Crystallization-Driven Formation of Diversified Assemblies for

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Crystallization-Driven Formation of Diversified Assemblies for Supramolecular Poly(lactic acid)s in Solution Xiaohua Chang, Jianna Bao, Guorong Shan, Yongzhong Bao, and Pengju Pan Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b00013 • Publication Date (Web): 28 Mar 2017 Downloaded from http://pubs.acs.org on March 30, 2017

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Crystal Growth & Design

Crystallization-Driven Formation of Diversified Assemblies for Supramolecular Poly(lactic acid)s in Solution

Xiaohua Chang, Jianna Bao, Guorong Shan, Yongzhong Bao, Pengju Pan*

State Key Laboratory of Chemical Engineering, College of Chemical and Biological Engineering, Zhejiang University, 38 Zheda Road, Hangzhou 310027, China

*Corresponding author. Tel.: +86-571-87951334; email: [email protected]

ABSTRACT: Precipitation (or solvent displacement) method has been a simple yet efficient way to prepare the micro- and nanoparticles of polymers. However, control over the particle morphology of semicrystalline polymer is extremely challenging in the precipitation method, due to the interplay of polymer crystallization with liquidliquid phase separation in solution. This limits the preparation of polymer particles with well-controlled morphology. Herein we report on the preparation of flowershaped and spherical biodegradable polymer particles by precipitating the ureido4[1H]-pyrimidione (UPy)-functionalized supramolecular poly(lactic acid) (PLA) from a good solvent to an anti-solvent. Morphology of PLA particles was successfully manipulated by the solution crystallization, molecular weight, and intermolecular interactions of polymer precursors. Homocrystallization of supramolecular poly(Llactic acid) yielded the follower-shaped particles in precipitation; yet stereocomplex

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crystallization of PLA supramolecular copolymers led to the formation of sphere particles. The underlying mechanism for crystallization-driven formation of various particles was proposed. The prepared sphere particles can be used as the carriers of hydrophobic drug. Degradation and drug release rates of the prepared PLA particles decreased with increasing the content of stereocomplexes. Our study paves a way to the biodegradable PLA particles with controllable morphologies that may find potential applications in biomedical field.

INTRODUCTION Micro- and nanoparticles of biodegradable polymers have shown great potentials for use as drug-delivery vehicles.1−3 They have been widely used as controlled drug release and delivery vehicles for therapeutic substances such as drugs, proteins, genes, and vaccines. Many methods including emulsion, salting out, precipitation, and spray-drying have been employed to make biodegradable polymeric micro- and nanoparticles.1,2 However, most of these processes have some common limitations such as the use of toxic additives (e.g., surfactant) that were difficult to remove from the final particles, the thermal degradation of processed materials due to high temperature employed, and the difficult control over particle size and distribution. Among these methods, the precipitation method, also known as solvent displacement method, is a relatively easy technique to prepare the biodegradable micro- and nanoparticles. The precipitation method can be realized in different ways such as (i) the addition of an anti-solvent into polymer solution, (ii) the addition of polymer solution into an anti-solvent, and (iii) the dilution of polymer solution by an antisolvent. The precipitated particles can be attained by filtration, centrifugation, or removing the solvent from the suspension under reduced pressure. If a semicrystalline polymer is treated by the surfactant-free precipitation method,


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polymer will crystallize during precipitating from the solution. The crystallization occurred in this process was rather complicated, which generally interplayed with the liquid-liquid phase separation.4 When the good solvent of semicrystalline polymer is gradually displaced by an anti-solvent in solution, three scenarios can occur: (i) the solution phase-separates and forms polymer-rich droplets; (ii) the polymer crystallizes within the droplets to form assemblies (or particles); (iii) the crystallized assemblies precipitate from solution.4,5 To that end, the precipitation method could attain the polymer particles or assemblies with diversified shapes and sizes if the liquid-liquid phase separation, crystal nucleation (e.g., homogeneous or heterogeneous nucleation) and growth rates were well controlled.4,6,7 Polyethylene (PE) particles with various morphologies have been successfully obtained via controlling the liquid-liquid phase separation and crystallization of polymer in solution.4,8−11 Poly(lactic acid) (PLA) is a biodegradable, biocompatible, and nontoxic polymer and has been used widely for various biomedical applications.12−14 It is a promising polymer matrix to prepare the biodegradable and biocompatible micro- and nanoparticles.1,15 Moreover, lactic acid is a chiral molecule; different stereoisomers of PLA, i.e., poly(L-lactic acid) (PLLA) and poly(D-lactic acid) (PLLA), can be synthesized by altering the stereoisomeric form of lactic acid or lactide monomer. An interesting crystallization behavior of PLA is the stereocomplexation between PLLA and PDLA, which affords the materials higher melting temperature (Tm), better hydrolytic and chemical resistance.16,17 Stereocomplexation of PLLA and PDLA is driven

by

the

hydrogen

bonding

interactions

between

complementary

enantiomers18−20 and thus has stronger driving force than the conventional homocrystallization of enantiopure PLLA. Stereocomplex crystallization can take place during precipitating PLLA and PDLA from the solution, allowing the



