Synthesis and Characterisation of fluorescent pyrene end capped polylactide fibres

Aula Alwattara,b, Athir Haddada,b, Quan Zhouc, Tatiana Nascimentoc,d, Ryan Greenhalghc,e,

Eliton Medeirosd, Jonny Blakerc, Adam Parrya , Peter Quaylea, Stephen Yeatesa

a School of Chemistry, the University of Manchester, Oxford Road, Manchester, M13 9PL,

UK.

b Chemistry Department, College of science, University of Basrah, Garmat Ali, 61004,

Basrah, Iraq.

c Bio-Active Materials Group, School of Materials, MSS Tower, The University of

Manchester, Manchester, M13 9PL, UK

d Materials and Biosystems Laboratory (LAMAB), Department of Materials Engineering

(DEMat), Federal University of Paraíba (UFPB), João Pessoa, PB CEP 58051-900, Brazil

e Now at Department of Physics, Cavendish Laboratory, Cambridge University, CB3 0HE,

Cambridge, UK

Abstract

Fluorescent markers are critical for tracking the position and movement of molecules both in

vivo and in vitro. Conventionally, synthetic dyes are non-covalently added to polymers for

fluorescent tracking but often diffuse away. Here we demonstrate, for the first time, a facile

method for the synthesis of fluorescent polylactic acid PLA nano/-microfibres for biomedical

applications using solution spin blowing (SBS). Pyrene end capped poly(L-lactide) (PLLA)

derivatives were synthesised using the ring opening polymerisation of L-lactide and their

characterisation using spectroscopic and thermal analysis. Submicron-sized fluorescent fibres

were produced from these PLLA derivatives using solution blow spinning techniques

generating polymer blends and core-shell fibres. Such system could be further exploited to

incorporate electrically conductive carbon allotropes via the pyrene aromaticity, producing

fluorescent and electrically active fibres for in vitro monitoring and electrical stimulation.

Keywords: Poly L-lactide, pyrene, polyaromatic synthesis, fluorescence marker, solution

blow spinning, nanofibres.

1. Introduction

Biodegradable polylactide (PLA) has important applications in regenerative medicine1 and

tissue engineering2 owing to its flexibility, bioresorbability, and biocompatibility.3 The

conjugation of fluorophores to PLLA generates a range of useful biological tools whose

preparation usually proceeds via the introduction of the marker in a non-covalent manner in

order to permit intracellular tracking but is often found to be ineffective due to poor solubility

and diffusion in the biological medium. These issues can be overcome through covalent

attachment of the fluorophore to the poly-lactide whilst recognising the fact that the

fluorescent marker mobility will increase upon hydrolysis of the polymer backbone.4,5

Notwithstanding the simplicity of mixing dyes with the polymers in order to generate

fluorescent aggregate structures,6 the labelling of PLLA with pyrene-derived

fluorophores,7,8,9,10 particularly PEGylated pyrene, through covalent attachment9,10,11 (e.g. via

an ester linkage) has attracted some attention of late. These constructs prevent the seepage of

the dye into the surrounding biological environment and enables the tracking of the poly-

lactide through the characteristic fluorescence emission of the pyrene moiety.12,13,14 It has

recently been shown that the side chain incorporation of pyrene into a polymer can promote

the dispersion of carbon allotropes,9 rendering the organic polymer composite electrically and

thermally conductive.15 Such electrically active polymers have been exploited for use tissue

engineering, generation of cell cultures, and more recently in the development of wearable

sensors.16,17,18

Cells are surrounded by an extracellular matrix (ECM) which is essentially a fibrous

interconnected network with pores sufficiently sized to permit cell migration.19,20 By

mimicking the natural ECM, nano/-microfibres can provide topographic and mechanical cues

to dictate cell behaviour. Fibres with small diameters and large surface areas are suitable for

mimicking natural ECM to enhance cell growth.21 Hence, nano/-microfibres are desirable

materials for producing tissue engineered scaffolds.

