Self-assembly, doxorubicin-loading and antibacterial activity of well-defined ABA-type amphiphilic poly(N-vinylpyrrolidone)-b-poly(D,L-lactide)-b-poly(N-vinyl pyrrolidone) triblock copolymers

Abstract

A series of ABA type well-defined amphiphilic poly(N-vinylpyrrolidone) (PNVP)-b-poly(D,L-lactide)-b-PNVP triblock copolymers have been synthesized via the combination of ring opening polymerization and xanthate-mediated reversible addition–fragmentation chain transfer polymerization, and analyzed by ¹H NMR spectroscopy and gel permeation chromatography. Aggregation properties of these amphiphilic triblock copolymers have been revealed by fluorescence spectroscopy, transmission electron microscopy and dynamic light scattering, and supported by ¹H NMR spectroscopy. Doxorubicin (DOX) has successfully been loaded into the block copolymer micelles with a loading efficiency of 37.5%. DOX-loaded PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁ block copolymer showed sustained release within 36 h. Antibacterial properties of DOX-loaded micelles have been found to be significantly effective with respect to free DOX in terms of minimum inhibitory concentration, disk diffusion assay, growth curve, bacterial reduction and enzymatic assay based on in vitro studies.

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Introduction

Self-assembly of amphiphilic block copolymers (ABCs) in an aqueous solution lead to the formation of polymeric micelles^1,2 and vesicles.^3–5 Potential applications of the polymeric micelles in the pharmaceutical and biomedical field as nanocarriers for drug delivery and tissue engineering are well known.^6–10 For drug delivery applications, hydrophobic drugs can easily be encapsulated in the core hydrophobic structure of polymeric micelles and the hydrophilic shell stabilizes the micelles in aqueous medium, with improved solubility. Such drug-loaded micelles have the unique enhanced permeability and retention (EPR) effect.^11,12 These advantages of polymeric micelles make them very promising as carriers of hydrophobic drugs for targeted drug delivery and prolonged blood circulation time.^13–16

The synthesis of biocompatible well-defined amphiphilic block copolymers using controlled radical polymerization techniques has attracted great attention in the field of biomaterials.^17,18 Recently, we have reported that the controlled synthesis of well-defined amphiphilic diblock and star block copolymers of ε-caprolactone (CL) and N-vinylpyrrolidone (NVP) by combining the controlled ROP of CL and the controlled xanthate-mediated reversible addition–fragmentation chain transfer (RAFT) polymerization of NVP.^19,20 Interestingly, amphiphilic block copolymers containing a hydrophobic poly(D,L-lactide) (PDLLA) were found very useful from the point of its pharmaceutical and biomedical applications owing to its biocompatibility and biodegradability.^21–24 On the other hand, amphiphilic block copolymers containing a hydrophilic poly(N-vinylpyrrolidone) (PNVP) were widely used in industries as a formulation additive in coatings, pharmaceutical and medical devices. N-Vinylpyrrolidone (NVP), as having the non-conjugation of its amide keto group with the vinyl group, can only be polymerized by radical polymerization.^25–34 An amphiphilic block copolymer containing a well-defined hydrophobic PDLLA segment and a well-defined hydrophilic PNVP segment will be very useful for the delivery of hydrophobic drugs. Recently, we have reported the well-defined amphiphilic diblock copolymers of D,L-lactide (DLLA) and NVP by combining the controlled ROP of DLLA and the controlled xanthate-mediated RAFT polymerization of NVP.³⁵ Very recently, we have also reported the synthesis of four-arm star poly(D,L-lactide-b-PNVP) and use of the micelles of the same as an efficient nano-carrier for the delivery of anticancer drug methotrexate.³⁶ However, there are very few reports on the synthesis of PDLLA-based ABA-type double hydrophilic amphiphilic triblock copolymers. Kang and Leroux³⁷ have reported synthesis of linear PVP-b-PDLLA-b-PVP and poly[N-(2-hydroxypropyl) methacrylamide]-b-PDLLA-b-poly[N-(2-hydroxypropyl) methacrylamide] as well as four-arm star (BA)₄ amphiphilic block copolymers by conventional free radical polymerization of NVP or N-(2-hydroxypropyl) methacrylamide using PDLLA dithiol chain transfer agent. The resultant polymers were of broad molecular weights, but self-assembled in aqueous solution. They also loaded two hydrophobic drugs indomethacin and paclitaxel by dialysis method in their polymeric micellar cores, but they did not study the drug release properties. Moreover, they did not report the study of any biological activity using the same. Recently, Hu et al.³⁸ have reported synthesis of novel well-defined PNIPAAm-b-PDLLA-b-PNIPAAm triblock copolymers prepared by combination of ROP and atom transfer radical polymerization (ATRP) under mild condition. Later on Gheybi et al.³⁹ have reported the synthesis of novel amphiphilic block copolymers poly(citric acid)-b-poly(L-lactide)-b-poly(citric acid) (PCA–PLA–PCA) via melt polycondensation reaction of citric acid and hydroxyl telechelic poly(L-lactide). To the best of our knowledge, there is no report on the synthesis of well-defined, biocompatible, ABA-type double hydrophilic, amphiphilic PNVP-b-PDLLA-b-PNVP triblock copolymers via RAFT polymerization and ROP methods. Here, HO–PDLLA–OH was synthesized via ROP using ethylene glycol as initiator (Scheme 1). The –OH end-group was then converted to the corresponding –Br end group (Br–PDLLA–Br) through a reaction with 2-bromopropionyl bromide. This –Br end-group was then converted to the corresponding O-ethyl xanthate end group X–PDLLA–X through an ionic substitution reaction with potassium O-ethyl xanthate. After that, the controlled/living radical polymerization of NVP was performed to synthesis a series of well-defined amphiphilic PNVP-b-PDLLA-b-PNVP triblock copolymers using the macro chain transfer agent X–PDLLA–X. The resultant polymers have been characterized by ¹H NMR and GPC studies. Further, the self-assembly behavior of the resultant amphiphilic block copolymers has been studied using ¹H NMR, fluorescence spectroscopy, transmission electron microscopy and light scattering study. DOX-loaded polymeric micelles of such amphiphilic triblock copolymers were successfully prepared and characterized by TEM and DLS. Antibacterial activities of these DOX-loaded polymeric micelles have been explored.

