Amphiphilic Glycopolypeptide Star Copolymer-based Crosslinked

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Amphiphilic Glycopolypeptide Star Copolymer-based Crosslinked Nanocarriers for Targeted and Dual Stimuli-Responsive Drug Delivery Bhawana Pandey, naganath Patil, Govind Sudhakar Bhosle, Ashootosh V Ambade, and Sayam Sen Gupta Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.8b00831 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on December 31, 2018

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Bioconjugate Chemistry

Amphiphilic Glycopolypeptide Star Copolymer-based Crosslinked Nanocarriers for Targeted and Dual Stimuli-Responsive Drug Delivery

Bhawana Pandeyac, Naganath G. Patilac, Govind S. Bhoslebc, Ashootosh V. Ambadea* and Sayam Sen Guptad*

aPolymer

Science and Engineering Division, bOrganic Chemistry Division, CSIR-

National Chemical Laboratory, Dr. Homi Bhabha Road, Pune - 411008, India.

cAcademy

of Scientific and Innovative Research, (AcSIR), New Delhi 110025, India

dDepartment

of Chemical Sciences, Indian Institute of Science Education and Research, Mohanpur, Kolkata- 741246, India. 1 ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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*Corresponding Author: [email protected]; [email protected]

ABSTRACT Glycopolypeptide-based nanocarriers are an attractive class of drug delivery vehicles due to the involvement of carbohydrates in receptor-mediated endocytosis process. To enhance their efficacy towards controlled and programmable drug delivery, we have prepared stable glycopolypeptide-based bioactive dual stimuli-responsive (redox and enzyme) micelles for delivery of anticancer drugs specifically to the cancer cells. Amphiphilic biocompatible miktoarm star copolymer which comprises two hydrophobic poly(ε-caprolactone) blocks, a short poly(propargyl glycine) middle block and hydrophilic galactose glycopolypeptide block was designed and synthesized. The star copolymer is initially self-assembled into uncrosslinked (UCL) 2 ACS Paragon Plus Environment

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micelles and free alkyne groups at the core-shell interface of the UCL micelles were crosslinked by bis-(azidoethyl) disulfide (BADS) via click chemistry to form interface crosslinked (ICL) micelles. ICL micelles were found to be stable against dilution. BADS imparted redox-responsive properties to the micelles while PCL rendered them enzyme-degradable. Dual stimuli-responsive release behaviour with Dox as model drug was studied individually as well as synergistically by applying two stimuli in different sequences. The galactose containing UCL and ICL micelles were shown to be non-toxic. Intracellular Dox release from UCL and ICL micelles was demonstrated in liver cancer cells (HepG2) by time-dependent cellular uptake studies and controlled release from ICL micelles compared to UCL micelles was observed. The present report opens a new approach towards targeted and programmable drug delivery in tumour tissues via specifically targeted (receptormediated), dual-responsive and stable crosslinked nanocarrier system.

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INTRODUCTION

Synthetic glycopolypeptides, are potential mimics of the natural glycoproteins that regulate various cellular processes through polyvalent interaction with carbohydratebinding receptor proteins (lectins) present on the cell surface.1-4 They are biocompatible, biodegradable, a desirable substitute to other glycopolymers5-7 and thus are suitable for various biological applications.8 The fact that several carbohydrate receptors are over-expressed on the surface of cancer cells as compared to normal cells9-11 for example, galactose binding asialoglycoprotein receptor (ASGPR) on liver cancer cells,12-14 has stimulated the development of carbohydrate-displaying nanocarriers obtained from glyco-macromolecules including glycopolypeptides for targeted drug delivery.15-18 Currently, several carbohydratecontaining nanocarrier-based delivery systems (viz. micelles, vesicles, nanorods, etc.) are being explored for such applications.19-25 Amphiphilic glycopolypeptides also undergo self-assembly process to form micellar nanostructures displaying bioactive carbohydrates on their surface.26-31 However, there are very few literature reports wherein glycopolypeptide-based micelles have been evaluated for drug delivery by