Methods to produce nano/-microfibres have gained increasing attention due to their

large surface area to volume ratios and flexibility in surface functionalities, when fibre

diameters are decreased to submicron sizes.22,23 Electrospinning has been investigated to

generate fibres as this technique can produce continuous fibre mats with submicron

diameters.24 However, solution blow spinning (SBS) offers a more efficient method to

fabricate nano/-microfibres at higher production rates when compared to conventional

electrospinning 25,18 and critically does not require the use of electric field and fibres can be

visually deposited on any surface in a 3-D fashion.23 In the SBS technique high pressure gas

is injected into the gap between two concentric nozzles while, at the same time, a solution

containing a polymer is fed into the inner nozzle. Most importantly, it has been found that

fibre diameters of the blown fibre can be finely tuned25 by judicious control of process

variables such as polymer injection rate, nozzle geometry, temperature, and gas pressure.

Here we demonstrate, for the first time, a facile method for the synthesis of fluorescent

PLA nano/-microfibres for biomedical applications using SBS; the fluorescent characteristics

of the PLLA-pyrene nano/-microfibres are also presented. We detail the synthesis of three

pyrene-based PLA polymers as fluorescent markers, all of which are readily accessible from

the ROP reaction between suitably hydroxylated pyrene derivatives with L(-)-lactide (L-LA)

mediated by catalytic quantities of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).18,26

2. Experimental

2.1. Characterisation

APCI mass spectra were acquired using an Agilent 6120 Quadrupole LCRS or a Thermo

Exactive Plus EMR System instrument. MALDI mass spectra were acquired with a Shimadzu

Axima Confidence instrument using a DCTB, dithranol or TCNQ matrix as stated. High

resolution mass spectra were determined using a Thermo Exactive Plus EMR or Thermo

Finnigan MAT95XP mass spectrometers.

1H NMR (500 MHz) spectra were recorded using a Bruker AvanceII+ spectrometer

referencing to the residual protons in CDCl3 (7.27 ppm) or DMSO-d6 (2.50 ppm). 13C NMR

spectra (126 MHz) were recorded using a Bruker AvanceII+ spectrometer referencing to the

residual solvent peak as appropriate: CDCl3 (77.00 ppm respectively), DMSO-d6 (39.51 ppm

respectively). All coupling constants (J) are recorded in Hz and chemical shifts (δ) are

reported in ppm. Signal multiplicities are designated as singlet (s), doublet (d), triplet (t),

quartet (q), multiplet (m) or any collection of those. IR spectra were recorded using a Thermo

Scientific Nicolet iS5 spectrometer with an iDS ATR accessory in the solid state.

UV-visible spectra were recorded using a Varian Eclipse 5000 spectrophotometer

between 200 – 800 nm using quartz cuvettes with a path-length of 1 cm. Fluorescence spectra

were recorded on a Varian Eclipse fluorescence spectrometer. Samples were irradiated (ex =

320 nm.) in quartz cuvettes (10 mm) and the emission wavelength was recorded between 300

– 600 nm.

GPC was used to determine the molecular weight of polymers in THF with a flow rate

of 1 mL min-1. [1−2 mg mL−1] at 40 °C using a Viscotek GPCmax VE2001 solvent/sample

module with 2 × PL gel 10 μm MIXED-B and a 1 × PL gel 500A column, a Viscotek

VE3580 RI detector. The system was calibrated with low polydispersity polystyrene

standards, in the range 200 to 6 × 106 g. DSC measurements were obtained on a Perkin-Elmer

Diamond DSC under nitrogen at a heating rate of 10 °C min-1.

2.2. Solution blow spinning

The concentration of PLLA, air pressure, and volumetric flow rate were optimised to tune the

fibre diameters. 150 mg, 180 mg, and 210 mg of PLLA were dissolved in 3 mL of chloroform

to obtain solutions with concentrations of 5, 6 and 7 w/v% for SBS. To obtain core-shell

fibres, PLLA and Py-La (AH1) were dissolved in chloroform at 7 and 7.7 w/v%, respectively.

For fibre blends different ratios PLLA: Py-La were investigated for each of the blends PLLA

and Py-La powder were dissolved in 3 mL chloroform. Solutions were stirred at 200 RPM for

at least 2 hrs at room temperature.

