Branched poly (0-amino ester) polymers multifunctionalized with silane and modified with amino acid end groups and the use of these polymers as gene carriers

Abstract

The present invention relates to branched poly(|3-amino ester) polymers (PBAE-Siaa) having the chemical structure of Formula X, multifunctionalized with silane and modified with amino acids, and the synthesis method of these polymers. A more effective gene delivery is achieved with the amino acid histidine, isoleucine, serine, methionine, phenylalanine or arginine and silane used during said modification.

Description

[0001] BRANCHED POLY (β-AMINO ESTER) POLYMERS MULTIFUNCTIONALIZED WITH SILANE AND MODIFIED WITH AMINO ACID END GROUPS AND THE USE OF THESE POLYMERS AS GENE CARRIERS

[0002] Field of the Invention

[0003] The present invention relates to multifunctionalized and modified branched poly(p-amino ester) polymers (PBAE) prepared for use as gene carriers and the synthesis method of these polymers. In the invention, the transfection property and intracellular targeting capacity of PBAE-Siaa structures obtained by multifunctionalizing and modifying PBAEs with silane and amino acid end groups are increased. In this way, an effective gene transfection is realized.

[0004] State of the Art

[0005] Gene therapy is the delivery of genetic material (DNA or RNA) into a person's cells to treat or prevent genetic disorders or diseases [1], This treatment approach was developed to correct, replace or complement faulty or missing genes in cells. Gene therapy aims to alleviate or completely eliminate the symptoms of diseases by targeting the genetic mutations that lie at the root of diseases. The methods used in gene therapy ensure the safe and effective delivery of therapeutic genetic material to target cells. Commonly used methods in gene therapy include the use of viral vectors and non-viral vectors. While viral vectors (e.g. retroviruses, adenoviruses, AAVs and lentiviruses) are preferred due to their high carrying capacity and efficiency; non-viral vectors (liposomes and polymers) offer a lower risk of immune response. Viral and non-viral vectors are gene delivery vehicles used in gene editing techniques such as CRISPR / Cas9, TALEN, etc. Treatments are applied by selecting the most appropriate method according to the type of disease to be treated, target cells and the patient's condition.

[0006] Gene delivery is at the core of gene therapy, ensuring that therapeutic genetic material reaches the right cells, becomes functional and cures the disease. For this process to be successful, the vectors used must be effective, safe, targeted and support longterm gene expression. Optimizing gene delivery is critical so that gene therapy can be used more broadly in clinical applications and treat more diseases. Transfection plays a critical role in gene delivery and offers an efficient, flexible and controllable method of delivering genetic material into cells [2], As a fundamental tool for researching, developing and optimizing gene delivery techniques in the laboratory environment, transfection provides significant advantages in areas such as the use of non-viral vectors, provision of temporary or permanent gene expression and target cell specificity.

[0007] The use of polymers in transfection processes is of great importance to ensure the safe and effective delivery of genetic material into cells. Polymers form complexes with genetic material, protecting the material and facilitating its passage through the cell membrane. This method has the advantage of creating a lower immune response compared to viral vectors, which increases the chance of success in treatment. Additionally, since polymers can be designed to be biodegradable and biocompatible, the risk of creating toxic effects in cells and tissues can be minimized. The chemical structures and surface properties of polymers can be modified to ensure efficient delivery of genetic material to target cells, thereby increasing target cell specificity. Therefore, polymers are of great importance and are widely used as non-viral gene delivery vehicles, especially in gene therapy and cell engineering applications.

[0008] Polymers used in transfection include polyethyleneimine (PEI), polylysine, dendrimers and polycaprolactone (PCL), each offering different advantages and disadvantages in terms of gene delivery efficiency, biocompatibility and toxicity. Although PEI stands out with its high gene delivery efficiency, it has problems such as cell toxicity and lack of biodegradability. Polylysine is less toxic and provides better biocompatibility than PEI but shows lower transport efficiency. Dendrimers allow surface modifications while providing high transport efficiency by forming strong complexes with the help of their branched structure but have limitations due to their complex synthesis process and potential for toxicity. PCL is a biodegradable polymer and is used for controlled gene release; however, it can be ineffective in some applications due to its low transport efficiency and limited surface modification capabilities. In general, the major shortcomings of these polymers include low transfection, low transport efficiency, difficulties in ensuring target specificity, and production costs. Poly(P-amino ester) (PBAE) is used as an important polymer in non-viral gene delivery systems and ensures the safe transfer of genetic material into cells. PBAEs facilitate passage through the cell membrane by forming electrostatic complexes with genetic material and are taken into the cell by endocytosis [3], Genetic material is released through pH changes within the cell. The ester bonds in the backbone structure of PBAEs undergo hydrolysis in aqueous environments, making PBAEs less toxic than PEI, which is more widely used as a non-degradable cationic polymer and has been investigated as a nucleic acid transport vehicle. The relatively low toxicity of these polymers allows higher amounts of polymers to be used in nanoparticle formulations without the toxicity concerns seen with non-biodegradable polymers. Hydrolytic degradation of ester bonds also facilitates the rapid release of the molecule it carries. Additional modifications are being applied to make these properties of PBAEs, especially transfection, more effective.

[0009] In the state of the art, studies on the modification of PBAEs are described in the article published by Zhou et al. [4], The article states that highly branched PBAEs are more effective at key stages such as DNA binding, DNA condensation, endosomal escape and cellular uptake. However, it seems that said polymers have a risk of gelling during the polymerization process in some cases. Although these polymers have the potential to be used in broad clinical applications, there are still shortcomings that need to be eliminated.