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preparation of stereocomplexed PLA particles with well-defined structure.21−29 These stereocomplexed particles showed great potentials as the matrices for controlled release.28,29 Because of the different liquid-liquid phase separation degree, growth kinetics and chain folding manners in the stereocomplexation and homocrystallization of PLA from solution, controlling the crystallization process and crystalline structure of PLA in the precipitation method can be a simple yet efficient way to prepare the particles with various morphologies. Crystalline structure and morphology are also critical factors influencing the degradation rate of biodegradable particles in potential biomedical applications.30−32 For example, Chen et al.21 have prepared the flower and cake-shaped stereocomplexed PLA particles by precipitating PLLA/PDLA stereo multiblock copolymers from a good solvent to an anti-solvent because of the stereocomplex crystallization of polymer during precipitation; such special morphologies cannot be easily prepared by other methods. This implied that the crystalline structure and crystallization process were essential for the morphologies of resulted particles in precipitation. Because the chain association and crystallization kinetics of polymers in solution are strongly influenced by molecular weight (MW) and intermolecular interactions, which would be key factors for the morphologies of resulted assemblies during precipitation. It has been found that the morphology and size of PLA particles can be tailored by the intermolecular interactions and polymer architecture.24,25 The combination of intermolecular interactions and stereocomplexation of PLLA and PDLA has allowed the preparation of well-defined biodegradable microparticles in the precipitation method.24,25 Brzezinski and Biela have obtained the microspheres (~1 µm) and microfibers by precipitating the 2-ureido-4[1H]-pyrimidinone (UPy)-


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functionalized PLLA and PDLA precursors from solution into an anti-solvent.25 Two UPy groups can dimerize through the self-complementary quadruple hydrogen bonding interactions,33 and thus the interactions between PLLA and PDLA chains and the morphologies of precipitated objects could be tuned by the number of UPy groups in the chain ends.25 They also found that the linear PLLA/PDLA mixture with ionic liquid end groups precipitated in larger microspheres from dioxane (~2 µm);23 yet the mixture of linear PDLA with imidazolium ionic end groups and the hydroxylterminated 6-arm star-shaped PLLA yielded the colloidal crystals under the same conditions.24 Despite recent progress, the interplay between crystalline structure, formation mechanism and morphology of PLA particles in the solution and precipitation process still remained unclear. In this study, we report a novel approach to prepare the flower-shaped and sphere particles of PLA by precipitating the 3-arm star-shaped PLLA or PDLA precursors containing self-complementary UPy end groups from a good solvent to an antisolvent. These UPy-bonded supramolecular PLAs were able to non-covalently assemble or disassemble by the reversible hydrogen bonding interactions. Interestingly, the morphology of particles could be well controlled and the flowershaped, sphere particles were attained by controlling the crystalline structure of PLA or varying the PLLA/PDLA ratio in the mixture. Morphology, crystalline state, enzymatic degradation, and drug release behavior of PLA microparticles with various morphologies were further investigated. Formation mechanisms of flower-shaped particles in PLA homocrystallization and sphere particles in stereocomplexation were also discussed and proposed.

EXPERIMENTAL SECTION



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Materials. L- and D-lactide (> 99.9%) were purchased from Purac Co. (Gorinchem, the Netherlands) and purified by recrystallization from ethyl acetate. (6isocyanatohexylaminocarbonyl-amino)-6-methyl-4[1H]pyrimidinone

(UPy-NCO)

was synthesized according to a published method.34 Tin(II) 2-ethylhexanoate [Sn(Oct)2, Aldrich-Sigma], 1,6-hexanediol (Aldrich-Sigma), trimethylolpropane (Aldrich- Sigma), and rifampicin (RIF, J&K chemical) were used as received. Toluene was dried by sodium and distilled after being refluxed for 48 h. Linear highmolecular-weight (HMW) PLLA (Mw = 64.1 kg/mol, Mw/Mn = 1.30) and PDLA (Mw = 63.6 kg/mol, Mw/Mn = 1.24) were synthesized by the bulk ring-opening polymerization (ROP) of L and D-lactide using 1,6-hexanediol as initiator and Sn(Oct)2 as catalyst, respectively.35 Low-molecular-weight (LMW) hydroxyl- and UPy-terminated telechelic and 3arm star-shaped PLLAs and PDLAs, marked as 2L(D)-xk, 3L(D)-xk, 2L(D)-U-xk, and 3L(D)-U-xk, were synthesized according to a published method,35 as shown in Supporting Information. Chemical structures of UPy-terminated telechelic and 3-arm star-shaped PLAs are illustrated in Figure 1. In the sample codes, the prefixed numerals “2” and “3” denote the telechelic and 3-arm star-shaped architectures, respectively. L and D denote PLLA and PDLA, respectively, and x represents the Mn (in kg/mol) of PLLA and PDLA precursors derived from 1H NMR, respectively. “U” represents the end functionalization by UPy group. Preparation of PLA Particles. Figure 2 illustrates the preparation procedure of PLA particles by precipitation method. 50 mg of PLLA or PLLA/PDLA mixture were dissolved in 50 mL of dichloromethane and stirred at 25 °C for 2 h. In the preparation of RIF-loaded particles, 5 mg of rifampicin (RIF, J&K chemical) was added into the solution. 120 mL of ethanol was added into the polymer solution in dropwise over a


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period of 5 h under continuous stirring; during which the solution became turbid. Then, the suspension was stirred under ambient conditions for 24 h to slowly evaporate dichloromethane, allowing the gradual precipitation of polymer from the suspension. The precipitate was collected by centrifugation at 25 °C, followed by drying at 60 °C for 4 h.

