exhibits anti-cancer properties.

2. A nano-bioconjugate combination (1) according to Claim 1; characterized by the aprotinin which has the ability to carry small drug particles and acts as a nano-carrier for anti-cancer drugs.

3. A nano-bioconjugate combination (1) according to Claim 1 or 2; characterized by the aprotinin which is used for increasing the uptake of anticancer drugs by cancer cells by inhibiting the proteolytic degradation of any therapeutically active substance in the extracellular matrix (ECM), making it specifically available for uptake by cancer cells and decreasing the uptake of anticancer drugs by healthy cells.

4. A nano-bioconjugate combination (1) according to any one of the preceding claims; characterized by the aprotinin which is used as a nano-carrier for anticancer drugs in cancer treatment and is used to enable the chemotherapeutic drug dose used in cancer treatment to be 10 to 100 times less than the drug dose administered without the nano-carrier.

5. A nano-bioconjugate combination (1) according to any one of the preceding claims; characterized by the aprotinin whose structure enables the anti-cancer(chemotherapeutic) drug it carries therein to be released and to target the cancer cell by being opened at acidic pH, i.e. in an acidic tumour environment.

6. A nano-bioconjugate combination (1) according to any one of the preceding claims; characterized by the aprotinin which exhibits a synergistic effect in anticancer therapy.

7. A nano-bioconjugate combination (1) according to any one of the preceding claims; characterized by the anti-cancer drug which is loaded onto the nano-carrier aprotinin protein; and is taken up by cancer cells by the opening of the aprotinin structure in the acidic pH environment around the cancer cell and used in the treatment of these cells.

8. A nano-bioconjugate combination (1) according to any one of the preceding claims; characterized by the anti-cancer drug which is doxorubicin, paclitaxel, etoposide, taxol, vinblastine and any anti-cancer agent or any compound having anti-cancer properties or derivatives thereof.

9. A nano-bioconjugate combination (1) according to any one of the preceding claims; characterized by the nano-bioconjugate combination which is synthesized by using a microfluidic co-flow mixing platform with two 'Y' shaped inlet and one outlet microchannels.

10. A nano-bioconjugate combination (1) according to any one of the preceding claims; characterized in that during the one-step synthesis with the platform, aprotinin is first dissolved in PBS (phosphate buffered salt); doxorubicin is then dissolved in water; the prepared aprotinin and doxorubicin solutions are separately carried to the platform inlet by means of syringe pumps and fed into the inlet microchannels separately; aprotinin and doxorubicin solutions that are fed from separate inlet microchannels are combined as they move on the platform and the product is obtained from the outlet microchannel by synthesizing Apr-Dox nano-bioconjugate; then, dialysis is performed in order to remove the excess / unbound drug from the Apr-Dox nano-bioconjugate solution.

11. A nano-bioconjugate combination (1) according to any one of the preceding claims; characterized in that aprotinin and doxorubicin solutions prepared by dissolving Img-lOOOmg aprotinin in PBS (phosphate buffered salt) and dissolving O.OOlmM-lOOmM doxorubicin in water are fed into the inlet microchannels at a flow rate of 10-10000 ul / hour.

12. A nano-bioconjugate combination (1) according to any one of the preceding claims; characterized in that it is a chemotherapeutic combination targeting cancer cells; acts as a targeting agent and facilitates the release of doxorubicin in the acidic tumour microenvironment by enabling doxorubicin drug to be released only at acidic pH (acidic tumour environment).

13. A nano-bioconjugate combination (1) according to any one of the preceding claims; characterized in that it enables increased efficiency and sensitivity of doxorubicin molecules in cancer treatment compared to doxorubicin alone.

14. A nano-bioconjugate combination (1) according to any one of the preceding claims; characterized in that it specifically increases drug uptake from cancer cells compared to healthy cells by means of aprotinin, thus protecting healthy body cells.

15. A nano-bioconjugate combination (1) according to any one of the preceding claims; characterized in that it has a pH- sensitive property and the structure of aprotinin allows the drug to be encapsulated by being opened at acidic pH (tumour acidic microenvironment).

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