Scheme 1 Synthesis of PNVP-b-PDLLA-b-PNVP triblock copolymer via ROP and xanthate mediated RAFT polymerization methods.

Experimental section

Materials

Ethylene glycol (S.D. Fine, Mumbai, India, 99%) was dried over CaO and distilled under reduced pressure. (D,L-Lactide) (DLLA) (Aldrich, St Louis, USA, 99%) was recrystallized from ethyl acetate. 2-Bromopropionyl bromide (Fluka, Israel, >97%), stannous 2-ethylhexanoate [Sn(Oct)₂] (Aldrich, St Louis, USA, 99%), diethyl ether (S.D. Fine, Mumbai, India), triethylamine (Loba Chemie, Mumbai, India, 99%), hexane (CDH, Mumbai, India), methanol (Loba Chemie, Mumbai, India, 99%), sodium hydrogen carbonate (Loba Chemie, Mumbai, India), ammonium chloride (S.D. Fine, Mumbai, India), anhydrous magnesium sulfate (Loba Chemie, Mumbai, India) were used as received. N-Vinylpyrrolidone (Aldrich, St Louis, USA, 99%) was dried over anhydrous magnesium sulfate and distilled under reduced pressure. 2,2′-Azobis(isobutyronitrile) (AIBN) (Spectrochem, Mumbai, India, 98%) was recrystallized from methanol. Tetrahydrofuran (THF) (Loba Chemie, Mumbai, India) was dried and fractionally distilled from sodium and benzophenone. Potassium O-ethyl xanthate was prepared according to procedure given elsewhere.⁴⁰ Doxorubicin (DOX) (Adriamycin) was purchased from Selleckchem, USA.

The antibacterial experiments were carried out using various bacterial strains. Escherichia coli MTCC 739 (ATCC 10536) were originally procured from the Institute of Microbial Technology (Chandigarh, India). The Staphylococcus aureus NCIM 5021 (ATCC 25923) strain was obtained from the National Chemical Laboratory (Pune, India). L. B. media (Himedia Lab. Ltd., Mumbai) was used for evaluating bacterial growth in liquid broth culture, and was supplemented with a 2% bacteriological agar (Himedia Lab. Ltd., Mumbai) to prepare the solid media used in plate culture studies.

General methods

Characterization. ¹H NMR spectra were recorded on a 300 MHz NMR (JEOL AL300 FTNMR, Japan) at room temperature in CDCl₃ or, D₂O solvent, and are reported in parts per million (δ) from internal standard tetramethylsilane or, residual solvent peak. The number-average molecular weight (M_n) and its dispersity (M_w/M_n) were determined by Gel Permeation Chromatography (Younglin ACME 9000, South Korea) in DMF at 40 °C with flow rate 0.5 mL min⁻¹ on two polystyrene gel columns [PL gel 5 μm 10⁴ Å columns (300 × 7.5 mm)]. The columns were calibrated with seven poly(methyl methacrylate) (PMMA) standard samples (PMMA Calibration Kit, M-M-10, Polymer Lab, UK). Fluorescence measurements of a series of aqueous PNVP-b-PDLLA-b-PNVP triblock copolymer solutions with concentrations ranging from 5 × 10⁻³ to 1 mg mL⁻¹ were carried out using a spectrofluorimeter (Fluoromax-4, HORIBA SCI., Japan) to find out the critical micelle concentration (cmc). The cmc value was calculated as per the procedure described elsewhere.³⁵ Transmission electron microscopy (TEM) (Technai 12, FEI, Netherland) operated at an acceleration voltage of 200 kV. The TEM samples were prepared by putting a drop of aqueous block copolymer solution (0.1 mg mL⁻¹) on the carbon-coated copper grid followed by the removal of extra solution with a filter paper. The dynamic light scattering measurements were performed by using Malvern Instrument (ZETA SIZER NANO-ZS90 Malvern INC, United Kingdom) to study the hydrodynamic size (R_h) of the free micelles and DOX-loaded micelles of polymers using 0.1 mg mL⁻¹ solution at 90° angle. UV-visible spectra were recorded by SHIMADZU instrument (UV-1700 pharmaspec, Japan) using the measurement cell of 10 mm optical path length.

Synthesis of dihydroxyl-terminated poly(D,L-lactide) (HO–PDLLA₄₈–OH) [run 1, Table 1]. Dihydroxyl-terminated poly(D,L-lactide) (HO–PDLLA₄₈–OH) was synthesized through ROP of D,L-lactide using ethylene glycol as initiator and Sn(Oct)₂ as the catalyst. 5.0 g (3.46 × 10⁻² mol) of D,L-lactide was placed in a 100 mL Schlenk tube, heated at 80 °C for 4 h under vacuum and dried. After cooling to room temperature, 0.092 mL (0.10 g, 1.65 × 10⁻³ mol) of ethylene glycol was added to the flask. Then, the reaction mixture was purged with nitrogen for 30 min. The Schlenk tube was then tightly closed, and heated to 150 °C and 20 μL (0.025 g, 6.2 × 10⁻² mmol, 0.5% w/w ratio of lactide) Sn(Oct)₂ was injected into the reactor vessel. The reaction was continued at 150 °C for 15 h. The polymerization process was stopped by freezing the reaction mixture with liquid N₂. The crude product was dissolved in 10 mL THF and precipitated from 200 mL hexanes. The precipitated polymer was collected by centrifugation (10 000 rpm for 10 min). The precipitated polymer was again dissolved in THF and precipitated from hexane twice and finally dried under vacuum at room temperature for 24 h. Gravimetric yield (%) = 3.9 g (93.5%).