Figure 1. (A, B) Chemical structures of UPy end-functionalized (A) 3-arm starshaped and telechelic and (B) PLLA and PDLA precursors. (C) Schematic illustration of stereocomplexable SMP formation in the mixture of UPy end-functionalized PLLAs and PDLAs.

Figure 2. Schematic illustration for preparation of PLA particles by precipitation method. (A) Addition of anti-solvent, (B) evaporation of good solvent, (C) centrifugation and dry under reduced pressure. Dichloromethane and ethanol were used as the good and anti-solvents, respectively. 7 ACS Paragon Plus Environment


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To determine the RIF content in drug-loaded particles, 2.5 mg of particles was dissolved into 10 ml of dimethylformamide (DMF) and the solution was measured by UV-Vis spectrophotometer (UV-1800, Shimazu). Drug loading content (DLC) was calculated from the absorption at 340 nm according to the standard curve of RIF.36 DLCs of RIF-loaded particles were calculated by DLC (wt%) = (weight of RIF in particles/weight of particles) × 100%. Enzymatic Degradation of PLA Particles. 5 mg of particles were wrapped by a filter paper and immersed in a vial containing 15 mL of phosphate buffer saline (PBS, pH = 7.4, 50 mM), 2 mg of proteinase K, and 1.0 mg of NaN3. The vial was incubated at 37 °C in a shaker at a speed of 120 r/min. After a predetermined time interval, the particles were collected, washed by ionized water, and lyophilized. Degradation ratio was calculated from the weight loss of particles.

In Vitro Release of RIF-Loaded PLA Particles. 2.0 mg of RIF-loaded particles were dispersed in 5.0 ml of PBS (pH = 7.4, 50 mM) and the solution was transferred to a dialysis bag (MWCO = 3500). It was then immersed in 10 mL of PBS (pH = 7.4, 50 mM) in a shaking bath at 37 °C. After a predetermined time interval, PBS solution outside the dialysis bag was removed for UV-Vis measurement and also replaced by the fresh buffer. The amount of released RIF was calculated from the UV absorption at 335 nm by comparing with the calibrated absorption curve of RIF in PBS (pH = 7.4, 50 mM).36 All the release experiments were conducted as triplicates and the average results were used. Characterization. 1H NMR spectra were measured on a 400 MHz Bruker AVANCE II NMR spectrometer (Bruker BioSpin Co., Switzerland) with CDCl3 as the solvent. Fourier transform infrared (FTIR) spectrum was measured on a Nicolet iS50 spectrophotometer (ThermoElectron, Madison, USA) with 32 scans and a


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resolution of 2.0 cm−1. MW and its distribution were measured on a Waters gel permeation chromatography (GPC, Waters Co., Milford, MA, USA) consisted of a Waters degasser, a Waters 1515 isocratic HPLC pump, a Waters 2414 RI detector, and two PL-gel mix C columns at 30 °C. Tetrahydrofuran (THF) was used as the mobile phase and polystyrene used as the standards. Particle morphology was observed on a CorlzeisD Utral55 field emission scanning electron microscopy (SEM) at an accelerated voltages of 5 keV. Melting behavior of PLA particles was measured on a NETZSCH 214 Polyma differential scanning calorimeter (DSC, NETZSCH, Germany) equipped with an IC70 intracooler. Sample was loaded into an aluminum pan and heated from 0 to 170 (or 220) °C at a heating rate of 10 °C/min. The maximum temperatures of 170 and 220 °C were used for PLLA and PLLA/PDLA stereocomplexed particles, respectively. Crystallinities of stereocomplexes (SCs) and homocrystallites (HCs) (Xc,SC, Xc,HC) were calculated by comparing the melting enthalpies of SCs and HCs (∆Hm,SC,

∆Hm,HC) with the value of an infinitely large crystallite (∆Hm0 = 142 J/g for SCs37 and 93 J/g for HCs38), i.e., Xc = ∆Hm/∆Hm0. Wide angle X-ray diffraction (WAXD) patterns of PLA particles were measured on the beamline BL16B1 of Shanghai Synchrotron Radiation Facility (SSRF) with a X-ray wavelength of 0.124 nm and a sample-to-detector distance of 130 mm. Diffraction patterns were acquired by a Rayonix SX-165 CCD detector (Rayonix, Evanston, IL, USA) with a resolution of 2048 × 2048 pixels and a pixel size of 80 × 80 µm2. The acquisition time of each pattern was 30 s. Two-dimensional (2D) data was converted into the one-dimensional profile by integration with Fit2D software. For the PLLA/PDLA mixed particles, the relative fraction of SCs (fSC) in PLLA/PDLA crystalline phase was estimated by comparing the intensities of 9 ACS Paragon Plus Environment


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characteristic diffraction peaks of SCs (ISC) and HCs (IHC), i.e., fSC = ISC/(IHC +

