Synthesis and characterization of biocompatible monotyrosine-based polymer and its interaction with DNA

A novel tyrosine-based copolymer containing L-tyrosine (Tyr) and diglycidylether of bisphenol A(DGEBA) was synthesized and studied for its interaction with DNA for potential applications in biological systems. The synthesis of the polymer was optimized by varying monomer ratios using 4(dimethylamino)pyridine (DMAP) as catalyst to yield polymers with Mw of 7500-8000. Further characterization with FTIR, NMR and thermal analysis supported the formation of monotyrosine10 DGEBA polymer. The interaction of 1:1 DGEBA-tyrosine copolymer with DNA was investigated by gel electrophoresis, thermal melting, and fluorescence spectroscopy in ratios ranging from 0.5:1 to 12:1 polymer –DNA (w/w). The copolymer was seen to lend stability to the DNA without damaging it and demonstrated endonuclease resistivity that is conducive for biological applications. Scanning Electron Microscopy, Dynamic Light Scattering and Zeta Potential studies of the polymer-DNA complex also 15 established that the polymer is capable of encapsulating DNA leading to the formation of the DNApolymer polyplex nano-assembly. The potential of the polymer for biological applications was further reinstated by its non-cytotoxicity.


Introduction
Tyrosine, while classified as a non-essential amino acid, is used 20 as a building block for several important neurotransmitters, including norepinephrine, 1-3 epinephrine 3 and dopamine 4 as well as play an important role in photosynthesis. 5 Apart from its biological roles, researchers have used tyrosine as an amino acid of choice to create unique polymers and supramolecular 25 structures. 6-8 Such polymers have been researched for applications in various biomedical areas including tissue engineering, drug and gene delivery amongst others. 9-12 The choice of tyrosine for creating novel polymers stems from the unique structure of the neutral amino acid that harbours an 30 aromatic ring and three functional groups that can be made to react with various reagents after careful manipulation of the reaction conditions. Herein, we report the synthesis and characterization of a copolymer that incorporates tyrosine, with DGEBA as a linker 35 through generation of ester and amine functionalities at linkages. The use of L-tyrosine as one of the main constituents of a copolymer with DGEBA has not been reported before to the best of our knowledge. Our synthetic methodology leads to the formation of a polymer that incorporates the less reactive, poorly 40 soluble L-tyrosine with the highly hydrolysable DGEBA. The incorporation of DGEBA in the polymer is due to its ability to polymerize efficiently through its highly reactive epoxide moiety that can be easily broken in mild acidic or basic conditions. The π-π interaction between the aromatic rings of the constituents 45 have been found to facilitate the formation of nanostructures through the self-assembly of such polymers 13 which also prompted us to investigate tyrosine in the context of polymerization with DGEBA. DGEBA has been extensively polymerized to generate copolymers with lactones and amines. 14,50 15 Synthesis of DGEBA homopolymer is also reported 16 by anionic polymerization mechanism. Structure and properties of several copolymers have been discussed previously by one of the authors. 17,18 Peripheral coupling of tyrosine to polycationic polymers has been 55 achieved that displayed reduced cytotoxicity and stability for invitro and in-vivo applications. 8,13 Tyrosine polymerization resulting in the generation of dityrosine units in the polymer matrix have been reported with potential bio-application. 9, 10,19 As such, many tyrosine polymers have been found to be 60 biocompatible by common cell cytotoxicity assays and many researchers have pointed out their potential bioapplications including drug delivery, gene delivery and tissue engineering. 9, 11 However, low cytotoxicity essentially does not mean that the concerned polymer does not inflict any damage to biomolecules 65 like DNA and scores of proteins inside the cell at the molecular level. The interactions of the polymers with important cellular contents need to be evaluated in detail to ensure safety of the cellular contents for the desired in-vitro or in-vivo applications. In this context, the interaction of tyrosine-based polymers with 70 biomolecules like DNA and proteins at the molecular level is still largely unknown. Thus, apart from the synthesis and the meticulous characterization of the novel tyrosine-DGEBA polymer herein, we also report a detailed study of the interaction of the synthesized tyrosine-DGEBA with 5 various forms of DNA viz. calf thymus DNA (ctDNA), plasmid and DNA oligomer duplex of approximately 50 base pair long. Our study also discusses the non-cytotoxicity and the ability of the polymer to resist endonuclease activity on the DNA.