¹H NMR (300 MHz, CDCl₃): δ (ppm) = 1.65–1.70 (m, 6H_c), 1.52–1.60 (d, 3H_e), 5.1–5.3 (m, 2H_b), 4.2–4.4 (t, 4H_a + 2H_d).

M_n (NMR) = 3400 g mol⁻¹, M_n (GPC) = 5200, M_w/M_n = 1.32.

Synthesis of dibromo-terminated PDLLA[Br(CH₃)CHCO–PDLLA₄₈–COCH(CH₃)Br] (Br–PDLLA₄₈–Br) [run 2, Table 1]. In a dried and nitrogen purged 250 mL round-bottom flask, 3.5 g [1.018 × 10⁻³ mol, calculated on the basis of molecular weight (3400 g mol⁻¹) obtained from ¹H NMR] HO–PDLLA₄₈–OH was dissolved with a mixture of 25 mL of dry THF and 0.8 mL (5.089 × 10⁻³ mol) triethylamine under stirring in nitrogen atmosphere and cooled in an ice bath. 0.43 mL (4.07 × 10⁻³ mol) 2-bromopropionyl bromide was added drop by drop to the above-reaction mixture under stirring. Then, the reaction was continued for 72 h under stirring at room temperature. The precipitated byproduct Et₃N·HBr was removed by filtration and filtrate was evaporated to dryness. The residue was dissolved in dichloromethane and washed thoroughly with 5% (w/v) aqueous sodium bicarbonate solution (4 × 250 mL). The organic layer was further washed with water (4 × 300 mL) and then dried over anhydrous Na₂SO₄, and filtered. The filtrate was evaporated and dried under vacuum at room temperature. The residue was dissolved in THF and precipitated from hexane and then dried under vacuum at room temperature for 24 h. Gravimetric yield (%) = 2.8 g (80%).

¹H NMR (300 MHz, CDCl₃): δ (ppm) = 1.54–1.58 (m, 3H_c), 1.83–188 (d, 3H_g), 4.2–4.5 (m, 2H_f + 4H_a), 5.1–5.3 (m, 2H_b).

M_n (NMR) = 3800 g mol⁻¹, M_n (GPC) = 5700, M_w/M_n = 1.31.

Typical synthesis of dixanthate-terminated PDLLA[C₂H₅O(S)CS(CH₃)CHCO–PDLLA₄₈–C(O)CH(CH₃)SC(S)OC₂H₅]₂ (X–PDLLA₄₈–X) [run 3, Table 1]. In a dried and nitrogen purged 250 mL round-bottom flask, 2.5 g (7.26 × 10⁻⁴ mol) Br–PDLLA₄₈–Br and 0.7 g (4.36 × 10⁻³ mol) potassium O-ethyl xanthate were dried and degassed by three freeze–pump–thaw cycles. In another well dried and nitrogen purged 50 mL round-bottom flask, 6.5 mL (7.70 × 10⁻² mol) pyridine was dissolved in 30 mL CH₂Cl₂ by stirring under nitrogen atmosphere. This solution was added to the previous reaction mixture during stirring under nitrogen. The reaction mixture was stirred for 36 h at room temperature and diluted with 100 mL CH₂Cl₂. The solution was washed consecutively with saturated NH₄Cl solution (4 × 50 mL), saturated NaHCO₃, solution (4 × 50 mL), and water (4 × 100 mL). The organic layer was dried over anhydrous MgSO₄ and filtered. The filtrate was dried under vacuum at room temperature for 48 h. The residue was dissolved in THF and precipitated from hexanes followed by drying under vacuum at room temperature for 24 h. Gravimetric yield (%) = 2.1 g (84%).

¹H NMR (300 MHz, CDCl₃): δ (ppm) = 1.35–1.70 (m, 6Hc + 6Hg + 6H_i), 4.55–4.7 (q, 4H_h), 4.3–4.5 (m, 4H_a + 2H_f), 5.1–5.2 (m, 2H_b).

M_n (NMR) = 4200 g mol⁻¹, M_n (GPC) = 6900, M_w/M_n = 1.33.

Typical synthesis of the ABA-type double hydrophilic amphiphilic triblock copolymer PNVP₂₃-b-PDLLA₄₈-b-PNVP₂₃ (run 1, Table 2). In a dried and nitrogen purged 30 mL Schlenk tube, 0.1 g [2.36 × 10⁻⁵ mol, calculated on the basis of molecular weight (4200 g mol⁻¹) obtained from ¹H NMR] (X–PDLLA₄₈–X) was dissolved in 1 mL THF. To it, 0.25 mL (0.261 g, 2.36 × 10⁻³ mol) NVP and 1.93 mg (1.18 × 10⁻⁵ mol) AIBN were added. A homogeneous solution was obtained after stirring and degassed under nitrogen for 30 min. The Schlenk tube was then immersed in an oil bath preheated at 80 °C for 24 h. The reaction was stopped by freezing the reaction mixture with liquid nitrogen. A small portion of the polymerization mixture was used to determine the monomer conversion by ¹H NMR. The rest part of the polymerization mixture was dissolved in 5 mL THF, precipitated from 200 mL hexanes, and dried under vacuum at room temperature for 24 h. Observed gravimetric yield (%) = 0.232 g (50.5%).

¹H NMR (300 MHz, CDCl₃): δ (ppm) = 1.2–1.8 (m, 6H_c + 4H_j + 6H_g + 6H_i), 1.8–2.2 (m, 4H_p), 2.2–2.5 (m, 4H_q), 3.0–3.5 (m, 4H_p), 3.5–4.0 (m, 2H_l), 5.2 (m, 2H_b).