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studying cellular uptake of drug-loaded micelles.27,28,30 For example, reductionsensitive amphiphilic glycopolypeptide-poly(ε-caprolactone) (GP–SS–PCL) GP block copolymer micelles27 and glycopolypeptide-based micelle (GPM) from lactose modified P(LP10-co-ZLL11) copolymer were synthesized and investigated for targeting therapy of hepatic cancer.28 In the conventional polymeric micelles, there is a thermodynamic equilibrium between micelles and their unimers in the aqueous phase.32 Due to this, during intravenous administration, the micelles are likely to be diluted below critical micelle concentration (CMC) and vulnerable to dissociation that may lead to premature drug release. This is one of the central issues regarding the biological application of selfassembled micelles.33 Additionally, interaction with the plasma proteins (high and low-density proteins) may disrupt the micellar assembly.34All these factors reduce the therapeutic efficacy of drugs loaded in micellar carriers before reaching the target and may cause undesired side effects.35,36 To improve the stability of micelle-like nanostructures, Wooley and co-workers, in their pioneering work, have introduced the concept of covalently crosslinked intramicellar assemblies.37 Crosslinking in the micellar assemblies not only retains 5 ACS Paragon Plus Environment


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their 3D structure in the solution but also enhances their stability below a particular concentration i.e. CMC so that disruption is minimized during blood circulation allowing them to be successfully used for drug delivery applications. It provides robust character to the micelles and also creates a barrier for the external agents that are responsible for the degradation of micelles and thus reduce the possibility of burst release of drug molecules to a large extent.36,37 The crosslinking of micelles can be carried out on the hydrophilic shell,38,39 within the hydrophobic core,40,41 or at the core-shell interface (interfacial crosslinking).36,42-44 Out of these three, interfacial crosslinking is a unique and interesting approach that combines the advantage of core and shell crosslinking.38 It not only permits crosslinking at high micellar concentrations without inter-micellar crosslinking but also limits the alteration in the properties of micellar core that may occur during crosslinking.44,45 Another important advantage of the crosslinking approach is that it allows incorporation of several stimuli-responsive groups in polymeric micelles such as reducible disulfide bonds,40,43 pH-cleavable linkages,43,46 and hydrolyzable ester bonds.47 Utilizing this approach, a stable micellar system, which can respond to different types of stimuli and aid in the controlled release of cargo can be designed. 6 ACS Paragon Plus Environment

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For glycopolypeptide-based micellar systems, this crosslinking approach for enhanced micelle stability and stimuli-responsiveness for controlled release has not been explored yet. Herein, we report glycopolypeptide-based interface crosslinked (ICL) micellar nanostructures that exhibit dual stimuli-responsive (redox and enzymeresponsive) nature as potential candidates for targeted and controlled drug delivery (Scheme 1). Redox-responsive nanocarriers are of great interest because of the active intracellular drug release due to the presence of GSH concentration gradient in the intracellular environment.44,45,48 Enzymes play an important role in various biological processes inside the intracellular environment by catalyzing several chemical reactions.49,50 Also overexpression of enzymes tumour cells compared to normal cells creates a gradient which act as a trigger for the enzyme-responsive release in controlled drug delivery application.51,52 We have designed and synthesized amphiphilic biocompatible miktoarm star copolymer [(PCL50)2-b-Pr-gly6-

b-GP40] comprising two hydrophobic poly(ε-caprolactone) (PCL) blocks, a short poly(propargyl glycine) middle block and hydrophilic glycopolypeptide (GP) block containing galactose units for targeting liver cancer cells. PCL, well-known biocompatible and FDA-approved polyester,53 was chosen as the degradable 7 ACS Paragon Plus Environment


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enzyme-responsive block. The star copolymer initially self-assembled into uncrosslinked (UCL) micellar structures. The free alkyne groups of the middle block at core-shell interface of the UCL micelles were then utilized for crosslinking using click chemistry with bis-azide-terminated disulfide containing molecule to form ICL micelles to introduce redox-responsive groups. Higher stability of ICL micelles to dilution compared to UCL micelles was demonstrated and synergistic dual-stimuliresponsive release of anti-cancer drug doxorubicin (Dox) was shown. Intracellular Dox release from UCL and ICL micelles was demonstrated by time-dependent cellular uptake studies which showed the controlled release from ICL micelles in response to stimuli in the intracellular environment compared to UCL micelles.


