Multistimuli-Responsive Amphiphilic Poly(ester-urethane

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Multi-stimuli-responsive Amphiphilic Poly(ester-urethane) Nano-assemblies Based on L-Tyrosine for Intracellular Drug delivery to Cancer Cells Rajendra Aluri, Sonashree Saxena, Dheeraj Chandra Joshi, and Manickam Jayakannan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00334 • Publication Date (Web): 17 Apr 2018 Downloaded from http://pubs.acs.org on April 17, 2018

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Multi-stimuli-responsive Amphiphilic Poly(esterurethane) Nano-assemblies Based on L-Tyrosine for Intracellular Drug delivery to Cancer Cells Rajendra Aluri, Sonashree Saxena, Dheeraj Chandra Joshi and Manickam Jayakannan*1 Department of Chemistry Indian Institute of Science Education and Research (IISER) Pune Dr. Homi Bhabha Road Pune 411008, Maharashtra India *1

Corresponding Author: [email protected]

Keywords: L-Amino

acid

Polymers,

Poly(ester-urethane)s,

Thermo-responsiveness,

Enzyme-responsiveness, Drug Delivery, Cancer Therapy

Abstract Multi-stimuli-responsive L-tyrosine based amphiphilic poly(ester-urethane)s nano-carriers were designed and developed, for the first time, to administrate anticancer drugs at cancer tissue environment via thermo-responsiveness and lysosomal enzymatic-biodegradation from single polymer platform. For this purpose, multifunctional L-tyrosine monomer was tailormade with PEG-lated side chain at the phenolic position along with urethane and carboxylic ester functionalities. Under melt dual ester-urethane polycondensation, the tyrosine-monomer reacted with diols to produce high molecular weight amphiphilic poly(ester-urethane)s. The polymers produced 100±10 nm spherical nanoparticles in aqueous medium and they exhibited thermo-responsiveness followed by phase transition from clear solution into turbid solution in heating/cooling cycles. Variable temperature transmittance, dynamic light scattering, and 1H-NMR studies revealed that the polymer chains underwent reversible phase transition from coil-to-expanded chain conformation for exhibiting the thermo-responsive 1

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behaviour. The lower critical solution temperature (LCST) of the nano-carriers were found to be corresponding to cancer tissue temperature (at 42-44 °C) which was explored as extracellular trigger (stimuli-1) for drug delivery through disassembly process. The ester bond in the poly(ester-urethane) backbones readily underwent enzymatic-biodegradation at the lysosomal compartments that served as intracellular stimuli (stimuli-2) to deliver drugs. Doxorubicin (DOX) and camptothecin (CPT) drug loaded polymer nanocarriers were tested for cellular uptake and cytotoxicity studies in normal WT-MEF cell line, and cervical (HeLa) and breast (MCF 7) cancer cell lines. In vitro drug release studies revealed that the polymer nanoparticles were stable at physiological conditions (37 °C, pH =7.4) and they exclusively underwent disassembly at cancer tissue temperature (at 42 °C, pH ~ 5) and biodegradation by lysosomal-esterase enzyme to deliver 90 % of DOX and CPT. Drug loaded polymer nanoparticles exhibited better cytotoxic effects than their corresponding free drugs. Live cell confocal microscopy imaging experiments with lysosomal-tracker confirmed the endocytosis of the polymer nanoparticles and their biodegradation at the lysosomal compartments in cancer cells. The increment in the drug content in the cells incubated at 42 °C compared to 37 °C supported the enhanced drug uptake by the cancer cells under thermo-responsive stimuli. The present work create new platform for L-amino acid multiple-responsive polymer nanocarrier platform for drug delivery and the proof-of-concept was successfully demonstrated for L-tyrosine polymers in cervical and breast cancer cells.

Introduction Polymers from L-amino acid resources are important classes of synthetic nanoscaffolds for gene, anticancer drug delivery to cancer cells,1-3 and tissue engineering4-5 etc. Ring opening polymerization of NCA-monomers from L-amino acids produced well defined di- and triblock synthetic polypeptides6-7 and the self-assembled fibrillar networks, micelles and vesicles from these polypeptides were explored for diverse applications in material science and biomedical fields.8-10 The synthetic polypeptides are found to be excellent biocompatible materials;11 however, the inherent limitation of the biodegradation of high molecular weight polypeptide chains by intracellular enzymes has appeared to be one of the major concern for their long-term applications.12 In the last one decade, significant efforts have been taken to develop non-peptide polymer analogues from L-amino acid resources based on polycondensation approaches as an alternative strategy for synthesising biodegradable polymeric

nanomaterial.13

Poly(ester-amide)s,14-18

poly(ester-urea),19

2


poly(disulfide-

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urethane)s20 poly(ester-urea-urethane),21 and polycarbonates,22-26 are some of the most important non-peptide polymer structures explored for biomedical applications. Among, these polymers, poly(ester-amide)s are an interesting class of polymers since they possessed amide linkages for hydrogen bond induced self-assembly and ester-linkages for biodegradation under intracellular environment. Poly(ester-amide)s based on L-malic acid,27 lactic acid,28 hyperbranched polymers from gallic acid and glycine,29 L-phenylalanine were accomplished.30 Structure-property relationship was also established in poly(ester-amide)s from various diols, dicarboxylic acids and α-amino acids.31 L-Amino acid based amphiphilic poly(ester-amide)s were found to be excellent nanoscaffolds for delivering various drugs and genes in cancer treatment.15,17 These polymer nano-carriers were found to be biodegradable under presence of intracellular species like glutathione (GSH) and lysosomal enzymes such as chymotrypsin. L-Cysteine based poly(ester-amide)s were recently reported for the delivery of doxorubicin and docetexal.32 L-Tyrosine based polycarbonates were also reported as matrix for cell adhesion, tissue fillers and bone repair, etc.22,24,26 We have been exploring solvent free melt polycondensation approach to make poly(ester-urethane)s from L-amino acid monomer resources.33-34 This synthetic methodology opened up new opportunity to access few unexplored polymer structures such as L-aspartic acid and L-glutamic acid based functional polyesters,35 L-cysteine based redox degradable disulphide polyesters,36 and hyperbranched poly(ester-urethane)s from L-serine,37 etc. Among these polymer structures, amphiphilic L-aspartic polyesters and L-tyrosine poly(ester-urethane)s nano-assemblies were demonstrated as enzyme-responsive nanocarriers for doxorubicin (DOX), camptothecin (CPT), topotecan and curcumin, etc.38-39 Recently, luminescent L-aspartic acid based polyester was also accomplished to create FRET probe platform for real-time monitoring of enzymatic-digestion of polymer nanoparticles.40 It is important to mention that poly(esterurethane)s are completely different from poly(ester-amide)s both in structure and design which requires different synthetic methodology such as dual ester-urethane polycondensation approach developed from our laboratory. Thus, the poly(ester-urethane)s are relatively new classes of polymers having both hydrogen bonding interactions for polymer self-assembly and aliphatic-ester linkages for biodegradation under physiological conditions. Most of the above discussed L-amino acid based non-peptide polymeric nanocarriers have limitation since these nanocarriers could be largely dissociated or degraded under only one type of biological events like enzymatic-digestion. This single-stimuli approach restricts their wide