M_n (NMR) = 9800 g mol⁻¹, M_n (GPC) = 12 100, M_w/M_n = 1.35.

Drug loading. 30 mg of PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁ tri-block copolymer (run 2, Table 2) was dissolved in 2 mL of DMF, and 6 mg (0.01 mmol) of DOX·HCl with 4.6 μL (3.3 mg, 0.033 mmol) TEA (3 mol eq. to DOX·HCl) were added into the polymer solution. The mixture was stirred at room temperature for 24 h. Final mixture was then dialyzed using a dialysis membrane [molecular weight cut off (MWCO) = 3500 Da] against distilled water which was renewed every 3 h during the course of initial 12 h, next every 6 h to remove the unloaded drug for 24 h. After dialysis, dialyzed drug-loaded micellar solution was filtered and concentrated to 3.0 mL and lyophilized. Lyophilized drug-loaded micelle was then dissolved in DMF and analyzed by UV absorbance at 485 nm, using a standard calibration curve experimentally obtained with DOX/DMF solutions. Drug loading content (DLC) and drug loading efficiency (DLE) were calculated according to the following formula:

Drug loading content (DLC) (wt%) = (weight of loaded drug/weight of polymer) × 100%

Drug loading efficiency (DLE) (wt%) = (weight of loaded drug/weight of drug in feed) × 100%

Table 1 Synthesis of X–PDLLA–X macro-chain transfer agent

Run	Sample	Yield^d (%)	Conv^e (%) (NMR)	Mn^e (NMR) g mol^−1	Mn^f (GPC)	Mw/Mn^f (GPC)	Comments
a Bulk polymerization using (D,L-lactide) and Sn(Oct)2 in the presence of ethylene glycol at 150 °C for 15 h.b Using HO–PDLLA–OH : triethylamine : 2-bromopropinonylbromide: 1 : 5 : 4 in THF at room temperature for 72 h.c Using Br–PDLLA–Br : potassium O-ethyl xanthate : pyridine: 1 : 6 : 106 in dichloromethane (DCM) at room temperature for 36 h.d Determined gravimetrically.e Determined by ^1H NMR.f Determined by GPC (DMF, 0.5 mL min^−1, 40 °C) calibrated against PMMA standards.
1	OH–PDLLA48–OH^a	94	96	3400	5200	1.32	Unimodal
2	Br–PDLLA48–Br^b	80	100	3800	5700	1.31	Unimodal
3	X–PDLLA48–X^c	84	100	4200	6900	1.33	Unimodal

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Table 2 Characteristic data of PNVP-b-PDLLA-b-PNVP block copolymer^a

Run	Block copolymer	NVP^b (equiv.)	Convn^c (NMR)	Mn^d (theor.)	Mn^e (NMR)	Mn/dispersity^f (GPC)	XPNVP^g (GPC)	XPNVP^g (NMR)	CMC^h (mg L^−1)
a Using 0.5 equivalent AIBN with respect to X–PDLLA48–X macroinitiator in THF at 80 °C for 24 h.b With respect to X–PDLLA48–X macroinitiator.c Conversion was determined by using ^1H NMR comparing the peak area of the residual vinylic segments of the NVP monomer at ∼7.0–7.1 ppm (^1H) with that of the methylene proton of the PNVP block of the polymer at 3.0–3.5 ppm.d Mn(theor.) = ^1H NMR mol. wt of X–PDLLA–X + ([NVP]0/[X–PDLLA–X]0) × fraction conversion of NVP (NMR) × molecular weight of NVP.e Determined from ^1H NMR by comparing the peak area of the methylene protons of PDLLA block at ∼5.2 ppm with that of the methylene proton of PNVP block at ∼3.0–3.4 ppm.f Determined by GPC (DMF, 0.5 mL min^−1, 40 °C) calibrated against PMMA standards.g XPNVP = mol-fraction of PNVP.h CMC value determined by fluorescence spectrometer.
1	PNVP23-b-PDLLA48-b-PNVP23	100	38	8500	9800	12 000/1.35	0.51	0.57	2.10
2	PNVP51-b-PDLLA48-b-PNVP51	200	40	13 100	15 500	18 300/1.50	0.62	0.73	4.02
3	PNVP70-b-PDLLA48-b-PNVP70	300	42	18 400	22 100	22 500/1.52	0.69	0.79	6.30

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Drug release study. DOX loaded polymer sample (5 mg) was dissolved in 2.0 mL phosphate buffer solution (PBS) (pH = 7.4) and transferred into dialysis tubing (MWCO = 3500 Da). The tubing was placed into 20 mL PBS solution. The system was stirred at 37 °C. At predetermined interval, 2.0 mL PBS was taken out and volume of solution was held constant by adding 2 mL PBS solution after each sampling. The amount of DOX released from drug-loaded micelles at any interval was measured by UV spectroscopy at 485 nm.