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Scheme 1. Schematic representation of the synthesized amphiphilic star coglycopolypeptides and their self-assembly into uncrosslinked (UCL) and interface crosslinked (ICL) micelles for targeted and controlled drug delivery.

RESULTS AND DISCUSSION

Synthesis and characterization of miktoarm star copolymer :(PCL50)2-b-Pr-gly6-bGP40



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In our previous report, nGP-(PCLm)2 star copolymers with varying block lengths of PCL (25, 50) and GP (10, 20, 40) were prepared and shown to assemble into different types of nanostructures such as micelles, vesicles and nanorods.26 Although the block co-polymer 40GP-(PCL50)2 formed micellar aggregates in aqueous solution, it was not explored for biological applications due to limited stability in media under dilute conditions. In an attempt to develop stable micellar systems from glycopolypeptide copolymers, an amphiphilic miktoarm star copolypeptide [(PCL50)2b-Pr-gly6-b-GP40] was designed. This construct contains an additional short poly(propargylglycine) block at the junction of hydrophobic and hydrophilic blocks, which could be used to form interface crosslinked micelles through click chemistry.


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(a) DMF, RT; (b) Proton sponge (1.0 eq.), 48 h, RT; (c) THF, hydrazine hydrate (25 eq.), 12 h. Scheme 2: Synthesis of amphiphilic miktoarm star copolymer (PCL50)2-b-Pr-gly6-bGP40.

Initially, amine end-functionalized branched macroinitiator (PCL50)2-NH2 was prepared via ring opening polymerization (ROP) of ε-caprolactone from the azidefunctionalized tetraethylene glycol (TEG)-bis-MPA diol initiator. Free terminal -OH groups of both PCL chains were acetylated to prevent the side reactions during NCA polymerization and then the azide group was reduced to amine. (Scheme S1 and 11 ACS Paragon Plus Environment


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S2). Molecular weight (Mn) of (PCL50)2-NH2 was calculated using

1H

NMR

spectroscopy by integrating the peak at 2.02 ppm for acetyl group (−CO−CH3) with respect to that for TEG protons (−CH2−CH2) at 3.63-3.65 ppm (Figure S13).

To obtain the final amphiphilic mikto-arm star copolymer architecture, sequential ROP of two conventional N-carboxyanhydrides (NCAs) viz. DL-propargylglycine NCA and α-galacto-O-lys NCA was carried out in order to minimize purification efforts (Scheme 2). DL-propargylglycine NCA was prepared by following the method of Heise. et al.11 and α-galacto-O-lys NCA was synthesized by following a previous methodology.13 The polymerization of propargylglycine NCA was initiated by (PCL50)2-NH2 (5) as a macroinitiator (M/I = 7) in dry DMF. The second monomer, α-

galacto-O-lys NCA (I:M1:M2 = 1:7:40) and ‘‘proton sponge’’ (0.5 eq.) were added to the above reaction mixture after completion of the first stage polymerization (10 min) as monitored by FT-IR spectroscopy. The completion of polymerization of second monomer was ascertained by disappearance of characteristic NCA anhydride stretching at 1785 cm−1 and 1858 cm−1 in the FT-IR spectra (Figure S1). Molecular weight of the star copolymer (P1) was calculated by

1H

NMR analysis using


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combined integration of the peaks for terminal –CH2 proton of PCL block (‘e’), methylene (-CH2) of galactose (‘b’) at 4.04-4.30 ppm and –CH proton of lysine (‘d’) (combinedly denoted as ‘b,d,e’) with respect to the characteristic anomeric proton of the GP segment at 5.67 ppm denoted as ‘a’ (Figure 1a). The Mn values determined from NMR spectra closely matched with that expected from M/I ratios used in the ROP. In the