The custom made SBS setup consists of two concentric nozzles: the inner nozzle

through which PLLA solutions were pumped by a syringe pump (World Precision

Instruments, model AL-1000, USA) at fluxes 100 μL min-1 and 200 μL min-1. The inner

nozzle protruded 2 mm beyond the coaxial outer nozzle and the diameter of the inner nozzle

was 1 mm. The pressurised air was delivered through the outer nozzle and was controlled by

a pressure regulator. A working distance of around 50 cm was used in all experiments. For

core-shell fibres, two syringe pumps were used with three concentric nozzles. The innermost

nozzle through which Py-La solution was pumped at 1.2 μL min -1, 2.5 μL min-1, and 5 μL

min-1. The PLLA solution was injected through the middle nozzle via a syringe pump with a

flow rate of 120 μL min-1.

2.3. Scanning electron microscopy (SEM)

Fibres were collected on carbon tabs and mounted on aluminium specimen stubs. Before

SEM analysis, each sample was coated with 10 nm of Au using a Phenom Pro2

(Phenomworld, Netherlands). SEM was performed on a Phenomworld Scanning Electron

Microscope G2 Pro (Phenomworld, Netherlands) and a Hitachi Scanning Electron

Microscope S-3000N (Hitachi, Japan) operated at a voltage of 5 kV. The diameters of fibres

were measured by using ImageJ (National Institutes of Health, USA). To obtain the mean

fibre diameters at least 50 random fibres were measure using ImageJ.

2.4. Materials

All reactants, reagents and dry solvents were purchased from Sigma Aldrich, Acros Organics

and Fisher Scientific and were used without further purification. PLLA, Purasorb® PL 18

was obtained from Corbion PURAC® (Corbion, Netherlands, Mn of 43,882 g mol-1 and

polydispersity index 1.872). Polymer solutions were prepared by solubilising the polymers in

chloroform. Column chromatography was performed using Davisil grade 636 (60Å pore size,

40-63 mesh) silica gel. TLC was carried out using DC-Fertigfolie POLYGRAM® SIL

G/UV254 precoated TLC sheets with substrate detection by UV light (254 and 365 nm).

2.5. Synthesis and characterisation of PLA derivatives

2.5.1. Synthesis of 2-(2-(2-(pyren-1-ylmethoxy)ethoxy)ethoxy)ethan-1-ol

Triethylene glycol (1.1 mL, 8 mmol) in dry tetrahydrofuran (THF, 25 mL) was added to NaH

(192 mg, 8 mmol; ex 60% dispersion in mineral oil) in a two-necked round bottom flask

under an atmosphere of nitrogen at ambient temperature. To this mixture was then added a

solution of 1-(bromomethyl)pyrene (295.2 mg, 1 mmol) in dry THF(10 mL) over a period of

ten minutes. The resulting reaction mixture was then brought to reflux, under dry nitrogen,

for a period of 24 hours. After cooling to room temperature, water (20 mL) was added to

quench the reaction mixture which was then concentrated in vacuo. The residue was

triturated (CH2Cl2, 3 × 50 mL) and the combined extracts washed with brine (2 × 75 mL) and

then dried over MgSO4. The solvent was removed in vacuo and the crude product was

purified by column chromatography (silica gel; gradient elution: 100% CH2Cl2 to methanol:

CH2Cl2 [5:95]) to afford 2-(2-(2-(pyren-1-ylmethoxy)ethoxy)ethoxy)ethan-1-ol as a brown-

coloured oil. Yield 273.3 mg (75%). 1H NMR (500 MHz, DMSO-d6) 3.39 - 3.44 (2 H, m,

CH2-O-CH2-CH2), 3.45 - 3.5 (2 H, m, -CH2-O), 3.51 - 3.58 (4 H, m, -CH2-O), 3.61 - 3.66 (2

H, m, -CH2-O), 3.70 - 3.76 (2H, m, -CH2-O), 4.57 (1 H, t, J = 5.4 Hz, O-H), 5.22 (2 H, s,

CH2-Ar), 8.06 - 8.14 (2 H, m, Ar-H), 8.24 - 8.29 (2 H, m, Ar-H), 8.32 (2 H, t, J = 6.9 Hz, Ar-

H), 8.40 (1 H, d, J=9.2 Hz, Ar-H) ppm. 13C NMR (126 MHz, DMSO-d6) δ 30.9, 60.4, 69.9,