Materials
Diglycidyl ether of bisphenol A(DGEBA), 4-(dimethylamino) pyridine (DMAP), L-Tyrosine (Tyr), calf thymus DNA (ctDNA), agarose, Ethidium bromide (EtBr) and chemicals for buffer preparation were purchased either from Sigma or Merck and used 15 as received. The pUC19 plasmid and Hind III restriction enzyme were purchased from New England Biolabs, USA. All other materials and solvents were used as received without further purification. All experiments of polymer-DNA interactions were done in triplicate and error bars were generated after calculating 20 the mean SD from the average value wherever applicable.

Synthesis of DGEBA-Tyrosine polymer
The optimized synthetic route is reported herewith (Scheme 1). Variations to solvent, catalyst, time, temperature and concentration of catalyst and monomers were also employed. 25 Copolymers were synthesized by adding DGEBA to L-Tyrosine in varying monomer ratios of 1:0.5 (Bp-Ty 1), 1:1 (Bp-Ty 2) and 1: of DGEBA spot after 48 hours. The product was precipitated in water after solvent evaporation. A sticky brown solid separated out of the solution that was washed with water (5 X 10 mL) and ether (5 X 5 mL) to remove DMF, DMAP and unreacted monomers. Crude product thus obtained was further purified by 45 dissolving in DMF and precipitating with water in an ice bath (0°C), following water and ether washes and drying under vacuum for 24 hours. The method for re-precipitation was repeated twice to obtain ultrapure compound for polymer characterization and further downstream studies with DNA. 50 Alternatively, the reaction mixture in DMF was concentrated under vacuum to a volume of 5 mL. To this, 20 mL cold methanol was added in an ice bath with stirring to precipitate out unreacted tyrosine. The extract was filtered and solvent evaporated to remove methanol and DMF. The polymeric product 55 was then precipitated out in water as discussed above. 1

Particle Size and Zeta Potential Determination
The hydrodynamic diameter and zeta potential of the polymer-DNA complexes were measured by Dynamic Light Scattering (DLS) experiments on a Delsa NanoC Particle Analyser (Beckman-Coulter). Individual samples of ctDNA (15 µg/mL) in 40 1 mM NaPi at pH 7.0 (2 mL) filtered through a membrane filter (PVDF, 0.2 µm) were prepared and aliquots of polymer solutions in DMSO were added to achieve different polymer-DNA (w/w) ratios with an overall 5% DMSO (v/v) in 1 mM NaPi buffer at pH 7.0. The DLS measurements were performed after 30 minutes 45 and as well as after 12 hours incubation in duplicate at 25°C and a scattering angle of 165°. The average particle size of each sample was obtained by using CONTIN analysis as the mean hydrodynamic diameter (standard deviation of five determinations including polydispersity). For zeta potential 50 measurements, polymer-DNA complexes at ratios of 1:1, 1:5, 1:10 and 1:20 containing 2.5µg/mL ctDNA were prepared in distilled water with 5% DMSO (v/v). These were incubated for 12 hours and the zeta potential was measured across electric field of 16.3 V/cm, scattering angle of 15° and cell positions of 0 mm, 55 ±0.35 mm and ±0.7 mm.

MTT Assay for Cell Activity
The MTT assay was performed to measure the metabolic activity of cells. 1 mg of Bp-Ty-2 was dissolved in 0.5 mL of 5% DMSO to make 2 mg/mL stock solution. The stock solution was diluted 60 to 100 µg/mL, 75 µg/mL, 50 µg/mL, 25 µg/mL and 10 µg/mL concentration solutions in the culture medium with serum. Cells cultured in normal medium were considered as cell control and 5% DMSO in culture medium as reagent control (0.5% DMSO). Equal volume (100 µL

