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Poly(ethylene glycol) conjugated Poly(lactide) based Polyelectrolytes: Synthesis and Formation of Stable Self-Assemblies Induced by Stereocomplexation Zibiao Li, Du Yuan, Xiaoshan Fan, Beng H. Tan, and Chaobin He Langmuir, Just Accepted Manuscript • DOI: 10.1021/la504860a • Publication Date (Web): 09 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Poly(ethylene glycol) conjugated Poly(lactide) based Polyelectrolytes: Synthesis and Formation of Stable Self-Assemblies Induced by Stereocomplexation

Zibiao Li,b Du Yuan,a Xiaoshan Fan,a Beng H. Tan,b,* Chaobin He a,b,*

a

Department of Materials Science and Engineering, National University of Singapore, Singapore 117574, Singapore b

Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 3 Research Link, Singapore 117602, Singapore

* correspondence to [email protected]; [email protected]

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Abstract: A series of pH-responsive amphiphilic poly(N,N-dimethylaminoethyl methacrylate)-block-poly(D-lactic acid)-block-poly(N,N-dimethylaminoethyl methacrylate) conjugated with poly(ethylene glycol) (DPDLA-D@PEG) and D-PLLA-D@PEG copolymers were synthesized using a combination of ringopening polymerization and atom-transfer radical polymerization followed by sequential quaternization of PDMAEMA chains and azide-alkyne click reaction with alkyne-end PEG. Gel permeation chromatography, nuclear magnetic resonance and fourier transform infrared spectroscopy results demonstrate the successful synthesis of the copolymers, and the conjugated PEG percentages in the copolymers can be tuned by the feeding ratios in the quaternization reaction. Conjugating PEG onto the PDMAEMA segments also successfully facilitated the D-PDLA-D@PEG, D-PLLA-D@PEG and its corresponding 1:1 D/L mixtures to be dissolved directly in aqueous solution at the desired concentration range without using any organic solvents unlike the copolymers without PEG conjugation (D-PDLA-D and D-PLLA-D). We demonstrate control over micellar size, charge and stability via three different preparation pathways, i.e., solution pH, percentages of PEG conjugation in the copolymers and formation of PLA stereocomplex in micellar core. Static and dynamic light scattering studies demonstrate that the size of the core-shell micelles increases when the solution pH is reduced due to the protonation of the PDMAEMA segments that caused the osmotic pressure within the micelle to increase until the micelles reached a maximum size. It is interesting to note that the micelles formed by 1:1 D/L mixtures have larger swelling ratios as well as aggregation number and hydrodynamic radius that do not change significantly with pH and dilution respectively as compared to micelles formed from individual D or L forms of the copolymers. The enhanced stability of the pH responsive micelles prepared by direct dissolution of the 1:1 D/L mixtures of the PEG conjugated PLA based polyelectrolyte in aqueous medium is attributed to the stereocomplex formation between PLLA and PDLA in the micellar core as confirmed by wide-angle X-ray scattering measurements. Keywords: Stimuli-responsive, stereocomplex poly(lactide), self-assembly, quaternized PDMAEMA, PEG conjugation.


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1. Introduction Stimuli-responsive micelles have been a well-researched topic in polymer science, primarily because of its application in producing a reliable system for targeted drug and gene delivery in physiological conditions.1-4 Poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) has gained significant attention among researchers in this area due to its hydrophilicity, pH sensitivity, ease of quaternization, and availability of the functional amine/ammonium moiety for complexation with acidic/anionic substances. It has a pKa of ∼7.0 and therefore behaves as a weak polybase at higher pH values, whereas at lower pH values, the amine groups are protonated, and the polymer behaves as a cationic polyelectrolyte.5-10 It is nontoxic, can be absorbed by endocytosis, and can be used as a non-viral DNA vector.11 In addition, it was found to have antibacterial, hemostatic, and anticancer activity,12-13 therefore rendering it an attractive system for biological applications. It is well known that co-polymerizing, grafting or forming amphiphilic networks (APN) of PDMAEMA with other hydrophobic polymers, can potentially enhance its affinity for more hydrophobic substrates.5, 14-19 Among these, the self-assembly of amphiphilic copolymers in a selective solvent above a threshold concentration, referred to as the critical aggregation concentration(CAC), to form polymeric micelles have been investigated extensively for numerous applications, mainly in the biomedical field.2022

However, polymeric micelles are susceptible to infinite dilution, a condition arising from their

administration. For example, micelles prepared from poly(ethylene glycol)-block-poly(D,L-lactide)(PEGb-PDLLA) have been shown to dissociate after intravenous injection and are rapidly excreted in urine.23 It is crucial that micelles retain their integrity, especially in vivo, to meet requirements such as long circulation times, accumulation at targeted sites, and controlled drug release. Strategies to yield micelles with improved stability mostly rely on the chemical cross-linking of either core24 or shell25 segments. In practice, such approaches are not optimal as the encapsulated guest molecule or biodegradability of the system may be altered by the cross-linking procedures. An alternative strategy, which is highlighted in the present work, consists of stabilizing the core by introducing physical cross-links through stereocomplex formation. A well-studied example is PLA26-28 where it has been shown that 3 ACS Paragon Plus Environment

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stereocomplex interactions between enantiomeric PLA could result in the formation of more stable micelles and nanoparticles in solution2, 29-33 and in terms of DFT modeling.34 However, majority of these amphiphilic PLA copolymers are not soluble directly in aqueous solution due to the high hydrophobicity of PLA. Instead the formation of stereocomplex micelle systems involved the dissolution of the enantiomeric PLA amphiphilic copolymers in a common good solvent (organic solvent) for both the PLA and hydrophilic segments followed by the gradual drop into aqueous solution and evaporation of the organic solvent.2, 32 It is expected that the gradual removal of the common good solvent would allow the self-assembly of the PLA copolymers to stable micellar structures. Besides the solvent evaporation method, stereocomplex micellar solutions were also prepared by dialyzing the organic solvent solutions of the copolymers against water.35 Despite the formation of stable micelles in water, the presence of residual organic solvents in the micelles are harmful to the human body and could pose serious compliance issues since the PLA micelle systems are being investigated for biological applications. To overcome this shortcoming, we proposed to incorporate hydrophilic polymers onto the PLA based polyelectrolytes for facilitating direct dissolution of the obtained water-soluble PLA based polyelectrolytes in an aqueous medium without using any organic solvents. In this contribution, the tertiary amino groups of the PDMAEMA blocks in the pH-responsive PDMAEMA-b-PLA-b-PDMAEMA triblock copolymers were quaternized by an alkyl halide to produce a series of concentrations of quaternary ammonium groups followed by PEG (which is a common hydrophilic polymer) conjugation onto the quaternized PDMAEMA segments. Micelles were then prepared by direct dissolution of the obtained water-soluble PEG conjugated PLA based polyelectrolyte in an aqueous medium without using any organic solvents. Since PDMAEMA is a pH-responsive polymer,7-9 the solubility of the micelles can be tuned by external stimuli. At low pH (pKa of ∼7.0), the corona chains become protonated and positively charged, leading to additional stabilization in solution. The effect of the stereocomplex formation between the enantiomeric PLA based polyelectrolytes on the stability of the micelles in aqueous solution against pH and dilution were investigated by static and dynamic light scattering. To the best of our knowledge, we demonstrate for the first time that, water 4 ACS Paragon Plus Environment

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soluble PEG conjugated PLA based polyelectrolytes can crystallize in a stereocomplex configuration in aqueous environment, which gives rise to pH-responsive micelles with enhanced kinetic stability. 2. Experimental Section 2.1. Materials 2-Bromoethanol (95%), Sodium azide (NaN3, >99.5%), 2-Bromopropionyl bromide (97%), poly(ethylene glycol) methyl ether (MPEG, Mn = 550), propargyl bromide solution (80 wt. % in toluene), Sodium hydride (NaH, 60% dispersion in mineral oil), α-Bromoisobutyryl bromide (98%), triethylamine (>99%),