70.0, 70.1, 70.7, 72.5, 123.8, 124.0, 124.2, 124.7, 125.4, 126.4, 127.2, 127.4, 127.5, 127.6,

128.8, 130.5, 130.7, 130.9, 130.9 and 132.1 ppm. MS (m/z (MALDI-dithranol) 364. ([M]+,

100%). HRMS (APCI+) C23H28NO4 ([M + NH4]+) requires 382.2013; found 382.2004. v

max/cm-1 3400 O-H (br), 3047 (Ar C-H), 2918 and 2871.2 (C-H), 1588 and 1499 (Ar C=C),

1114 and 1057 (C-O). The 1H NMR data for this compound was identical to that reported in

the literature.27

2.5.2. Synthesis of 3-(pyren-1-yloxy)propan-1-ol

1-Bromopyrene (218 mg, 1.0 mmol), caesium carbonate (977 mg, 3.0 mmol) and CuCl (6.7

mg, 5 mol%) were added to a two neck RBF (50 mL) and purged with N2. 1,3-Propanediol

(3 mL, 18 mmol) was then added via syringe and the reaction mixed heated at 130 °C for 24

hrs. On cooling to ambient temperature the reaction was quenched by the addition of 1M

HCl, adjusting the reaction mixture to pH 3. The mixture was then extracted with Et2O (2× 50

mL) organic extracts washed (H2O, 2 × 50 mL; brine, 75 mL) and dried (MgSO4). The

solvent was removed in vacuo and the crude product purified by column chromatography

(silica gel; gradient elution: CH2Cl2: pet. ether [3:7] to ethyl acetate: petroleum ether [3:2])

to afford 3-(pyren-1-yloxy)propan-1-ol as an off-white solid. Yield 133.3 mg (28%).); mp

230 °C (dec). 1H NMR (500 MHz, chloroform-d) 1.24-1.3 (1 H, m, O-H), 2.29 (2 H, quin, J

= 5.95 Hz, C-C-H), 4.06 (2 H, m, HO-C-H), 4.50 (2 H, m, Ar-O-C-H), 7.59 (1H, d, J = 8.39

Hz, Ar-H), 7.88 - 7.94 (1 H, m, Ar-H), 7.94 - 8.01 (2 H, m, Ar-H), 8.05 (1 H, d, J = 9.15 Hz,

Ar-H), 8.08 - 8.16 (3 H, m, Ar-H), 8.44 (d, J=9.15 Hz, 1 H, Ar-H) ppm. 13C NMR (126 MHz,

Chloroform-d) δ 32.3, 60.4, 66.4, 109.2, 120.4, 120.9, 124.2, 124.3, 124.9, 125.1, 125.4,

125.5, 125.8, 126.1, 126.5, 127.2, 131.6, 131.7, 152.8 ppm (accidental equivalence results in

the observation of only 16 resonances). MS (m/z) 276.1 ([M]+, 100%). HRMS (ESI+)

C19H17O2 ([M + H]+) requires 277.1223, found 277.1220. vmax/cm-1 3298 O-H (br), 2929 and

2920 (Ar C-H), 2359 (C-H), 1598 and 1575 (Ar C=C), 1253 (C-O), 1114 and 1071 (C-C).

2.5.3. Preparation of pyrene-poly(L-lactides)

To a solution of pyrene-1-methanol (0.7 g, 3.0 mmol) and L-lactide (L-LA) (1.3 g, 9.0 mmol)

in dry CH2Cl2 (15 mL) was added DBU (24 µL. 1% of the total monomer weight) and the

resultant reaction mixture was stirred for 24 hrs at 25 °C under N2. The reaction mixture was

then diluted with CH2Cl2 (50 mL) and purified column chromatography (silica gel; eluent

100% CH2Cl2 then 100% MeOH). The solvent was removed in vacuo at 25 °C and then the

final product was dried under vacuum (1 Torr) at 25 °C for 24 hrs. The reaction was worked

up by the slow addition of reactants to 500 mL diethyl ether to precipitate the polymer which

was then collected by vacuum filtration. The crude polymer was washed with diethyl ether (5

x 20 mL) and then dried under vacuum (1 Torr) at 25 °C for 24 h.