[0010] In the state of the art article published by Wu et al. [5], the synthesis of a new hyperbranched poly(amino ester) (HPAE) class and their DNA transport potential are described. These HPAEs are produced by Michael addition polymerization of trimethylolpropane triacrylate (TMPTA) and 1-(2-aminoethyl)piperazine (AEPZ) monomers. The polymerization process is carried out by strategically combining amine groups with different reactivities and the resulting HPAEs contain both secondary and tertiary amines in their core and primary amines in their periphery. The resulting structure, like poly(ethyleneimine) (PEI), shows positive effects on DNA condensation, endosomal escape and gene transport. However, since research on increasing the transfection properties of HPAEs is limited, further studies are required for transition to clinical use. The limitations and inadequacies of the solutions in the state of the art, the insufficiency of the modifications of the polymers commonly used in the known state of the art to improve transfection, the observation of problems such as gelation and / or rapid dissolution of the polymers, etc., made it necessary to develop in this field.

[0011] Brief Description and Objects of the Invention

[0012] The present invention discloses branched poly(P-amino ester) polymers (PBAE-Siaa) multifunctionalized with silane and modified with amino acids and the method of synthesizing these polymers. The amino acids histidine, isoleucine, serine, serine, methionine, phenylalanine or arginine used during modification and silane used during multifunctionalization enhance the transfection effects of PBAE-SiaaS and thus make them more effective in gene delivery. Branched poly(p-amino ester) polymers (PBAE-Siaa) functionalized with silane and amino acids subject to the invention are shown with the chemical formula Formula X.

[0013]

[0014] Here; R= is selected from the group His, lie, Met, Ser, Phe or Arg.

[0015] The object of the invention is to provide a polymer with high transfection efficiency to be used in gene delivery. In the invention, PBAE-Siaa polymers obtained by multifunctionalizing and modifying the synthesized branched poly(|3-amino ester) polymers with silane and amino acids have the chemical formula Formula X. With the structures used during the terminal group modification of these polymers, the transfection efficiency of PBAE-SiaaS is increased. In addition, with the silane groups, the transported genetic material is effectively protected during this transportation. The advantages gained in all these steps increase the efficiency of the entire transfection process. The amino acid (histidine, isoleucine, serine, methionine, methionine, phenylalanine or arginine) used in the modification of PBAE-Siaa optimizes the interaction of the final structure, PBAE-Siaa polymers (Formula X), with the target cells and increases the transfection efficiency.

[0016] Description of the Figures

[0017] Figure 1: FTIR analyzes of PBAE-Siaa polymers

[0018] Figure 2:1H NMR analysis of PBAE, PBAE-Si, PBAE-SiHis(Formula I) polymers Figure 3:1H NMR analysis of PBAE-SiArg(Formula VI), PBAE-SiPhe(Formula V), PBAE-SiIle(Formula II), PBAE-SiMet(Formula III), PBAE-SiSer(Formula IV) polymers Figure 4: Proton buffering capacities of PBAE-Siaa polymers

[0019] Figure 5: Size, PDI and Zeta potential values of nanoparticular systems from PBAE, PBAE-Si, PBAE-SiArg(Formula VI), PBAE-SiPhe(Formula V), PBAE-SiHis(Formula I), PBAE-SiIle(Formula II), PBAE-SiMet(Formula III), PBAE-SiSer(Formula IV) polymers (A: Size-week graph, B: PDI-week plot and C: Zeta potential-week plot)

[0020] Figure 6: Gel Electrophoresis Result of PBAE-Siaapolymers (A: PBAE, B: PBAE-Si, C: PBAE-SiHis(Formula I), D: PBAE-SiIle(Formula II), E: PBAE-SiMet(Formula III), F: PBAE-SiSer(Formula IV), G: PBAE-SiArg(Formula VI), H: PBAE-SiPhe(Formula V)) Figure 7: DNA protection efficiency analysis of PBAE-Siaa polymers

[0021] Figure 8: MTT Results of nPBAE polymers, A: HEK293T cells and B: cells. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

[0022] Figure 9: Cell viability and transfection efficiency results of nPBAEs (A: HeLa, B: HEK293T cells)

[0023] Figure 10: Fluorescence microscope images of transfection efficiency of different nPBAEs in HEK293T cells (1: PBAE, 2: PBAE-Si, 3: PBAE-SiHis(Formula I), 4: PBAE-SiIle(Formula II), 5: PBAE-SiMet(Formula III), 6: PBAE-SiSer(Formula IV), 7: PBAE-SiPhe(Formula V), 8: PBAE-SiArg(Formula VI))

[0024] Figure 11: Fluorescence microscope images of transfection efficiency of different nPBAEs in HeLa cells (1: PBAE, 2: PBAE-Si, 3: PBAE-SiHis(Formula I), 4: PBAE-SiIle(Formula II), 5: PBAE-SiMet(Formula III), 6: PBAE-SiSer(Formula IV), 7: PBAE-SiPhe(Formula V), 8: PBAE-SiArg(Formula VI))

[0025] Detailed Description of the Invention The invention relates to branched poly(|3-amino ester) polymers (PBAE-Siaa) multifunctionalized with silane and modified with amino acids and to the method of synthesizing these polymers. The branched poly(|3-amino ester) polymers multifunctionalized and modified with these silanes and amino acids are denoted by PBAE-Siaa and have the chemical structure of Formula X. The amino acids histidine, isoleucine, serine, methionine, methionine, phenylalanine or arginine and silane used during the aforementioned modification increase the transfection effect of PBAEs and thus make them more effective in gene delivery.