1,1,4,7,10,10-hexamethyltriethylenetetramine

(HMTETA,

99%),

N,N,N′,N′′,N′′-

Pentamethyldiethylenetriamine (PMDETA, 99%), copper(I) bromide(CuBr, 99%), stannous octoate [Sn(Oct)2] (95%), anhydrous 1,4-Dioxane (99.8%), anhydrous toluene (99.8%), and anhydrous N,Ndimethylformamide (99.8%) were obtained from Sigma-Aldrich. 2-(Dimethylamino)ethyl methacrylate (DMAEMA) stabilized with hydroquinone monomethyl ether was obtained from Merck and used as received. Ethylene glycol (99.8%, Sigma-Aldrich) was distilled over CaH2 before use. L-lactide (L-LA) and D-lactide (D-LA) (Purac Biochem, The Netherlands) were used without further purification. Alkyneended MPEG was prepared according to the previous procedures and the details are given in the Supporting Information.36 2.2. Synthesis of PDMAEMA-b-PLLA-b-PDMAEMA (D-PLLA-D) and D-PDLA-D Copolymers PLLA-diBr and PDLA-diBr macroinitiators were synthesized using ring opening polymerization (ROP) followed by terminal groups modification based on a previously described method with some modification as detailed in Scheme 1 and in the Supporting Information.32 PDMAEMA-b-PLLA-bPDMAEMA triblock copolymers were prepared by atom transfer radical polymerization (ATRP). Molar feed ratio of [PLLA-diBr] : [DMAEMA] : [CuBr] : [HMTETA] = 1 : 1000 : 1 : 2 was applied for all polymer synthesis. As a typical example, PLLA-diBr was first introduced into a nitrogen filled round bottom flask (RBF) followed by successive addition of 1,4-dioxane and DMAEMA monomer through syringe injection. Later, the RBF was purged and refilled with nitrogen using vacuum-nitrogen-cycling system three times. HMTETA and CuBr were added quickly under nitrogen atmosphere. Polymerization 5 ACS Paragon Plus Environment

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was allowed to proceed under continuous stirring at 60 °C for a desired reaction time. The molecular weight was monitored by gel permeation chromatography (GPC) analysis. After polymerization, the reaction was stopped by diluting the reaction mixture with THF and exposing it to ambient atmosphere for 1 h. Catalyst complex was removed by passing the reaction mixture through a short neutral Al2O3 column. After concentrating the filtrates, the solutions were precipitated into excess ether and the final product PDMAEMA-b-PLLA-b-PDMAEMA (D-PLLA-D) was obtained through centrifugation. By using PDLA-diBr as the macroinitiator, another series of D-PDLA-D triblock copolymer in the similar molecular weight range was prepared (Table 1). 2.3 Synthesis of PEG Conjugated D-PLLA-D (D-PLLA-D@PEG) and D-PDLA-D@PEG D-PLLA-D@PEG was synthesized through one-pot approach using 2-Azidoethyl-2-bromopropanoate (AEBP), synthesized as described in the Supporting Information, as coupling agent. In a typical procedure, D-PLLA-D (1.0 g) was dissolved in DMF (10 mL) and AEBP (0.072 mL) was then introduced into the mixture. The [AEBP]/[DMAEMA] molar ratio was fixed at 1 : 10, targeting at a 10% quaternization degree of D-PLLA-D@N3. After stirring at 50 °C for 48 h, the mixture was cooled to room temperature. Next, 0.55 g of propargyl-terminated PEG and 0.09 g of PMDETA were added and the flask was purged with N2 for 30 min. CuBr (0.07 g) were added quickly under nitrogen atmosphere. After stirring for 24 h at ambient temperature, the solution was dialyzed against deionized water using a dialysis membrane (Spectrum dialysis membrane, MWCO 1000) for 48 h to remove the excess PEG (change the dialysis water every 6 h), and the final product (D-PLLA-D@PEG) was subsequently collected through lyophilization. D-PDLA-D@PEG copolymers with various compositions were prepared with a similar method. 2.4. Characterization Techniques Nuclear Magnetic Resonance (1H-NMR) spectra were recorded on a Bruker AV-400 NMR spectrometer at room temperature. Chemical peaks are reported in ppm with reference to solvent peaks (DMF:δ 8.03, 2.92 and 2.75 ppm; CHCl3: δ 7.3 ppm; andH2O: δ4.8 ppm). Chemical compositions of the 6 ACS Paragon Plus Environment

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copolymers were evaluated from the proton integral regions as assigned in Figure 1. Gel Permeation Chromatography (GPC) (Shimadzu SCL-10A and LC-8A system) equipped with a Shimadzu RID-10A refractive index detector was used to determine the molecular weight and polydispersity of the assynthesized copolymers. DMF (0.1M LiBr) was used as the eluent at a flow rate of 1.0 mL/min at 40°C. Monodispersed poly(methylmethacrylate) (PMMA) standards were used to obtain a calibration curve. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) of the synthesized D-PLLA-D@PEG and D-PDLA-D@PEG copolymers were obtained on a Shimadzu TR100 spectrometer using a single reflection horizontal ATR accessory. The sample was mounted on top of the ATP crystal and lightly pressed with a premounted sample clamp. Each spectrum was collected in the range of 4000-400 cm-1 with a resolution of 4 cm−1 and a scan number of 64 at room temperature. Dynamic Light Scattering (DLS) measurements were made with a Brookhaven BI-200SM multi-angle goniometer equipped with a BI-APD detector. The light source was a 35 mW He-Ne laser emitting vertically polarized light of 632.8 nm wavelength. From the expression Γ = Dapp q 2 , the apparent translational diffusion coefficients, Dapp, were determined where Γ is the decay rate and q is the scattering vector. The apparent hydrodynamic radius, Rh can be determined by the Stokes-Einstein relationship:37-38 Rh =

kT 6πη D app

, where k, η and T are the Boltzmann constant, viscosity of solvent, and the absolute

temperature, respectively. The scattering intensity of each concentration of the copolymer in deionized water was measured and plotted against the polymer concentration. The concentration at which the scattering intensity increases sharply was defined as the critical micelle concentration, CMC. Static light scattering (SLS) measurements were performed to determine the weight-average molecular weight (Mw), z-average radii of gyration (Rg), and second virial coefficients (A2) of the micelles in aqueous solution

q 2 R g2 Kc 1 = [1 + ] + 2 A2 c 3 according to the relation; ∆Rθ M w , where K is the optical constant, which depends on the refractive index increment (dn/dc) of the polymer solution, c is the concentration of the polymer solution and Rθ is the excess Rayleigh ratio.39-40 The scattering angles, θ ranged from 50° to 120° at 10° 7 ACS Paragon Plus Environment

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intervals while the copolymer concentration ranged from 0.5 to 1.0 mg/mL. The refractive index increment (dn/dc) of each copolymer solution as a function of pH was measured using a BI-DNDC differential refractometer at a wavelength of 620 nm. The instrument was calibrated primarily with potassium chloride (KCl) in aqueous solution. In the dilution experiments (Figure 4(d)), the concentration-dependent Rg of the micelles at each dilution were obtained from a plot of Kc/Rθ against sin2(θ/2) where the slope yields the apparent Rg (Rg, app).39-40 Wide angle X-ray scattering (WAXS) was carried out on Bruker GADDS X-ray diffractometer with an area detector, operating under Cu-Kα (1.5418 Å) radiation (40kV, 40mA) at room temperature. Samples prepared at 3.0 mg/mL in aqueous solution were frozen rapidly using liquid nitrogen, followed by freeze drying under high vacuum (0.02 mBar) for three days. PLLA-diBr in powder was used as control. The dried samples were scanned from 5 to 40° (2θ). 2.5

Sample preparation in Aqueous Solution

Prior to DLS, SLS and WAXS measurements, the individual D-PDLA-D and D-PLLA-D copolymers with and without PEG conjugation as well as 1:1 D/L mixture solutions were prepared in 10 mM sodium chloride (NaCl) aqueous solution. 10 mM NaCl solution was used as the solvent in order to maintain a constant ionic strength in all the polymer solutions. Since the D-PDLA-D and D-PLLA-D copolymers and its corresponding 1:1 D/L mixture solution were unable to be dissolved directly in NaCl solution at the desired concentration range (~1 mg/mL), the following preparation method was adopted: first, individual 2.0 mg/ml stock solutions of D and L copolymers were prepared by dissolving 20 mg of copolymer in 10 mL of DMF. The stock solutions were filtered through PTFE filters before the desired amounts of individual D and L copolymers as well as D/L mixture were added dropwise into 2 ml of 10 mM NaCl solution under stirring. The samples were then stirred under room temperature for 48 h to ensure that the DMF has fully evaporated and stable nanoparticles were formed in NaCl solution. The pH of the aqueous solution obtained is approximately 7-8. The individual D and L copolymer solutions and its corresponding 1:1 D/L mixture solutions without PEG conjugation are labeled as Sample 1, Sample 2 and Sample 1+2 respectively (see Table 2). In contrast, when PEG is conjugated onto the quaternized triblock copolymers to produce D-PDLA-D@PEG and D-PLLA-D@PEG copolymers, the individual D 8 ACS Paragon Plus Environment