3. Results and discussion

3.1. Fluorescent PLA derivatives

The synthesis of 2-(2-(2-(pyren-1-ylmethoxy)ethoxy)ethoxy)ethan-1-ol has been reported by

Duhamel and co-workers27 however, in our hands, a modified Williamson ether synthesis,

similar to that reported by Ward,28 proved highly effective and obviated the use of

stoichiometric quantities of silver oxide. We find that the use of an excess (8 eq.) of

triethylene glycol in this reaction minimized bis-alkylation and enabled the synthesis of the

mono-ether in good yields (75%), Fig. 1A.

Figure 1. A) Synthesis of 2-(2-(2-(pyren-1-ylmethoxy)ethoxy)ethoxy)ethan-1-ol; B)

synthesis of 3-(pyren-2-yloxy)propan-1-ol; C) 1H NMR spectrum (500 MHz, chloroform-d)

of 3-(pyren-2-yloxy)propan-1-ol; D) 13C NMR spectrum (126 MHz, chloroform-d) of 3-

(pyren-2-yloxy)propan-1-ol.

The synthesis of 3-(pyren-2-yloxy)propan-1-ol was accomplished via an SNAr reaction

between 1-bromopyrene and 1,3-propanediol, a reaction that was mediated by caesium

carbonate as base in the presence of CuCl as a catalyst. Although the isolated yield of the

desired product from this reaction was only moderate (28%) its synthesis, in a single step,

from readily available starting materials was judged to be of greater importance in this

instance. The 1H and 13C NMR spectra for 3-(pyren-2-yloxy)propan-1-ol are shown in Fig.

1C and 1D respectively.

The PLLA-pyrene polymers were synthesised using a simple protocol, which involved

the ring opening polymerisation (ROP) of L-lactic acid using a suitable hydroxylated pyrene

derivative as initiator in the presence of DBU as a catalyst at ambient temperature. The use of

DBU allows a high degree of polymerisation, very narrow controlled molecular weight and

low catalyst concentration of 1 mol%. This methodology is preferable to that employing

stannous octanoate (Sn(Oct)2), one of the most commonly used catalysts currently employed

for the promotion of these polymerisation reactions and is experimentally easy to adopt.27,29,30

Most notably, the DBU-catalysed process is free from the toxicity associated with the tin-

based systems which limits their potential utility, especially for biomedical use. The

structures of the polymers generated in our study were characterised spectroscopically (IR, 1H

NMR, and 13C NMR) and by GPC. The structures of AH1, AH2 and AH3, and reactant

quantities are listed in Error: Reference source not found; these polymers have similar Mn

values to those recently reported for those derived from the ROP of L-LA with

pyrenebutanol.27

Table 1: Structures of the polymers AH1-AH3, reactant quantities, and molecular weight

distributions.

Structure/Abbreviation Alcohol (g):L-Lactide (g):DBU (µl) Mw/Mnc

(Da)Mnd

(Da) ÐM

Pyrene-1-methanol =0.232: 3.603 : 112 5500/3200 4600 1.72

3-(pyren-1-yloxy)propan-1-ol = 0.100: 1.304: 25 5700/3100 6700 1.83

2-(2-(2-(pyren-1-ylmethoxy) ethoxy)ethoxy) ethan-1-ol =

0.200: 1.977: 257700/4600 5700 1.67

c Determined by GPC analysis, d Determined by the 1H NMR ( Fig. 2 (A)).

Fig. 2A shows the 1H NMR spectra of the polymers, highlighting the (-CH) and (-CH3)

protons in PLLA. The resonance at 4.3 ppm corresponds to the (-CH) of the chain end

group and that at 5.2 ppm corresponds to the (-CH) proton in the polymer backbone. The

resonances between 1.3 - 1.7 ppm are characteristic of the (-CH3) groups in the backbone

of the polymer and the end group of the PLLA. The (-CH2) protons attached to the pyrene

have a chemical shift at 5.95 ppm. The 13C NMR spectra of AH1, AH2 and AH3 are

presented in Fig. 2B. These spectra indicate the presence of the lactide carbonyl, methyl (-

CH3) and methine (-CH) groups at 168, 18 ppm and 68 ppm respectively; the

remaining methylene resonances (-CH2) appear between 30 – 72 ppm while the pyrene

carbons appear in the region typical of aromatic compounds ( 120 – 132 ppm). All of the

polymers possessed absorptions at 1740 cm-1 confirming the presence of a saturated ester

moiety.