Synthesis of Tyrosine-DGEBA polymers
Polymerization of tyrosine and DGEBA is hypothesized to proceed via nucleophilic attack of DMAP on tyrosine to facilitate its activation and the ring opening of the DGEBA epoxide moiety (Scheme 1). Tyrosine, having low solubility in most organic 85 solvents, is appreciably soluble in aqueous solvents depending on pH. It exists primarily in zwitterionic form, and hence is not readily reactive. The reaction between tyrosine and DGEBA was carried out in an organic medium as the epoxide moiety of DGEBA gets hydrolyzed in aqueous solution. Initial reactions 90 were performed using tetrahydrofuran as solvent. However, low solubility of tyrosine in THF led us to employ DMF as the solvent.
The reaction in DMF with 1:1 monomer ratio (Bp-Ty 2) was monitored until the consumption of at least one of the monomers. 95 The reaction was followed for 48 hours at 70°C when disappearance of DGEBA spot on TLC and no sedimentation of unreacted tyrosine were observed. The reaction at 16 hours and 24 hours at 10% DMAP concentration showed DGEBA spot on TLC and sedimentation of excess tyrosine indicative of 100 incomplete reaction. It is apparent from Figure 1A that the tyrosine consumed in the reaction is linearly proportional with time up to 50 hours. The higher the catalyst concentration, the higher is the slope or higher is the reaction rate ( Figure 1A, 1B). 5 mole percent DMAP concentration gave only 15% yield 105 compared to 58% with 10 mole percent DMAP at 48 hours and 70°C. DMAP was not used at concentrations above 10% to avoid its embedding in the polymer matrix. With 5% catalyst, excess tyrosine sediments out of the reaction and TLC confirmed presence of 10 excess DGEBA in the solution, indicating that 10 mole percent DMAP was required for complete consumption of the reagents ( Figure 1A). The reaction between tyrosine and DGEBA was initially performed without any catalyst, with variations to temperature 15 from 25, 40 to 70°C without any yield. Excess tyrosine was seen to sediment in the flask at the end of each reaction. Thus, an initiator/catalyst was employed such that it may catalyze the reaction. NaOH and triphenyl phosphine (TPP) were also used, at 10 mole percent in the basic pH range. Both failed to digest 20 tyrosine in the reaction ( Figure 1B) unlike DMAP, which demonstrated consumption of tyrosine and change in colour of solution from a white colloidal mixture to dark brown clear solution indicating reaction between tyrosine and DGEBA.
Temperature variation was carried out to ascertain the 25 dependence of the reaction on temperature. As seen from Figure  1C, the reaction proceeds faster on increasing the temperature. At room temperature, the reaction failed to proceed, indicating the need to heat to activate the reaction. On going from 70 to 100°C, complete consumption of tyrosine was observed at the end of 16

Gel Permeation Chromatography
The molecular weights of the synthesized polymers were determined using GPC as tabulated below ( Table 1) and DGEBA as an AB type random copolymer, which is further supported, by NMR, ESI-MS and thermal studies.

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The ESI-MS spectra for Bp-Ty 2 (Supporting Information, Figure S1) copolymer shows major fragments at 495 Da (m/z) corresponding to 1 unit each of DGEBA and tyrosine with the loss of a CO moiety, 521 Da (m/z) for a complete DGEBA-Tyr unit and 227 Da (m/z) for bisphenol A moiety of DGEBA. The 70 data show that the linkage between tyrosine and DGEBA results due to the ring opening of the epoxide moiety and its reaction with the carboxyl and amine groups of tyrosine without the loss of any water molecule. This supports the absence of any peptide linkage (between two tyrosine units) in the copolymer. This data 75 also confirms that the major repeating unit in the polymer is Tyrosine-DGEBA.

Fourier Transform Infrared Spectroscopy
The IR spectrum for Bp-Ty 2 copolymer (Figure 2A) shows absorption peaks at 3400 cm -1 (medium, broad, hydroxyl groups and secondary amines), 1653 cm -1 (strong, carbonyl group-ester or amide group with tyrosine), 1605 cm -1 (strong, aromatic C=C), 10 1580 cm -1 (medium, aromatic C=C and N-H bend from tyrosine), 1505 cm -1 (strong, aromatic C=C), 1243 cm -1 (strong, broad, C-N stretch for tyrosine) and 1235 cm -1 (C-O-C bend for DGEBA). Disappearance of the characteristic peak of DGEBA at 914 cm -1 for epoxide bending indicates ring opening of the DGEBA 15 moiety. The strong peak appearing at 1653 cm -1 in the product is an indication of the formation of a carbonyl group in the copolymer.
The peak at 1653 cm -1 was normalized with respect to the 1605 cm -1 peak (C=C stretching in aromatic compounds) for the 20 synthesized Bp-Ty copolymers. As depicted in Figure 2B, this peak had high transmittance for Bp-Ty 2 and Bp-Ty 3. As expected, the transmittance decreased for 1:0.5 DGEBA-Tyr (Bp-Ty 1) copolymer due to low amount of tyrosine incorporated in the polymer. 25