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and L copolymer solutions and its corresponding 1:1 D/L mixture solutions were able to be dissolved directly in 10 mM NaCl solution at desired concentrations. The samples were then stirred under room temperature for 24 h followed by filtration with the polyvinylidene fluoride (PVDF) 0.45 micron syringe filter. The pH of the aqueous solution obtained is also approximately 7-8. The individual D-PDLAD@PEG and D-PLLA-D@PEG copolymer solutions and the corresponding D/L mixture solutions are labeled as Sample 3-6 and Sample 3+4 and Sample 5+6 respectively (see Table 2). Next, the pH of all the individual as well as mixture solutions in 10mM NaCl were adjusted to the required values with sodium hydroxide (NaOH) and hydrochloric acid (HCl) solutions to investigate the change in morphology of the aggregates with varying pH. The samples were then stirred under room temperature for another 24 h to allow complete equilibration before being transferred into light scattering test tubes and sealed. For WAXS measurements, the pH adjusted samples in 10 mM NaCl (individual D and L copolymers as well as 1:1 D/L mixture) were freeze dried to obtain the solids.

3. Results and Discussion 3.1. Synthesis and Characterization of D-PLLA-D@PEG and D-PDLA-D@PEG Copolymers Previously, we reported the synthesis of amphiphilic copolymers consisting of hydrophobic enantiomeric PLA and different hydrophilic components such as poly(acrylic acid) (PAA), poly(ethylene glycol) (PEG) and poly(N-isopropylacrylamide) (PNIPAAM)) in various architectures, and investigated their stereocomplex effect on the self-assemblies in aqueous solutions.32, 41-42 The results demonstrate that the stereocomplex formation of enantiomeric PLA can be used as an interesting vehicle to trigger the morphology transformation of the copolymer self-assembled nanoparticles, tune the thermoresponsiveness of the polymer solution and modulate the hydrogel rheology properties. However, in the current study, we described the synthesis of PEG conjugation of enantiomeric PLA based polyelectrolytes (D-PDLA-D and D-PLLA-D), and investigated the stabilization effect induced by stereocomplexation in the aqueous dispersions at different pH. During the synthesis, a bifunctional AEBP linker with bromopropionyl and azide group at opposite chain termini was designed to facilitate the PEG 9 ACS Paragon Plus Environment

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conjugation through sequential quaternization of PDMAEMA chains and azide-alkyne click reaction with alkyne-end PEG. The synthetic route of D-PDLA-D@PEG and D-PLLA-D@PEG copolymers are presented in Scheme 1. First, the starting PLLA-diBr and PDLA-diBr macroinitiator for ATRP were prepared from the reaction of terminal hydroxyl end groups in PLLA-diol and PDLA-diol with 2-bromoisobutyryl bromide according to our previous report.32 The Mn (NMR) of the obtained starting macroinitiators were approximately 3.33 × 103 Da and 3.14 × 103 Da for PLLA-diBr and PDLA-diBr, respectively (Table 1), which is within the optimal range of molecular weights for strong stereocomplex interaction.28 The typical 1H NMR spectra for PLLA-diBr is presented in Figure 1(a). The degree of substitution of the hydroxyl groups can be obtained by calculating the ratio of the signal at 5.16 ppm (-CO-CH-, PLLA) and the signal at 1.94–1.98 ppm from the methyl protons of the 2-bromoisobutyryl fragment. From this method, the degree of hydroxyl group substitution that produces PLLA-diBr and PDLA-diBr were estimated to be 95.3% and 97.5% respectively. In addition, the peaks associated with the methyl proton (1.57 ppm) of PLLA and methylene groups from the initiator residues (4.20 ppm) were also clearly identified in Figure 1(a), attesting the successful preparation of ATRP macroinitiator. Next, D-PLLA-D and D-PDLA-D triblock copolymers were synthesized in 1,4-dioxane at 60 °C for 24 h via ATRP of DMAEMA from the respective PLLA-diBr and PDLA-diBr macroinitiators. The copolymers were recovered by repeated precipitation in hexane and diethyl ether. The chemical structure of the assynthesized triblock copolymers were characterized by 1H NMR spectroscopy. As a typical example, Figure 1(b) shows the 1H NMR spectrum of the D-PLLA-D copolymer. Signals corresponding to methyl protons (C–CH3) of the backbone of the PDMAEMA blocks are observed in the region of 0.90–1.10 ppm, and signals associated with the methylene protons (C–CH2) of the backbone of the PDMAEMA block can be identified in the region of 1.82–1.91 ppm. The chemical shift at 2.28 ppm is assigned to the methyl (N–CH3) protons of the DMAEMA units and the chemical shift at 2.56 ppm is assigned to the methylene (N–CH2) protons of the DMAEMA units. The peak at 4.06 ppm corresponds to the methylene protons adjacent to the oxygen moieties of the ester linkages on the PDMAEMA block (CH2–O–C=O).43 On the other hand, the peaks associated with PLLA as assigned in Figure 1(a) were also observed in 10 ACS Paragon Plus Environment

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Figure 1(b), confirming the D-PLLA-D triblock architecture. From 1H NMR, the chemical composition of D-PLLA-D triblock copolymer was calculated based on intensity ratio of PLLA methine proton (δ 5.16 ppm) and PDMAEMA methylene proton (δ 2.56 ppm) and are summarized in Table 1. The results showed that D-PLLA-D and D-PDLA-D triblock copolymers with similar block length were successfully prepared through this ATRP method, which could possibly facilitate the complete stereocomplexation of the enantiomeric PLA blocks in aqueous self-assemblies.44 Finally, D-PLLA-D@PEG and D-PDLA-D@PEG copolymers were synthesized using a sequential reactions consisting of quaternization of PDMAEMA chains in D-PLLA-D and D-PDLA-D triblock copolymers, and subsequent azide-alkyne cycloadditions with alkyne-end PEG through a one-pot approach (Scheme 1(c)). To facilitate these reactions, a bifunctional AEBP linker with bromopropionyl and azide group at opposite chain termini was designed. AEBP was prepared according to the reaction sequence shown in Scheme S1(A), and the chemical structure was confirmed by 1H NMR (Figure S1(A)). The quaternized D-PLLA-D@N3 and D-PDLA-D@N3 copolymers with two different chemical compositions were synthesized by varying the AEBP feed. These two copolymers were used as intermediate products for following PEG conjugation. The chemical structures of D-PLLA-D@N3 and DPDLA-D@N3 copolymers were also investigated by 1H NMR. As shown in Figure S1(C), the respective peak intensities of –N–CH2–signal and the –N–CH3–signal of PDMAEMA at 2.56 and 2.28 ppm reduced after quaternization. On the other hand, new signals were observed at 3.34 ppm and 3.63 ppm, corresponding to the methyl groups (–N+–CH3–) and methylene protons (–N+–CH2–) of the quaternized compound.45 Another new peak was also observed at 4.64 ppm due to the shift of methylene protons (– C(O)–O–CH2–) from 4.1 ppm.46 In addition, the characteristic of the –O–CH2–peaks at 4.32 ppm, and the methyl peaks of AEBP chain at 1.55 ppm, confirmed the presence of AEBP on the pendant amino groups of the PDMAEMA (Figure 1(b) and Figure S1(C)). PEG conjugation was performed via Cu(I)-catalyzed azide-alkyne click reaction between alkyneend PEG and azide groups in the freshly prepared D-PLLA-D@N3and D-PLLA-D@N3 copolymer solutions (Scheme 1(c)). The terminal hydroxyl group of MPEG was esterified to introduce alkyne according to the protocol described in the experimental section (S1.1, Scheme S1(B)). In the 1H NMR of 11 ACS Paragon Plus Environment