1H

NMR spectrum, the characteristic peaks of protons in

poly(propargylglycine) block in P1 copolymer were not visible due to overlap with other peaks, hence to confirm their presence in the copolymer, P1 was reacted with benzyl azide using Cu(I) catalyzed azide-alkyne ‘‘click chemistry’’(Scheme S3). The number of benzyl groups incorporated was estimated from 1H NMR spectra by integrating the characteristic anomeric proton of the GP segment at 5.67 ppm (‘a’) with respect to the peak for aromatic protons of benzyl group at 7.3-7.43 ppm (‘f’), which suggested the incorporation of ~6 units of propargylglycine (Figure 1b). A broad signal for triazole proton (‘g’) was also seen in the spectrum at 8.2 ppm indicating formation of click product. The overall ratio of (PCL50)2 segment, DLpropargylglycine and α-galacto-O-lysine units in the star copolymer P1 was found to be 1:6:40 as expected. GPC analysis showed monomodal curve for the resulting 13 ACS Paragon Plus Environment


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acetyl protected star copolymer (P1) with a dispersity (Đ) value of 1.18 (Mn = 50.8 KDa) (Figure 2a).

Figure 1: 1H NMR spectrum of (a) protected miktoarm star copolymer (PCL50)2-b-Prgly6-b-AcGP40 and (b) polymer P1 after click reaction with benzyl azide.


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Further, acetyl groups in the GP segment of P1 copolymer were removed by using hydrazine hydrate under mild conditions and deprotection was confirmed by the absence of peaks for acetyl protons in

1H

NMR spectra of the deprotected

amphiphilic star copolymer (PCL50)2-b-Pr-gly6-b-GP40 (P2) (Figure S20). GP segment in the star copolymer is synthesized by the polymerization of enantiomerically pure α-galacto-O-lys NCA after the polymerization of racemic DL-propargylglycine NCA to afford a polypeptide backbone comprising racemic and enantiomerically pure blocks. Hence, the secondary structure of block copolypeptide in deionized water was determined by circular dichroism (CD) spectroscopy analysis of P2, which showed minima at 208 and 222 nm, characteristic of α-helical polypeptide chain (Figure 2b). Thus, it was revealed that helical nature of the GP arm in the star copolymer was not disturbed by incorporating a random and short poly(propargylglycine) block.



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Figure 2: (a) GPC chromatograms of amine-terminated branched (PCL50)2-NH2 and protected star copolymer P1 and (b) CD spectrum of deprotected star copolymer P2 in DI water.

Self-assembly and preparation of interface crosslinked (ICL) micelles The synthesized three-arm star copolymer P2 was dissolved in DMSO and water/DMSO mxture (1:1) was added to it so that the amphiphilic block copolymer made up of chemically distinct blocks undergoes self-assembly process in the selective solvent (a good solvent for one block and a poor solvent for another block).54 During this process, diffusion of common solvent (DMSO) in the aqueous medium forces the PCL (hydrophobic) block to undergo microphase separation and leads to the formation of aggregated structures. The obtained clear solution was


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dialyzed thoroughly against deionized water to remove DMSO, which completes the self-assembly process. The morphology of the self-assembled structures was determined by transmission electron microscopy (TEM) analysis using uranyl acetate (0.2 wt %) as stain. Well-dispersed particles with spherical morphology of an average size of ~140 nm were observed (Figure 3c and Table 1). These are called uncrosslinked (UCL) micelles hereafter. The morphology of self-assembled structures was also determined by atomic force microscopy (AFM) analysis (Figure 3d) which corroborated with the TEM results. A hydrodynamic diameter (Dh) of ~163 nm for the UCL micelles was observed in dynamic light scattering (DLS) measurements (Figure 3b and Table 1). In the previous study on GP-PCL star copolymers, wherein different types of morphologies were obtained by tuning molecular parameters of the copolymer, the formation of final morphological structures was explained as being governed by combination of three important parameters viz. Hydrophilic weight fraction (Wphilic), helicity of GP block, and crystallinity of branched PCL chains instead of only by Wphilic.26 The extent of helicity has a strong correlation with the self-assembly of GP based block copolymers. For example, a short GP segment present in 10GP-(PCL25)226 and 12GP-b-PPO55 17 ACS Paragon Plus Environment


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systems was shown to be roughly helix-coil or weak helix system exhibiting about ~23%

and ~21%

of helicity, that leads to formation of nanorods and vesicles

respectively. As the length of GP segment increases, the percent helicity increases (~52% for P2) and leads to formation of assembly with higher curvature such as micelles.26,56 Crystalline behaviour of PCL depends on the type of block copolymer and polymer architecture, that is , linear or branched.57