Figure 2. A) 1H NMR (500 MHz, chloroform-d) spectra of AH1, AH2, and AH3; B) 13C

NMR 1H NMR (126 MHz, chloroform-d) spectra of AH1, AH2, and AH3.

The main aim of this research was to prepare lactide polymers which possess covalently

attached fluorescent domains so that their fluorescent properties could be investigated. The

UV-vis spectra of AH1 and AH3 in CH2Cl2 (0.6 mg mL-1) show absorbance bands at = 345

nm and 342 nm respectively which can be assigned to S0-S2 transitions of the pyrene nucleus;

a low intensity, higher energy, S0-S1 transition at = 375 nm is also apparent (Fig. 3A). This

data is similar to that reported by Shubert7 for graft co-polymers derived from the ROP of L-

lactide with 1-pyrenebutanol. In comparison to AH1 and AH3, the UV-vis spectrum for AH2

is broadened and red-shifted and exhibits an absorption at = 345 nm with two additional

absorptions at =364 nm and 384 nm. This data is in accord with that previously reported

for alkoxypyrenes where the red shift has been attributed to the electronic effect of the

oxygen substituent.31 The topology of pyrene-containing polymers has been probed

extensively32,33,34 using pyrene excimer fluorescence spectroscopy where the appearance of a

broad signal between 470-530 nm in the emission spectrum (ex at the S02 transition, c.a. 340

n.m. for 1-(alkoxymethyl)pyrene derivatives) is indicative of excimer formation. In the case

of AH1, AH2 and AH3 fluorescence spectra were measured at five different concentrations

between 0.6 – 0.006 mg mL-1 in CH2Cl2. All three polymers show three emission signals

between 370 – 428 nm (Fig. 3B-D) whose values were essentially concentration independent;

these spectra are also essentially devoid of the longer wavelength, excimer emissions typical

of aggregated structures and are more in keeping with “monomer” transitions.

Figure 3. A) UV-VIS Spectra of AH1, AH2, and AH3, showing fluorescence emission

intensity at a range of concentrations for B) AH1, C) AH2, and D) AH3. (ex = 320 n.m.;

spectra recorded in CH2Cl2 as solvent).

The Thermal properties of the polymers described above were studied by

thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The

degradation behaviour 4.3 - 8.5 mg samples of these polymers was measured under nitrogen

atmosphere when heated from 30 °C to 600 °C at constant heating rates of 10 °C min-1. The

initial decomposition temperature tangential (Ti), the temperature of the final decomposition

(Tf), the degree of decomposition temperature at 50 % loss of the sample weight (T50%), and

char content (%) at 300 °C were determined and are outlined in Table S1 (ESI). The rate of

decomposition (%wt min-1) was calculated from the slope of the degradation curve between

(20 – 80) % weight loss. Fig. 4A shows that the decomposition process consists of one stage

starting at 208 °C, 179 °C, and 195 °C for AH1, AH2, and AH3 respectively. The char

content varied between 7.67 - 9.11 wt% due to the pyrene structure and was in agreement

with other studies.29,30,35

Figure 4. Showing A) TGA thermogram of AH1, AH2, and AH3, DSC thermograms for B)

AH1, C) AH2, and D) AH3.

The low glass transition temperature between 40 °C to 56 °C for all three polymers is

apparent from the data depicted in Fig. 4B-D. The reduced glass transition temperature for

polymers AH2 and AH3 is due to the plasticizing effect of the etheric group that leads to

enhanced polymer flexibility. The molecular weight of AH3 is higher than the other two

polymers (AH1 and AH2) but the presence of three, conformationally mobile, ethylene oxide

groups reduces the glass transition temperature. The polymers also show exothermic peaks

that correspond to crystallisation temperatures of 129 °C, 102 °C, and 117 °C for AH1, AH2,

and AH3, respectively (Table S2, ESI).

It is clear from the data presented in Figures 4B, C and D that there is no substantial change

in either the crystallinity or thermal decomposition for the polylactide which incorporates the

pyrene when compared to polylactide itself (Fig. 4A).