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alkyne-end PEG, the signals of the protons in the propargyl groups were observed at 2.44 and 4.2 ppm, respectively (Figure S1(B)).47 Alkyne-end PEG was then conjugated with D-PLLA-D@N3 and D-PLLAD@N3 copolymers with two different quaternization ratios to afford four types of copolymers, D-PLLAD@PEG-1, D-PLLA-D@PEG-2, D-PDLA-D@PEG-1, and D-PDLA-D@PEG-2. The

1

H NMR

spectrum of a typical specimen, D-PLLA-D@PEG-2, was presented in Figure 1(c). As anticipated, all the signals belonging to the three components are well resolved and can be clearly identified. Comparing with the precursors before conjugation, a new signal at 3.6 ppm emerged as a characteristic single peak of the methylene protons in PEG, implying the successful conjugation of PEG in D-PLLA-D@PEG copolymers.48-49 The relative molecular weight and polydispersity of the as-synthesized copolymers were calculated from the GPC curves as shown in Figure S2 and presented in Table 1. As shown in Figure S2, all the traces show a nearly symmetrical and unimodal peak of the molecular weight distributions reveals the high purity of tested samples rather than a mixture with its precursors. In addition, the molecular weight of D-PLLA-D@PEG increases and experiences a shorter elution time as comparison to D-PLLAD and PLLA-diBr traces. This clearly illustrates the occurrence of conjugation reaction between alkyne groups in PEG terminals and the azide in the pendant amino groups of the PDMAEMA. The molecular weight difference between D-PLLA-D and D-PLLA-D@PEG copolymers, mainly contributed from the PEG click reaction, were used to approximately evaluate the conjugation percentage (%) of PEG attached to the PDMAEMA segments. As presented in Table 1, the percentage of PEG chain conjugated to PDMAEMA is about 11.3% and 22.1% for D-PLLA-D@PEG-1 and D-PLLA-D@PEG-2, respectively, whereas these numbers were found to be 13.7% and 23.9% for respective D-PDLA-D@PEG-1and DPDLA-D@PEG-2 copolymers. These results are in good agreement with the quaternization ratio as evaluated from the residual intensities of –N–CH2– peak at 2.56ppm in PDMAEMA (Table 1). In addition, the ATR-FTIR spectroscopy was used to monitor and confirm the synthesis reactions of DPDLA-D@PEG and D-PLLA-D@PEG copolymers as detailed in the Supporting Information and Figure S3. NMR, GPC and ATR-FTIR results demonstrate the successful synthesis of D-PLLA-D@PEG and D-


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PDLA-D@PEG copolymers, and the conjugated PEG percentages in the copolymers can be tuned by the feeding ratios in the quaternization reaction.

3.2. Formation of Stereocomplex from Mixture of D-PDLA-D@PEG and D-PLLA-D@PEG Copolymers in Aqueous Solution Wide angle X-ray scattering (WAXS) was used to indicate the formation of stereocomplex interactions between enantiomeric pairs of the synthesized D-PDLA-D@PEG and D-PLLA-D@PEG copolymers in aqueous solution based on the crystalline structures of the polymers and their enantiomeric mixtures. Figure 2 depicts the WAXS curves of PLLA-diBr macroinitiator, D-PLLA-D, D-PLLAD@PEG-1 and the equimolar mixtures of the corresponding L and D enantiomeric polymers. The asprepared PLLA-diBr macroinitiator exhibits prominent diffraction peaks at 2θ = 14.7°, 16.5°, 19.0°, and 22.3°, which is ascribed to the α-form homocrystallities PLLA (Figure 2(a)).50 These homocrystallities PLLA peaks become broader in D-PLLA-D and D-PLLA-D@PEG-1 copolymers, albeit with other peaks attenuated (Figures 2(b) and (c)), which is not unexpected due to a lower crystallinity of the copolymers in the presence of PDMAEMA and low molecular weight PEG. On the other hand, the WAXS spectra of all the equimolar mixed samples (Figures 2(d), (e) and (f)) demonstrate distinct changes compared to the corresponding individual samples (Figures 2(a), (b) and (c)). Figures 2(d), (e) and (f) depict that new diffraction peaks appear at 2θ approximately 11.8°, 20.6°, and 23.8° for all the equimolar mixed samples, which could suggest the formation of stereocomplex interactions between PLLA and PDLA as described in previous studies.30-31, 41, 51-52 3.3. Self-assembly of D-PDLA-D@PEG and D-PLLA-D@PEG Copolymers in Aqueous Solution Micelles with PLA segments as the core and PDMAEMA and PEG brushes as the corona are expected to form in aqueous solution. NMR spectroscopy was used to investigate the solvent effect on the micelle structure as shown in Figure S4(a) in the Supporting Information.48, 53 d-DMF is a good nonselective solvent for PLA, PDMAEMA, and PEG, while D2O is a good selective solvent for PEG and PDMAEMA but poor for PLA. In d-DMF, the peaks due to PLA, PDMAEMA and PEG segments are 13 ACS Paragon Plus Environment

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sharp and well-defined unlike in D2O, the peak assigned to PEG is still sharp while the PLLA peaks disappear, and the peaks ascribed to PDMAEMA become broad (Figure S4(b)).These changes in the sharpness of the peaks demonstrate that the molecular motion of PLLA is much slow in water, indicating the formation of an aggregated hydrophobic core structure made up of PLLA with the hydrophilic PEG and PDMAEMA as the outer corona, thus confirming a core–shell type of micelle formation.48, 53 Dynamic light scattering (DLS) was used to determine the critical micelle concentration (CMC) of the individual D and L copolymer solutions (Samples 1-6) at neutral pH (~7) as shown in the Supporting Information (Figure S5) and the values are tabulated in Table 2. As anticipated, Samples 1 and 2 possess low CMC values (~ 0.02mg/mL). When a fraction of the PDMAEMA segment in the copolymer is quaternized and grafted with PEG (~13%), the CMC values increased as depicted in Samples 3 and 4, as compared to samples without PEG. In addition, the CMC values increased further with increasing quaternization and PEG content (~25%) in the copolymers (see Samples 5 and 6) suggesting that the higher amount of quaternization on the PDMAEMA segment and higher PEG content disfavours micellization due to a decreased in hydrophobicity of the copolymers. When comparing the effect of stereocomplex formation on the CMC of the copolymer solutions, samples having 1:1 D/L mixture (Samples 1+2, 3+4 and 5+6) posses approximately five times lower CMC values compared to their individual counterparts as revealed in Table 2. The lower CMC values in the mixture solutions is most probably due to stronger interactions between the PLLA and PDLA segments to form stereocomplex aggregates which favours micellization promoted by an increased in hydrophobicity. The hydrodynamic radius, Rh of the micelles in aqueous solution formed by the individual D and L copolymer and 1:1 D/L mixture solutions at different pHs were measured using DLS for copolymer concentrations ranging from 0.5 to 1.0 mg/mL, well above the CMC, to ensure micelle formation. Figures 3(a) and (b) demonstrate the particle size distribution of the micelles formed from Sample 3 and Sample 3+4 respectively for pH ranging from 2 to 10. In the individual solution (Figure 3(a)), the distribution is unimodal at high pH and becomes bimodal when the pH decreases beyond ~ 5. At high pH (> 6) having unimodal particle size distribution, the peaks correspond to aggregates comprising of a PLA core surrounded by either PDMAEMA only for Samples 1 and 2 or PDMAEMA and PEG corona for 14 ACS Paragon Plus Environment