The preparation of crosslinked micelles for the production of stable nanostructures requires a combination of self-assembly and covalent bond formation between the polymer chains. The star copolymer, (PCL50)2-b-Pr-gly6-b-GP40, was designed in such a way that alkyne groups of middle poly[Pr(gly)] block can be covalently crosslinked via click chemistry, while simultaneously introducing redox-responsive groups by using dithiol containing diazide crosslinker, viz. bis-(azidoethyl) disulfide (BADS). It is assumed that the alkyne groups-containing middle block is present at the core-shell interface of the UCL micelles, and hence the crosslinked micelles prepared therefrom are called as interface crosslinked (ICL) micelles (Figure 3a). DLS analysis of the amphiphilic polymer in DMSO/water (1:1) mixture showed Dh of ~153 nm (Figure S2), which indicates that UCL micelles are formed before crosslinking reaction 18 ACS Paragon Plus Environment

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since the size is similar to the micelles obtained after dialysis (163 nm). Therefore, it can be said that UCL micelles are first assembled and then subsequently crosslinked to give ICL micelles. The UCL micelles were reacted with cross-linker BADS in DMSO: water (1:1) in the presence of copper sulfate and sodium ascorbate to crosslink the middle block. The obtained ICL micelles were analyzed by TEM (Figure 3e) and AFM (Figure 3f) techniques, which showed average particle size of ~105 nm whereas DLS analysis gave Dh of ~129 nm (Figure 3b and Table 1). Moreover, all particles were well dispersed, with no aggregation, implying that there were negligible intermicellar reactions during crosslinking. This is the major advantage of interface crosslinked micelles similar to core crosslinked micelles as has been seen in the previous studies wherein outer coronal hydrophilic part prevents crosslinkable inner shell to undergo intermicellar crosslinking due to steric hindrance.45 Measurements from different techniques showed that ICL micelles have a lower diameter compared to UCL micelles, which indicates high degree of crosslinking resulting in the formation of a fixed covalent network between the randomly oriented free propargyl groups of poly[Pr(gly)] block that may lead to decrease in size. In the



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literature, shrinkage in size by 10-15 nm was observed after photo-crosslinking of PEG-PAC-PLA/FA-PEG-PLA block copolymer micelles.42

Figure 3: (a) Schematic for the synthesis of interface crosslinked (ICL) micelles via click chemistry, (b) DLS size distribution of UCL and ICL micelles, (c) TEM image and (d) AFM image of UCL micelles; (e) TEM image and (f) AFM image of ICL micelles.

Dye encapsulation and CMC determination Micellar structure of assemblies from P2 was confirmed by encapsulation of hydrophobic rhodamine B octadecyl ester (RBOE) dye in UCL and ICL micelles (Figure S3). UV-Vis spectra of RBOE encapsulated UCL micelles and ICL micelles 20 ACS Paragon Plus Environment

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(λmax ≈ 565 nm) show a clear blue shift compared to water (λmax ≈ 575 nm), which indicates that RBOE is predominantly located in a relatively nonpolar environment i.e. hydrophobic core of the micelles.55 It was evidenced by a visual change in colour of the solution and from fluorescence emission spectrum, which showed a broad peak with λmax at 578 nm when excited at 554 nm. This result suggests that these bioactive micellar assemblies are capable of encapsulating hydrophobic guest molecules. Further, supporting evidence for formation of ICL micelles could be obtained by determination of CMC value of the micelles. Fluorescence emission spectra of the encapsulated RBOE dye at different polymer concentrations was recorded by exciting at 554 nm and λem was plotted versus concentration of polymer to obtain the typical CMC curve. CMC value of 1.56 µM was obtained for UCL micelles (Figure 4a), however, ICL micelles did not have detectable CMC value (Figure 4b). At concentration below CMC, UCL micelles would dissociate into unimers whereas ICL micelles would not. Due to core-shell interface crosslinking, RBOE dye was stabilized within the hydrophobic core of ICL micelles even at very low concentration.58,59



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Figure 4: Plot of fluorescence intensity versus the logarithm of the polymer concentration (µM) in aqueous solution (a) UCL micelles (b) ICL micelles. Inset: Fluorescence emission spectra of RBOE in aqueous solution of polymer at different concentrations.