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applicability in biomedical applications; hence, new polymer design is required to make multipurpose L-amino acid polymer nano-carriers to maximize their therapeutic efficacies. The “smart polymeric nano-carriers” are required to be multi-stimuli-responsive so that they could be employed to exploit maximum number of biological events or stimuli that occur both at the tissue and intracellular level.41-42 The temperature and pH of the cancer tissue environment (40-42 °C and pH ≤ 6.8) are different from normal tissues (37 °C and pH = 7.4).43 Thermo-responsive polymeric materials have ability to undergo phase separation at cancer tissue environment by which the nano-carrier concentration could be enhanced many folds compared to normal tissues.44-45 Further, polymer nanocarriers have ability to selectively accumulate (a passive way) at the cancer tissue environment through enhanced permeability and retention (EPR) effect;46 thus, the introduction of thermo-responsiveness in polymers provides additional advantage as active target at cancer tissues along with EPR effect.47 PNIPAAM48-49 and polyacrylates47,50-51 are most intensively studied thermoresponsive polymers in the literature for biomedical applications. In these polymers, the nonbiodegradable C-C backbone in polyacrylates is a main concern with respect to their digestion at the intracellular compartments and clearance from organism during the drug delivery. We have reported enzyme-degradable PEG-lated amphiphilies (not polymers) based on renewable resource 3-pentadecyl phenol for drug delivery to the cancer cells.52-53 PCL based biodegradable thermo-responsive nanocarriers were also reported for drug delivery.54-55 The above discussion emphasized that creating new polymer design by merging the enzymeresponsive biodegrading properties of non-peptidic L-amino acid polymers with additional stimuli of thermo-responsiveness would open up new opportunities in cancer drug delivery. The introduction of additional thermo-responsive features in these polymer structures would result new smart multi-stimuli-nano-carrier that are capable of exploiting both extracellular (temperature) and intracellular (enzymatic-degradation) in single polymer nano-scaffold. Up to our knowledge, there is no report in the literature for L-amino acid based nano-carriers in drug delivery applications that are responsive to both temperature and enzymes. The lack of non-availability of suitable synthetic methodology is one of the limitations to merge both the thermo-responsive and enzyme-biodegradation concepts together. To address this important task, here we utilized our expertise in melt polycondensation chemistry for L-amino acid polymers33-40 with thermo-responsive polymer materials52-53 to develop the first generation of enzyme and thermo-responsive L-amino acid polymer nano-vectors for drug delivery applications. For this purpose, L-tyrosine platform was chosen and new amphiphilic L-

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tyrosine poly(ester-urethane)s nanocarriers were tailor made to deliver anticancer drugs in cancer cell lines. This new concept in shown in figure 1.

Figure 1: (a) Development of multi-stimuli responsive L-tyrosine based amphiphilic poly(ester-urethane)s. (b) Drug delivering capabilities of polymer nanocarriers under thermo-responsiveness and lysosomal enzymatic-biodegradation in cancer cells. Here, we report the design and development of new classes of thermal and enzymeresponsive

L-tyrosine

based

biodegradable

amphiphilic

poly(ester-urethane)s

and

demonstrate their anticancer drug delivery abilities in cancer cells. The polymer structure was build with following features to accomplish the above target: (i) the L-tyrosine was suitably modified as multi-functional monomer to yield poly(ester-urethane)s, (ii) the phenolic unit was substituted with long polyethylene glycol chains (hydrophilic) to impart appropriate amphiphilicity in the hydrophobic backbone so that the resultant polymer chains could selfassemble into < 200 nm nanoparticles in water, (iii) the selective phase separation of PEGchains in water upon heating (or reversible in cooling) produced the first thermo-responsive L-amino acid polymer nano-carriers, (iv) the LCST of the polymer nano-carriers were found to be closer to the cancer tissue temperature (40-42 °C) which is very useful to enrich the 5



polymer-drug concentration at the cancer tissue site, and (v) the polymer backbone has aliphatic ester and urethane (or carbamate) chemical linkages; thus, the lysosomal enzymes could readily biodegrade the backbone to bring the disassembly exclusively at the intracellular compartment. Anticancer drugs such as doxorubicin (DOX) and camptothecin (CPT) and the in vitro studies confirmed that these polymeric nanoparticles are responsive to both esterase-enzyme and temperature. The drug release patterns established that the polymer nanoparticles are stable at physiological conditions (pH 7.4 PBS, 37 °C) and they undergo bio-degradation in the presence of esterase enzyme (stimuli-1). The thermoresponsive drug release studies has confirmed that at 37 °C (healthy tissue temperature) polymeric nano particles are highly stable and they undergo selective disassembly at 42 °C (cancer tissue temperature, stimuli-2). Cytotoxicity studies and cellular internalization studies were carried out in normal wild type mouse embryonic fibroblast cell lines (WT-MEFs) and human breast cancer cell lines (MCF 7). The drug loaded polymeric nano particles showed excellent cell killing in the cancer cells compared to free drugs. The cellular internalization was confirmed by confocal microscope and flow cytometry analysis. The present investigation opens up new opportunities to design L-amino acid based biodegradable and thermo-responsive nanocarriers and the proof-of-concept was demonstrated more exclusively on L-tyrosine based amphiphilic poly(ester-urethane)s.

Experimental procedure Materials: L-Tyrosine, PEG-mono methyl ether-350, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, 1,12-dodecanediol, 1,4-cyclohexane dimethanol, tosyl chloride, titanium tetrabutoxide (Ti(OBu)4), DAPI, DOX.HCl, Camptothecin and horse liver esterase enzyme, Tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), DMSO, and 4% paraformaldehyde, fluoromount were purchased from Aldrich chemicals and used without further purification. Other reagents and solvents were purchased locally and purified prior to use. WT-MEF, human breast cancer cells (MCF 7) and cervical cancer cells (HeLa) were maintained in DMEM (Gibco) which contained 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin−streptomycin. Cells were maintained at 37 °C under a 5% CO2 atmosphere. 40% DPBS (Gibco) were used for cell washing, 0.05% trypsin was obtained from Gibco. 96- well or 6-well with flat bottomed plastic plates were obtained from Costar. Lab TEK 8 well cover glass chamber were obtained from Nunc159 Lab Tek. Lysotracker Green DND-26 was purchased from Cell 160 Signaling Technologies (CST). The C-methyl 6


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ester N-methyl carbamate of tyrosine ( compound 1) was synthesized as reported earlier.39 Instrument model, method of analysis, and other details are given in the supporting information.

Synthesis of Polyethylene glycol monomethyl ether-tosylate (2): PEG-350 monomethyl ether (15.0 g, 42.8mmoL) and triethyl amine (18.0 mL, 128.0 mmoL) were stirred for 10 minutes in dichloromethane (50 mL). p-Toluene sulfonylchloride (16.28 g, 85.7mmoL) in dichloromethane (50 mL) was added drop by drop to the above reaction mixture at ice cold condition. The reaction continued for 12 hours at 25 ˚C. Solvent was evaporated by rota evaporator and the crude product was purified by column chromatography using 10 % chloroform in methanol as mobile phase. Yield: 18.2 g (87 %). 1H-NMR (400 MHz, CDCl3) δ ppm: 2.44 (s, 3H, -C6H5-CH3), 3.37 (s, 3H, -O-CH2-CH2-O-CH3), 3.58-3.68 (m, 26H, -OCH2-CH2-O-), 4.15 (t, 2H, -CH2-CH2-O-SO2-C6H5-), 7.78-7.80 (d, 2H, -C6H5-), 7.33-7.35 (d, 2H, -C6H5-). 13C-NMR (100 MHz, CDCl3) δ ppm: 144, 132, 129, 127, 71, 70, 69, 68, 58, 21. FT-IR. 829, 985, 1054, 1177, 1243. 2850, 2933.