[0026] The invention relates to highly transfectional branched poly(|3-amino ester) polymers (PBAE-Siaa), multifunctionalized with silane and modified with amino acids, used as gene carriers, having a chemical structure of Formula X,

[0027]

[0028] Here; R= is selected from the group His, lie, Met, Ser, Phe or Arg.

[0029] In one embodiment of the invention, the branched poly(|3-amino ester) polymer of the present invention is a polymer having the chemical structure of Formula I (PBAE-SiHis).

[0030]

[0031] In one embodiment of the invention, the branched poly(P-amino ester) polymer of the present invention is a polymer having the chemical structure of Formula II (PBAE- Sille).

[0032]

[0033] In one embodiment of the invention, the branched poly(|3-amino ester) polymer of the present invention is a polymer having the chemical structure of Formula III (PBAE- SilVIet).

[0034]

[0035] In one embodiment of the invention, the branched poly(3-amino ester) polymer of the present invention is a polymer having the chemical structure of Formula IV (PBAE- Siser).

[0036]

[0037] In one embodiment of the invention, the branched poly(P-amino ester) polymer of the present invention is a polymer having the chemical structure of Formula V (PBAE- Siphe).

[0038]

[0039] In one embodiment of the invention, the branched poly(P-amino ester) polymer of the present invention is a polymer having the chemical structure of Formula VI (PBAE- SiArg).

[0040]

[0041] Synthesis method of branched poly(β-amino ester) polymers (PBAE-Siaa) with high transfection, multifunctionalized with silane having Formula X chemical structure and modified with amino acids, comprising the process steps of;

[0042] i. obtaining poly(p-amino ester) (PBAE) polymer by reacting bisphenol A ethoxylate diacrylate monomer and diethylenetriamine monomer with tetrahydrofuran (THF),

[0043] ii. Reaction of the methylol groups of N-[3-(Trimethoxysilyl) propyl]ethylenediamine monomer with the amino acid histidine, isoleucine, serine, methionine, phenylalanine or arginine in distilled water (dH2O) in a nitrogen atmosphere,

[0044] iii. Reacting the PBAE polymer obtained in process step (i) and the structure obtained in process step (ii) in nitrogen atmosphere to obtain PBAE-Siaa polymers with Formula X chemical structure

[0045]

[0046] An embodiment of the synthesis method of branched poly(P-amino ester) polymers (PBAE-Siaa) with high transfection, multifunctionalized with silane having Formula X chemical structure and modified with amino acids, comprising the process steps of;

[0047] i. Reacting 0.1-10 mmol of bisphenol A ethoxylate diacrylate monomer and 0.1-10 mmol of diethylenetriamine monomer with tetrahydrofuran (THF) at 0-100° for 1-24 hours to obtain poly(β-amino ester) (PBAE) polymer, ii. Reaction of the methylol groups of 0.1-10 mmol N-[3-(Trimethoxysilyl) propyl]ethylenediamine monomer with the various amino acid as 0.1-10 mmol amino acid histidine, isoleucine, serine, methionine, phenylalanine or arginine in distilled water (dH2O) in a nitrogen atmosphere at room temperature for 1-24 hours,

[0048] iii. Reacting the PBAE polymer obtained in process step (i) and the structure obtained in process step (ii) in nitrogen atmosphere at room temperature for 1-24 hours to obtain PBAE-Siaa polymers with Formula X chemical structure

[0049]

[0050] Another embodiment of the synthesis method of branched poly(P-amino ester) polymers (PBAE-Siaa) with high transfection, multifunctionalized with silane having Formula X chemical structure and modified with amino acids, comprising the process steps of;

[0051] i. Reacting 1.2 mmol of bisphenol A ethoxylate diacrylate monomer and 1 mmol of diethylenetriamine monomer with tetrahydrofuran (THF) at 50°C for 24 hours to obtain poly(|3-amino ester) (PBAE) polymer,

[0052] ii. Reaction of the methylol groups of 0.2 mmol N-[3-(Trimethoxysilyl) propyl]ethylenediamine monomer with the various amino acid as 0.6 mmol amino acid histidine, isoleucine, serine, methionine, phenylalanine or arginine in distilled water (dH2O) in a nitrogen atmosphere at room temperature for 24 hours,

[0053] iii. Reacting the PBAE polymer obtained in process step (i) and the structure obtained in process step (ii) in nitrogen atmosphere at room temperature for 1-24 hours to obtain PBAE-Siaa polymers with Formula X chemical structure

[0054]

[0055] In one embodiment of the method of synthesis of PBAE-Siaa polymers subject to the invention, the reaction of 1.2 mmol bisphenol A ethoxylate diacrylate monomer and 1 mmol diethylenetriamine monomer in tetrahydrofuran (THF) is carried out at 50°C for 48 hours and PBAE polymer is obtained (Reaction 1.).

[0056]

[0057] Bisphenol a ethoxylate diacrylate Diethylene triamine

[0058]

[0059] PBAE

[0060] Reaction 1. PBAE synthesis reaction Elsewhere, aminosilane (Siaa) is obtained by reacting the methylol groups of 0.2 mmol N-[3-(Trimethoxysilyl) propyl]ethylenediamine monomer with 0.6 mmol histidine, isoleucine, serine, methionine, phenylalanine or arginine acid in distilled water (dH2O) in a nitrogen atmosphere at room temperature for 24 hours (Reaction 2.). At this stage, what is meant by Siaa expression is the amino acid-linked silane structure, i.e. aminosilane.

[0061]

[0062] Reaction 2. Siaa synthesis reaction

[0063] Then, PBAE and Siaa are reacted with PBAE polymer at room temperature under nitrogen atmosphere for 24 hours to obtain branched PBAE-Siaa polymers with the end group multifunctionalized with silane and modified with amino acid (Reaction 3.). These PBAE-Siaa polymers have the chemical structure of Formula X.