Stability studies UCL and ICL micelles The main objective of this work is to prepare glycopolypeptide-based stable micellar systems by introducing crosslinking at the interface of core and shell. Therefore, the stability of micelles was evaluated by performing four experiments, viz. dilution assay, size determination below CMC, time dependent dye release and time dependent change in particle size on both UCL and ICL micelles. Firstly, for dilution assay, aqueous solutions of ICL and UCL micelles were diluted with 10-fold volume of DMF and analyzed by DLS technique. Disappearance of particles in the size


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range of 100-200 nm and appearance of the new curve around ~10 nm was observed for the UCL micelles, which confirmed their disruption probably due to the dissolution of the polymer in DMF. On the other hand, particle size in the same range (100-200 nm) as before was observed for ICL micelles after dilution (Figure 5a); similar observation was made in previous reports.40,41 This confirms that ICL micelles prepared here retain their structural integrity after dilution in DMF, which is a good solvent for both the blocks.

Figure 5. Stability studies of UCL and ICL micelles (a) DLS analysis of UCL and ICL micelles upon dilution by 10-fold volume of DMF (b) DLS analysis of UCL and ICL micelles below CMC.



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Further, when DLS measurements were carried out above and below CMC, UCL micelles showed a huge change in polydispersity and particle size (both on higher and lower side) while ICL micelles showed similar value as that above CMC confirming their stability (Figure 5b). Here, the change in size dispersity of UCL micelles upon dilution can be explained by re-aggregation of individual amphiphilic block copolymer chains into meta-stable micellar assemblies (< 100 nm and ~500 nm) eventually leading to precipitated polymer particles. In a report, when the crosslinked micelles prepared from PEG-DTT-PCL based block copolymer were diluted by addition of 1000-fold water (C 95% consumption of the first monomer, the second NCA monomer, α-galacto-O-lysNCA (40 eq.) along with the proton sponge (0.5 eq. of the second monomer) in dry DMF (100 mg mL-1) was added and the reaction progress was monitored by FT-IR spectroscopy. After completion of reaction (generally 48 h), aliquots were removed from the reaction mixture for GPC


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analysis and solvent was removed under reduced pressure to obtain a solid residue that was re-dissolved in DCM, washed with 1N HCl to remove “proton sponge” and precipitated in diethyl ether. The precipitated polymer was centrifuged and dried to afford block co-glycopolypeptides (P1) as white solid in 80% yield. 1H

NMR (400 MHz, CDCl3): δ (ppm): 1.36-1.80 (m, 12H), 1.82-1.95 (m, 2H), 1.98-

2.10 (m, 12H), 2.90-3.24 (m, 3H), 3.70-4.0 (m, 1H), 4.0-4.15 (m, 1H), 4.17-4.35 (m, 3H), 4.9-5.15 (m, 2H), 5.2-5.5 (m, 1H), 5.6-5.80 (m, 1H).

Deprotection of acetyl-protecting group of star block copolymer: [(PCL50)2-b-Pr-gly6b-AcGP40] The acetyl-protected block copolypeptide (PCL50)2-b-Pr-gly6-b-AcGP40 was dissolved in THF and hydrazine monohydrate (25 eq.) was added. The reaction mixture was allowed to stir for 12 h at room temperature. Reaction was quenched by adding acetone and solvent was removed under reduced pressure to obtain solid residue that was dissolved in deionized water/DMSO (1:1) mixture and filtered through 0.45 µm membrane filter. The filtered solution was dialyzed against DI water using



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dialysis membrane of 12 kDa MWCO for 2 days by changing water every 6 h. Dialyzed block copolymer (P2) was lyophilised to obtain white fluffy solid (70% yield). 1H

NMR (400 MHz, DMSO-d6): δ (ppm): 0.90-1.80 (m, 10H), 2.08 (m, 1H), 2.20-2.30

(m, 1H), 2.75-3.15 (m, 2H), 3.20-3.50 (m, 5H), 3.66 (s, 1H), 3.93-4.10 (m, 1H), 4.22 (s, 1H), 4.49 (s, 1H), 4.65 (s, 1H), 4.84 (s, 1H), 5.17-5.22 (m,1H).