Nuclear Magnetic Resonance Spectroscopy
The peaks corresponding to the functionalities are shown in the NMR spectra ( Figure 3).
In the 1 H-NMR, the protons are accounted for in the 4-9 ppm 30 range. For amide functionality, a peak between 4-6 ppm is expected 21 that is absent here, indicative of ester bonding between tyrosine and DGEBA, which correlates with the mass spectrometry result discussed previously. The monomeric ratios in the synthesized copolymers were calculated from the NMR 35 spectra by taking a ratio of the number of aliphatic methyl protons of DGEBA to aromatic protons of DGEBA and Tyrosine. With a substrate ratio of DGEBA-Tyr as 1:1, the ratio of aliphatic methyl protons to aromatic protons was calculated as 6:12 or 1:2. The NMR spectrum for the synthesized polymer Bp-Ty 2 ( Figure   40 3B), displays this aliphatic to aromatic proton ratio close to 1:2.     Ty 1), the synthesized copolymer contains monomers in the same ratio, which is supported by the fact that DGEBA undergoes homopolymerization in the above method. This also indicates the 30 presence of copolymer units and DGEBA homopolymer within the polymer matrix of Bp-Ty 1. The copolymer Bp-Ty 2 has a T g centred at 77°C after which the copolymer starts to melt beyond 120°C (Supporting Information, Figure S3).

35
Agarose gel assay was conducted to determine the effect of copolymer on DNA structure. The supercoiled form of pUC19 plasmid DNA after incubation with the polymer does not show the presence of any linear or nicked form even after incubation for 24 hours ( Figure 5A). This  Figure S4, Supplementary  Information). Additionally, mobility shift of the DNA bands incubated with Bp-Ty was not observed whereas DNA incubated with PEI shows mobility shift at concentration ratio of 0.75:1 polymer-DNA (w/w). This suggests that PEI tends to neutralise 60 the peripheral charge of DNA at lower concentrations due to its polycationic nature. Since the agarose gels were stained with EtBr after completion of the gel run, the decrease in the intensity of the plasmid bands is attributed to insufficient intercalation of the EtBr into DNA due to the shielding effect of the polymer on introduced by the Bp-Ty polymer.  The absence of any nuclease activity and unwinding of the DNA in presence of the polymer is a good indication that the polymer does not inflict any damage or modification to the DNA, which is 15 desired for any biological application. The effect of the restriction enzyme, Hind III on the polymer-DNA complex to determine endonuclease activity is shown in Figure 6. Plasmid DNA, after incubation with the polymer at higher concentrations resists endonuclease activity of the enzyme Hind III to an appreciable 20 degree ( Figure 6A). As seen from Figure 6A, the free DNA (Lane 7) is completely linearized in the presence of Hind III. While up to polymer to DNA ratio of 2:1, the nuclease activity of Hind III remained unhindered, thereafter a gradual decrease in the nuclease activity of Hind III with increase in polymer 25 concentration was observed. The nuclease activity of Hind III was reduced to ca. 50% at polymer concentration 8 times more than the plasmid concentration ( Figure 6B). The decrease in nuclease activity suggests that at higher polymer concentrations, the DNA strands are not exposed to the 30 surrounding due to shielding and encapsulation effect of the polymer that prevents recognition of DNA bases by the endonucleases giving rise to insufficient amount of the linear form of the plasmid. 23 To check whether the polymer has any effect on Hind III enzyme, the enzyme was co-incubated with the 35 polymer at higher concentration followed by dialysis of the solution and incubation of the purified enzyme with DNA. No effect on the endonuclease activity of Hind III was observed, which further confirms the fact that interaction of the polymer with DNA is responsible for inhibition of endonuclease activity. 40 This result is noteworthy since in the light of the possible use of any DNA cargo to be transported to a particular cellular location, the presence of numerous endogenous nucleases can degrade the DNA even before being released from the polyplex, thus rendering the delivery ineffective. In this case, the polymer can 45 offer some resistance to endonuclease activity to the DNA cargo, albeit at higher concentrations.