[0064]

[0065] Reaction 3. PBAE-Siaa synthesis reaction

[0066] The synthesis of PBAE-Si polymer containing only silane groups is carried out by reacting 0.2 mmol of N-[3-(Trimethoxysilyl) propyl]ethylenediamine monomer with PBAE under similar conditions without modification with amino acids.

[0067] The structures of the synthesized PBAE-Siaa polymers were analyzed by Fourier Transform Infrared Spectrophotometer (FTIR). Using tablets prepared by diluting the polymer / KBr ratio to 1 / 200 mg, spectra were recorded in the wave number range 400-4000 cm-1(Figure 1). The broad absorption band with a maximum value of 3428 cm-1is due to the N-H asymmetric and symmetric stretching vibrations of the primary amine groups and the secondary amine N-H stretching vibrations. The small peak at 3100-3000 cm-1with a maximum at 3059 cm-1is due to C-H bonds in aromatic rings. The peaks at 3000-2750 cm-1with peaks at 2964, 2937 and 2870 cm-1are interfered with the asymmetric and symmetric stretching vibrations of -CH2 in the -CH2-NH2structure and the vibrations of -CH3 groups in the diacrylate structure. The band with a maximum of 2825 cm-1in the 2835-2800 cm-1region originates from tertiary amine structures. The peak with a maximum point of 1733 cm-1arises from C=O stretching vibrations in the acrylate ester structures. In the maximum absorption band of 1636 cm-1, N-H inplane deformation vibrations of primary amine structures and C=C stretching vibrations of vinyl groups are found interfering. In the maximum absorption band of 1609 cm-1, N-H in-plane deformation vibrations of secondary amine structures and C=C stretching vibrations of vinyl groups are found interfering. The 1577 cm-1peak occurs from the shear vibrations of primary amine groups. The long and sharp peak with a peak of 1505 cm-1arises from the skeletal, stretching and in-plane deformation vibrations of the C=C bonds in the aromatic rings. The peaks with peaks at 1458 cm-1and 1410 arise from -CH2- deformation and asymmetric bending vibrations and =CH2 deformation and shear vibrations in the diacrylate structure, respectively. The small peaks located in the 1390-1300 cm-1region are formed by deformation and shaking vibrations of -CH2 groups in -CH2-NH2 structures. In the band with a maximum of 1293 cm-1, bending vibrations of -CH2 groups in -CH2-NH2 structures and vibrations originating from p-substituted phenol structures are interfering. The peaks at 1244 cm’1and 1182 cm-1arise from the characteristic stretching and deformation vibrations of p-substituted phenol structures. In the peak with a maximum of 1060 cm-1, vibrations arising from primary amine C-N stretching and phenol structures are interfering. The peaks at 922 cm’1and 827 cm’1are related to the shaking and deformation vibrations of -CH2 and -NH2 groups. According to the FTIR spectra of PBAE, it is seen that polymer chains with both primary amine and acrylate terminations, containing secondary and tertiary amine groups in their structure, are formed. When the FTIR spectra of the realized PBAE-Si modification are examined, it is seen that some of the existing peaks in PBAE shift slightly, some disappear and new absorption bands are formed. The intensity of the absorption bands in the 3000-2800 cm’1region increased with the effect of -CH2- groups added to the polymer structure. The intensity of the peak, which has a maximum of 1604 cm’1, increases due to the N-H deformation vibrations of the secondary amine formed / incorporated into the structure by modification. In the PBAE spectrum, the band with a maximum of 1410 cm’1, resulting from =CH2deformation and shear vibrations of acrylate vinyl groups, has disappeared. The absorption band at approximately 1397 cm’1consists of vibrations arising from Si-C structures. The intensity of the absorption band, which has a maximum of 1244 cm’1, increased due to the interference of Si-C deformation vibrations. A new absorption band was formed at approximately 1075 cm’1, resulting from the asymmetric stretching vibrations of the -Si-O-C bond. The peak with a maximum of 890 cm’1was formed by the Si-0 stretching vibrations of Si-OH groups. In the FTIR spectrum of PBAE-Si modification, it is seen that both new absorption bands belonging to silane groups are formed and the intensity of the bands belonging to secondary amine groups increases. This shows that the intended reaction has occurred. In the FTIR analysis obtained as a result of modification of PBAE-Si polymer with amino acids, a significant increase in the absorption bands of primary and secondary amines (1640-1580 cm-1, 1380-1360 cm-1regions and approximately 1060 cm-1) showed that the modification was realized. The chemical structure and composition of the polymers were analyzed by1H NMR. Additionally, Mestrenova v14.1.2-25024 software (Mestrelab Research SL, Spain) was used to perform phase and elemental correction of the1H NMR spectrum to support the accuracy and sensitivity of the analysis. The relevant1H NMR peaks of all synthesized polymers are shown in Figure 2 and Figure 3. Numerical expressions indicating the peaks are marked on the molecule. PBAE polymer comprises the peaks; 6=1.55 (s, -H3C-C-CH3); 6=2.43 (s, -HN-CH2-CH2-NH), 6=2.71 (s, O=C-CH2-CH2-NH), C-CH2-CH2-NH), 6=6.81

[0068]

