Technical Field

The invention relates to the synthesis of new generation paramagnetic agents developed for use in applications in cell biology applications, tissue engineering, artificial tissue and organ technologies, drug screening and regenerative medicine and their use in cell analysis and separation of different cells with magnetic levitation.

State of the Art (Background)

The synthesis of many polymeric paramagnetic agents used or not used in the magnetic levitation method, which have different chemical structure and content, is known in the state of the art.

In their study, Shen, Y. et al. synthesized and characterized a stabilized polymeric gene carrier, a polyethylene glycol)-co-poly(ethylenimine) (PEG-PEI) copolymer, using isophorone diisocyanate (IPDI) as the coupling reagent.

In the study conducted by Ratzinger, G. et al., the preparation of particulate contrast agents for magnetic resonance imaging (MRI) based on biodegradable poly(D,L-lactide-co- glycolide) (PLGA) based nanocarriers is reported. Leading chelating ligands di ethylenetriaminepentaacetic acid (DTP A) and 1,4, 7,10-tetraazacyclododecane- 1,4, 7,10- tetraacetic acid (DOTA) were modified on gadolinium, PLGA nanospheres up to 236 mg per mg by separator-assisted covalent surface inoculation.

The studies in the market and literature show that paramagnetic agents such as Manganese (Il)chloride, Gadolinium (Ill)chloride, Zinc chloride and Gadobutrol (Gadavist) are used for both medical imaging and separation studies with magnetic levitation technology, but these paramagnetic agents kill cells above a certain concentration. High sensitivity is required for the separation of biological materials such as cells using the magnetic levitation method, and an increase in concentration is used to increase the sensitivity, but the expected separation sensitivity cannot be achieved due to the increase in cell death directly proportional to the increased concentration. Therefore, there is a need for a new generation of paramagnetic solutions with high biocompatibility and high separation sensitivity in the magnetic levitation method.

Brief Description and Objects of the Invention

Although polymeric paramagnetic agents have been used in the studies known in the art, the content and chemical formulas of the materials used as paramagnetic agents completely differ from the invention.

The present invention relates to the synthesis of new generation polymer structured paramagnetic agents and their use in cell analysis and separation of different cells by magnetic levitation.

In this invention, new generation polymer structured paramagnetic agents containing lysine diisocyanate and gadolinium binding groups (Dota-NHS ester) as well as polyethylene glycol and polyethylenimine in a different structure from the Gd ³⁺ salt based paramagnetic agents (Gadobutrol, Gadodiamide and Gadoteric acid) generally used in the magnetic levitation method were synthesized, and the usability of the synthesized paramagnetic agents in cell separation analyses was demonstrated.

Due to the presence of polyethylene glycol and lysine groups containing new generation polymeric paramagnetic agents, the invention has a higher biocompatibility compared to its counterparts such as Manganese (Il)chloride, Gadolinium (III) chloride and Zinc chloride, and shows higher sensitivity in cell separation since it carries more Gd ³⁺ ions thanks to its branched polymeric structure.

The synthesized polymeric paramagnetic agents mPEP4-D0TA-Gd and mPEP16-D0TA-Gd have been shown to be 7.8 to 7.3 times more sensitive when used for cell discrimination in the magnetic levitation method compared to similar paramagnetic agents in the literature and on the market. Low toxicity and high separation sensitivity in the magnetic levitation method are the most important advantages of this invention. Definitions of Figures Describing the Invention

Figure 1: NIH-3T3, MDA MB-231, SAOS-2 and BCE C/D-lb cells separated using mPEP4- DOTA-Gd and mPEP16-DOTA-Gd polymeric paramagnetic agents synthesized depending on levitation heights in the microcapillary canal.

Detailed Description of the Invention

Paramagnetic agents synthesized by the invention include polyethylene glycol and polyethyleneimine, lysine diisocyanate and gadolinium (Gd) binding groups.

The structural formulas of the obtained paramagnetic agents are shown below.

The method of synthesis of the paramagnetic agent comprises the following process steps: a) DBTL catalyzed reaction of ethyl lysine diisocyanate (ELDI) and methoxy polyethylene glycol (mPEG) in a 4: 1 molar ratio, b) DBTL catalyzed reaction of mPE polymer and mPE:Polyethylene imine (PEI) in molar ratios of 8: 1 and 44: 1, respectively, c) Reaction of mPEP3 or mPEP5 copolymers with DOTA-NHS ester in a molar ratio of 1 :4 and 1 : 16, respectively, followed by gadolinium loading

Synthesis of paramagnetic agents The first step of the method is the synthesis step of the polymeric paramagnetic agents. The synthesis was initiated by the preparation of the isocyanate-tipped-precursor ELDI-mPEG (mPE) polymer. It was synthesized by the reaction of ethyl lysine diisocyanate (ELDI) and methoxy polyethylene glycol (mPEG) (molar ratio ELDEmPEG 4: 1) with catalysis of Dibutyltin dilaurate (DBTL) as shown in Diagram 1 (Stage 1). After the reaction, the mixture was precipitated using petroleum ether and a methoxy polyethylene glycol ELDI (mPE) polymer with an active NCO functional group was obtained for the next step.