DGEBA-Tyrosine copolymer lends stability to the DNA
Increase in DNA melting temperature (Tm) followed by addition of a compound or polymer is indicative of increase in the thermal 50 stability of the DNA because of intercalation, groove binding, etc. The melting temperatures of the complexes formed between ctDNA and polymer at different ratios is shown in Table 2. The Tm of native ctDNA was observed at 66°C, and a gradual increase in the Tm was documented, up to 71°C with 1:1 weight 55 ratio of polymer to DNA. A reference thermal melting data obtained to determine the effect of DMSO on Tm (not shown) showed a decrease in Tm to 65.5°C. However, the highest increase in melting temperature on complexation was found with the DNA-polymer ratio of 1:1 that confirmed the stabilisation of 60 the DNA in the presence of the the copolymer (Table 2). However, sharp Tm was unavailable at higher polymer concentrations, possibly due to formation of higher order structures and aggregates.

65
The EtBr replacement assay was performed to measure the degree of association of the polymer with DNA (Figure 7). The decrease in fluorescence of EtBr intercalated into DNA results from the quenching of EtBr after it comes out from the DNA into the solvent. 24 A gradual decrease in the fluorescence of EtBr was 70 observed upon the addition of the polymer.  Figure 7, EtBr fluorescence is seen to decrease by nearly 30% only when the DNA and the polymer ratio is 12:1. 5 This decrease in fluorescence is attributed to partial intercalation, thereby releasing bound EtBr in solution. The presence of other non-covalent interactions between polymer and DNA may also introduce change in DNA conformations, thereby releasing bound EtBr. Insertion of aromatic residues of the polymer between base 10 pairs of DNA may lead to bending of the helix at the point of intercalation, essentially releasing EtBr and hence the decrease in fluorescence. 25 Most notably, the replacement of intercalated EtBr is not substantial at comparable DNA and polymer ratio. This observation points to the fact that at lower concentrations of 15 the polymer, its intercalation into DNA is negligible, which means that the conformational distortion leading to DNA damage is minimal under such condition. The data correlate well with the agarose gel assay data ( Figure 5) wherein a gradual decrease in band intensity is observed on 20 increasing amount of the polymer. Thus, the DNA-copolymer complex formed is shown to be stable, with efficient binding and ability to restrict endonuclease degradation as desirable for a biocompatible material. 25