In vitro antimicrobial activity assay.
Cell preparation. Eischherichia coli (E. coli)/Staphylococcus aureus (S. aureus) cells were grown overnight in LB medium at 37 °C and then harvested at the exponential growth phase. Cultures were centrifuged at 6000 rpm for 10 min to pellet cells, and pelleted cells were washed twice to remove residual macromolecules and other growth medium constituents and then resuspended in sterile isotonic saline solution [0.8 wt% NaCl]. The cells were quantified via optical density (OD) measurement at 600 nm to obtain cell samples containing 10⁶ CFU mL⁻¹.
Determination of minimum inhibitory concentration (MIC). The minimum inhibitory concentration (MIC) was considered as the lowest concentration of an antibacterial compound studied that inhibits the visible growth of a microorganism after overnight incubation.⁴¹ MIC of normal amphiphilic triblock copolymer PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁, DOX, and DOX-loaded amphiphilic triblock copolymer micelles were determined by LB broth method against Gram negative bacteria E. coli and Gram positive bacteria S. aureus. Miller (Luria-Bertani; LB) was used as diluent for the bacterial strains. Inoculates were prepared by suspending the overnight culture growth in sterile LB media. The prepared samples were dispensed into 10 mL of sterile 0.8 wt% NaCl saline water to make the bacterial cell concentration of about 10⁶ CFU mL⁻¹ and then shaken at 37 °C. 200 μL aliquots having bacterial suspension of 10⁶ CFU mL⁻¹ and amphiphilic triblock copolymer/DOX/DOX-loaded polymeric micelles of different concentrations in the range of 0.5–15 μg mL⁻¹ were dispersed in 96-well plate and the plate was incubated at 37 °C for 24 h. The microwell plates were read at 600 nm using an ELISA reader to determine the MIC value.
Study of antimicrobial activity using disc diffusion method. The antimicrobial activity of the triblock copolymer PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁, DOX, and DOX-loaded triblock copolymer micelles against E. coli and S. aureus bacterial cells was determined by the disc diffusion method.⁴² For E. coli cells, the concentrations of triblock copolymer, DOX, and DOX-loaded triblock copolymer micelles used were 8.3, 1.1 and 0.7 μg mL⁻¹, respectively. For S. aureus cells, the concentrations of triblock copolymer, DOX, and DOX-loaded triblock copolymer micelles used were 11.8, 1.4 and 0.9 μg mL⁻¹, respectively. These pathogenic test organisms were grown in Mueller–Hinton broth (Hi-media) at room temperature on a rotary shaker at 150 rpm. Bacterial pathogens (10⁶ CFU mL⁻¹) were spreaded over the Mueller–Hinton Agar plates. Inoculates were applied to the plates along with control disc. Amphiphilic triblock copolymer, DOX, and DOX-loaded polymeric micelles along with a control were delivered on 6 mm sterile discs. The plates were pre-incubated at 4 °C for half an hour to facilitate uniform diffusion and further incubated at 37 °C for 24 h and the zones of inhibitions were observed.
Growth curves of exposed bacterial cells. For E. coli cells, the concentrations of triblock copolymer PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁, DOX, and DOX-loaded triblock copolymer micelles used were 8.3, 1.1 and 0.7 μg mL⁻¹, respectively. For S. aureus cells, the concentrations of triblock copolymer, DOX, and DOX-loaded triblock copolymer micelles used were 11.8, 1.4 and 0.9 μg mL⁻¹, respectively. The bacterial cell concentration was adjusted to 10⁶ CFU mL⁻¹ and incubated in a shaking incubator up to 6 h at 37 °C. Growth curves of bacterial cell cultures were monitored at different time interval through the measurement of the optical density (OD) at 600 nm.
Cell viability test. The anti-bacterial activity of normal amphiphilic triblock copolymer PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁, DOX, and DOX-loaded triblock copolymer micelles were determined by micro-dilution method⁴³ in comparison with control. Inoculates were prepared by suspending the overnight culture growth in sterile LB media. The new prepared samples were dispensed into 10 mL of a sterile 0.8 wt% saline water containing about 10⁶ CFU mL⁻¹ of bacterial cells (E. coli or, S. aureus), and then shaken at 37 °C. After 6 h contact, 0.1 mL of the suspension was taken out from the test tube and diluted to a certain volume (to ensure the bacterial colonies grown could be counted easily and correctly) by 10-fold dilution. The diluted solution was plated on LB agar plates in triplicate and incubated at 37 °C for 24 h. The killing rate (η) is relative to the viable bacteria counts as follows: η = [(Y − X)/Y] × 100%, where Y is the number of microorganism colonies on the control tube (sterile 0.8 wt% saline water without sample) and X is the number of microorganism colonies on the samples.
Evaluation of anti-bacterial mechanism. The anti-bacterial effect of amphiphilic triblock copolymer PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁, DOX, and DOX-loaded triblock copolymer micelles was determined by measuring the activity of β-D-galactosidase. A modified O-nitrophenol-β-D-galactopyranoside (ONPG) test procedure was used in this experiment.^44,45 Standard solution containing a cell suspension of 1 mL with a concentration of 10⁶ CFU mL⁻¹ and 1 mL of ONPG solution with a concentration of 25 mM were added simultaneously into 5.5 mL PBS buffer solutions (pH 7.4) and kept at 37 °C incubator for 15 min with shaking. Then, 100 μL of each copolymer PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁, DOX, and DOX-loaded micelle with concentration of 8.3, 1.1 and 0.7 μg mL⁻¹, respectively, for E. coli and 11.8, 1.4 and 0.9 μg mL⁻¹, respectively, for S. aureus were added into a 96-well plate and labeled separately. 100 μL of the above standard solutions containing the cell suspensions and ONPG in PBS buffer were added in the respective wells and labeled. At different time interval, the changes of optical density at 420 nm were measured by using a Multi-label Microplate Reader (Perkin Elmer VICTOR 3). At the same time two type control samples, bacterial strain with ONPG and block copolymers with ONPG were also measured.

Results and discussion

Synthesis of PNVP-b-PDLLA-b-PNVP amphiphilic triblock copolymers

Amphiphilic triblock copolymers with poly(D,L-lactide) as a hydrophobic core and poly(N-vinylpyrrolidone) as a hydrophilic shell were synthesized via combination of ROP and xanthate-mediated RAFT polymerization as shown in Scheme 1. In the first step, HO–PDLLA–OH has been synthesized in bulk with 94% yield via ROP of DLLA at 150 °C using ethylene glycol as initiator and Sn(Oct)₂ as catalyst (run 1, Table 1). Its formation is confirmed from ¹H NMR [Fig. 1(A)].³⁵ GPC chromatogram [Fig. 2(a)] depicts unimodal nature with M_n (GPC) = 5400 with dispersity = 1.32. The HO–PDLLA–OH was then converted to the corresponding Br–PDLLA–Br on reaction with 2-bromopropionyl bromide in the presence of triethyl amine (run 2, Table 1). It's formation is confirmed from ¹H NMR [Fig. 1(b)] as revealed from the appearance of the characteristic methine ‘f’, and methyl ‘g’ protons of 2-bromopropionyl end group at 4.72 and 1.80 ppm, respectively. GPC chromatogram [Fig. 2(a)] conforms unimodal nature with M_n (GPC) with dispersity of 5700 and 1.31, respectively.