ISC).39,40

RESULTS AND DISCUSSION Synthesis of Hydroxyl and UPy End-Functionalized PLLAs and PDLAs. Synthetic conditions and MWs of hydroxyl- and UPy-terminated PLAs are shown in Table 1. MWs of PLAs were manipulated from the lactide/initiator feed ratio. As shown in Table 1, high yields ( ≥ 93%) were achieved in the ROP of L- and D-lactide. MWs of polymers, denoted as Mn,th, were theoretically calculated from the yield and lactide/initiator feed ratio. As shown in the NMR spectrum (Figure S1), the resonance peaks corresponding to the methyl, methine, and terminal methine protons of hydroxyl-terminated PLA are seen at ~1.5 (peak b), 5.2 (peak a), and 4.3 ppm (peak a′), respectively.41 MWs of polymers, marked as Mn,NMR, were also calculated from the NMR spectra by comparing the resonance intensity of terminal methine protons to all methine protons. MWs of PLAs measured by NMR were similar to Mn,ths. The synthesized polymers showed narrow elution peaks in the GPC curves (Figure S2), corresponding to the low polydispersity index (PDI < ~1.16). Notably, the MWs measured by GPC were larger than Mn,th and Mn,NMR; due to the different solution behavior of PLA from the standard samples in GPC mobile phase. Table 1. Molecular characteristics of LMW telechelic and 3-arm star-shaped PLLA and PDLA precursors.

2L-9.6k

lactide/initiator feed ratio (mol/mol) 56/1

2D-10.3k

56/1

98

8020

10280

14360

1.12

3L-9.9k

56/1

97

7960

9920

10680

1.07

3D-10.6k

56/1

96

7880

10620

12380

1.16

Sample

a

Yield (%)

Mn,thb (g/mol)

Mn,NMRc (g/mol)

Mnd (g/mol)

PDId

96

7860

9650

16440

1.13


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a

Numerals in sample codes denote the Mn,NMR (g/mol) measured by 1H NMR. bMn

calculated theoretically, M n,th = M n,initiator +

[lactide] × 144.13 × yield , where Mn,initiator [initiator]

is the molecular weight of initiator and [lactide]/[initiator] is the molar feed ratio of lactide to initiator. cMn derived from 1H NMR. dMn and polydispersity index (PDI) measured by GPC. Afterward, the terminal hydroxyls of PLA were converted to UPy by reacting with excess of UPy-NCO.34 End functionalization of PLAs by UPy motifs was confirmed by the presence of characteristic N-H resonances of UPy at 13.1, 11.9, and 10.2 ppm42 (Figure S1) and the characteristic FTIR absorptions of UPy at 1663, 1587, and 1522 cm−1 (Figure S3).43 Resonance peaks for the terminal methine protons of hydroxyl-terminated PLAs at 4.3 ppm disappeared completely after the end functionalization by UPy motifs, demonstrating the nearly quantitative conversion of terminal hydroxyls to UPy groups. The results demonstrated the successful synthesis of hydroxyl and UPy end-functionalized PLLA and PDLAs with well-controlled MWs and chain architectures. Because the quadruple hydrogen bonding between UPy groups, the UPy end-functionalized PLLA and PLLA/PDLA mixture would form the homocrystalline (HC) and stereocomplexable (SC) supramolecular polymers (SMPs) in the solvent or solid state, respectively,35 as illustrated in Figure 1C. Viscosity measurement has demonstrated that UPy end-functionalized PLLA or PDLA oligomers formed SMPs through UPy dimerization.35,44 Assembled Structures of Homocrystalline PLA. We selected dichloromethane as a good solvent and ethanol as an anti-solvent and prepared the PLA particles by the solvent/anti-solvent displacement method. As illustrated in Figure 2, the polymers were first dissolved in good solvent to achieve the homogenous solution; this was followed by the gradual addition of anti-solvent. Size and morphology of assemblies



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formed from different PLAs were analyzed by SEM. Figure 3 shows the SEM images for the assembled objects of various PLLA. Unexpectedly, the HC-SMP composed of UPy-functionalized 3-arm PLLA (3L-U-9.9k) precursors assembled in flower aggregates with the sizes of 20~40 µm; these flowers were packed by self-organized, splayed, and smooth lamellae petals (Figure 3A,B). Similar flower aggregates were also formed for the UPy-functionalized 3-arm PDLA precursors (Figure S4A). The flowers were somewhat analogous to the assembled structure of PE during the slow crystallization from solution.4 Lamellae petals grew from the boot and towards the radial direction of flower assemblies. Branching and interpenetrating of lamellae petals were present in the growth of flower assemblies (Figure 3C). This suggested that the growth of individual lamellae was topologically restricted by the neighboring lamellae in such multilamellar objects. The thickness of lamellae petals was estimated to be ~ 100 nm by measuring the standing petals in SEM images; corresponding to the 5~6 times of the long period of PLLA homocrystallites.45

Figure 3. SEM micrographs of formed assembles for homocrystalline PLLA during precipitation.