Self-assembly of deprotected [(PCL50)2-b-Pr-gly6-b-GP40] copolymer in (P3) aqueous solution (a) Preparation of uncrosslinked (UCL) micelles Fully deprotected polymers [(PCL50)2-b-Pr-gly6-b-GP40] were dissolved in DMSO and water/DMSO mixture (1:1) was added slowly under stirring, to maintain the final polymer concentration at 0.5 mg mL-1. The solution was allowed to stir for 4 h, filtered through 0.45 μm membrane filter and dialyzed against DI water (MWCO = 12 kDa) for two days. DI water was changed every 6 h to remove DMSO. The as prepared micelles are labelled as UCL micelles.

(b) Preparation of interface crosslinked (ICL) micelles


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Deprotected block copolymer (2.0 mg, 0.082 μmol) and cleavable cross-linker bis(azidoethyl) disulfide (60 μg, 0.295 μmol, 0.6 mol eq. of alkynyl group) were dissolved in DMSO and water/DMSO (1:1) mixture was added slowly under stirring to maintain the final polymer concentration at 0.5 mg mL-1. The solution was allowed to stir for 4 h. Aqueous solution of CuSO4, (7.2 μL, 5 mg/mL, 0.25 mol eq. of alkynyl group) and sodium ascorbate (5 μL, 5 mg/mL, 0.25 mol eq. of alkynyl group) were added to the above mixture and stirred overnight at room temperature. The solution was filtered using 0.45 μm membrane filter paper and subsequently purified by dialysis against DI water (MWCO = 12 kDa) for three days, changing water every 6 h to remove DMSO, copper salt and sodium ascorbate. The as prepared micelles are labelled as ICL micelles.

Doxorubicin encapsulation (a) Preparation of Dox-loaded UCL micelles Dox.HCl was neutralised with triethylamine in DMSO and mixed with solution of the block copolymer in DMSO. Further water/DMSO mixture (1:1) was added slowly under stirring to maintain the final polymer concentration at 0.5 mg mL-1 and further



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stirred for 4 h. The solution was then transferred to a dialysis bag (MWCO = 12 kDa) and purified by extensive dialysis against deionized water for 48 h. The solution was filtered through a 0.45 μm membrane filter and used for further studies. The as prepared micelles are labelled as Dox-UCL micelles.

(b) Preparation of Dox-loaded ICL micelles Block copolymer solution and cleavable crosslinker taken in DMSO were mixed with deprotonated Dox and water/DMSO (1:1) mixture was added slowly under stirring to maintain the final polymer concentration at 0.5 mg mL-1. The above solution was stirred for 4 h to form UCL micelles, to which copper sulphate and sodium ascorbate were added and stirred overnight at room temperature. The solution was dialysed against DI water for 48 h using a dialysis bag (MWCO = 12 kDa). The Dox encapsulated micellar solution was filtered using a 0.45 μm membrane filter for further studies. The as prepared micelles are labelled as Dox-ICL micelles. Using similar method, Dox-loaded crosslinked micelles crosslinked using a non-cleavable crosslinker (1,6-diazidohexane) and the mixture of cleavable and non-cleavable crosslinker (1:1) were prepared. They are labelled as Dox-NICL and Dox-ICL1 micelles, respectively. The concentration of Dox encapsulated in Dox-loaded 46 ACS Paragon Plus Environment

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micelles was calculated using UV-Vis spectroscopy, and drug loading efficiency (DLE) was determined using the equation: Drug Loading Efficiency (DLE) = (We/ Wf) x 100 where We is the amount of encapsulated Dox in micelles, Wf is the amount of feed Dox.