Fig. 1 ¹H NMR spectra (A) HO–PDLLA–OH, (B) Br–PDLLA–Br and (C) X–PDLLA–X in CDCl₃.

Fig. 2 (a) Gel permeation chromatograms of HO–PDLLA₄₈–OH (run 1), Br–PDLLA₄₈–Br (run 2) and X–PDLLA₄₈–X (run 3) (Table 1). (b) Gel permeation chromatograms of macro-chain transfer agent X–PDLLA₄₈–X and the resulted block copolymers PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁ (run 2) and PNVP₇₀-b-PDLLA₄₈-b-PNVP₇₀ (run 3) (Table 2).

Br–PDLLA–Br was then end-functionalized using potassium-O-ethyl xanthate to yield X–PDLLA–X (run 3, Table 1) and was confirmed by ¹H NMR [Fig. 1(c)] with the appearance of new peak characteristic of the methylene ‘h’ protons of the xanthate end group at 4.6 ppm. GPC chromatogram [Fig. 2(a)] confirms unimodal nature with M_n (GPC) and dispersity of 6, 900 and 1.33, respectively.

X–PDLLA₄₈–X was used as a macro-chain transfer agent for the xanthate mediated RAFT polymerization of NVP in THF using [X–PDLLA₄₈–X] : [AIBN] = 1 : 0.5 at 80 °C for 24 h. The results of the synthesis and characterization of PNVP-b-PDLLA₄₈-b-PNVP block copolymers are shown in Table 2. Runs 1, 2 and 3 correspond to 100, 200, and 300 equivalents of NVP monomer loadings with respect to X–PDLLA₄₂–X macro-chain transfer agent, respectively. Molecular weight and PNVP content of the resulted block copolymers are increased with the increase in the monomer loading as expected [Table 2 and Fig. 2(b)]. The presence of tailing in the low molecular weight part of the gel permeation chromatograms of the triblock copolymers indicates the presence of ∼2–6% [calculated from the difference of the experimental and theoretical values of PNVP content] PNVP homopolymers as all these copolymers are completely soluble in water.

The typical ¹H NMR spectrum (in CDCl₃) [Fig. 3(A)] of the block copolymer, prepared in run 1 (Table 2), revealed, in addition to the characteristic peaks of the PDLLA block, the presence of the characteristic peaks of the PNVP backbone methine proton ‘k’ at ∼3.5–4.0 ppm, the methylene protons ‘l’, ‘q’, and ‘p’ of the pyrrolidone ring at ∼3.0–3.5, 2.2–2.5, and 1.8–2.2 ppm, respectively, apart from methylene protons ‘j’ of the PNVP block overlapped between ∼1.2–1.8 ppm range with methyl protons ‘c’ of PDLLA block. The ‘f’ methine peak at 4.72 ppm of X–PDLLA–X is shifted in the block copolymer to ∼2.3 ppm which is overlapped with the methylene peak ‘q’ of the pyrrolidone ring at ∼2.2–2.5 ppm. All these results indicate the successful occurrence of hetero chain-extension.

Fig. 3 ¹H NMR spectra of PNVP₂₃-b-PDLLA₄₈-b-PNVP₂₃ triblock copolymer in (A) d-chloroform, and (B) D₂O at room temperature.

Self-assembly of amphiphilic PNVP-b-PDLLA-b-PNVP triblock copolymers in water

The ¹H NMR spectrum of PNVP₂₃-b-PDLLA₄₈-b-PNVP₂₃ triblock copolymers in D₂O is shown in Fig. 3(B). Here, the peaks attributed to PDLLA are suppressed in comparison with the ¹H NMR spectrum obtained in d-chloroform [Fig. 3(A)]. This observation indicates the possible formation of micellar aggregates in aqueous solution with PDLLA blocks as the core and PNVP blocks as the shell. In order to study the critical micelle concentration (cmc) of such triblock copolymers in water, fluorescence spectroscopy is used with pyrene as the probe. Typical fluorescence excitation spectra (300–360 nm) of pyrene (6 × 10⁻⁷ M) at different PNVP₂₃-b-PDLLA₄₈-b-PNVP₂₃ [run 1, Table 2] concentrations recorded at an emission wavelength of 394 nm are shown in Fig. 4(a). Fig. 4(b) shows the corresponding plot of the I₃₃₇/I₃₃₃ intensity ratio (from fluorescence measurements) vs. the log of the PNVP₂₃-b-PDLLA₄₈-b-PNVP₂₃ block copolymer concentration (mg mL⁻¹) in water. The observed cmcs of the block copolymers PNVP₂₃-b-PDLLA₄₈-b-PNVP₂₃, PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁ and PNVP₇₀-b-PDLLA₄₈-b-PNVP₇₀ are ∼2.10 × 10⁻³, 4.02 × 10⁻³, and 6.30 × 10⁻³ mg mL⁻¹, respectively [Table 2]. These values indicate that the cmc value of such amphiphilic block copolymers increases with the increase in the chain length of PNVP block. Similar type of results is also reported in the literature.^19,20,35 From the TEM image (Fig. 5(a)) the average diameter of PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁ micelles is observed at 27 nm. But the DLS measurement (Fig. 6) revealed that the average hydrodynamic diameter (R_h) and the corresponding PDI of PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁ micelle are 75.3 nm and 0.423, respectively. The observed smaller size of the micelle in TEM measurement is probably arising from the dehydration and shrinkage of the micelles during drying in the TEM measurement.