Synthesis of L-Tyrosine monomer (3): To a suspension of tyrosine monomer (1) (8.25 g, 32.6 mmoL) in dry acetonitrile (80.0 mL), K2CO3 (14.39 g, 104.3mmoL) was added. The reaction mixture was purged with N2 gas for 15 minutes and it was refluxed for 30 minutes. To this solution, PEG-350 tosylate (13.68 g, 27.1 mmoL) in dry acetonitrile (20.0 mL) was added drop wise and reaction was refluxed for 48 hours under the nitrogen. The reaction mixture was filtered through sintered funnel, solvent was evaporated by rota evaporator, and the crude product was purified by column chromatography using 20 % methanol in chloroform.Yield: 9.0 g (94 %). 1H-NMR (400 MHz, CDCl3) δ ppm: 3.00-3.04 (m, 2H, -ArCH2), 3.37 (s, 3H, -OCH3), 3.53-3.56 (t, 2H, O-CH2-CH3), 3.67 (s, 3H, -NH-COOCH3), 3.71 (s, 3H, -COOCH3), 3.66 (t, 2H, O-CH2-CH2-O-), 3.68 (t, 2H, O-CH2-CH2-O-), 3.73 (t, 2H, O-CH2-CH2-O-), 3.84 (t, 2H, O-CH2-CH2-O-), 4.08-4.10 (t, 2H, O-CH2-CH2-O-), 4.60 (s, 1H, Ar-CH2CH), 5.14 (s, 1H, NH), 6.81-6.83 ( m, 2H, Ar), 7.01-7.03 ( m, 2H, Ar). 13C-NMR (100 MHZ, CDCl3) δ ppm: 172, 157, 156, 130, 127, 114, 71, 70, 69, 67, 58, 54, 52, 37.FT-IR. (cm−1): FT-IR 742, 826,986, 1056, 1176, 1244, 1358, 1399, 1445, 1517, 1616, 1697, 1759, 2867, 2935.

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Synthesis of L-tyrosine poly(ester-urethane)s (P-12): Detailed procedure for the synthesis of L-Tyrosine based poly(ester-urethane)s was described for P-12 polymers. L-Tyrosine monomer (3) (0.5 g, 0.86 mmoL), 1,12-dodecane diol (0.17 g, 0.86 mmoL, for polymer P-12) and titanium tetrabutoxide (0.003 g, 0.008 mmoL, 1 mole % as catalyst) were taken and melted in a cylindrical shaped polymerization reactor having provision of nitrogen purge. The polymerization mixture was immersed at 100 °C oil-bath and degassed with nitrogen and vacuum at least three times each. The polymerization reactor was immersed in oil-bath at 150 °C and purged with nitrogen gas for 4 h with constant stirring. The viscous melt was subsequently subjected to stirring under vacuum (0.05 bar) for 2h at 150 °C. At the end of the polymerization white viscous solid mass was obtained as polymer product. The polymers were purified by dissolving it in hot tetrahydrofuran, filtered and precipitated into methanol. The purification procedure was repeated at least twice to obtain pure polymer samples. Yield: 0.59 g (97 %). 1H-NMR (400 MHz, CDCl3) δ ppm: 1.27 ( bs, 18H, -CH2-CH2-), 1.60 (m, 4H, -CH2-CH2-), 1.83 (m, 4H, -CH2-CH2-), 3.00-3.04 (m, 2H, -C6H5CH2), 3.38 (s, 3H, -OCH3), 3.56 (m, 2H, -OCH2CH2O-), 3.60-3.67 (m, 24H, -OCH2CH2O-), 3.70-3.74 (m, 2H, OCH2CH2O-), 3.84 (m, 2H, -OCH2CH2-), 4.08-4.13 (m, 6H, -OCH2CH2-, –COOCH2-, and NHCOOCH2-), 4.56 (s, 1H, C6H5CH2CH-), 5.12 (s, 1H, NH-), 6.81-6.83 (m, 4H, C6H5-), 7.01-7.03 ( m, 4H, C6H5-).

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C-NMR (100 MHZ, CDCl3) δ ppm: 171, 157, 155, 130, 127,

114, 71, 70, 69, 67, 65, 58, 54, 37, 32, 29, 25. All other polymers P-6, P-8, P-10 and P-CHDM were produced by following above procedure using 1,6-hexadiol; 1,8-octanediol; 1,10-decadiol and 1,4-cyclohexanedimethanol, respectively. The yield and NMR date are provided in the supporting information.

Optical transmittance measurement: Optical transmittance of aqueous solutions of all the polymers was measured using Perkin Elmer Lambda 45 UV-visible spectrophotometer equipped with temperature controlled peltier system. In LCST measurement concentration of the polymer were maintained as 3 mg/mL. Heating and cooling cycles were recorded by heating and cooling the samples from 30 °C to 80 °C. The sample was equilibrated at each temperature for 10 minutes prior to recording their spectra.

Drug encapsulation and in vitro release: The drug loading experiment was performed with the anticancer drugs DOX and CPT employing the dialysis method. The procedure was adopted from the previously reported protocol.39-40 The dialyzed solution was filtered, 8


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lyophilized and stored in dark at 4 °C. The drug loading content (DLC) and drug loading efficiency (DLE) were determined by absorption spectroscopy using the molar extinction coefficients as 11500 L mol-1 cm-1 and 11250 L mol-1 cm-1 for DOX and CPT, respectively, their formulae have been mentioned in supporting information under the experimental section. The release profiles of drug loaded polymer nanoparticles were studied by dialysis method using absorbance spectroscopy. The typical experimental protocol was followed as reported earlier..[1,2] In order to study the thermo-responsive trigger for drug release, the polymer concentration was maintained as 3 mg/mL since the minimum concentration required to show thermo responsiveness is 2 mg/ml and the release studies were carried out at 44 °C. The cumulative drug release was calculated using the following equation.33,34 Cumulative drug release = {[Amount of drug released at time‘t’] / [Total amount of drug in nanoparticles taken in dialysis tube]} × 100.

Cell viability assay and Cellular Uptake by Confocal microscopy and Flow Cytometry: These studies were carried out following our reported procedure40 and the details are given in the supporting information. The cytotoxicity of nascent polymer and drug loaded polymer nanoparticles was studied in MCF 7, HeLa and WT-MEF cell lines using the MTT assay. The protocol for cell viability assay and cellular uptake was same as that reported earlier34. The absorbance from the purple formazan crystals formed as a result of MTT reduction by mitochondrial dehydrogenase enzyme was the measure of viable cells. LSM 710 confocal microscope was used for imaging and flow cytometry was carried out on the BD LSR Fortessa SORP cell analyzer. The 561 nm laser was used for the excitation of DOX and the band pass filter chosen was 610 ± 10 nm. The fluorescence histograms were recorded for a population of 10, 000 cells. Internalization of drugs inside the cells were monitored using live cell imaging where in 8 well containing live cell chambers were used to grow HeLa cells at a density of 25000 cells per well.35 The cells were exposed to P-12 nanoparticles loaded with DOX and free DOX for 4 h and the cells were treated with lyso-tracker green DND 26 and imaged using confocal microscope. Lyso tracker green was excited using 488 nm laser and imaged under green channel. The concentration of DOX was maintained at 2 µg/ml. The images were processed using Image J software.