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In a control experiment, the assembled structures of high, low-MW (HMW, LMW) PLLAs without UPy functionality and the HC-SMP of UPy-functionalized telechelic PLLA (2L-U-9.6k) were investigated. As shown in Figure 3D, similar flower assemblies were obtained for the HMW PLLA without UPy functionality. However, the LMW 3-arm PLLA (3L-9.9k), LMW 3-arm PDLA (3D-10.6k), and 2LU-9.6k HC-SMP did not form the well-defined assemblies but the randomly stacked lamellae (Figure 3E,F,S4B), even though the 2L-U-9.6k HC-SMP exhibited larger lamellae than LMW PLLA (3L-9.9k). To verify the interplay between polymer crystallization and assembled morphology, we investigated the crystalline structure of flower assemblies by WAXD and DSC, as shown in Figures 4 and 5. The flower assemblies of enantiopure HCSMP exhibited characteristic diffraction at 2θ =13.5° in the WAXD patterns (Figure 4A), which corresponded to the 110/200 diffraction of α-form homocrystallites of PLLA,46 and a lower Tm at 146.2 °C in the DSC heating curve (Figure 5A). We note that the X-ray used herein had a different wavelength (0.124 nm) from the common X-ray based on Cu Kα radiation (0.154 nm). Therefore, the angles of diffraction peaks were different from those used reported in literatures46 but the calculated lattice dimensions were the same. As shown in Figure 5B, the crystallinity of enantiopure flower-like particles was as high as 62%, suggesting that the better diffusion ability of polymer chains in precipitation facilitated to form the more perfect crystallites.


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B 1.0 SC220

0.8

mD 0.5 0.4

fSC

Intensity (a.u.)

HC110/200

SC110

A

0.6 0.4

0.3 0.2

0.2

0.1 0

8

12

16

0.0 0.0 0.1 0.2 0.3 0.4 0.5

20

mD

2θ (°)

Figure 4. (A) WAXD patterns of particles made from 3L-U-9.9k HC-SMP and 3L-U9.9k/3D-U-10.6k SC-SMPs with different mDs. (B) Plots of SC fraction (fSC) as a function of mD.

A mD 0.5

Tm,SC

B

XC,SC

Tm,HC

0.4 0.3 0.2

0.4 0.2

0.1 0

80

XC,HC

0.6

XC

Heat flow endo up

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SC300/030


0.0

120

0.0 0.1 0.2 0.3 0.4 0.5

160

200 Temperature (°C)

mD

Figure 5. DSC results of particles made from 3L-U-9.9k HC-SMP and 3L-U9.9k/3D-U-10.6k SC-SMPs with different mDs: (A) DSC curves in the first heating scan at 10 °C/min; (B) Plots of Xc,HC and Xc,SC as a function of mD. Formation Mechanism of Homocrystalline Flower Assemblies. The formation mechanism of PLA particles or assemblies with different morphologies were proposed and illustrated in Figure 6A. The formation of crystalline assemblies described above is associated with the occurrence of two different physical processes, namely a liquid-liquid phase separation which creates a large number of domains, followed by crystal nucleation and growth in these domains. We consider that (i) slow 14 ACS Paragon Plus Environment

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crystallization rate, (ii) small quantity of nucleation site, and (iii) high MW of polymer or strong intermolecular interactions are prerequisite to generate the large and well-organized flower assemblies. As illustrated in Figure 6A, when the antisolvent was gradually added into the isotopic polymer solution, the solution separated into polymer-rich and polymer-poor phase which were dynamically unstable. The polymer-rich phase separated into the form of small droplets, giving rise to a large number of concentrated domains, which acted as the nuclei for polymer crystallization in liquid-liquid phase separation. The size of polymer-rich droplets and the concentration of polymer within droplets would increase as the volume fraction of anti-solvent enhanced. When the concentration of polymer within droplets exceeded a critical value, polymer would crystallize in the droplet. Similar as the slow growth polymer single crystal in solution,47 the homocrystallization of PLLA was rather slower in solution; this facilitated the formation of fine crystalline lamellae.

Figure 6. Schematic representation of formation process of PLA particles during precipitation: (A) homocrystalline flower-shaped particles; (B) stereocomplexed 15 ACS Paragon Plus Environment


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sphere particles. For clarity, UPy units in the polymers were omitted in schematic figure. Previous paper4 has reported the different nucleation mechanisms for PE crystallization in biphenyl solution at various temperatures; the slow and fast crystallizations at higher and lower temperatures were stemmed from heterogeneous and homogeneous nucleations, respectively. We hypothesized that the slow homocrystallization of PLLA in the droplets was mainly induced by heterogeneous nucleation. As shown in SEM images (Figure 3A), most of flowers grew from the boot (or edge of assemble) but not from the center, implying that the heterogeneous nucleation was mainly originated from the a few active foreign heterogeneities accumulated near the droplet/solvent interfaces (Figure 6A). The quantity of active foreign heterogeneities would be small in our system; this allowed the slow growth of large assemblies. Because the simultaneous occurrence of liquid-liquid phase separation and polymer crystallization, the polymer chains outside the droplets could enter the droplets as the crystallization progressed; this ensured the continuous growth of crystalline lamellae. During crystallization, the crystal growth could continue in the dilute polymer phase after exhaustion of polymer in the droplet; this also facilitated the growth of large flowers with well-splayed petals (Figure 6A). By comparing the images shown in Figure 3, it is concluded that high MW and stronger intermolecular interactions are essential for forming the large flower assemblies. Due to the high content of UPy functionality, 3L-U-9.9k would have the UPy-linked networked structure in solution, possessing a similar solution behavior as HMW PLLA. HMW polymers would have the stronger association ability and entangle (or interact) more chains inside the droplets. Furthermore, the long polymer chains of HMW PLLA and SMP can interpenetrate into different lamellae, resulting in