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Samples 3-6 respectively. The size of the micelles increases when the pH of the samples is reduced up to pH 3, beyond which the size remains a plateau suggesting that the protonation of the PDMAEMA segments caused the osmotic pressure within the micelle to increase until the micelles reached a maximum size. No further change in the micelle size was observed beyond pH 7 as the osmotic pressure within the PDMAEMA segments reached its minimum. In addition to the increase in particle size when pH is reduced, an additional peak appears in the fast mode region (Rh~2.3 ± 0.2 nm) which probably corresponds to the free individual block copolymers. The appearance of the fast mode peaks will be further verified by static light scattering measurements in the later section. In contrast, the 1:1 D/L mixture samples demonstrate unimodal particle size distribution throughout the whole pH range investigated as demonstrated in Figure 3(b) for Sample 3+4 where the Rh at peak maximum of the micelles increases continuously from ~85 ± 4 nm to ~140 ± 8 nm when the pH is reduced from ~8 to ~3 and then slowly levels off to reach an apparent plateau. In addition, the dependence of decay rate Γ (the 2 reciprocal of relaxation time, τ) on q2 (according to Γ = Dapp q ), shown in Figure 3(c), exhibits a

linearity confirming that the observed peaks in Figures 3(a) and (b) originate from the translational diffusion of the copolymer micelles.37 A summary of the change in hydrodynamic radius, Rh of the individual D and L copolymer and 1:1 D/L mixture solutions at different pHs is depicted in Figures 4(a) and (b). In both figures, the increase in Rh with reduction in pH could be ascribed to the swelling of the aggregates due to enhanced osmotic pressure exerted by the counter ions trapped inside the polymeric network by the electrostatic attraction exerted from the protonated PDMAEMA. It is mainly the “hydrophobic interaction” among the hydrophobic PLA and the undissociated PDMAEMA constituents (more dominant at higher pH) which offers the attractive interaction and responsible for the aggregate/micelles formation in aqueous solution. When pH is reduced below 8, the aggregates swell until an equilibrium size is reached at approximately pH 4 and the size remained stable with a further reduction in pH, due to the balance between the hydrophobic attractive interactions of the PLA and undissociated PDMAEMA and the electrostatic repulsion between the dissociated positively charged PDMAEMA segments. The data given in Figure 15 ACS Paragon Plus Environment

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4(a) reveals that at a given pH, the Rh of Samples 3 and 4 are similar due to the similar chemical compositions of the two enantiomeric copolymers. When PLA stereocomplexation is introduced (Sample 3+4), the Rh increased significantly from that of the individual D or L forms suggesting stronger interactions in the mixture which is most probably due to interactions between the PLLA and PDLA blocks to form stereocomplex aggregates since this interaction is absent in the individual samples (Sample 3 and 4). Similar larger increment in Rh is also observed in Sample 1+2 and Sample 5+6 which have 1:1 D/L mixtures when compared to Sample 1 and Sample 5 respectively as depicted in Figure 4(b). Further, Figure 4(c) demonstrate that the apparent extent of swelling with reduction in pH (Rh (pH)/Rh (pH=8)),

is also higher in aggregates formed by 1:1 D/L mixtures as compared to individual D or L forms.

For instance, the Rh of Sample 3 increases continuously from ~73 ± 4 nm to ~110 ± 6 nm, corresponding to a swelling ratio of 1.5 when the pH is reduced from ~8 to ~3 as compared to a larger increment of ~85 to 140 nm, corresponding to a swelling ratio of 1.65 in Sample 3+4 for the same pH range. This interesting observation may indicate that the stronger stereocomplex interaction in the mixture enhances the osmotic swelling in the aggregates as the PDMAEMA is being protonated when pH is reduced. In addition, the swelling ratio of both Sample 5 and Sample 5+6 are lower when compared with Sample 3 and Sample 3+4 for the same pH range suggesting that manipulation of the quarternization and PEG content in the copolymers could affect the extent of swelling of the aggregates when pH is reduced. The stability of the micelles at high and low pH, formed from the individual D or L forms of the copolymers and 1:1 D/L mixtures towards dilution was also investigated by measuring the size of the micelles in Sample 3 and 3+4, prepared at 1.0 mg/mL and diluted to four different concentrations. Figure 4(d) shows that the hydrodynamic radius, Rh and apparent radius of gyration, Rg, app of the micelles are almost constant when Sample 3+4 at pH 4 and 8, are diluted by a dilution factor as high as 5 (from 1.0 to 0.2 mg/mL) which further prove that the aggregates are very stable with dilution. Note that the pH of the solutions was maintained at a constant pH throughout the dilutions. Bouteiller and coworkers examined the stability of the stereocomplex aggregates formed by enantiomeric PLA block copolymers in THF using small-angle neutron scattering (SANS) and SLS and reported similar findings to ours where the aggregates remained stable over months and are not sensitive to dilution.54-56 However, when similar 16 ACS Paragon Plus Environment

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stability experiments were performed on Sample 3, the Rh of the micelles decreases by approximately 20% , i.e., from approximately 76 to 62 nm and 114 to 95 nm, at pH 8 and 4 respectively, when diluted by a dilution factor of 5 as depicted in Figure 4(d). Likewise, the Rg, app of micelles formed from Sample 3 also decreases significantly (approximately 25%) when diluted at both pH 8 and 4, further indicating that the stability of the micelle core is susceptible to dilution. This finding clearly suggests that unlike the micelles having stereocomplex PLA as the core (e.g., Sample 3+4), the hydrophobically driven micelles formed from the individual D or L forms of the copolymers (e.g., Sample 3) are dynamic and reversible in nature and kinetically unstable (not frozen). Static light scattering (SLS) experiments was further used to elucidate the effect of stereocomplexation and pH on the aggregation behaviour of the PEG conjugated PLA copolymers in aqueous solution. The refractive index increment of the samples, (dn/dc) as a function of pH is shown in Figure S6 in the Supporting Information for Sample 3 and 3+4. In both samples, the dn/dc values decreased when pH is reduced due to the presence of more positive protonated PDMAEMA segments which caused the micelles to have lower dn/dc values as compared to micelles in the higher pH region (> pH 6). For instance, the dn/dc values of Sample 3+4 at pH 8, 5 and 2.5 are 0.150 mL/g, 0.130 mL/g and 0.126 mL/g respectively. The molecular weight of the micelle (Mw,micelle), together with the radius of gyration (Rg) were determined by a Zimm plot in SLS as a function of pH. Note that the Mw,agg values in aqueous solutions are much larger than the Mw,single values of the individual D-PLA-D and D-PLAD@PEG copolymers obtained by GPC, further confirming the formation of micelles in aqueous solution. Subsequently the apparent aggregation number, Nagg (Nagg = Mw,micelle/Mw,single) of the individual D or L forms and 1:1 D/L mixtures of micelles in aqueous solution were calculated as a function of pH. Figure 5(a) clearly demonstrates that the individual D or L forms of the micelles (Sample 1, 3 and 5) exhibit a large Nagg in the high pH regime which gradually decreases when pH is reduced suggesting that in addition to particle swelling, there is substantial expulsion of polymer chains from the micelle during protonation of the PDMAEMA segments, thus resulting in formation of micelles with lower Nagg. When pH is gradually reduced, the electrostatic repulsion between the dissociated positively charged PDMAEMA segments overcomes the “hydrophobic interaction” among the PLLA or PDLA which is 17 ACS Paragon Plus Environment

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responsible for the attractive interaction among the polymeric chains in the micelle. In contrast, the Nagg of the micelles formed from the 1:1 D/L mixtures remained almost unchanged when pH is reduced suggesting that the presence of hydrophobic stereocomplex interaction in the micelle is sufficiently strong to yield a stable swollen particle even when the micelle is fully protonated at low pH. The substantial expulsion of polymer chains from the individual D or L forms of micelle during protonation of the PDMAEMA segments can be further explained by the presence of an additional peak in the fast mode region (Rh~2.3 ± 0.2 nm) when pH gradually decreases which corresponds to the expelled copolymer chains, as observed in Figure 3(a). However, the additional peak in the fast mode region is not observed for the micelles formed from the 1:1 D/L mixture samples throughout the whole pH range (Figure 3(b)) indicating that no significant expulsion of copolymer chains occurred when stereocomplex interaction is present in the micelle. The absolute Nagg is in the order of Sample 1+2 > Sample 3+4 > Sample 5+6 suggesting that increasing the quaternization of the PDMAEMA segments and PEG content in the PDMAEMA-PLA copolymers reduces the hydrophobic interactions (both PLLA/PDLA and stereocomplex interactions) between the L and D form of the copolymers thus limiting the number of copolymer chains in the micelles. Figure 5(a) also clearly explains the smaller micelle size (Rh) and extent of swelling (swelling ratio) in Sample 5+6 when compared to Sample 3+4 as observed in Figure 4(b) and 4(c) respectively since there are less copolymer chains in the micelle formed from Sample 5+6, to begin with. In addition to the Mw,micelle and Nagg, the dimensionless ratio Rg/Rh which is indicative of the micelle structure,57-58 ranges from 0.43 to 0.76, depending on the pH, as depicted in Figure 5(b). The values of Rg/Rh for hard-sphere micelle, random coil, and rod-like structure are reported as 0.78, 1.78, and ≥2, respectively.57-58 The deviation of Rg/Rh of our PEG conjugated PDMAEMA-b-PLA-bPDMAEMA copolymers from the hard sphere value suggests that the micelles are spherical in shape and possess a thick hydration layer (core-shell micelle structure). When comparing the micelles formed from the 1:1 D/L mixtures and the individual D or L forms of the copolymers, the strong stereocomplex interaction in the micelles formed from the mixtures of copolymers provides stronger driving force for self-assembly and larger micelles cores were formed in aqueous solution as manifested by the larger 18 ACS Paragon Plus Environment