Fig. 4 (a) Fluorescence excitation spectra (monitored at λ_em = 394 nm) of pyrene (6 × 10⁻⁷ M) in the presence of increasing concentration (mg mL⁻¹) of PNVP₂₃-b-PDLLA₄₈-b-PNVP₂₃ triblock copolymer (run 1, Table 1) solution in water and (b) the corresponding semilogarithmic plot of the fluorescence excitation intensity ratio (I₃₃₇/I₃₃₃) of pyrene vs. the concentration of polymer.

Fig. 5 The TEM image of (a) blank micelles and (b) DOX-loaded micelles of PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁ block copolymer.

Fig. 6 Plot of scattering intensity at 90° scattering angle vs. the effective hydrodynamic diameter of blank and DOX-loaded micelles of PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁ block copolymer in water at 0.1 mg mL⁻¹ concentration.

DOX loading and in vitro release

The DOX-loaded polymeric micelles are prepared using dialysis method. The amount of DOX encapsulated into polymeric micelle of PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁ is calculated from the absorbance of DOX at 485 nm of its UV spectrum on comparison with the corresponding calibration curve. The drug loading content is 7.1% and drug loading efficiency is 37.5%. TEM result [Fig. 5(b)] shows that the average diameter of the DOX-loaded micelles is 63 nm whereas DLS result [Fig. 6] reveals that hydrodynamic diameter (R_h) and its polydispersity are 150 nm and 0.176, respectively. Both TEM and DLS results show that the size of the drug-loaded micelles is larger than that of blank micelles. All these results confirm the successful loading of DOX in PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁ micelles.

In vitro release study were carried out from DOX-loaded micelles of PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁ at 37 °C in pH = 7.4 PBS solution. Fig. 7 shows that sustained DOX drug release behavior up to 36 h with a maximum of 15.60% release.

Fig. 7 Percentage of cumulative release of DOX from DOX-loaded micelles of PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁ block copolymer in pH = 7.4 PBS at 37 °C.

Antibacterial activity

The minimal inhibitory concentrations (MIC) of amphiphilic triblock copolymer PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁, DOX and DOX-loaded triblock copolymer micelles were determined in order to assess their antibacterial activity. DOX and DOX-loaded micelles showed lower MIC values compared to copolymers for both bacteria strains tested indicating their stronger anti-microbial activity (Table 3). The MICs of 8.3, 1.1 and 0.7 μg mL⁻¹ were observed for triblock copolymers, DOX and DOX-loaded micelles, respectively, against the E. coli. The corresponding observed MICs for S. aureus are 11.8, 1.4 and 0.9 μg mL⁻¹, respectively.

Table 3 Summary of minimal inhibitory concentrations (μg mL⁻¹) against bacterial strain

Bacterial strain	Amphiphilic triblock copolymer	DOX	DOX-loaded copolymer micelles
E. coli	8.3	1.1	0.7
S. aureus	11.8	1.4	0.9

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Disk diffusion assay

The antimicrobial activities of amphiphilic triblock copolymer PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁, DOX and DOX-loaded triblock copolymer micelles against E. coli and S. aureus are shown in Fig. 8 and Table 4. It was found that these samples showed effective antibacterial activity, although differences existed among them. In general, the DOX-loaded micelles showed more effective inhibition on both bacterial cells compared to DOX and copolymers. The inhibitory effect was measured based on clear zone surrounding circular disc (Fig. 8). If there is no surrounding clear zone, we assumed that there is no inhibitory zone, and furthermore, the diameter was valued as zero. In terms of the surrounding clearing zone, control did not show inhibitory effect against both bacterial cells. The triblock copolymer, DOX and DOX-loaded micelles showed a clear inhibitory zone at their different concentrations of 1, 2.5 and 5 μg mL⁻¹ against both bacterial cells. Without polymer was used as control, and the inhibition zone was included as 0 mm in diameter with triblock copolymers.

Fig. 8 Growth inhibition of bacteria E. coli [a–c] and S. aureus [d–f] on agar plates at incubated at 37 °C for 24 h, using (a and d) polymer, (b and e) doxorubicin and (c and f) DOX-loaded polymer. Spots 1, 2, and 3 represented different concentrations 1, 2.5, and 5 μg mL⁻¹ respectively, against both bacterial cells.

Table 4 Diameter of inhibitory zone against E. coli and S. aureus

	Sample concentration (μg mL^−1)
	1	2.5	5	1	2.5	5
	Diameter (mm) of inhibitory zone
	E. coli			S. aureus
Triblock copolymer	0	0	0	0	0	0
DOX	2.3	4.2	7.6	2.6	4.7	7.8
DOX-loaded micelle	5.0	7.8	12.8	5.2	8.1	13.0

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Growth curves of bacterial cells

The plots of the optical density (OD) at 600 nm versus the culture time for amphiphilic triblock copolymer PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁, DOX and DOX-loaded micelles with 8.3, 1.1 and 0.7 μg mL⁻¹ concentration, respectively, against E. coli [Fig. 9(a)], with 11.8, 1.4 and 0.9 μg mL⁻¹ concentration, respectively, against S. aureus [Fig. 9(b)] both at 10⁶ CFU mL⁻¹ concentration as measured by the shaking flask method. The smaller is the OD of the medium, as determined by the absorbance at 600 nm, the higher is the anti-bacterial activity of the tested material since fewer microbial cells were produced. Control and copolymer exhibited fast and almost similar OD increment throughout the 6 h incubation period showing that polymer itself had negligible inhibitory growth activity against both bacterial cells.

Fig. 9 Effect of triblock copolymer PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁, DOX, and DOX-loaded micelles (polymer + doxorubicin) on the growth of (a) E. coli and (b) S. aureus at 10⁶ CFU mL⁻¹ concentration.