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Results and Discussion

Synthesis of Amphiphilic Poly(ester-urethane)s L-Tyrosine is a unique L-amino acid with three different functional groups which are carboxylic acid, amine and phenolic-OH. The carboxylic acid and amine functional groups were converted into carboxylic methyl esters and methyl urethane, respectively to yield compound 1 as shown in scheme-1. Polyethylene glycol monomethyl ether with average molecular weight of 350 was converted into its corresponding tosylate (2). L-Tyrosine phenolic group in compound 1 was substituted with 2 to get PEG-lated L-Tyrosine monomer (3). The structure of the monomer was characterized by 1H, 13C- NMR and MALDI-TOF and it was found to be thermally stable up to 200 °C for melt polymerization. The MALDI-TOF spectrum of the L-tyrosine monomer 3 showed mass peaks having difference of 44 amu with respect to (OCH2CH2)x + Na+/K+ (see supporting information, SF-1). L-Tyrosine monomer (3) was subjected to melt polymerization with commercial aliphatic diols such as 1,6- hexane diol; 1,8- octane diol; 1,10-decane diol; 1,12-dodecane diol and 1,4-cyclohexane dimethanol (CHDM) to yield polymers as shown in scheme-1. Typically, 1:1 mole ratio of the monomer and diols were taken along with Ti(OBu)4 catalyst (1 mol %) in a test tube shaped polymerization reactor and subjected to melt polymerization at 150 °C. At 150 °C both the ester and urethane functional groups equally react with diols to produce polymers.33,37 The polymer synthesis is done in one-pot involving two step process by purging with nitrogen gas to produce 3-8 oligomeric chains which were further polycondensed under high vacuum to yield L-tyrosine based poly(ester-urethane)s. The variation of chain lengths in the aliphatic diols from C6 to C12 as well as cyclo-aliphatic rings (from CHDM) gave enough structural diversity to tune hydrophobicity of the backbone. In the present design each repeating units carry one long PEG chains as pendent; thus, the resultant polymer matrix have significant hydrophilic-hydrophobic balance (side chain and main chain) to self-assemble in aqueous medium. Further, the fact that polymers were produced under solvent free melt approach which is an added advantage for biomedical applications.

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Scheme 1. Synthesis of L-tyrosine monomer and amphiphilic poly(ester-urethane)s

Figure 2. 1H-NMR spectra of monomer 3 (a) and polymer P-12 (b) in CDCl3. 13C-NMR spectra of monomer 3 (c) and polymer P-12 (d) in CDCl3 (asterisks are solvent peaks). (e) GPC chromatograms of polymers in THF at 25 °C. (f) Table containing molecular weights and the glass transition temperature (Tg) of the polymers are given. The occurrence of the melt polymerization was confirmed by NMR spectroscopy. 1H and 13C-NMR spectra for polymer P-12 and monomer 3 are shown in figure 2 and the peaks are assigned with alphabets for different protons in the chemical structure (see Figure 2a). The methoxy protons in -COOCH3 and -NHCOOCH3 appeared at 3.66 ppm and 3.72 ppm 11



along with the (OCH2CH2)x in monomer (see protons a+b+h). In the 1H-NMR spectrum of P12, new peaks corresponding to the formation of new ester -COOCH2CH2- and new urethane -NHCOOCH2CH2- appeared at 4.04 ppm and 4.10 ppm, respectively (see Figure 2b). In the 13

C-NMR spectrum of the monomer 3 (see Figure 2c), the peaks corresponding to the

methoxy carbon atoms in -COOCH3 and -NHCOOCH3 appeared at 52.26 ppm and 52.27 ppm, respectively. These carbon atoms completely disappeared in the polymer spectrum (see Figure 2d, shown by arrow) and two new peaks at 65.52 and 65.54 ppm appeared corresponding to newly formed urethane and ester linkages -NHCOOCH2CH2-, COOCH2CH2-, respectively. Further, new peaks with respect to the -(CH2)10- aliphatic chain in the backbone were also clearly visible in the polymer spectra (see Figure 2b and 2d). Hence, both the 1H-NMR and

13

C-NMR analysis confirmed the occurrence of the melt

polymerization reaction and it may be concluded that both the ester and urethane units in the monomer 3 underwent polycondensation with diols to produce desired poly(esterurethane)s.36 A similar structural characterization was done for other polymers and their 1H and

13

C-NMR spectra are given in the supporting information (see figures SF-2 and SF-3).

The gel permeation chromatograms in the figure 2e showed mono-modal distributions and the polymers were produced in moderate molecular weights in the range of Mn = 4.0 to 14.4

× 103 g/mol and Mw = 7.5 to 24.8 × 103 g/mol (see table in figure 2f). It is observed that there is a linear increment in the molecular weights of the polymers with increase in the carbon atoms in diols employed in polymerization reaction. The newly synthesized L-tyrosine poly(ester-urethane)s consists of PEG-side chain as pendants. Thus, two distributions are possible in the polymer chains: (i) corresponding to the distribution arising from the main-chain backbone (where n =1,2, 3…) and (ii) additional distribution from the PEG chains ((OCH2CH2)x, where x= 3,4,…) as shown in scheme-1. The MALDI-TOF spectrum of high molecular weight polymer chains were difficult to obtain; thus, the polymer samples having low molecular weight chains at the nitrogen purge stage were subjected to MALDI-TOF analysis. MALDI-TOF mass spectrum of the polymer was recorded and provided in SF-4. The spectrum showed clear distribution of PEG chains (varying x-values) for repeating units n=1, 2 and 3. The molecular weights of the peaks for x= 3 and 4 are assigned in repeating units n=1, 2 and 3 and the polymer chains were predominantly found to have ester functional groups at the ends. Thus, this further confirmed the occurrence of the dual ester-urethane melt polycondensation process. Thermogravimetric analysis showed that the polymers were stable up to 300 °C (see SF-5). Differential scanning 12


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calorimetry studies revealed that all the polymers are in amorphous nature and the glass transition temperature showed little increment with increase in the carbon atoms in diol (see Tg values in table in Figure 2). This trend is attributed to the high degree of flexibility introduced by the PEG chains in the repeating unit which predominantly overcome on the structural changes created by the variation in the aliphatic diol segment in the chain backbone. Rheological measurements, storage modulus (G’), and loss modulus (G’’) are shown in SF-6. All the polymers were having higher storage modulus than the loss modulus which indicated the elastic behaviour of the polymers. Loss modulus was predominating at higher strain values (˃5-10%) indicating that at higher strains polymers were in viscous nature. At lower strains they behaved as elastic materials. With the increasing the shear rate, the viscosity of the polymers was found to decrease up to 10 s-1 followed by abrupt increase in the viscosity (see figure SF-6). This increase in viscosity with increase in shear rate is called shear thickening and it is an important characteristic of the engineering thermoplastic materials. The monomer 3 and P-12 polymer were subjected to circular dichroism (CD) spectroscopic analysis to study their ability to form secondary structures. The CD spectra in SF-7 showed peak at 227 nm with positive cotton effect with respect to the proline-type II type self-assembly in tetrahydrofuran.[34-35,37] Interestingly, the CD signal of the monomer exactly retained in the polymer indicating that the optical properties of the L-tyrosine part was not affected by the polymerization process.