In vitro drug release studies (a) Glutathione-mediated drug release

The release of Dox from ICL micelles was studied in the presence of glutathione using the dialysis method. Briefly, 2.0 mL of drug loaded ICL micelle solution was transferred to a dialysis bag (MWCO = 1,000 kDa), placed in release media (PBS, 10 mM, pH 7.4) of various GSH concentrations (0, 2 μM and 10 mM) and was shaken at 100 rpm at 37oC. At pre-determined time intervals, 3.0 mL of solution was taken out from the medium and replaced with 3.0 mL of fresh release medium. The release study of Dox from NICL and ICL1 micelles was performed in 10 mM GSH using a similar methodology. The amount of Dox released was calculated from fluorescence measurements using linear standard calibration curve with the concentration range



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of 0.001-10 µg mL-1. The excitation and emission wavelengths were 480 and 590 nm, respectively. The release experiments were performed in triplicate.

(b) Enzyme-mediated drug release The release of Dox from ICL micelles was studied in the presence of esterase enzyme using the dialysis method. Dox-loaded ICL micellar solution (1 mL) was incubated with 10 units of esterase enzyme, transferred to a dialysis membrane (MWCO =1,000 kDa) and placed in release media (PBS, pH 7.4, 10 mM) that was shaken at 100 rpm at 37oC. Periodically, 1.0 mL of aliquot was taken from the medium and replaced with 1.0 mL of fresh release medium. The amount of Dox released was calculated by using the method same as used for GSH-mediated release. Similarly, release of Dox from the ICL micelles was studied by the sequential application of dual stimuli (Case 1: Glutathione/enzyme and Case 2: enzyme/Glutathione).

In vitro cytotoxicity assay In vitro cytotoxicity assay of blank UCL and ICL micelles was done by seeding the HepG2 cells in 96-well plate (flat-bottomed) with a density of 10,000 cells per well in


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minimum essential medium (MEM) having 10% FBS. The cell-seeded plate was placed in a incubator (37°C with 5% CO2) for 24 h. Further, UCL and ICL micelles were prepared in the serum-free MEM with a final concentration of 10, 20, 40, 60, 80, 100, 200, and 300 μg mL-1 and further incubated for 4 h at 37 °C with 5% CO2. The cells were further incubated for another 40 h. After 40 h, the media was exchanged with 110 μL of solution of MEM containing 10% FBS. Then, filtersterilized MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent (0.45 mg/mL) was added to each well and further incubated for 4 h at 37 °C with 5% CO2. To dissolve the insoluble purple coloured formazan crystals formed after incubation for 4 h, media was exchanged with 100 μL of DMSO was added. The absorbance of the coloured solution was measured at 550 nm using a microtiter plate reader and the relative percent cell viability was obtained from the following equation: Relative percent cell viability = (Atest/Acontrol) × 100%. Where Atest is the absorbance of the sample treated cells, Acontrol is the absorbance of the untreated cells (control).



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Experiments were performed in triplicate and absorbance was measured as a mean of triplicate measurements. The cell viability data was plotted against the concentration of blank UCL and ICL micelles by representing the percentage relative to untreated cells (control). Similarly, MTT assay of free Dox, Dox-loaded UCL and ICL micelles were performed following the same procedure for 24 h.

In vitro drug release study using epifluorescence microscopy Cellular uptake studies on the HepG2 cells was done by seeding the cells in the 24well plate with a density of 50000 cells per well in minimum essential medium (MEM) having 10% FBS. Further, the cell-seeded plate was placed in an incubator (37°C with 5% CO2) for 24 h. After that, media was exchanged with MEM containing 100 μg/mL of Dox encapsulated UCL and ICL respectively, and incubated for 2, 12, 24 and 48 h respectively at 37 °C with 5% CO2. After incubation for specific hours, incubated cells were washed with PBS three times, and the fixation of cells was done by using 4% paraformaldehyde solution. For nucleus staining, DAPI was used. All the images for Dox containing micelles were taken at 500 ms exposure time using an epifluorescence microscope (Carl Zeiss).


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ASSOCIATED CONTENT Supporting Information: Additional experimental details for the synthesis, sample preparation of other experiments, characterization and NMR spectra of monomers and polymers (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *A.V.A.: [email protected]. *S.S.G.: [email protected]

ACKNOWLEDGMENT B.P acknowledges CSIR, New Delhi for fellowship; N.G.P and G.S.B acknowledge UGC for fellowship. We acknowledge Amit. K. Yadav for CD measurements.

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Graphical Abstract


Amphiphilic Glycopolypeptide Star Copolymer-based Crosslinked

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