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Rg/Rh values as compared to micelles formed from individual copolymers. Further, the micelles formed from both methods demonstrate a decreasing trend in Rg/Rh when pH is decreased mainly due to the phase transition of the PDMAEMA segments from the neutral state to the protonated state at high and low pH respectively. During this transition, PDMAEMA segments transform from a collapsed state near the micelle core to an expanded state away from the core resulting in a decrease in micelle core size and overall Rg/Rh values while maintaining the spherical shape of the micelles as indicated by the values of Rg/Rh< 0.78. However, Figure 5(b) demonstrates that the decrease in Rg/Rh values is smaller in micelles formed from the 1:1 D/L mixtures of copolymers compared to the individual D or L forms due to the higher stability of the former as reflected in the Nagg values (Figure 5(a)). For example the Rg/Rh ratio decreases from 0.74 to 0.60 in Sample 3+4 when pH is reduced from 8 to 3 as compared to the decrease from 0.71 to 0.48 in Sample 3 for the same pH range. In the unstable individual D or L forms of micelles, there occurs significant expulsion of copolymer chains in addition to particle swelling when the PDMAEMA segments are being protonated. As a consequence, the micelle core size and overall Rg/Rh values decreases much faster compared to the stereocomplex micelles for the same pH range. To summarize, the constant Nagg of micelles formed from the 1:1 D/L mixtures (Figure 5(a)) implies that stable micelles are formed in aqueous solution when stereocomplex interaction is present in the micelles and that the change in Rg/Rh ratios as a function of pH (Figure 5(b)) is purely due to the PDMAEMA segments adopting a more compact and extended conformation at high and low pH respectively. To further support the pH responsiveness of the PDMAEMA segments in the micelles, zeta potential experiments were conducted from pH 2 to 9 for both the micelles formed from the individual D or L forms of the copolymers and 1:1 D/L mixtures to examine the overall charge distribution on the surface of the micelle. As described in the Supporting Information and Figure S7, the zeta potential of the micelles increases when the pH of the samples is reduced up to pH 3, beyond which the zeta potential remains a plateau confirming that the PDMAEMA segments are protonated upon the addition of acid and maximum protonation is achieved at approximately pH 3. Thus, the zeta potential results provide another evidence of the pH responsiveness of the PEG conjugated PLA micelles.


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On the basis of the light scattering and WAXS results, the conformation of the pH responsive micelles formed by the individual PDLA or PLLA copolymers and 1:1 D/L mixture solutions in aqueous solution can be schematically illustrated as shown in Figure 6. We hypothesize that the individual DPDLA-D@PEG and D-PLLA-D@PEG copolymers self-assemble via weak solvophobic interactions to form smaller micelles unlike the stronger stereocomplex interaction between the PDLA (black solid lines) and PLLA (black dotted lines) blocks in the mixture solutions which forms larger micelles. In both scenarios, the PLA chains shrinks away from the solvent environment resulting in the formation of micelles with either individual PLA enantiomers or stereocomplex PLA as the core and PDMAEMA (pink solid lines) and conjugated PEG (green solid lines) as corona. When the solution pH is reduced, both the individual D or L forms of the micelles (Figure 6B) and stereocomplex micelles (Figure 6C) swell due to enhanced osmotic pressure exerted by the counter ions trapped inside the polymeric network by the electrostatic attraction exerted from the protonated PDMAEMA. In addition to particle swelling, the individual D or L forms of the micelles exhibit substantial expulsion of polymer chains from the micelle during protonation of the PDMAEMA segments due to the increasing electrostatic repulsion between the positively charged PDMAEMA segments which overcomes the “weak solvophobic interaction” among the PLLA or PDLA, thus resulting in formation of micelles with lower Nagg as demonstrated in Figure 6B. In contrast, no significant change in the aggregation number is observed for the micelles formed from the 1:1 D/L mixtures when pH is reduced (Figure 6C) suggesting that the presence of stereocomplex interaction in the micelle is sufficiently strong to yield a stable swollen particle even when the micelle is fully protonated at low pH.

4. Conclusion pH-responsive

PDMAEMA-b-PDLA-b-PDMAEMA@PEG

and

PDMAEMA-b-PLLA-b-

PDMAEMA@PEG copolymers with well-defined structure were successfully prepared using relatively simple and well-established polymerization methods and the micelle self-assemblies in aqueous solution was studied using DLS and SLS. The presence of conjugated PEG on the copolymers facilitate the individual D or L forms of the copolymers and its corresponding enantiomeric mixtures to be dissolved 20 ACS Paragon Plus Environment

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directly in aqueous solution without using any organic solvents. In dilute aqueous solutions, DLS demonstrate that the size of the micelles increases when the pH of the samples is reduced due to the protonation of the PDMAEMA segments which caused the osmotic pressure within the micelle to increase until the micelles reached a maximum size. Stereocomplex interactions formed between enantiomeric PLA segments of similar molecular weights in aqueous solution were confirmed with WAXS, while DLS and SLS studies revealed that the stereocomplex micelles have larger swelling ratios and enhanced stability against pH and dilution as compared to micelles formed from individual D or L forms of the copolymers. When compared to numerous stable micellar systems in aqueous solution, which involved the use of organic solvent and tedious post-processing, the water soluble PEG conjugated PLA based polyelectrolytes developed in this paper could give rise to pH-responsive micelles with enhanced kinetic stability simply by direct dissolution of equimolar mixture of the enantiomeric PLA copolymers. The copolymers could be further exploited in anti-adhesive and anti-bacterial applications. 5. Acknowledgements The authors gratefully acknowledge the financial support and technical assistance from the Institute of Materials Research and Engineering (IMRE) under the Agency of Science, Technology and Research (A*STAR).