[0069] i polymer comprises the peaks; 6=1.57 (s, -Si-CH2-CH2-CH2-NH), 6=2.56 (s, -Si-CH2-CH2-CH2-NH; -NH-C / 72-CH2-NH-CH2), 6=3.50 (tt, -NH-CH2-CH2-NH-CH2), 6=4.01 (m, -NH-CH2-CH2-NH-CH2), 6=4.11 (s, -Si-O-C / 73), 6=4.29 (s, -Si-CH2-CH2-CH2-NH), 72.8%. PBAE-SiHispolymer, which has the chemical structure of Formula I comprises the peaks; 6=3.94 (q, -CH2-C / - / -NH2), 6=4.10 (s, -CH2-CH-NH2), 6=6.82 (ddt, -C=CH-NH-CH=N), 6=7.10 (dq, -C=C / 7-NH-C / 7=N) 62.1%. PBAE-SiArg polymer, which has the chemical structure of Formula VI comprises the peaks; 6=1.57 (s, -NH-CH2-C / - / 2-C / - / 2-CH), 6=3.49 (m, H2N-CH-CH2), 6=3.70 (m, / 72N-C=NH; H2N-CH-CH2), 6=3.93 (m, / 7N-C-NH-C / 72), 6=6.82 (dq, HN-C-N / - / -CH2) 88.5%. PBAE-Siphe polymer, which has the chemical structure of Formula V comprises the peaks; 6=3.49 (m, -C-C / - / 2-CH-NH2), 6=3.94 (t, -C-CH2-CH-NH2), 6=4.04 (q, -C-CH2-CH-NH2), 6=6.83 (m, -HC=CH-CH=CH-CH), 6=7.10 (d, / 7C=CH-C / 7=CH-C / 7) 40.9%. PBAE-Siiie polymer, which has the chemical structure of Formula II comprises the peaks; 6=1.57 (s, H3C-CH2-CH-C / - / 3), 6=2.42 (s, H3C-CH2-C / - / -CH3), 6=3.93 (q, H2N-C / - / -CH), 6=7.08 (m, H2N-CH-CH) 86.5%. PBAE-Siwet polymer, which has the chemical structure of Formula III comprises the peaks; 6=1.57 (S, H3C-S-CH2-CH2-CH-NH2), 6=3.70 (dq, H3C-S-CH2-CH2-CH-NH2), 6=3.93 (q, H3C-S-CH2-CH2-CH-NH2), 6=4.11 (s, H3C-S-CH2-CH2-CH-NH2) 76.7%. PBAE-Siser, which has the chemical structure of Formula IV comprises the peaks; 6=3.68 (s, OH-CH2-C / - / -NH2), 6=3.94 (t, OH-CH2-CH-NH2), 6=7.09 (d, OH-CH2-CH-NH2) 50%.

[0070] The proton buffer capacity of PBAE-Siaa polymers with the chemical structure of Formula X was determined by acid-base titration. 6 mg of polymer was dissolved in 150 mM NaCI solution to a final concentration of 0.5 mg / mL and the pH of the solution was adjusted to pH 10 using NaOH. pH 2.5 was titrated to pH 2.5 using 0.1 M HCI by taking measurements with a pH meter every 20 pL. NaCI was used as negative control groups (Figure 4). The buffering capacity of the polymeric carrier system between pH 7.4 and 5 is important for determining the endosomal escape potential of the carrier system. It is observed that the buffer capacity of the PBAE-Siaa (Formula X) polymers of the present invention is increased compared to the polymers not multifunctionalized with end-group silane and without amino acid modification. PBAE-Sinis (Formula I) polymer modified with Histidine amino acid exhibits the highest buffering capacity.

[0071] Synthesis of nanoparticles from PBAE-Siaa (Formula X) polymers is carried out using the nanoprecipitation technique. According to this method, polymers were dissolved in dimethyl sulfoxide (DMSO) at concentrations of 10 mg / mL. Nanoparticles are obtained by taking these solutions (200 L), adding them to deionized water (1300 pL) and mixing with vortex for 30 seconds at 2400 rpm. Here, the size and zeta potential values of the nanoparticles obtained were measured. The 4-week stability of the nanoparticle formulations was monitored and the prepared nanoparticles were stored at 4(±1)°C. Particle size, size distribution and zeta potential of the nanoparticles were measured during storage. No significant changes in particle size or PDI values were observed during the 4-week stability studies of the nanoparticles. Nanoparticles maintain their overall colloidal stability. A decrease in the zeta potential of polymeric nanoparticles was observed during storage (Figure 5).

[0072] Nanoparticle-gene complexes were prepared by treating Green Fluorescent Protein-Coding Circular Plasmid DNA (pDNA; pEGFN1) with polymers at varying ratios. The ability of nanoparticle formulations to complex with pDNA was analyzed by agarose gel electrophoresis. Nanoparticle:pDNA complexes were loaded into the wells of the agarose gel and subjected to electrophoresis at 100V for 1 hour. Then, the gels examined under ultraviolet (UV) light were evaluated by taking photographs (Figure 6). The electrophoresis method was chosen to evaluate the conservation analysis of DNA.

[0073] Gene and polymer complexes were prepared and after 30 minutes of incubation, 1 pL of DNase I (1 lU / pL) dissolved in buffer solution containing 50 mM Tris, 10 mM MgCI2 was added to the indicated ratios to see if DNase I would have any effect. Tris-MgCl2 buffer was added to the wells where DNase I was not added. They were allowed to mix in a shaking incubator for 30 min at 37±1 °C. At the end of this period, 4 pL of 0.25 M EDTA was added to all wells and waited for 10 minutes. Then, 4 pL Heparin (5000IU / mL) was added and mixed for 2 hours in a 37±1 °C incubator. At the end of the period, the agar was loaded into the gel and run at 130V for 1 hour. Then, the gels examined under UV light were evaluated by taking photographs (Figure 7).