Thermo-responsiveness of amphiphilic poly(ester-urethane)s In the present investigation, the polymer geometry was designed with hydrophobic backbone and hydrophilic PEG chains as pendent. Upon dispersing in aqueous medium, the PEG chains would be highly hydrated and protruded outside whereas the backbone would be folded away from water. This selective chain folding of hydrophilic and hydrophobic units in the polymer backbone would result in folded hair-pin type amphiphiles. The aggregations of these amphiphiles produce polymer micelles in which the PEG chains occupy the outer-shell whereas the polymer backbone placed at the core of the micelles. This polymer self-assembly is schematically shown in figure 3a. It is very well reported in the literature that hydrated PEG chains would tend to undergo thermo-responsive phase transition to non-hydrated state above its lower critical solution temperature (LCST).42 In order to study the thermoresponsiveness of the current polymer design, the aqueous dispersed polymer nanoassemblies were subjected to transmittance studies. The polymers were dialyzed against 13



milli-Q water using semi permeable membrane with MWCO = 1000 daltons (see experimental section for more details). The resultant dialyzed polymer solution was found to be transparent at room temperature with 100% transmittance (see vials in figure 3b). Upon heating, the polymer solution turned into turbid solutions and it could be turned back to clear solution by allowing it to cool to ambient conditions. The temperature-induced phase transitions in the aqueous polymer solutions are captured and the photographs of polymer solution in vials are shown in figure 3b.

Figure 3. (a) Self-assembly of polymer micelles in aqueous medium. (b) Photographs of dialysed solutions of polymer samples in vials before and after LCST. (c) % Transmittance of polymer solutions at different temperatures in the heating cycle. (d) % Transmittance of P12 polymer at different concentrations in the heating cycle. (e) Plot of % transmittance versus polymer concentration. (f) Thermo-reversibility of phase transition in P-12 polymer in aqueous solution for 10 consecutive heating and cooling cycles. The concentrations of the polymers were maintained as 10mg/mL for figures (b), (c) and (f). The samples were equilibrated for 10 minutes at each temperature prior in recording their absorbance spectra. These aqueous solutions were subjected to transmittance study to investigate the thermoresponsive behaviour. The samples were typically heated to a particular temperature and equilibrated for 10 minutes using Peltier heating set-up attached in the absorbance spectrophotometer. The ten minutes equilibration ensured the stability of the % transmittance data. In the cooling cycle, a similar protocol is followed at each temperature to record the % transmittance. For example in figure 3b, the % transmittance of the polymers showed thermoresponsiveness in three stages (or steps): (i) in the first stage, the polymers retained solubility 14


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with same % transmittance below LCST, (ii) in the second-stage, the polymer chains undergo sudden phase separation from highly transparent to highly opaque (or turbid) stage, and (iii) in the third-stage, the polymer chains retained their phase separated form above the LCST even at much higher temperatures near to the boiling point of water. All these three stages are perfectly reversible in the subsequent cooling cycles. The % of transmittance was studied as a function of temperature and the plots are shown in figure 3c for heating cycle (see SF-8 for cooling cycle). It was found that all the aqueous solutions of polymers changed from transparent to turbid solutions at LCST = 40-42 °C (see figure 3b, 3c). The LCST of the polymers decreased from 50 to 42 °C while increasing the aliphatic diol length from C8 to C12 (the polymer with C10 did not follow this trend). Interestingly, the extent of change in turbidity for all the polymers seem to be almost identical. The polymer P-10 did not follow the trend for decrease in LCST while increasing the aliphatic chain length in the diol segments. Indeed, the polymer P-10 was made more than three times to remove the doubts regarding any abnormality in the structure on LCST; however, all the three polymer batches showed similar LCST values. Thus, the reason for the unusual low LCST value of P-10 polymers compared to P-12 was not clear at present and it is rather difficult to provide structural correlation between the aliphatic chain lengths in the dial segments with the LCST temperatures. From these studies it was found that P-12 polymer with PEG-350 side chain and 1,12-dodecane diol in the main chain showed the phase transition near to the cancer tissue temperature (42-43 °C). Thus, the polymers P-12 was chosen for future studies. The concentration of P-12 polymer was varied to find out the minimum concentration required for complete phase transition for transparent to turbid. The LCST of the polymer solutions were found to be almost same for the concentration ranging from 1.0 to 10 mg/mL (see Figure 3d). The plot of 100 % turbidity versus the concentration of polymer showed that minimum 3.0 mg/mL polymer concentration is required to notice a visible change in the 100 % turbidity (see Figure 3e). The reversibility of the thermo-responsive phase transition was further investigated for 10 consecutive heating cooling cycles between 30 and 60 °C. The plot in figure 3f showed very good reproducibility in the phase transition between the transparent and turbid state in the heating and cooling cycles.

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Figure 4. (a) DLS histogram, (b) FE-SEM and (c) AFM images of P-12 polymer nanoparticles. (d) Variable temperature DLS histograms of P-12 nanoparticles. (e) Plot of DLS size versus the temperature for P-12 nanoparticles in heating and cooling cycles. The concentrations of the polymers were maintained as 10 mg/mL for experiments in (d) and (e). To study the phase separation phenomena in detail the P-12 polymer aqueous assemblies was subjected to dynamic light scattering studies. DLS histograms of P-12 showed the formation of 100 ± 10 nm nano-aggregates. The nano-assemblies, the aqueous polymer sample was subjected to field emission scanning electron microscope (FE-SEM) and atomic force microscope (AFM) at 25 °C. FE-SEM images (see Figure 4b) showed the existence of isolated spherical nanoparticles of < 50 nm and aggregated nanoparticles of 100±20 nm. The formation of these spherical particles was further confirmed by AFM images. AFM images of P-12 polymer (see figure 4c) confirmed the existence of spherical nanoparticles. To study the thermo-reversibility in P-12 polymer, the DLS histograms recorded in the heating and cooling cycles as a function of temperature (see figure 4d). Data in the figure 4d confirmed that at ambient temperature polymer was completely dispersed in the aqueous medium with the formation of nanoparticles of the size around 100 ± 10 nm. Upon heating the nanoparticles underwent phase separation which resulted in the formation of nano assemblies of around 600 ± 10 nm. To strengthen the above hypothesis variable 16