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7. Betthausen, E.; Drechsler, M.; Fortsch, M.; Schacher, F. H.; Muller, A. H. E. Dual stimuliresponsive multicompartment micelles from triblock terpolymers with tunable hydrophilicity. Soft Matter 2011, 7 (19), 8880-8891. 8. Plamper, F. A.; Ruppel, M.; Schmalz, A.; Borisov, O.; Ballauff, M.; Müller, A. H. E. Tuning the Thermoresponsive Properties of Weak Polyelectrolytes: Aqueous Solutions of Star-Shaped and Linear Poly(N,N-dimethylaminoethyl Methacrylate). Macromolecules 2007, 40 (23), 8361-8366. 9. Plamper, F. A.; Schmalz, A.; Penott-Chang, E.; Drechsler, M.; Jusufi, A.; Ballauff, M.; Müller, A. H. E. Synthesis and Characterization of Star-Shaped Poly(N,N-dimethylaminoethyl methacrylate) and Its Quaternized Ammonium Salts. Macromolecules 2007, 40 (16), 5689-5697. 10. Plamper, F. A.; Ruppel, M.; Schmalz, A.; Borisov, O.; Ballauff, M.; Müller, A. H. E. Tuning the Thermoresponsive Properties of Weak Polyelectrolytes: Aqueous Solutions of Star-Shaped and Linear Poly(N,N-Dimethylaminoethyl Methacrylate). Macromolecules 2007, 40 (23), 8361-8366. 11. van Steenis, J. H.; van Maarseveen, E. M.; Verbaan, F. J.; Verrijk, R.; Crommelin, D. J. A.; Storm, G.; Hennink, W. E. Preparation and characterization of folate-targeted pEG-coated pDMAEMAbased polyplexes. Journal of Controlled Release 2003, 87 (1–3), 167-176. 12. Huang, J.; Murata, H.; Koepsel, R. R.; Russell, A. J.; Matyjaszewski, K. Antibacterial polypropylene via surface-initiated atom transfer radical polymerization. Biomacromolecules 2007, 8 (5), 1396-1399. 13. Yancheva, E.; Paneva, D.; Maximova, V.; Mespouille, L.; Dubois, P.; Manolova, N.; Rashkov, I. Polyelectrolyte complexes between (cross-linked) N-carboxyethylchitosan and (quaternized) poly 2(dimethylamino)ethyl methacrylate : Preparation, characterization, and antibacterial properties. Biomacromolecules 2007, 8 (3), 976-984. 14. Karanikolopoulos, N.; Zamurovic, M.; Pitsikalis, M.; Hadjichristidis, N. Poly(DL-lactide)-bpoly(N,N-dimethylamino-2-ethyl methacrylate): Synthesis, Characterization, Micellization Behavior in Aqueous Solutions, and Encapsulation of the Hydrophobic Drug Dipyridamole. Biomacromolecules 2010, 11 (2), 430-438. 15. Mao, J.; Ji, X.; Bo, S. Synthesis and pH/Temperature-Responsive Behavior of PLLA-bPDMAEMA Block Polyelectrolytes Prepared via ROP and ATRP. Macromolecular Chemistry and Physics 2011, 212 (7), 744-752. 16. Mei, A.; Guo, X.; Ding, Y.; Zhang, X.; Xu, J.; Fan, Z.; Du, B. PNIPAm-PEO-PPO-PEOPNIPAm Pentablock Terpolymer: Synthesis and Chain Behavior in Aqueous Solution. Macromolecules 2010, 43 (17), 7312-7320. 17. Kryuchkov, M. A.; Detrembleur, C.; Jerome, R.; Prud'homme, R. E.; Bazuin, C. G. Synthesis and Thermal Properties of Linear Amphiphilic Diblock Copolymers of L-Lactide and 2-Dimethylaminoethyl Methacrylate. Macromolecules 2011, 44 (13), 5209-5217. 18. Munier, S.; Messai, I.; Delair, T.; Verrier, B.; Ataman-Onal, Y. Cationic PLA nanoparticles for DNA delivery: Comparison of three surface polycations for DNA binding, protection and transfection properties. Colloids and Surfaces B-Biointerfaces 2005, 43 (3-4), 163-173. 19. Iván, B.; Feldthusen, J.; Müller, A. H. E. Synthesis strategies and properties of smart amphiphilic networks. Macromol. Symp. 1996, 102 (1), 81-90. 20. Savic, R.; Luo, L. B.; Eisenberg, A.; Maysinger, D. Micellar nanocontainers distribute to defined cytoplasmic organelles. Science 2003, 300 (5619), 615-618. 21. Shuai, X. T.; Merdan, T.; Unger, F.; Wittmar, M.; Kissel, T. Novel biodegradable ternary copolymers hy-PEI-g-PCL-b-PEG: Synthesis, characterization, and potential as efficient nonviral gene delivery vectors. Macromolecules 2003, 36 (15), 5751-5759. 22. Torchilin, V. P.; Lukyanov, A. N.; Gao, Z. G.; Papahadjopoulos-Sternberg, B. Immunomicelles: Targeted pharmaceutical carriers for poorly soluble drugs. Proceedings of the National Academy of Sciences of the United States of America 2003, 100 (10), 6039-6044. 23. Burt, H. M.; Zhang, X. C.; Toleikis, P.; Embree, L.; Hunter, W. L. Development of copolymers of poly(D,L-lactide) and methoxypolyethylene glycol as micellar carriers of paclitaxel. Colloids and Surfaces B-Biointerfaces 1999, 16 (1-4), 161-171.


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Controlled Synthesis, Characterization, and Stereocomplex Formation. Biomacromolecules 2009, 10 (5), 1217-1223. 45. Meyer, F.; Minoia, A.; Raquez, J. M.; Spasova, M.; Lazzaroni, R.; Dubois, P. Poly(aminomethacrylate) as versatile agent for carbon nanotube dispersion: an experimental, theoretical and application study. J. Mater. Chem. 2010, 20 (33), 6873-6880. 46. Roy, D.; Knapp, J. S.; Guthrie, J. T.; Perrier, S. Antibacterial Cellulose Fiber via RAFT Surface Graft Polymerization. Biomacromolecules 2007, 9 (1), 91-99. 47. Han, J.; Zhu, D.; Gao, C. Fast bulk click polymerization approach to linear and hyperbranched alternating multiblock copolymers. Polym. Chem. 2013, 4 (3), 542-549. 48. Li, Z.; Zhang, Z.; Liu, K. L.; Ni, X.; Li, J. Biodegradable Hyperbranched Amphiphilic Polyurethane Multiblock Copolymers Consisting of Poly(propylene glycol), Poly(ethylene glycol), and Polycaprolactone as in Situ Thermogels. Biomacromolecules 2012, 13 (12), 3977-3989. 49. Li, Z.; Li, J. Control of Hyperbranched Structure of Polycaprolactone/Poly(ethylene glycol) Polyurethane Block Copolymers by Glycerol and Their Hydrogels for Potential Cell Delivery. The Journal of Physical Chemistry B 2013, 117 (47), 14763-14774. 50. Sun, Y.; He, C. Synthesis and Stereocomplex Crystallization of Poly(lactide)–Graphene Oxide Nanocomposites. ACS Macro Letters 2012, 1 (6), 709-713. 51. Sun, Y.; He, C. Synthesis, stereocomplex crystallization, morphology and mechanical property of poly(lactide)-carbon nanotube nanocomposites. RSC Advances 2013, 3 (7), 2219-2226. 52. Sun, Y.; He, C. Biodegradable “Core–Shell” Rubber Nanoparticles and Their Toughening of Poly(lactides). Macromolecules 2013, 46 (24), 9625-9633. 53. Loh, X. J.; Ong, S. J.; Tung, Y. T.; Choo, H. T. Dual responsive micelles based on poly[(R)-3hydroxybutyrate] and poly(2-(di-methylamino)ethyl methacrylate) for effective doxorubicin delivery. Polym. Chem. 2013, 4 (8), 2564-2574. 54. Portinha, D.; Belleney, J.; Bouteiller, L.; Pensec, S.; Spassky, N.; Chassenieux, C. Formation of Nanoparticles of Polylactide-Containing Diblock Copolymers: Is Stereocomplexation the Driving Force? Macromolecules 2002, 35 (5), 1484-1486. 55. Portinha, D.; Boué, F.; Bouteiller, L.; Carrot, G.; Chassenieux, C.; Pensec, S.; Reiter, G. Stable Dispersions of Highly Anisotropic Nanoparticles Formed by Cocrystallization of Enantiomeric Diblock Copolymers. Macromolecules 2007, 40 (11), 4037-4042. 56. Portinha, D.; Bouteiller, L.; Pensec, S.; Richez, A.; Chassenieux, C. Influence of preparation conditions on the self-assembly by stereocomplexation of polylactide containing diblock copolymers. Macromolecules 2004, 37 (9), 3401-3406. 57. Peiqiang, W.; Siddiq, M.; Huiying, C.; Di, Q.; Wu, C. Laser Light-Scattering Study of Poly(sulfoalkyl methacrylate)s in 0.1 M NaCl Aqueous Solution. Macromolecules 1996, 29 (1), 277-281. 58. Burchard, W. Combined static and dynamic light scattering [Online]; Clarendon Press: Oxford, 1996; pp. 439-476.


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Scheme and Figures

Scheme 1. Synthesis route of PEG conjugated PDMAEMA-b-PLLA-b-PDMAEMA@PEG (D-PLLAD@PEG) and D-PDLA-D@PEG copolymers.


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Figure 1. 1H NMR spectra of (A) PLLA-diBr and (B) D-PLLA-D in CDCl3, and (C) D-PLLA-D@PEG2 in d-DMF. The peak assignments in Figure 1(B) are also applicable to Figure 1(C).


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Figure 2. XRD spectra of (a) PLLA-diBr, (b) D-PLLA-D, (c) D-PLLA-D@PEG-1, (d) SC-PLLAdiBr/PDLA-diBr, (e) SC-D-PLLA-D/D-PDLA-D and (f) SC-D-PLLA-D@PEG-1/D-PDLA-D@PEG-1.