[0074] Cervical cancer cell line (HeLa) and embryonic kidney cell line (HEK293T) cells were used to determine the transfection efficiency of synthesized PBAE-Siaa (Formula X) nanoparticles. HeLa and HEK293T cells were grown in 75 cm2culture dishes using DMEM medium containing 10% FBS, 100 U / ml penicillin, 2mM L-Glutamine. The cytotoxic effect of PBAE-Siaa nanoparticles was determined by the 3-(4,5-dimethylthiazole-2,5-diphenyltetrazolium method (MTT Method). This method is based on the conversion of MTT into water-insoluble formazan crystals as a result of mitochondrial dehydrogenase activity in living cells and the measurement of the absorbance at 570 nm wavelength in a spectrophotometer by dissolving these crystals with dimethyl sulfoxide (DMSO). Cells were seeded in 96-well petri dishes and incubated for 24 hours. After the incubation period, different concentrations of PBAE-Siaa nanoparticles were added to the cells. After incubation for 4 hours, the medium containing the nanoparticles is removed, MTT solution is added and left for 3 hours. The formed formazan crystals were dissolved with DMSO and the absorbance was measured at a wavelength of 570 nm in a spectrophotometer. The viability of the cells was calculated as a percentage by comparing them with the control group cells (Equation 1).

[0075] Absorbance of the experimental group

[0076] Viability% — - — -; - - -; - -xl00 Absorbance of control

[0077] Equation 1. %Viability calculation method The effectiveness of gene delivery systems is based on their ability to safely transfer genetic material without harming target cells, and cytotoxicity is a key determining factor in this process. The structure of surface functional groups and their modification with different molecules have a significant effect on cytotoxicity by changing the surface charge of nanoparticles. Especially cationically charged nanoparticles show higher interaction with cells compared to neutral or negatively charged nanoparticles, which may increase the risk of cytotoxicity. However, modifications to the surface of nanoparticles can be designed to increase or decrease cytotoxicity depending on the functional groups added, which is of great importance in terms of optimizing the safety and efficiency of gene delivery systems.

[0078] MTT test was applied on HEK293T and HeLa cell lines to determine the cytotoxicity properties of different nanoparticle PBAE-Siaa (nPBAE-Siaa). The effects of nPBAE-Siaa samples at different concentrations (10, 50, 100, 250 and 500 pg / mL) on cell viability were examined at the end of a 24-hour period and the results are shown in Figure 8. According to the MTT results in HEK293T cells, cell viability is slightly affected depending on the modified amino acid structure at 10 and 50 pg / mL concentration values. At the concentration value of 100 pg / mL, nPBAE-Siviet was most affected, and the cell viability was 55%, for nPBAE this value was 65% and for other modified products it was approximately 70-75%. At 250 and 500 pg / mL concentration values, cell viability is around 55% in all nanoparticle formulations. When we examined the HeLa cell line MTT results, no cytotoxic effect on cell viability was observed in PBAEs modified at 10 and 50 pg / mL concentration values, while nano-branched poly(P-amino ester) polymers (nPBAE) showed a significant difference (cell viability 45%) compared to control cells. This means that the modification shows a more moderate cytotoxicity in HeLa cells. When the concentration value reached 100 pg / mL, no significant cytotoxic effect was observed in the other nanoformulations, with nPBAE, nPBAE-Sinis and nPBAE-Siiie affected nanoformulations being the most affected. In these analyses, at the highest concentrations of 250 and 500 pg / mL, the cell viability of all nanoparticle formulations is seriously affected. By evaluating the MTT results and gel electrophoresis data in both HEK293T and HeLa cell lines together, the complex ratios in the transfection application were determined as 80:1. This value is the value at which viability is high in both cell lines. According to all analysis results, the transfection efficiency of polymers with suitable properties was analyzed. Cells were seeded in 24-wells and incubated for 24 hours. Nanoparticular systems that form a complex with the gene were added to the cells and incubated for 4 hours, and at the end of the 4th hour, the medium on the cell was discarded and the medium was added again and incubated for 72 hours. The transfection efficiency of nPBAE-Siaa carriers was analyzed by examining the expression of green fluorescent protein in cells after 72 hours under 460-480 nm fluorescence light in an inverted microscope. Transfected cells were identified based on the green fluorescence displayed by the cells. In addition, transfection efficiency was determined by measuring the transfection efficiency in flow cytometry at a wavelength of 488 nm (Figure 9).

[0079] The most important element in gene therapy applications of PBAE-Siaa (Formula X) polymers is to establish the balance between high transfection efficiency and low cytotoxicity. Within the scope of the invention, modification of nPBAE-Siaa polymers with silane-functionalized end groups with various amino acids was carried out. The transfection efficiency of nanoparticles obtained from modified products was examined in HEK293T and HeLa cell lines. According to gel electrophoresis results, transfection efficiency was examined at rates that encapsulated pDNA 100%. The results obtained are given in Figure 9. Fluorescence microscope images are given in Figure 10 for HEK293T cells and Figure 11 for HeLa cells.