3.2. Fluorescent fibres

Fibres were produced using a core-shell SBS set up in which Py-La was used as the core and

PLLA as the shell. The simple, yet effective, apparatus used in these SBS experiments was

constructed using readily available laboratory equipment, as shown below (Fig. 5) in

diagrammatic form.†

Figure 5. Schematic of the SBS apparatus used for preparation of nano fibers by the core-

shell method used in this study.

Fig. 6 A-B shows representative SEM images of the core-shell fibres (other images are

shown in Fig. S1A-B, ESI). The fibres have random orientations and there are beads present.

† Details available from J. B. upon request.

The volumetric flow rate of the shell (PLLA) was held constant and the core flow rate (Py-

La) was modified to control the composition. Due to the higher relative flow rate of the shell

(PLLA 120 µL min-1) the mean fibre diameter was relatively constant varying between 233 –

264 nm (flow rates and diameter distributions are shown in table S3 and Fig. S2 (ESI),

respectively).

PLLA and Py-La blends were also spun into fibres and representative images are shown in

Fig. 5C-F. The blended fibres have a random orientation and exhibited less beading in

comparison to the core-shell fibres. In addition, they had a mean fibre diameter varying

between 111 – 170nm (Fig. S3, ESI), which is considerably less compared to the core-shell

fibres

Figure 6. SEM images of core-shell fibres generated from flow rates of PLLA/Py-La at A)

120/2.5 μL min-1 and B) 120/1.2 μL min -1. SEM images of PLLA:Py-La blends with C)

70:30, D) 80:20, E) 90:10, and F) 95:5. Insets show higher magnification SEM images.

The flow rate, air pressure, and diameter of inner nozzle were held constant at 200 mL min -1,

344 kPa, and 2 mm to investigate how increasing the concentration of Py-La influenced the

fibre diameter. At higher Py-La concentrations the fibre diameter was larger and the smallest

diameter fibres were obtained using blends with a higher PLLA concentration (95:5,

PLLA:Py-La).

In agreement with the fluorescence emission spectra shown in Fig. 3B-D, the polymer

solutions fluoresced under UV illumination (Fig. 7A-B). Further, the subsequent fibres

produced by SBS also fluoresced under UV illumination (Fig. 6C, right). Therefore, we have

demonstrated that by exploiting SBS parameters, including composition and flow rates, we

can produce fibres with diameters close to 100 nm that exhibit fluorescent emission from

PLLA derivatives. Such structures provide a fibrous material for in vivo or in vitro tracking.

Figure 7. A) Solutions of AH1, AH2, and AH3 in CH2Cl2 (0.6 mg mL-1) under ambient

lighting, B) under UV illumination at 365 nm. C) Representative images of fluorescent

PLLA:Py-La fibres under ambient lighting (left) and under UV illumination at 365 nm

(right).

4. Conclusions

In summary, we have presented the synthesis of PLLA pyrene end capped derivatives via a

ring opening polymerisation; these materials have been fully characterised by spectroscopic

techniques and by thermal analysis. We have successfully fabricated fluorescent fibres using

PLLA pyrene end capped derivatives using the operationally robust and flexible SBS

technique. By careful control of experimental parameters (including concentration, air

pressure, and volumetric flow rate) SBS and enables the preparation fluorescent fibres with

diameters ~ 100 nm. The incorporation of fluorescent tags within the polylactide framework

may provide structural insights of the polymer itself 31 or enable the synthesis of

biodegradable polymeric materials whose dispersion can be tracked in vivo. Biological

applications of these polymers are now under active investigation within our group.

Acknowledgements

The authors thank Corbion PURAC® (Corbion, Netherlands) for kindly supplying PLLA

sample. The University of Manchester thanks the EPSRC (EP/ K039547/1) for the provision

of Bruker NMR spectrometers. Aula Alwattar kindly acknowledges the Iraqi Ministry of

Higher Education and the University of Basrah (Chemistry Department) for the provision of a

research scholarship. JJB, ESM and TDLN acknowledge support from CNPq and CAPES

(Brazil), Grant Numbers PVE 304898/2014-7, and CAPES-PDSE 8881.132372/2016-01,

respectively.

Appendix A. Supplementary data

Supplementary data related to this article can be found at

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