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temperature DLS studies was carried out by sweeping the temperature from 30 °C to 80 °C and cooled back to 30 °C. The size of the nano-aggregates from this experiment in Figure 4e showed the phase transition of polymer nanoparticles from nano-meter sized assemblies below LCST to sub-micrometer sized larger particles above LCST. The reversibility of this phase transition behaviour was examined by measuring the size of the nanoparticles in 10 consecutive heating and cooling cycles (see SF-9). It was found that the polymer retained the LCST behaviour even after 10 cycles and the size of the nanoparticles oscillated between nano-metre to sub-micrometer in both heating and cooling cycles. There was no hysteresis observed in either % transmittance (see Figure 3f) or size of the nanoparticles (see Figure 4e) in heating cooling cycles indicating that the phase transition followed almost identical pathways. To study the structural changes of poly(ester-urethane)s from the transparent to turbid phase transition process, the P-12 polymer was subjected to variable temperature 1H-NMR studies in D2O (see figure 5a). Polymer sample was heated from 30 °C to 80 °C at 10° intervals and then cooled back to 30 °C. In the hydrated form (below LCST), the protons corresponding to ethylene glycol side chains at 3.65 ppm showed broad peaks from 30 to 45 °C and no peaks were observed for hydrophobic aliphatic polymer chain backbone or phenyl protons. As the temperature increased above LCST, the signals of ethylene glycol units were sharpened at 3.65 ppm and the peaks for the aliphatic part in the hydrophobic and phenyl ring also appeared at 1.0-2.0 ppm and above 7.00 ppm, respectively. This trend was attributed to the following arrangements of the polymer nanoparticles in the aqueous medium. Below LCST, the PEG units projected at the periphery of the nanoparticles which interacted with D2O solvent. At this condition, the hydrophobic chains at the core of the nanoparticles were not accessible by the solvent; thus, no signals were observed in the spectra.56-57 Above LCST, the polymer nanoparticles underwent more open-chain conformation in which both the PEGs and the hydrophobic backbone came in contact with D2O solvent molecules. As a result, the signals were observed for both hydrophilic and hydrophobic parts of the polymers in the nanoparticles. The NMR signals were perfectly reversible in the heating and cooling cycles confirming their reversible self-assembly process (see SF-10 for cooling cycle).

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Figure 5: (a) 1H-NMR spectra of P-12 polymer in D2O solvent in the heating cycle at various temperatures. (b) Mechanism for the phase separation of the polymers above and below LCST. The plausible mechanism for this temperature dependent phase transition is shown in figure 5b. At ambient temperature, inter-molecular hydrogen bonding between the oxygen of ester and urethane carbonyl with water molecules are favourable. This makes the PEG part in the polymer chains at the highly hydrate state. PEG chains behaves as more hydrophilic in nature and the inter-molecular hydrogen bonding between water molecules and PEG chains facilitates the complete dispersion of the polymer chains in aqueous medium. Thus, below LCST the polymer chains adopted more globular conformation and existed as < 100 nm sized nanoparticles. At higher temperatures, however, the PEG chain water molecules interaction became weak which made the polymer chains less hydrate. As a result, the polymer chains precipitated out in the aqueous medium. The increase in the NMR signals for the

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hydrophobic segments above LCST revealed that the polymer chains probably underwent disassembly from the globular nature to open-chain confirmation. The aggregation of these open-chain produced micrometer-sized polymer aggregates (as shown Path-1 in figure 5b). Another possible aggregation mechanism could be drawn for the precipitation of tiny nanoparticles together above LCST to produce micro-meter sized particles (path-2 in Figure 5b). The path-2 is ruled out in the present investigation since this process would not expose the hydrophobic part of the polymers chains to give signals in the NMR spectra above LCST. Thus, the variable NMR experiments revealed that the polymer chains were expected to undergo globular to more open-chain conformation changes above LCST which in turn produced micro-meter sized aggregates and account for the turbidity of the solution.

Figure 6. (a) Fluorescence spectra of pyrene fluorophore in various concentration of P-12 polymer in water (λex = 334 nm). (b) Plots of PL intensity of pyrene at I1 and I1/I3 versus the polymer concentration. The break point (shown by arrow) represents the critical micelle concentration of polymer. (c) Fluorescence spectra of pyrene fluorophore at various temperature in the heating cycle (λex= 334 nm). The samples were equilibrated at each temperature for 10 minutes prior to recording the emission spectra. (d) Plots of PL intensity of pyrene at I1 versus temperature in the heating and cooling cycles. For the temperature dependent studies the polymer concentration was maintained as 500.0 µg/mL.

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The critical micelle concentration (CMC) of the polymer nanoparticle was determined by pyrene fluorophore. For this purpose, the pyrene concentration was maintained as 0.6 µM and the polymer P-12 concentration was varied from 0.2 µg/mL to 16.0 µg/mL. The resultant polymer solution was subjected to fluorescence spectroscopic analysis by exciting the pyrene fluorophore at 334 nm. The fluorescence spectra of the polymer solution are shown in figure 6a. It is clear from the emission spectra that the pyrene encapsulation increased with increase in the polymer concentration indicating that pyrene was more solubilized in the polymer nanoparticle environment. The plot of PL intensity (at 372 nm with respect to I1 peak) versus polymer concentration in figure 6b showed continuous increase in the intensity with a break point ∼ 3.0 µg/mL. The ratio of the peak intensities I1/I3 is the measure of the micropolarity of the pyrene probe in hydrophobic pocket of the polymer nanoparticles. The plot of I1/I3 versus polymer concentration showed break point at 2.5 µg/mL. Both the PL intensity plot and the I1/I3 plot showed break points at 2.5 µg/mL which was attributed to the CMC of the amphiphilic polymer nanoparticles. To study the role of the thermo-responsiveness polymer nanoparticles and to support the NMR observation in Figure 5; pyrene encapsulated polymer at 500 µg/mL concentration (above CMC) was subjected to temperature dependent fluorescence studies in heating and cooling cycles. The fluorescence spectra of the pyrene emission in the heating cycle are shown in Figures 6c (for cooling cycles, see SF 11). The emission spectra showed reduction in pyrene PL intensity with increase in temperature with respect to the dis-assembly of polymer nanoparticles and release of pyrene in the aqueous environment. The plot of PL intensity for heating and cooling cycles is shown in figure 6d. The pyrene intensity decreased drastically with increase in temperature and the process was noticed completely irreversible in the subsequent cooling cycles. This implied that once pyrene was released from the polymer hydrophobic pocket to the aqueous environment, it did not return back to the original site in the cooling cycles. This trend clearly supports the path-1 shown in figure 5b based on NMR studies. This observation is just opposite to the Chung et al

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report on the pyrene encapsulation in PIPAA thermo-responsive polymer which

followed the path-2 process of aggregated to isolated nanoparticles transformation as shown in Figure 5b. The % transmittance measurements, DLS data and variable temperature NMR, and pyrene encapsulation studies indicate that the L-tyrosine amphiphilic polymers possessed a unique ability to undergo thermo-responsive phase separation closer to cancer tissue temperature.

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Encapsulation of Drugs and Thermo-responsive Release

Figure 7. DLS histogram, FE-SEM and AFM images of (a) CPT loaded P-12 nanoparticles and (b) DOX loaded P-12 nanoparticles. (c) Absorbance spectra of P-12 polymer and its CPT and DOX loaded nanoparticles. (d) Fluorescence spectra of P-12 polymer and its CPT and DOX loaded nanoparticles. (e) % Transmittance of CPT and DOX loaded P-12 nanoparticles in heating and cooling cycles.