The synthesis was continued with the synthesis of methoxy polyethylene glycol to ELDI polyethyleneimine3 (mPEP3) and methoxy polyethylene glycol to ELDI polyethyleneimine 5 (mPEP5) by changing the mPE molar ratio of branched poly(ethyleneimine) (bPEI) in the reaction mixture and named according to the number of mPE groups bound on the structure. mPEP3 and mPEP5 synthesis was carried out for 16 hours under DBTL catalyst, under reflux and nitrogen atmosphere, as shown in mPE and bPEI and mPE and bPEI Diagram 1 (Stage 2), respectively. As in the first step, a DBTL catalyst solution was added to the reaction medium. After the reaction, mPEP3 and mPEP5 were precipitated in diethyl ether and mPEP polymers with the NH2 functional groups required for the next step were obtained.

In the last stage of the synthesis, the free-NEE functional groups of the mPEP products were reacted with l,4,7,10-Tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), and then the DOTA groups were allowed to bind the Gadolinium (Gd) magnetic agents, thus synthesizing the mPEP4-Dota-Gd and mPEP16-Dota-Gd polymeric magnetic agents. mPEP4-Dota-Gd and mPEP16-Dota-Gd were performed at pH 8.5 as shown in Diagram 1 (Stage 3) with the reactions of mPEP3 and Dota-NHS ester (mole ratio 1 :4) and mPEP5 and Dota-NHS ester (mole ratio 1 : 16), respectively. After the reaction, the suspension was dialyzed with a dialysis tube (cellulose membrane 6-8 kDa), after which gadolinium was bound to mPEP4-Dota and MPEP16-Dota products. Accordingly, the molar ratio for GdCL6H2O: DOTA was adjusted to 10:1 and carried out at room temperature for three days. After this step, the dialysis phase was continued for 5 days to remove excess gadolinium.

Diagram 1 : Synthesis diagram for new polymer structured paramagnetic agents (mPEP4-Dota-Gd and mPEP16-Dota-Gd).

Use of new generation paramagnetic agents in cell separation The magnetic levitation assembly consists of two Neodymium (NdFeB) magnets placed in an anti-Helmholtz configuration (with the same poles facing each other); a micro-capillary channel placed between the magnets; and four mirrors that allow the inside of the channel to be observed with the help of a microscope. Cells suspended in individual mPEP4-DOTA-Gd or mPEP16-DOTA-Gd polymeric magnetic agent solutions are loaded into the assembly from the entrance of the micro-capillary glass channel. Said polymeric paramagnetic agents are usable in cellular studies and do not show toxicity. Different types of cells with different densities can be separated by balancing the cells in the microcapillary canal as a result of the magnetic and buoyancy forces on them within the magnetic field created by the magnets (Figure 1). As shown in Figure 1, with the preparation and later use of mPEP4-Dota-Gd or mPEP16-Dota-Gd paramagnetic agents as 80 mg ml ^-1 , the cells in the capillary canal remained suspended at different heights due to their different cell densities. It was determined that NIH-3T3, MDA MB-231, SAOS-2 and BCE C/D-lb cells were suspended at an average height of 390.25, 155.66, 126.83 and 288.80 pm in the cell medium containing mPEP4-DOTA-Gd, respectively, and at an average height of 804.08, 449.99, 240.47 and 723.84 pm in the cell medium containing mPEP16-DOTA-Gd, respectively. Therefore, it has been shown that cells can be separated easily and with high sensitivity in magnetic levitation method by using synthesized mPEP4-DOTA-Gd or mPEP16-DOTA-Gd polymeric magnetic agents. In particular, due to the high amount of bound Gd contained in the paramagnetic agent mPEP16-DOTA-Gd, its separation capacity and sensitivity are higher and lower density differences can be easily distinguished.

This method is a fast and simple method that can be used in single cell analysis studies. In this method, by analyzing the images of different suspended cell types to be obtained from the microcapillary canal with the help of a microscope, the cells can be separated from each other thanks to the balance bands in which they will be positioned depending on their density. Thus, cell separation applications can be carried out quickly and cost-effectively.