[0080] A decisive role in the formation of an efficient gene delivery system is the hydrophilic / hydrophobic balance in the structure that maintains the stability of pDNA before its release into the cytosol and determines the efficiency of pDNA encapsulation. It is known in the state of the art that hydrophobic amino acid modifications give better results than hydrophilic amino acid modifications. The reason for this is that hydrophobic groups interact more easily with phospholipids in the cell membrane, thus facilitating cell internalization. On the other hand, in order to increase transfection efficiency, high amounts of modification of hydrophobic groups may cause cytotoxicity. Studies in the state of the art show that amino acid modifications with hydrophilic / hydrophobic groups such as amine, guanidine, imidazole, carboxyl, hydroxyl, thiol, amide, lipid or aromatic ring from the molecular structure increase transfection success. In the invention, a multifunctional structure is given to the end groups by silane modification, and amino acid modification is carried out through them. Within the scope of the invention, arginine and histidine from the cationic amino acid group; phenylalanine, methionine and isoleucine from the hydrophobic amino acid group; serine is used from the hydrophilic amino acid group. When the values given in Figure 9 are examined, synthesized nPBAE showed a limited transfection efficiency in both HeLa and HEK293T cell lines (15.6 ± 3.13 for HeLa; 48.1 ± 2.65 for HEK293T); silane modification slightly increased this success (24.08 ± 3.22 for HeLa; 65.74 ± 0.24 for HEK293T). With amino acid modifications, the transfection success of nPBAE increases in varying amounts depending on the type of amino acid conjugated to the structure. In particular, arginine, phenylalanine, and methionine modifications appear to exhibit the highest transfection success (approximately 92%) in both HeLa and HEK293T cell lines. This success of arginine is due to the fact that a-amino, e-amino and guanidinium groups in its structure act as cationic groups and provide a positive charge, which in turn affects the surface charge of the molecule and facilitates cell internalization. It is observed that phenylalanine and methionine, which are selected as hydrophobic amino acid group modifications, also cause an increase in transfection efficiency. It is reported in the state of the art that hydrophobic compounds have potential fusion activity and that the hydrophobic interaction between these compounds and phospholipids exerts a significant effect on cellular uptake and endosomal escape. It is also known that modifications with aromatic amino acids are easier to release pDNA after cellular uptake compared to modifications with other hydrophobic amino acids. In conclusion, the higher transfection efficiency of nanoparticle systems designed with aromatic amino acids is explained by higher hydrophobicity, higher cellular uptake and easier intracellular DNA dissociation of the polyplexes. Although methionine and phenylalanine modifications show very close transfection efficiency, 100% encapsulation of pDNA occurs at a lower polymer amount for phenylalanine. This shows that the hydrophobicity of phenylalanine forms a more effective complex with pDNA. The cell spread of these three modifications is approximately 85% in the HeLa cell line and 15% in the HEK293T cell line (Figure 9). Another modification tool selected from the cationic amino acid group, histidine conjugation, yielded the highest buffer capacity results, but transfection efficiency did not match the success of arginine, phenylalanine and methionine. Histidine conjugation facilitates endosomal escape but does not appear to be superior to other amino acid modifications in cell internalization. It is thought that only the a-amino group of histidine is theoretically protonated under neutral pH conditions, and this is due to the fact that the nitrogen in the imidazole ring is not protonated. This situation affects the surface charge of the molecule and constitutes an important element in cell internalization. As the data in Figure 9 confirms this situation, the transfection success in HeLa and HEK293T cell lines is higher and creates a significant difference compared to pure PBAE. Another amino acid modification selected from the hydrophobic group gives results close to methionine and phenylalanine in the transfection efficiency of isoleucine-conjugated PBAEs in the HEK293T cell line, but remains at a lower level in the HeLa cell line. It is thought that the main reason for this is directly related to the fact that the molecular structure does not contain a special structure such as an aromatic ring and, accordingly, the transfection efficiency remains at a lower value. For the serine amino acid chosen as the hydrophilic group modification, a transfection beyond what is known in the state of the art is observed. While it showed transfection at a value close to cationic or hydrophobic amino acid modifications (95%) in the HEK293T cell line, this value was obtained as 72.5% in the HeLa cell line and remained below arginine, phenylalanine and methionine modifications.

[0081] Statistical analysis and comparable data sets were evaluated by two-way ANOVA test. Probability values of p < 0.05 were considered statistically significant (Figure 8).

[0082] In the present invention, end group modified branched poly(P-amino ester) (PBAE) based polymers containing silane groups are synthesized to be used as gene carriers. Then, these polymers were characterized and their transfection efficiency was evaluated. Branched PBAE polymers are synthesized using bisphenol A ethoxylate diacrylate, diethylenetriamine and N-[3-(Trimethoxysilyl)propyl] ethylenediamine, and the amino acid histidine, isolosine, serine, methionine, phenylalanine or arginine is added for end group modification. The structures of the polymers were confirmed by nuclear magnetic resonance (1H NMR), Fourier transform infrared spectroscopy (FTIR) analysis. Nanoparticle structures of these polymers were prepared using the nanoprecipitation technique, and their plasmid DNA complex formation and protection capacities and endosomal escape abilities were determined. Finally, the cytotoxicity of the nanoparticles on HeLa and HEK293T cell lines was tested by MTT assay, and their transfection efficiencies were tested by flow cytometry and fluorescence microscopy. All tested nanoparticles show lower toxicity in HeLa cells compared to HEK293T cells. Transfection efficiency followed an increasing order starting from linear PBAE to silane modification followed by modifications by histidine, isoleucine, serine, methionine, phenylalanine, and arginine. The invention highlights the potential of silane- and amino acid-functionalized PBAE polymers as gene carriers and the impact of these modifications on transfection efficiency.

[0083] In this invention, N-[3-(Trimethoxysilyl)propyl]ethylenediamine compound, which has not been used in PBAE modification before according to the state of the art, is used for terminal group modifications of PBAE polymers. In this way, the chemical functionality of the polymer is increased by creating different side groups in the polymer chains and the transfection efficiency of gene delivery systems is increased. In addition, modifications with amino acids optimize the interaction of polymer nanoparticles with cells, resulting in higher transfection efficiency in targeted cell lines.