Nanoparticles of these L-tyrosine polymer was loading water insoluble drugs such as doxorubicin (DOX ) and camptothecin (CPT). Loading capabilities of these poly(esterurethane)s were investigated by dialysis method using semi permeable membrane of the MWCO = 1000 daltons. The resulted drug loaded transparent polymer nanoparticles were subjected to absorbance spectroscopy to determine the amount of encapsulated drugs. It was found that the P-12 polymer nanoparticles showed the drug loading efficiency (DLE) as 50 % and 14% for DOX and CPT, respectively. The drug loading content (DLC) was obtained as 5.0 % and 1.4% for DOX and CPT, respectively. These samples were named as P-12-DOX and P-12-CPT. DLS histogram of the DOX and CPT loaded P-12 polymer has shown in figures 7a and 7b. DLS histogram showed size of drug loaded nanoparticles as < 200 nm and it suggested that the size of the nanoparticles was retained even after the encapsulation of the anticancer drugs. FESEM images and AFM images showed spherical nanoparticles with size of 98 % DOX released in the presence of esterase enzyme. Degradation behaviour polymer nanoparticles was tested by DLS studies and the details are shown in figures 9c. The initial histograms showed monomodal distribution with average sizes of 200±15 nm. As the time progressed, the esterase enzyme reacted and cleaved the ester bonds in these poly(ester-urethane)s and disassembled

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the nanoparticles. As a result, the polymers chains tend to phase separate from the aqueous medium due to their poor solubility which produced larger size aggregates. In the absence of esterase enzyme in pH=7.4 PBS (not shown), the nanoparticles retained their stable selfassembled structures even after 48 hours. DOX loaded polymer nanoparticles also showed the same trend in their DLS histograms in the presence of esterase enzyme and no appreciable change was observed in the absence of enzymes. The DLS sizes of the drug loaded polymer nanoparticles were plotted against incubation time and showed in figure 9d. From the release studies, it can be concluded that these poly(ester-urethane) nano scaffolds are very stable at physiological conditions and selectively undergoes cleavage in the presence of esterase enzyme or cancer tissue temperature to release the loaded cargoes. Hence, the custom designed L-tyrosine nanoparticles are unique classes of dual-responsive biomaterials having enzymatic-biodegradability as well as responsive to temperature-stimuli in a single polymer system which is yet to be reported in the literature.

Figure 9. (a) Schematic representation of cellular internalisation of polymer nanoparticles. (b) Cumulative drug release profiles of CPT and DOX loaded P-12 nanoparticles in pH 7.4 24


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PBS in the presence and absence of esterase enzyme at 37 °C. (c) DLS histograms of CPT loaded polymer nanoparticles at various time intervals of incubation in presence of esterase enzyme. (d) The plots of DLS size of CPT and DOX loaded nanoparticles at various time intervals in the presence or absence of esterase enzyme in PBS at 37 °C.

Cytotoxicity and cellular uptake

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The cytotoxicity of these poly(ester-urethane) scaffolds was investigated in non cancerous wild-type mouse embryonic fibroblasts (WT-MEFs) cell lines, breast cancer MCF 7 cell lines and cervical cancer HeLa cell lines using MTT assay. The cytotoxicity of the nascent polymer scaffold was investigated by treating the cell lines with different concentrations of polymer ranging from 1.0 µg/mL to 60.0 µg/mL and the data has been shown in the figures 10a. These polymer nanoparticles were found to be non-toxic and confirmed biocompatible nature of the L-tyrosine poly(ester-urethane) nanoparticles to the cell lines. Further to investigate the drug internalization and cell killing ability of the drug loaded polymer nanoscaffolds, P-12-CPT and P-12-DOX polymer samples were administered to the cell lines. Drug concentrations were maintained as 0.1 µg/mL to 1.0 µg/mL and the MTT assay data is shown in figures 10b and 10c. In normal WT-MEF cell lines, free drugs showed much more killing than the drug loaded polymer nano scaffolds at all concentrations (see Figures 10b and 10c). The free CPT and DOX became less effective and the drug loaded polymer scaffolds accomplished enhanced cell killing (see figures 10d to 10f). From these results, it is clear that th enanoscaffolds have capability to perform better in killing cancer

cells over the normal cells (see SF-12 for HeLa). Figure 10. Cytotoxicity of P-12 nascent nanoparticles (a), free CPT and CPT loaded P-12 (b), and free DOX and DOX loaded P-12 (c) in WT-MEF cell line. Cytotoxicity of P-12 nascent nanoparticles (d), free CPT and CPT loaded P-12 (e), and free DOX and DOX loaded P-12 (f) in MCF 7 cell line. (Incubation time = 72 h). 26


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Cellular internalization were visualized using confocal laser scanning microscope (CLSM) DOX and the dapi-stained nuclei were visualized in red channel and blue channels, respectively and the details are shown in figure 11a. From the image, it can be confirmed that the free DOX is predominantly internalized to nucleus of the cells and it was further confirmed from magenta colour of the merged image. DOX loaded polymer nanoparticles were internalized throughout the cell, nucleus as well as cytoplasm of the cells supporting the cleavage of nanoparticles in the cytoplasm by enzymes and subsequent delivery of the DOX to the nuclei. From this study it was confirmed that P-12 polymer nanoparticles were successfully internalized and released DOX inside the cells. P-12-CPT administered cells were visualized by exciting at λ = 405 nm and visualized through blue channel (no DAPI staining). The images corresponding to free CPT and P-12-CPT showed bright blue fluorescence which confirmed the successful internalization of the CPT (see SF- 13). The uptake of the drug loaded polymer nano particles were further confirmed by flow cytometry analysis. The plot in the figure 11b revealed that free DOX administered cells were showing the DOX intensity of 200-300 a.u where as the cells incubated with DOX in polymer scaffold were showing 1500-2000 a.u DOX intensity. From this flow cytometry data, it was confirmed that when DOX was administered from polymer scaffold it was taken up by the cells by 6-8 times more compared to free drugs. The cellular uptake of the nanoparticles is energy driven process; thus, at low temperature (4 °C), the ATP to ADP hydrolysis is slowed down at the intracellular level which indirectly influences the energy requirements for the cellular uptake across the cell membranes.59-60 To test the role of temperature on the endocytosis process, the cellular uptake studies were carried out for DOX loaded nanoparticles at 4 °C and the data is compared for 37 °C in figure 12. The DOX intensity was found to be significantly high for the cells incubated at 37 °C compared to 4 °C. Hence, the reduction of DOX intensity at 4 °C is attributed to the reduction in the cellular uptake of DOX loaded nanoparticles. This provided direct evidence for the increased DOX uptake at 37 °C and supported the endocytosis process of the DOX loaded polymer nanoparticles as shown in scheme-1. From the above three independent experiment i.e., MTT assay, confocal microscope images and flow cytometry it may be concluded that these poly(ester-urethane) nano scaffolds synthesized from L-tyrosine amino acid are compatible to the cells and excellent vehicles for delivering the anti cancer drugs.

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Figure 11. (a) CLSM images and (b) flow cytometry histograms of free DOX and DOX loaded P-12 nanoparticles in MCF 7 (9 h incubation, [DOX]=2.0 µg/mL and 10,000 cells were used).

