*2.2. Self-Assembled Nanoparticle*

Stereocomplexes can build nanoparticles by self-assembly, apart from micelle formation. Non-covalent interactions, such as electrostatic interactions, hydrogen bonding, and hydrophobic-hydrophobic interactions are driving forces to induce self-assembly of amphiphilic block copolymers. The stability of the self-assembly can be influenced by several environmental factors, including the pH, temperature, polymer concentration, and ionic strength [22–25]. Bishara et al. synthesized stereocomplex particles composed of an enantiomeric triblock copolymer (PLA-PEG2000-PLA) by blending in acetonitrile solutions [26]. Figure 2a shows that the stereocomplex nanoparticles have smooth surfaces resulting from PEG segments and sizes ranging from the nanometer to micrometer range. The size of stereocomplex particles increased with increasing concentration of PLA in the triblock copolymer (Figure 2b). This size of biodegradable particles affects the drug release rate from a particular carrier. Smaller-sized stereocomplex particles have a faster degradation rate owing to their larger surface area, as shown in Figure 2c. In the in vitro release profile test, stereocomplex particles with triblock copolymer could encapsulate 80% of the water-soluble drug dexamethasone, which was completely released for 30 days (Figure 2d). Similar to the previous degradation results, particles with a higher PLA concentration could release drugs at a slower rate. Biodegradable particles were fully degraded during the two months after complete release of dexamethasone phosphate. The hydrophilic drug was assumed to accelerate the infiltration of water into the polymer material compared to the original polymer.

Liu et al. produced pH-sensitive stereocomplex nanoparticles consisting of methoxy poly(ethylene glycol)-poly(L-histidine)-polylactide (mPEG45-PH30-PLA82) tri-block copolymer by self-assembly [27] (Figure 3a). In this study, the mPEG45-PH30-PLLA82/mPEG45- PH30-PDLA82 stereocomplex stably maintained a mean diameter of 90 nm at pH 6.8, whereas the diameter of mPEG45-PH30-PLA82 increased to the micrometer scale under the same conditions. The mean diameter of the stereocomplex nanoparticles slightly decreased when the pH changed from 5.0 to 7.9, as shown in Figure 3b,c. It was considered that lower pH conditions caused swelling of the nanoparticles with protonation of poly(L-histidine) in the tri-block copolymer [28]. Transmission electron microscopy (TEM) revealed that the

stereocomplex particles retained their spherical shape, ranging from pH 5.0 to 7.4, and the TEM image confirmed a reduction in the particle size at pH 7.9 (Figure 3d,e). Furthermore, the cell viability of the stereocomplex nanoparticles was 90 % higher than that of homopolymer nanoparticles in co-culture with mouse 3T3 fibroblasts. This was attributed to the reduction of cytotoxic PDLA segments through stereocomplexation. Numerous studies have demonstrated that stereocomplex particles are capable of loading not only proteins, but also drugs.

**Figure 2.** (**a**) Scanning electron microscopy (SEM) images of stereocomplex nanoparticles composed of PLLAx-PEG2000-PLLAy with D-PLA (x + y = 25, left) and PLLAx-PEG2000-PLLAy with 10% *w*/*w* dexamethasone phosphate with D-PLA, respectively (right). (**b**) Stereocomplex particle size at different concentrations. Closed and open squares depict PLLAx-PEG2000-PLLAy with D-PLA (x + y = 25) and PLLAx-PEG2000-PLLAy with D-PLA (x + y = 50) specimens, respectively. (**c**) HPLC analysis for lactic acid release from copolymers and stereocomplexes. Open and closed circles depict PLLAx-PEG2000-PLLAy (x + y = 25) and PLLAx-PEG2000-PLLAy (x + y = 50), respectively. Closed squares and triangles depict PLLAx-PEG2000-PLLAy (x + y = 25) with D-PLA specimen and same material containing 10% *w*/*w* dexamethasone phosphate, respectively. (**d**) HPLA analysis for in vitro release of dexamethasone from stereocomplexes. Closed and open triangles depict specimens composed of PLLAx-PEG2000-PLLAy (x + y = 25) with D-PLA and PLLAx-PEG2000- PLLAy (x + y = 50) with D-PLA, respectively. This experiment was conducted in phosphate buffer (pH 7.4) at 37 ◦C [26], Copyright 2005. Reproduced with permission from WILEY-VCH Verlag GmbH & Co.

Lim and Park (2000) synthesized stereocomplex microspheres based on the solventcasting method, after polymerization of PLLA-PEG-PLLA and PDLA-PEG-PDLA of a tri-block ABA copolymer [29]. Then, a bovine serum albumin (BSA) protein could be encapsulated in the stereocomplex by the double emulsion solvent evaporation method. Their study reported that stereocomplex microspheres showed a more sustainable and predictable release pattern of the protein than that of the homopolymer. This can be attributed to the hydrophilic PEG unit in the tri-block microspheres preventing aggregation and non-specific adsorption of the protein. In the in vitro release profiles of BSA, PEG stereocomplex microspheres and PEG tri-block copolymer microspheres exhibited a higher initial burst effect than that of PLLA microspheres, but the microspheres based on PEG showed a larger cumulative release (Figure 4). This is attributed to the water uptake capacity of the microspheres that increased owing to the PEG of the hydrophilic unit. After the burst release in the initial stage, the microspheres showed a relatively sustained release by a diffusion-controlled mechanism over 50 days.

**Figure 3.** (**a**) Formation process schematic of mPEG45-PH30-PLLA82/mPEG45-PH30-PDLA82 stereocomplex nanoparticles. Mean diameters of mPEG45-PH30-PLLA82 (**b**) and mPEG45-PH30- PLLA82/mPEG45-PH30-PDLA82 stereocomplex nanoparticles (**c**) with various pH conditions. TEM images of the stereocomplex nanoparticles at pH 7.4 (**d**) and pH 5.0 (**e**) [27], Copyright 2012. Reproduced with permission from WILEY-VCH Verlag GmbH & Co.

**Figure 4.** In vitro BSA release profile from three specimens [29], Copyright 2000. Reproduced with permission from John Wiley & Sons, Inc.