[0084] As a result, the invention includes PBAE-silane-amino acid modifications that are not included in the state of the art, and this combination has the potential to increase the efficiency of gene carrier systems. With the invention, significant progress is made in gene therapy approaches in the state of the art, contributing to the development of more effective and safe gene carrier systems. REFERENCES

[0085] [1] Naldini, L. (2015). Gene therapy returns to centre stage. Nature, 526(7573), 351-360.

[0086] [2] Yin, H., Kanasty, R. L., Eltoukhy, A. A., Vegas, A. J., Dorkin, J. R., & Anderson, D. G. (2014). Non-viral vectors for gene-based therapy. Nature Reviews Genetics, 15(8), 541-555.

[0087] [3] Lynn, D. M., & Langer, R. (2000). Degradable poly(β-amino esters): Synthesis, characterization, and self-assembly with plasmid DNA. Journal of the American Chemical Society, 122(44), 10761-10768.

[0088] [4] Zhou, D., Cutlar, L., Gao, Y., Wang, W., O'Keeffe-Ahern, J., McMahon, S., Duarte, B., Larcher, F., Rodriguez, B. J., Greiser, U., Wang, W. 2016a. "The transition from linear to highly branched poly(beta-amino ester)s: Branching matters for gene delivery", Sci Adv, 2(6), e1600102.

[0089] [5] Wu, D., Liu, Y., Jiang, X., Chen, L., He, C., Goh, S. H., Leong, K. W. 2005. "Evaluation of hyperbranched poly(amino ester)s of amine constitutions similar to polyethylenimine for DNA delivery", Biomacromolecules, 6(6), 3166-3173.

Claims

CLAIMS1. Branched poly(β-amino ester) polymer multifunctionalized with silane having Formula X chemical structure and modified with amino acids to be used as gene carrier,wherein; R= is selected from the group His, lie, Met, Ser, Phe or Arg.

2. A branched poly(p-amino ester) polymer according to claim 1, wherein it has the following chemical structure of Formula I3. A branched poly(β-amino ester) polymer according to claim 1, wherein it has the following chemical structure of Formula II4. A branched poly(β-amino ester) polymer according to claim 1, wherein it has the following chemical structure of Formula III5. A branched poly(β-amino ester) polymer according to claim 1, wherein it has the following chemical structure of Formula IV6. A branched poly(β-amino ester) polymer according to claim 1, wherein it has the following chemical structure of Formula V7. A branched poly(β-amino ester) polymer according to claim 1, wherein it has the following chemical structure of Formula VI8. A branched poly(β-amino ester) polymer according to claim 1, wherein it is used as a gene carrier in a cervical cancer cell line (HeLa) and an embryonic kidney cell line (HEK293T).

9. Synthesis method of branched poly(β-amino ester) polymers (PBAE-Siaa) with high transfection, multifunctionalized with silane having Formula X chemical structure and modified with amino acids to be used as gene carriers, comprising the process steps of;i. obtaining poly(β-amino ester) (PBAE) polymer by reacting bisphenol A ethoxylate diacrylate monomer and diethylenetriamine monomer with tetrahydrofuran (THF),ii. reacting the methylol groups of N-[3-(Trimethoxysilyl) propyl]ethylenediamine monomer with the amino acid histidine, isoleucine, serine, methionine, phenylalanine or arginine in distilled water (dH2O) in a nitrogen atmosphere,iii. reacting the PBAE polymer obtained in process step (i) and the structure obtained in process step (ii) in nitrogen atmosphere to obtain PBAE-Siaa polymers with Formula X chemical structure10. A method of synthesizing a branched poly(p-amino ester) polymer according to claim 9, comprising the process steps of;i. reacting 0.1-10 mmol of bisphenol A ethoxylate diacrylate monomer and 0.1-10 mmol of diethylenetriamine monomer with tetrahydrofuran (THF) at 0-100° for 1-24 hours to obtain poly(β-amino ester) (PBAE) polymer, ii. reacting the methylol groups of 0.1-10 mmol N-[3-(Trimethoxysilyl) propyl]ethylenediamine monomer with the various amino acid as 0.1-10 mmol amino acid histidine, isoleucine, serine, methionine, phenylalanine or arginine in distilled water (dH2O) in a nitrogen atmosphere at room temperature for 1-24 hours,iii. reacting the PBAE polymer obtained in process step (i) and the structure obtained in process step (ii) in nitrogen atmosphere at room temperature for 1-24 hours to obtain PBAE-Siaa polymers with Formula X chemical structure11. A method of synthesizing a branched poly(p-amino ester) polymer according to claim 9, comprising the process steps of;i. reacting 1.2 mmol of bisphenol A ethoxylate diacrylate monomer and 1 mmol of diethylenetriamine monomer with tetrahydrofuran (THF) at 50°C for 24 hours to obtain poly(β-amino ester) (PBAE) polymer,ii. reacting the methylol groups of 0.2 mmol N-[3-(Trimethoxysilyl) propyl]ethylenediamine monomer with the various amino acid as 0.6 mmol amino acid histidine, isoleucine, serine, methionine, phenylalanine or arginine in distilled water (dH2O) in a nitrogen atmosphere at room temperature for 24 hours,iii. reacting the PBAE polymer obtained in process step (i) and the structure obtained in process step (ii) in nitrogen atmosphere at room temperature for 24 hours to obtain PBAE-Siaapolymers with Formula X chemical structure12. A branched poly(β-amino ester) polymer having high transfection, multifunctionalized with silane and modified with amino acids, synthesized by a method according to any one of claims 9-11.

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