Figure 12. (a) CLSM images of DOX loaded P-12 incubated in MCF7 cells at at 4 °C and 37 °C. (b) DOX intensity in the cells were estimated as CTCF (corrected total cell fluorescence using imaje J software) and plotted for DOX loaded P-12 incubated in MCF7 cells at 4 °C and 37 °C. DOX concentration=2.0 µg/mL. The cells were incubated for 30 minutes at 4 °C and 37 °C.

Internalization of drugs inside the cells were monitored using live cell imaging where in 8 well containing live cell chambers were used to grow HeLa cells at a density of 25000 cells per well. The cells were exposed to P-12 nanoparticles loaded with DOX and free DOX for 4h and were treated with lyso-tracker green DND 26 and imaged using confocal microscope. Lyso tracker green was excited using 488 nm laser and imaged under green 28


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channel. The concentration of DOX was maintained at 2 µg/ml. As can be seen in the Figure 13a, the live cells exhibited the presence of lysosomes showing bright green luminescence in the cytosol of the cells. The live cell imaging of the free DOX in Figure 13b showed the DOX predominantly localized in the nucleus and not in the cytosol or lysosomes. The polymer nano-carriers assisted delivery in Figure 13c images clearly showed the DOX colocalization in the lysosomes evident from the resultant yellow colour due to overlay of red and green fluorescence together in the lysososmes. This technique was extremely comprehensive in tracing the pathway by which the nanoparticles are taken into the cells. The co-localization of DOX loaded nanoparticles and lysotracker made it evident that the nanoparticles are indeed taken up by the process of endocytosis and not merely diffusion. The enzymes in the lysosomal compartment trigger their degradation and subsequent DOX

released to the nucleus.

Figure 13: Live cell CLSM images of control (a), free DOX (b), and DOX loaded P-12 (c) in HeLa cells using lyso-tracker (green color) (4h incubation, DOX concentration=2.0 µg/mL).

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Figure 14: CLSM images of free CPT and CPT loaded P-12 incubated in MCF 7 cells at two different temperatures. (a) 37 °C healthy tissue temperature, and (b) 42 °C closer to tumour tissue temperature (Incubation time 4h). To further evaluate the temperature responsive drug release of polymer nano particles, cellular internalization studies were carried out at two different temperatures 37° and 42 °C. After administering the CPT drug loaded nanoparticles, the cells were incubated at two different temperatures 37 °C (normal tissue temperature) and 42 °C (cancer tissue temperature) for 4h and the internalization of the drugs were visualized through confocal microscopy (see figure 14). The results confirmed that at 42 °C more internalization of the DOX occurred compared to normal tissue temperature which confirmed the thermoresponsive drug delivery of polymer nanoparticles. Efforts to test the cytotocity of CPT loaded or DOX nanoparticles at 42 °C was not successful. The cells were not able to survive at this temperature for more than 16 h incubation; thus, 72 h MTT was not possible to estimate the cytotoxicity effect. Therefore, the comparisons of cell growth inhibition at thermo-responsive conditions at 42 °C was not able to establish in the present studies. The thermo-responsive effect along with EPR enhancement could be predominantly visible in the in vivo studies. Nevertheless, the present approach has opened new opportunity for structural engineering in L-amino acid polymer for the development of thermo-responsive nano-carriers based on L-tyrosine. The nano-carrier is also enzymatic biodegradable at the intracellular lysosomal compartments to deliver both DOX and CPT and the proof of concept is demonstrated in cervical and breast cancer cell lines. 30


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Conclusion In summary, a new class of amphiphilic multi-stimuli-responsive poly(esterurethane)s were designed and synthesized from L-tyrosine amino acid resources and their multiple drug delivering capabilities were demonstrated in cervical and breast cancer cell lines. The polymers were in-build with thermo-responsiveness and enzymatic-biodegradation as multi-stimuli and the proof-of concept was successfully demonstrated for the delivery of DOX and CPT. PEG-lated L-tyrosine monomer was custom designed through organic reactions and it was subjected to solvent free melt condensation polymerization to produce amphiphilic poly(ester-urethane)s. In the aqueous medium, these amphiphilic poly(esterurethane)s were self-assembled into < 200 nm sized nanoparticles through selective phase separation of polymer backbone and PEG-chains as core and shell of the spherical nanoparticle geometry. These polymer nanopartciles exhibited unique thermo-responsive phase transition having lower critical solution temperature (LCST) closer to cancer tissue temperature (at 42 °C). The thermo-responsiveness was further studied in detail using optical transmittance, DLS and variable temperature NMR spectroscopy. These studies revealed that the polymer nano-assemblies underwent reversible phase transition from coil-to-expanded chain conformation and exhibited turbid or clear solution in the heating and cooling cycles, respectively. These L-tyrosine poly(ester-urethane)s nanoparticles were capable of loading hydrophobic anticancer drugs such as DOX and CPT. In vitro drug release studies revealed that these polymer nanoparticles were very stable at normal physiological conditions (37 °C, pH = 7.4 PBS) and released the loaded drugs at cancer tissue temperature (42 °C, pH = 7.4 PBS) via disassembly process (thermo-responsiveness). Once the polymer nano-carriers were taken up by the cells, it underwent biodegradation by lysosomal esterase enzymes (enzymeresponsiveness) to deliver the DOX and CPT at the intracellular compartments. Cytotoxic studies revealed that the nascent poly(ester-urethane)s nanoparticles were highly biocompatible and their drug loaded polymer nanoparticles showed better killing compared to free drugs. The cytotoxicity studies were done in normal cells (WTMEFs), cervical (HeLa) and breast (MCF 7) cell cells. CLSM and flow cytometry revealed the enhanced uptake of the drug loaded polymer nanoparticles over free drugs. Live cell imaging using lyso-tracker staining clearly evident for the digestion of polymer nano-carriers in lysosomal compartments. Though the present investigation has demonstrated the thermo- and enzyme responsive multi-stimuli polymer nano-carrier approach exclusively in L-tyrosine system; the

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approach is not restricted to this example alone and in principle it may be expanded to other L-amino acid polymers for wide range of biomedical applications. Supporting Information: Biology experimental details, stack plots of 1H-NMR,

13

C-NMR,

spectra of polymers, TGA, DSC and rheology data of polymers, variable temperature NMR data, DLS data, cytotoxicity data for HeLa cells, and confocal microscopy images, and NMR details of monomer intermediates are provided in supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgments: The authors thank research grant from Department of Science and Technology (DST) for the project SB/S1/OC-37/2013. Authors thank the confocal microscopy facilities built at IISER Pune Campus for all the bio-imaging experiments. Sonashree Saxena thanks IISER Pune for Ph.D research fellowship. Dheeraj Joshi thanks CSIR-UGC for research fellowship. Author

Information:

Corresponding

Author:

Prof.

Manickam

[[email protected]]. The authors declare no financial interest.

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Jayakannan

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Table of Contents

Multi-stimuli-responsive Amphiphilic Poly(ester-urethane) Nano-assemblies Based on L-Tyrosine for Intracellular Drug delivery to Cancer Cells

Rajendra Aluri, Sonashree Saxena, Dheeraj Chandra Joshi and Manickam Jayakannan*1

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Multistimuli-Responsive Amphiphilic Poly(ester-urethane

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