presence of DNA
The size and the surface charge of the polymer in presence of the DNA were investigated with DLS. As depicted in Figure 8A, good correlation was not obtained with only DNA or the polymer. However, upon addition of the polymer to DNA, the 30 correlation function gets stabilized that indicate formation of stable particles of finite size. The same trend was observed with the intensity distribution, where only the DNA or the polymer had broad distributions that consequently narrowed down when co-incubated at respective concentrations ( Figure 8B). Polymer to DNA ratio after 12 hour incubation 5 Hydrodynamic diameter of DGEBA-Tyr copolymer-DNA complexes at various copolymer to DNA w/w ratios was measured using DLS after incubation for 30 min (Supporting Information, Table S1) as well as after 12 hours ( Figure 8C). Our results indicate possible formation of polyplex between the 10 polymer and the DNA. A gradual increase in the hydrodynamic diameter of the polyplex was observed until polymer concentration was 5-6 times that of DNA. Our study shows that even after incubation of the DNA with the polymer for 12 hours, the polyplexes are stable as evident from the correlation and the 15 particle size distribution. This is in contrast with PEI incubated DNA where aggregation is more prominent after 12 hours of incubation that can deter data acquisitions. In the present case of Bp-Ty polymer, there is a gradual increase in the average hydrodynamic radii whereas with PEI the hydrodynamic radii 20 increases with increase or decrease in polymer concentration after 2:1 PEI-DNA ratio (w/w) 26 . It is to be noted that DLS data with PEI and DNA could only be obtained after addition of relevant concentration of NaCl unlike Bp-Ty polymer. Nevertheless, the average hydrodynamic radii of DNA condensates obtained by 25 incubating with Bp-Ty polymer and PEI (Mn 10000) were found to be similar (~300 nm in the concentration range 5:1 w/w). The best results in terms of correlation and intensity distribution was obtained when the concentration of polymer is between 1 to 5 times greater than the DNA. The sudden increase of the 30 diameter at polymer concentration 10 times more than the DNA suggests formation of aggregates at such concentrations. This is hypothesised to be due to the condensation of polymeric sheets with DNA leading to a more linearized complex. At 0.5:1 polymer to DNA ratio, the hydrodynamic diameter is around 219 35 nm with standard deviation and polydispersity of 3 nm and 0.2 respectively indicating a uniform entity. With increasing polymer ratio to 1:1, the polydispersity and scattering intensity remain the same, but the diameter increases, which is in agreement with literature. 20 At higher ratios of polymer to DNA such as 10:1, the 40 polydispersity is seen to increase significantly indicating a higher degree of aggregation. Overall, stable particle formation was observed within a certain range of polymer concentration with DNA that shows appreciable interaction between the polymer and DNA. Comparison of the DLS data obtained after incubation of the DNA with the polymer for 30 minutes and 12 hours revealed 5 some interesting differences (Supporting Information, Table S1). On incubation for 30 minutes, the hydrodynamic radii of the particles formed with a certain ratio of polymer to DNA, are smaller as compared to those incubated for 12 hours, indicating slow aggregation with time. It is also to be noted that the 10 diameter observed in DLS is the average hydrodynamic diameter and not the actual diameter of the polyplex, which is proven to be much lesser than that of the hydrated form. 27 Zeta potential was recorded for copolymer-DNA complexes to gain insight about the complexes' surface charge and its potential stability. Effective 15 charge density is considered as a crucial parameter in determining the structure and morphology of DNA in the condensed state. DNA condensation occurs when ~90% of the surface negative charge of DNA is neutralized in an aqueous solution and that is irrespective of the side chain functionality of the condensing 20 agent. 28 The observed pKa of the polymer at 7.6 and 11.3 (Supporting Information, Table S2) confirms 80-100% ionization of the polymer according to Henderson-Hasselbalch equation, thereby supporting DNA condensation leading to the formation of the polyplex. The zeta potential results show that the free 25 polymer has a zeta potential of 17 mV, possibly due to the basicity of the amine groups of tyrosine. The positive zeta potential for the polymer is also indicative of few free hydroxyl and carboxyl moieties in the polymer matrix. The surface charge for free DNA was observed to be -23.7 mV which is expected 30 due to the phosphate groups on DNA. Zeta potential for the polymer-DNA complexes became negative after incubation indicating that the phosphate groups on DNA interact with the free amine groups thereby making the whole entity negative. It has also been reported earlier that the condensed DNA may well 35 exist as partially dissociated polyanion, effectively bringing down the zeta potential. 29 As evident from Figure 8D, the zeta potential stabilized for 5:1 and 10:1 polymer to DNA ratios at a negative value, indicating the presence of free phosphate groups for binding. With further 40 increase in polymer concentration, a decrease in zeta potential was observed due to the availability of fewer free phosphate groups coupled with the fact of higher degree of aggregation and low stabilisation of the polyplex. The 20:1 complex was observed to be least stable as expected.