14

14

pH 9.5

12

pH 8.4

10

pH 7.2

(b)

pH 6.4 8

pH 5.6 6

pH 4.8

4

pH 3.8

pH 8.4 pH 7.5

10

pH 6.8 8

pH 6.1 6

pH 5.0 pH 3.7

4

pH 2.6

pH 2.9

2

pH 9.8

12

Normalized Intensity

(a) Normalized Intensity

2

pH 1.9

pH 2.0

0

0

1 1

10

100

10

1000

100

1000

Hydrodynamic Radius, Rh(nm)

Hydrodynamic Radius, Rh(nm)

3.5

(c)

3.0

Γ x 10-3 (m2 / s)

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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y = 0.5445x R² = 0.9976

2.5

Sample 3 2.0 1.5

Sample 3+4

1.0

y = 0.4667x R² = 0.9938

0.5 0.0 0

1

2

3

4

q2

x 10-14

(m-2)

5

6

Figure 3. Distribution of hydrodynamic radius, Rh of micelles in (a) Sample 3 and (b) Sample 3+4 for pH ranging from 2 to 10. (c) Dependence of decay rate Γ on q2 for Sample 3 (open symbols) and Sample 3+4 (closed symbols), both at pH 8.4. The solid lines are included to guide the eye. All samples were prepared at polymer concentration of 0.5 mg/mL and Rh is calculated from Eq. (1). 27 ACS Paragon Plus Environment

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(a)

140

120

100

80

60

40 0

2

4

6

8

10

12

pH 1.7

130

(c)

1.6

Rh (pH) / Rh (pH=8)

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

Hydrodynamic radius, Rh (nm)

Langmuir

1.5

110

1.4

Rh and 90 Rg, app (nm) 70

1.3 1.2 1.1

50 1.0

(d) 0.9

30 0

2

4

6

8

10

0

pH

0.2

0.4

0.6

0.8

1

1.2

Concentration (mg/mL)

Figure 4. Hydrodynamic radius, Rh of micelles formed by individual D or L forms of copolymers and 1:1 D/L mixtures as a function of pH for (a) samples having similar conjugated PEG percentages in the copolymers and (b) samples having varying conjugated PEG percentages in the copolymers. (c) Swelling ratio (Rh (pH)/Rh (pH=8)) of micelles as a function of pH for samples having varying conjugated PEG percentages in the copolymers. Sample 1 (white triangle), Sample 3 (white circle), Sample 4 (grey circle), Sample 5 (white square), Sample 1+2 (black triangle), Sample 3+4 (black circle) and Sample 5+6 (black square). All samples were prepared at polymer concentration of 0.5 mg/mL. (d) Rh (black symbols) and Rg,app (white symbols) of aggregates as a function of polymer concentration in Sample 3 at pH 4 (square) and pH 8 (diamond) and Sample 3+4 at pH 4 (circle) and pH 8 (triangle) which were prepared at polymer concentration of 1.0 mg/mL and diluted to four different concentrations while maintaining the respective pH of the solution. For all the figures, the solid lines are included to guide the eye. Measurements were performed at scattering angle of 90° and Rh is calculated from Eq. (1).


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0.8

200

(b)

(a) 180

0.7 160

Rg / Rh

Aggregation Number, Nagg

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140

0.6

120

0.5 100

0.4

80 0

2

4

6

8

10

0

12

2

4

6

8

10

12

pH

pH

Figure 5. (a) Aggregation number, Nagg and (b) dimensionless ratio Rg/Rh of micelles formed by individual D form of copolymers and 1:1 D/L mixtures as a function of pH determined from static light scattering (SLS) within the concentration range of 0.5 to 1.0 mg/mL. Sample 1 (white triangle), Sample 3 (white circle), Sample 5 (white square), Sample 1+2 (black triangle), Sample 3+4 (black circle) and Sample 5+6 (black square). The solid lines are included to guide the eye.

Figure 6. Schematic illustration of (A) enantiomeric D-PLLA-D@PEG and D-PDLA-D@PEG copolymers and (B) the proposed self-assembly mechanism of pH responsive D-PLLA-D@PEG copolymer in aqueous solution. The increased electrostatic repulsion at lower pH between charged PDMAEMA overcomes the hydrophobic interaction between PLLA, resulting in formation of micelles with lower aggregation number. (C) the proposed self-assembly mechanism of D-PLLA-D@PEG and DPDLA-D@PEG mixture at molar ratio of 1:1 in aqueous solution. Formation of stereocomplex between PLLA and PDLA in the micelle core enhances the micelle stability even at low pH, thus no significant change in the aggregation number is observed. 29 ACS Paragon Plus Environment

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Tables Table 1.Molecular characteristics of D-PDLA-D@PEG and D-PLLA-D@PEG copolymers and their prepolymers

Samples

a

Block Length (Da) b

c

PDMAEMA@Q d (%)

PEG Conjugation e (%)

-

-

-

2.94

1.1

g PEG Branch [Mn]/kDa f No.

PDI

PDLA-diBr

PLA 3140

PDMAEMA -

D-PDLA-D D-PDLA-D@PEG-1

3140 3140

8730 7360

15.4

13.7

10.9

10.52 16.56

1.1 1.4

D-PDLA-D@PEG-2

3140

6650

25.8

23.9

19.1

21.03

1.5

PLLA-diBr

3330

-

-

-

-

3.01

1.2

D-PLLA-D

3330

8470

-

-

-

11.41

1.1

D-PLLA-D@PEG-1

3330

7430

12.3

11.3

8.8

16.23

1.2

D-PLLA-D@PEG-2

3330

6260

26.2

22.1

17.2

20.86

1.4

a

g

PEG conjugated enantiomeric PDMAEMA-b-PDLA-b-PDMAEMA and PDMAEMA-b-PLLA-b-PDMAEMA triblock copolymers are denoted as D-PDLA-D@PEG and D-PLLA-D@PEG, where D represents PDMAEMA. b Estimated from 1H NMR spectroscopy based on intensity ratio of PLA methine proton (δ 5.16 ppm) and the methylene proton (δ 4.15 ppm) in the ROP initiator of ethylene glycol. c Calculated based on intensity ratio of PLA methine proton (δ 5.16 ppm) and PDMAEMA methylene proton (δ 2.56 ppm). d Quaternizatino ratio as obtained from the residual intensities of –N–CH2– peak at 2.56 ppm in PDMAEMA. e Evaluated from GPC, by calculating the molecular weight difference before and after PEG conjugation. f Calculated based on GPC results. g Determined by GPC.

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Langmuir

Table 2.DLS and SLS data of D-PDLA-D, D-PLLA-D, D-PDLA-D@PEG and D-PLLA-D@PEG micelles formed from direct dissolution in aqueous solution without any pH adjustment, i.e., pH of solution is approximately 8.

Designation

Samples

a

CMC (mg/mL)

Rha(micelles) (nm)

Rg/Rh

1

D-PDLA-D

0.020

2

D-PLLA-D

1+2

Mw, agg x 10-6 (g/mol)

59±3

0.75

2.0 ±0.1

154

0.025

58±4

-

-

-

D-PDLA-D + D-PLLA-D

0.005

68±4

0.76

2.5±0.1

191

3

D-PDLA-D@PEG-1

0.150

73±4

0.71

2.5±0.2

132

4

D-PLLA-D@PEG-1

0.120

73±3

-

-

-

3+4

D-PDLA-D@PEG-1 + DPLLA-D@PEG-1

0.030

85±4

0.73

3.1±0.2

161

5

D-PDLA-D@PEG-2

0.200

63 ±3

0.69

3.5±0.2

117

6

D-PLLA-D@PEG-2

0.200

65 ± 4

-

-

-

5+6

D-PDLA-D@PEG-2 + DPLLA-D@PEG-2

0.050

72±3

0.71

4.3±0.3

142

a

c b

c

Nagg

determined by DLS measurements at room temperature, bRg, Mw,agg and Nagg were determined by SLS measurements at room temperature. All samples for DLS and SLS analyses were prepared by direct dissolution in 10mM NaCl aqueous solution in the concentration range of 0.5 to 1.0 mg/mL except for Sample 1, Sample 2 and Sample 1+2 which were first prepared in DMF (organic solvent) followed by gradual drop into 10mM NaCl aqueous solution and finally evaporation of the organic solvent.

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For Table of Contents Use Only

Poly(ethylene glycol) conjugated Poly(lactide) based Polyelectrolytes: Synthesis and Formation of Stable Self-Assemblies Induced by Stereocomplexation

Zibiao Li,b Du Yuan,a Xiaoshan Fan,a Beng H. Tan,b,* Chaobin He a,b,*

a

Department of Materials Science and Engineering, National University of Singapore, Singapore 117574, Singapore

b

Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 3 Research Link, Singapore 117602, Singapore

* correspondence to [email protected]; [email protected]

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