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the growth of more lamellae in a same assemble and the interpenetration between neighboring lamellae during crystallization (Figure 3C). The interpenetration between lamellae could also increase the dimensional stability and self-standing ability of assembles and thus avoided the destroying and disassembling of assembled flowers. Even though the 2L-U-9.6k SMP also contained UPy hydrogen bonds, the insufficient UPy functionality and linear topology may restrict the precursors forming HMW SMP. Therefore, the linear 2L-U-9.6k SMP formed similar disordered assembles as LMW PLLA (3L-9.9k) in precipitation. Assembled Structures of Stereocomplexed PLA. In order to prepare the stereocomplexed assemblies, we mixed hydroxyl or UPy-functionalized PLLA and PDLA precursors equivalently in solution and analyzed the morphologies of formed assemblies. Crystalline structure of polymers in precipitation governed the morphology of formed assemblies. Different from the HC-SMP, the SC-SMP composed of equivalent 3-arm PLLA and PDLA precursors (e.g., 3L-U-9.9k/3D-U10.6k) assembled into the sphere particles with the diameter of 2~4 µm; yet the surfaces of these sphere particles were invaginated (Figure 7A). We note that some of particles conglutinated during precipitation or the evaporation of solvent. HMW PLLA homopolymer did not form the well-defined assemblies but interconnected fibrils (Figure 7B), analogous to the assembled structure of HMW isotactic polystyrene in solution48 and the microporous structure of polymer membranes prepared by phase inversion.49 Small particles (size < 1 µm) were obtained for the LMW 3-arm PLLA/PDLA 1/1 mixture (3L-9.9k/3D-10.6k, Figure 7C) and the SCSMPs composed of equivalent linear PLLA and PDLA precursors (2L-U-9.6k/2D-U10.3k, Figure 7D). The particles of 3L-U-9.9k/3D-U-10.6k racemic SC-SMP exhibited diffractions at 2θ = 9.6°, 16.7° and 19.3° in the WAXD patterns (Figure 4A).



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These diffractions are characteristic of PLLA/PDLA SCs.50 Stereocomplexed particles exhibited higher Tm (203 °C) in the DSC heating curve (Figure 5A).

Figure 7. SEM micrographs of assembled structures formed from racemic SC-SMPs and PLLA/PDLA mixtures. Overall, the 3-arm UPy-functionalized PLLA and PDLA precursors were able to form diversified assemblies by controlling the crystalline manner. Both the flowershaped and spherical particles could be obtained from the enantiopure HC-SMP and racemic SC-SMP based on the UPy-functionalized 3-arm PLLA, PDLA precursors. Because the contents of PLLA and PDLA are equal in SCs, the crystalline structure of formed particles is controllable via varying the mixing ratio of PLLA and PDLA precursors. Changing the mixing ratio of 3-arm UPy-functionalized PLLA and PDLA precursors allowed well controlling the fractions of flower-shaped and spherical particles. Figure 8 shows the SEM images of particles prepared from the 3L-U9.9k/3D-U-10.6k SC-SMPs with different PLLA/PDLA ratios. When mD (mass fraction of PDLA blocks in SC-SMP) was 0.1, smaller sphere particles existed in the 18 ACS Paragon Plus Environment

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edge of flowers, which was due to the coexistence of HCs and SCs. As the PLLA/PDLA ratio approached to 1/1 or the mD increased from 0 to 0.5, the fractions of sphere particles increased and that of flower-shaped particles decreased; this was due to the increased fraction of SCs (as discussed below).

Figure 8. SEM micrographs of particles prepared from 3L-U-9.9k/3D-U-10.6k SCSMPs with different mDs. The particles of SC-SMPs with non-equivalent PLLA and PDLA contents contained both SCs and HCs. Both the characteristic diffractions of SCs and HCs were present in the WAXD patterns of the particles formed from the 3L-U-9.9k/3DU-10.6k SC-SMPs with mD = 0.1-0.4 (Figure 4A). In the DSC heating curves, two melting regions were observed for the particles of SC-SMPs with mD = 0.1−0.4 at 130−145 and 200−210 °C, corresponding to the melts of HCs and SCs, respectively (Figure 5A). As mD increased from 0 to 0.5, SC diffractions and melting peaks of SCSMP particles gradually increased, with companied by the decrease of the HC



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diffractions and melting peaks (Figures 4A, 5A). Meanwhile, fSC, Xc,SC of SC-SMP particles enhanced and their Xc,HCs decreased as the contents of L- and D-enantiomers approached to equal in the SC-SMPs (Figure 4B, 5B). As the mD of SC-SMP increased from 0 to 0.5, the increased stereocomplex crystallization led to the enhanced contents of sphere particles (Figure 8). Formation Mechanism of Stereocomplexed Particles. We consider that (i) the fast crystallization and (ii) low MW or cleavable polymer chains are prerequisite for PLA to form spherical assemblies in the stereocomplex crystallization from solution. Compared to HCs, SCs possess the intermolecular hydrogen bonding interactions and have highly decreased solubility in the solvents such as CH2Cl2, CH3Cl, THF, and DMF. Therefore, stereocomplexation of PLLA and PDLA has the stronger driving force and occurs faster in solution crystallization. We observed that the PLLA/PDLA SC-SMP solution became turbid at much lower anti-solvent content than the HC-SMP solution, suggesting the faster crystallization rate and the ease of crystallization of PLLA/PDLA mixture in solution (Figure S5). As illustrated in Figure 6B, PLLA and PDLA will phase separate from the dilute solution upon the addition of anti-solvent. Meanwhile, PLLA and PDLA will form the helical pairs (i.e., nuclei precursors) through the intermolecular hydrogen bonds inside the droplets or in the solution, as demonstrated for the PLLA/PDLA crystallization in melt state.51 These nuclei precursors can lead to the fast homogeneous nucleation of PLLA and PDLA in solution crystallization. Due to the stronger driving force and fast crystallization rate, stereocomplexation may take place immediately after the phase separation of PLLA and PDLA from the dilute solution; this would retard the formation of large polymer-rich droplets.