Bacterial growth was at much slower rate in the experimental groups containing doxorubicin with the late exponential phase being reached after 3 h. For the experiments with DOX-loaded micelles, bacterial growth was slowest after 2 h of incubation period. Then, after about 3 h, the optical density dropped to almost zero. So, DOX-loaded micelles showed most efficient cell growth inhibition for both bacteria reaching ∼100% compared to the non-treated control within 3 h, whereas that for free DOX is ∼75%.

Colony counting method

The antibacterial activity (which was expressed in terms of decrease in number of colonies) of the amphiphilic triblock copolymer PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁, DOX and DOX-loaded micelle against E. coli and S. aureus was tested in 96-well round-bottom plates by CFU assay [Fig. 10(a) and (b), respectively]. Exponentially grown bacteria were incubated with above-mentioned growth curve concentration of block copolymer, DOX and DOX-loaded micelle, and the number of CFUs was analyzed through harvesting bacteria at different time points by plating serial dilutions on LB plates. The surviving colonies were enumerated after 24 h. The results were compared with bacteria grown in LB medium (control) and the reaction mixture containing all the components except block copolymer, DOX and DOX-loaded micelle.

Fig. 10 (a) E. coli and (b) S. aureus (10⁶ CFU mL⁻¹) cells were treated with amphiphilic triblock copolymer PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁, DOX and DOX-loaded micelles at 37 °C for 3 h at 200 rpm shaking speed. Cell-viability rates were determined by the colony-counting method and expressed as a percentage of control.

The control and the corresponding concentration of block copolymer showed almost no antibacterial activity over the span of the study [Fig. 10], suggesting no toxicity. In comparison, DOX and DOX-loaded amphiphilic triblock copolymer micelle showed significant toxicity towards E. coli. After 3 h of exposure to DOX and DOX-loaded micelle, more than 82 and 97%, respectively, of the bacteria were killed relative to the control.

Antibacterial mechanism

β-D-Galactosidase enzyme has specific hydrolyzing activity for galactose β-glycosidic bond of galactose unit in sugar, fat, and lactose.⁴⁶ When the E. coli/S. aureus cell suspension containing O-nitrophenol-β-D-galactopyranoside (ONPG) was treated with antibacterial agents, after cell damaged, β-D-galactosidase leaked out of the cells, and catalyzed the hydrolysis of ONPG in the solution to O-nitrophenol (ONP).⁴⁷ Because ONP, the product of ONPG hydrolysis, has a characteristic absorption at 420 nm, the magnitude of cell death was measured quantitatively by monitoring the increase in optical density (OD) value at 420 nm with time. The changes of absorption with time were measured for triblock copolymer PNVP₅₁-b-PDLLA₄₈-b-PNVP₅₁, free DOX and DOX-loaded micelles along with two control samples, one bacterial strain with ONPG and another triblock copolymer with ONPG. The results are summarized in Fig. 11. The results show that the absorptions of the control systems are nearly zero and unchanged with time. Negligible increase in OD compared to that of controls was observed for triblock copolymer indicating that it has no considerable antibacterial property. For both cell suspensions into which DOX and DOX-loaded micelles were added, the OD at 420 nm increased considerably with time. DOX-loaded micelle was found to be most effective as revealed by the faster and greater increase of OD value than others.

Fig. 11 Plots of the optical density (OD) at 420 nm corresponding to the concentration of O-nitrophenol from different well plates at different time periods with (a) E. coli and (b) S. aureus cells at 10⁶ CFU mL⁻¹ concentration.

Conclusions

Synthesis of amphiphilic ABA-type triblock copolymers PNVP-b-PDLLA-b-PNVP of varying PNVP chain lengths has successfully carried out via the combination of ROP and xanthate-mediated RAFT polymerization. The polymers were characterized by ¹H NMR and GPC. The critical micelle concentrations of these triblock copolymers were determined by fluorescence spectroscopy using pyrene as probe. Self-assembly of these block copolymers lead to the formation of micelles in water as revealed by TEM, DLS and supported by ¹HNMR. DOX has efficiently been loaded into the micelles of PNVP₅₁-PDLLA₄₈-PNVP₅₁ triblock copolymer with a loading efficiency of 37.5%. Interestingly, DOX-loaded micelle has showed a sustained release up to 36 h. Antibacterial properties of DOX-loaded micelles have been studied and compared with free DOX and block copolymer in terms of MIC, growth curve, disk diffusion assay, bacterial reduction and enzymatic assay based in vitro studies. In each case, DOX-loaded micelles shown better activity than free DOX. These results clearly show potential efficiency of PNVP-b-PDLLA-b-PNVP as nanocarrier for the better delivery of the therapeutics.

Acknowledgements

Authors acknowledge the financial supports of Department of Science Technology, New Delhi, India through the sanction no. SR/S1/PC-25/2006, and Department of Biotechnology (DBT), New Delhi, India, through the sanction no. BT/PR889/NNT/28/570/2011. K. Ramesh gratefully acknowledges Indian Institute of Technology (BHU), Varanasi, India for teaching assistantship. RKG wants to thank D. S. Kothari Post-Doctoral Fellowship, University Grants Commission, Government of India (No. F. 4-2/2006 (BSR)/13-618/2012(BSR)) for the grant of Post-Doctoral Fellowship for financial support and thanks to Dr Sunit Kumar Singh Head of the Department Molecular Biology Unit, Institute of Medical Sciences, BHU, Varanasi, India, for providing laboratory and technical support. SS thanks BHU for providing JRF fellowship under UGC RFSMS scheme. We are thankful to Dr Ida Tiwari, Department of Chemistry, BHU, Varanasi, India, for providing the fluorescence facilities. SS acknowledge BHU for the research fellowships.

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Footnote

† Present address: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas-72701, USA.

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