Imaging studies with DNA via SEM
The morphology of the copolymer-DNA complexes formed at ratio of 0.5:1 was examined using FE-SEM. The images of pure polymer were also obtained for reference ( Figure 9A and 9B). The Bp-Ty 2 polymer had a sheet-like appearance with 50 protrusions (50-100 nm) to the single-layered epoxy sheets. After incubation with DNA, the polymer sheets start aggregating around the DNA. In the SEM image for 0.5:1 polymer-DNA complex ( Figure 9C and 9D), dense globular DNA fragments are observed. The morphology of the polymer sheet also changes 55 after DNA interaction, showing smooth and rough patches. The smooth patches are attributed to free polymer. The rough patches as seen in Figure 9D are clusters of DNA spread throughout the polymer. The SEM images for 0.5:1 polymer-DNA polyplex display DNA on the surface of the polymer as well. For 1:1 60 polyplex ( Figure 9E and 9F), the DNA could hardly be seen on the surface of the polymer sheet possibly due to its encapsulation by the polymer. As seen in Figure 9F, the DNA is much more discreetly spread out than in 9D. Thus, the SEM images of copolymer-DNA show DNA clusters and free polymer indicating 65 that there are portions on the polymer that are not engaged in DNA interaction, which increase with polymer concentration. This also supports the agarose gel and EtBr experiments portraying the entrapment of DNA within the polyplex with increasing polymer concentration. 70 The interaction of DNA with the polymer at the interface and as a whole entity is pictorially depicted in Scheme 2. In summary, the polymer does not show any noteworthy nuclease activity and even protects plasmid DNA from endonuclease activity. Partial 75 displacement of EtBr from DNA and elevated Tm were observed following incubation of the polymer with ctDNA. As deduced from SEM, the DNA is assembled in clusters and entrapped within the polymer matrix. DLS studies showed concentration dependant stable particle formation with ctDNA in the presence 80 of the copolymer.

Cell viability
Tyrosine-based polymers have been previously reported to be non-cytotoxic. 9 In the present study, the cell viability of L929 mammalian cells (mouse fibroblast) incubated with the copolymer Bp-Ty 2 was evaluated by assessment of the cell 10 metabolic activity (MTT assay). The metabolic activity of the cells in the presence of Bp-Ty 2 in the concentration range 10-100 µg/mL is seen to remain high (Figure 10). At 100 µg/mL concentration of Bp-Ty 2, the metabolic activity is close to the reagent control and significantly higher than the 15 negative control phenol, which is indicative of low cytotoxicity of the polymer at this concentration. At lower concentrations of 10-75 µg/mL, the metabolic activity is observed to be greater, even higher than the reagent control that does not contain any polymer. This signifies that in the presence of the polymer at relevant 20 concentrations, the normal functioning of the cells are not interrupted. Overall, the synthesized polymer is observed to be non-cytotoxic at reasonable concentrations. The biocompatibility of the polymer is noticeable even in the presence of DGEBA moieties, probably owing to the occurrence of the tyrosine units. 25 Incase the polymer find applications in vector delivery in future, its non-cytotoxicity may be an advantageous property since commonly used few PEIs have been reportedly found to be cytotoxic 26,30 .

Conclusions
DGEBA and Tyrosine derived Bp-Ty copolymers with different monomer ratios were synthesized successfully using DMAP as a catalyst. Characterization of the polymer was done by FTIR spectroscopy that indicates presence of characteristic wavelengths 5 for both the starting materials as well as the formation of carbonyl bonds. 1 H and 13 C NMR indicated presence of carbonyl linkages and the former also displayed aliphatic to aromatic proton ratio close to 1:2. Thermal degradation studies confirmed the monomer ratios as 0.49:1, 1:1 and 1.1:1 in the synthesised 10 polymers Bp-Ty 1, 2 and 3 with an equimolar content of Tyrosine with DGEBA as the maximum incorporated in the polymers. Cell viability studies performed using the MTT assay confirmed the non-cytotoxic nature of the polymer. The Bp-Ty 2 polymer was thus studied with DNA to ascertain its 15 employability in biological applications. Our experiments indicate possible formation of polyplex at relevant concentration of the DNA and the polymer. The polymer was found to increase the melting temperature by 4°C, thereby lending stability to the DNA after polyplex formation. Agarose gel assays proved that 20 the polymer does not damage the DNA and even offer resistance to endonuclease activity, a fact promising for its potential bioapplications. Fluorescence assay conducted with the intercalated EtBr suggested partial intercalation or groove binding of the polymer with the DNA helix at higher polymer 25 concentrations. DLS experiments showed stable particle formation at polymer-DNA ratios of 1:1 till 5:1 and a decrease in zeta potential of the polymer upon complexation with DNA. The polymer-DNA conjugate was imaged on SEM that displayed clusters of DNA spread discreetly on the polymer sheet. Overall, 30 a novel and biocompatible polymer with DGEBA and tyrosine was created and characterized. The interaction of the polymer with DNA was evaluated that gave important insights regarding its potential to be used in biological applications. In particular, our studies indicate that the polymer has potential biological 35 applications including but not limited to scaffolding and vector delivery.