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With referring to the crystallization of PE in biphenyl at low temperature,4 the homogeneous nucleation of PLLA/PDLA chains in droplets seemed to take place near the droplet/solution interfaces (Figure 6B). The surfaces of stereocomplexed particles did not show the radial growth of polymer lamellae as that observed in the homocrystalline flowers. This suggested that the lamellae grew parallelly along the droplet/solution interfaces and the chain direction was radial relative to the center of droplets, as illustrated in Figure 6B. Therefore, the assembled structure was nearly spherical and closed particles. We note that the assembled particles were likely to be hollow, because the lamellae growth would stop if the polymer chains inside the droplets were exhausted. The hollow structure was clearly seen for the assembled objects of 2L-U-9.6k/2D-U-9.3k SC-SMP (Figure 7D). As shown in the following experiment, we indeed found that the drug could be loaded inside the particles of 3LU-9.9k/3D-U-10.6k SC-SMP; which also indicated the hollow structure of stereocomplexed particles. The surface of 3L-U-9.9k/3D-U-10.6k SC-SMP particles was not smooth and exhibited invaginated groove. The invagination might be resulted from the depletion of polymer chains during crystallization and the elimination of solvent inside the spheres. We note that MW and intermolecular interaction are also essential for the assembled structure of stereocomplexed PLA. Because of the long chains of HMW PLLA and PDLA, a PLLA chain could form helical pairs with different PDLA chains and vice versa; this made PLLA and PDLA physically-crosslinked in the solution. Thus, HMW PLLA/PDLA mixture assembled in the interpenetrated fibrils but not the dispersed particles during solution crystallization. In the case of 3L-U-9.9k/3D-U10.6k SC-SMP, parts of UPy hydrogen bonds between precursors might dissociate during the formation of helical pairs and phase separation; this could facilitate the



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formation of polymer-rich droplets and dispersed particles. The content of UPy groups also influenced the morphology of stereocomplexed PLA particles, due to the effect of intermolecular interactions. Because the stronger intermolecular interactions of 3L-U-9.9k/3D-U-10.6k SC-SMP than the 3L-9.9k/3D-10.6k mixture and 2L-U9.6k/2D-U-9.3k SC-SMP, the former could associate more chains in a polymer-rich droplet and thus generated the larger particles. Enzymatic Degradation and In Vitro Drug Release. Enzymatic degradation of PLA particles with different crystalline structures and morphologies was assessed in PBS (pH = 7.4, 50 mM) at 37 °C with the presence of proteinase K. The weight loss after degradation for 48 h was used to indicate the degradation rate. As shown in Figure 9A, the degradation ratio of particles gradually decreased as the ratio of L and D-enantiomers in SC-SMPs approached to 1/1 or the fraction of SCs increased. 41 wt% of homocrystalline flower-shaped particles were degraded after 48 h; this degradation ratio decreased to 25 wt% for the stereocomplexed sphere particles made from the racemic SC-SMP (mD = 0.5). Different degradation behavior of particles are attributed to the effects of crystalline structure and morphology. First, due to the compact chain packing and stronger interchain interactions, SCs could not be easily attacked by enzyme and thus had the slower degradation rates than their homocrystalline counterparts. Similar results have been reported for the stereocomplexed films,30 micelles,31 and hydrogels.32 Second, as demonstrated by SEM (Figure 3A-C), the homocrystalline flower-shaped particles composed of bundles of thin lamellae had larger porosity and surface area than the stereocomplexed sphere particles; this porosity structure would promote the enzymatic and hydrolytic degradation of flowershaped particles.


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Drug loading content (%)

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Degradation rate (%)

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A

40 30 20 10 0

0.0 0.1 0.2 0.3 0.4 0.5

4

B

3 2 1 0

0.0 0.1 0.2 0.3 0.4 0.5

mD

mD

Figure 9. Enzymatic degradation ratio (in 48 h) and drug loading content for the particles prepared from 3L-U-9.9k HC-SMP and 3L-U-9.9k/3D-U-10.6k SC-SMPs with different mDs. RIF was used as a model drug to evaluate the drug loading and release behavior of PLA particles with different morphologies and crystalline structures. DLCs varied in a large range (0.3-4.1%) for the particles of HC-SMP and SC-SMPs with different PLLA/PDLA ratios. Because the homocrystalline flower-shaped particles had the very thin lamellae and the voids between lamellae were opened to the solvent, the encapsulated drugs would be removed in the washing process followed by precipitation. Therefore, DLC of the homocrystalline flower-shaped particles formed by HC-SMP was as low as 0.34% (Figure 9B). In contrast, the stereocomplexed particles had closed structure and the drugs could be encapsulated inside the sphere particles during polymer crystallization and precipitation. Besides, it was observed that some of stereocomplexed sphere particles aggregated during precipitation. The aggregation would generate additional voids or pores between particles, which also enhanced the capacity of drug loading. Possible interactions between SCs and drugs could also increase the drug encapsulation ability of stereocomplexed sphere particles. For these reasons, DLC of particles gradually increased as the ratio of L and D-enantiomers approached to 1/1 in SC-SMP. DLC of 23 ACS Paragon Plus Environment


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stereocomplexed particles made from racemic SC-SMP (mD = 0.5) was as large as 4.11%, which was more than 10 times higher than that of the homocrystalline flowershaped particles. In vitro release behavior of RIF-loaded PLA particles was investigated in PBS (pH = 7.4, 50 mM) at 37 °C. Figure 10 shows the cumulative release profiles of particles prepared from the HC-SMP and SC-SMPs with different PLLA/PDLA ratios. The drug release profiles were strongly influenced by the morphology and crystalline states of PLA particles. As shown in Figure 10A, the homocrystalline flower-shaped particles of enantiopure HC-SMP showed much faster release than the stereocomplexed sphere micelles of racemic SC-SMP. After release for 84 h, 62% of RIF in homocrystalline flower-shaped particles were released off; yet this value was 4.4% for the stereocomplexed sphere micelles. The opened structure, faster degradation rate, and weaker polymer/drug interactions of homocrystalline flowershaped particles may be responsible for their faster drug release. The similar relationship between drug release rate and PLA crystalline state has been reported for the PLA/poly(ethylene glycol) copolymer micelles.31 As expected, the drug release rate of SC-SMP particles with different PLLA/PDLA ratios decreased with increasing the fraction of stereocomplexed sphere particles (Figure 10B). After release for 84 h, the cumulative released drug fraction of SC-SMP particles increased from 4.4% to 18.6% as the mD of SC-SMP decreased from 0.5 to 0.1.


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A

60

Cumulative release (%)

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Cummulative release (%)

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40 HC-SMP Racemic SB-SMP

20

0

0

20

40

60

mD

SB-SMP

0.1

15 0.2

10

0.3 0.4

5

0.5

0

80

B

20

0

20

Time (h)

40

60

80

Time (h)

Figure 10. Cumulative release of RIF from the particles of 3L-U-9.9k HC-SMP and 3L-U-9.9k/3D-U-10.6k SC-SMPs: (A) comparison between HC-SMP and racemic SC-SMP particles, (B) SC-SMPs with different mDs.

CONCLUSIONS A simple yet efficient method was successfully achieved to prepare the PLA biodegradable particles by the solution crystallization of SMPs based on the UPyterminated 3-arm star-shaped PLLA and PDLA precursors. Morphology of assembled particles was strongly influenced by MW, stereostructure of polymers and the crystallization manner in solution. Both the homocrystalline flower-shaped and sphere particles could be attained by varying the contents of PLLA and PDLA in SMPs. These morphologies reflected the interplay of liquid-liquid phase separation and nucleation of polymer crystals in solution. The attained PLA particles showed controllable degradation rates, DLCs, and drug release behavior, which strongly depended on the particle morphology and crystalline structure. The spherical morphology and stereocomplexed structure afforded the racemic SC-SMP particles higher DLC, slower degradation and drug release rates. This study opens up a simple method to prepare the PLA particles with diversified morphologies and crystalline structures through controlling the polymer crystallization in solution. The particles



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prepared herein are biocompatible and biodegradable, which have good promise to serve in biomedical applications.

ASSOCIATED CONTENT Supporting Information 1

H NHR, GPC, FTIR, and SEM data of telechelic and 3-arm star-shaped LMW

PLLAs or PDLAs precursors with or without the UPy end functionality. Images of 3L-U-9.9k HC-SMP and 3L-U-9.9k/3D-U-10.6k SC-SMP samples in solution with the different volume factions of anti-solvent.

AUTHOR INFORMATION Corresponding Author *Tel.: +86-571-87951334, e-mail: [email protected] (P.P.).

ACKNOWLEDGEMENTS We thank Prof. Bernard A. Lotz (Institut Charles Sadron, CNRS) for valuable discussion. This work was financially supported by the Natural Science Foundation of China (21674095), Natural Science Foundation of Zhejiang Province, China (LR16E030003), and State Key Laboratory of Chemical Engineering of China (SKLChE-16Z). WAXD was measured at the BL16B1 beamline of SSRF, China.

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For Table of Content Use Only Crystallization-Driven Formation of Diversified Assemblies for Supramolecular Poly(lactic acid)s in Solution Xiaohua Chang, Jianna Bao, Guorong Shan, Yongzhong Bao, Pengju Pan* Synopsis: A novel and simple approach is reported to prepare biodegradable micro- and nanoparticles with diversified morphologies through precipitating supramolecular poly(lactic acid) (PLA) from a good solvent to an anti-solvent. Homocrystallization of supramolecular PLA yielded the flower-shaped particles in precipitation; yet the stereocomplex crystallization of PLA supramolecular copolymers led to the formation of spherical-shaped particles.


Crystallization-Driven Formation of Diversified Assemblies for

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