*3.1. Anti-Cancer Therapy*

Recently, numerous studies have reported that biodegradable polymeric nanocarriers possess a superior stealth function for the detection of the reticuloendothelial system in the human body and exhibit an enhanced permeability and retention effect [56–61]. In particular, these polymeric nanoparticles are capable of having several beneficial properties, such as biomimetics, stimuli-sensitivity, easy modification, and exquisite target specificity compared to carriers composed of other materials. These advantages support the possibility that stereocomplex nanoparticles can be used as a promising approach in the field of anticancer therapy.

Goldberg proposed that oligomers of PDLA were used for the formation of stereocomplex crystals with L-lactate in the human body to induce lactate deficiency in cancer cells [62]. This study suggested that tumor growth could be inhibited and terminated by stereocomplexation through lactate deficiency to retain the electrical neutrality of tumors. In preliminary experiments, it was demonstrated that high concentrations of PDLA could induce stereocomplexation with lactate in the body, which exhibited cytotoxic effects on the tumor. Li et al. (2016) fabricated sc-PLA-coated nanoparticles with multiple functionalities for a highly tunable drug delivery system via simple LbL self-assembly [14]. TEM images show that the nanoparticles had a spherical shape with a core–shell structure and a diameter of approximately 190 nm (Figure 12b). The in vitro drug release profiles of doxorubicin (DOX)-loaded nanoparticles were analyzed at different pH and temperature conditions to demonstrate the ability to adjust the drug delivery of the sc-PLA-coated nanoparticles. As shown in Figure 12c, the drug release rate was significantly reduced with increasing pH from 3.5 to 7.4. The cumulative release of DOX was 73.1 vs. 55.6% at pH 3.5 and 7.4 after 12 h, respectively. This is because acidic conditions could stimulate protonation of the tertiary amine groups in the outermost layer of the sc-PLA-coated nanoparticles, resulting in the swelling of nanoparticles. Furthermore, a lower pH yields the nanoparticles pHresponsive properties through an increase in the solubility of DOX, which retains phenols and amines. In addition to pH, the cumulative release of DOX was 39.8 vs. 60% at 37 and 20 ◦C after 12 h, respectively. A lower temperature condition could facilitate a more rapid release via swelling of the outer layer of sc-PLA-coated nanoparticles. Consequently, the nanoparticles based on sc-PLA could tune the rate and amount of drug release, depending on the physiological conditions. To identify the cytotoxicity of DOX-loaded sc-PLA-coated nanoparticles to breast cancer cells, MCF-7 cells were incubated with free DOX and two types of DOX-loaded sc-PLA nanoparticles (Figure 12d). During the incubation of cancer cells with free DOX, most of the DOXs were localized in the cell nuclei, instead of the cytoplasm. In contrast, the DOXs released from all sc-PLA particles infiltrated the cells, and their accumulation was significantly increased. These results indicated that sc-PLAcoated nanoparticles could effectively act as anti-cancer drug carriers by improving the cell uptake efficiency.

**Figure 12.** (**a**) Schematic representation for highly tunable sc-PLA coated nanoparticles and drug delivery. (**b**) TEM image of Fe3O4@SiO2@SC-N nanoparticles. Scale bar: 200 nm. (**c**) In vitro drug release profiles of DOX-loaded Fe3O4@SiO2@SC-D nanoparticles in acidic condition of pH 3.5 (A), physiological condition of pH 7.4 (B), and Fe3O4@SiO2@SC-N nanoparticles at 20 ◦C (C) and 37 ◦C (D). (**d**) Confocal laser scanning microscope observations of MCF-7 cells incubated with free DOX (**i**), DOX-loaded Fe3O4@SiO2@SC-N (**ii**), and Fe3O4@SiO2@SC-D (**iii**) nanoparticles. Each panel named DIC, DAPI, Dox, and Merged depicts a differential interference contrast (DIC) image, cell nuclei staining by DAPI, DOX fluorescence in cells, and overlay of the all images, respectively. (iv) Comparison of fluorescence intensity in three specimens calculated by ImageJ [14], Copyright 2015. Reproduced with permission from the American Chemical Society.

Brzezi ´nski et al. synthesized DOX-loaded stereocomplexed microspheres using spontaneous precipitation after the polymerization of L-proline-functionalized PLLA and PDLA via coordination polymerization (Figure 13a) [63]. Therein, the size of the microparticles was dependent on whether the L-proline end groups were blocked or unblocked in the microspheres. Based on this correlation, they obtained spherical microspheres with various sizes ranging from 0.5 to 10 μm through adjusting the solvent and functionalization. In the in vitro release profiles of DOX, stereocomplexed microspheres with Boc-protected L-proline exhibited cumulative release within 10% of loaded contents for 100 h, while microspheres with unblocked L-proline showed a faster cumulative release of 41–81% of loaded contents for the same duration (Figure 13b). This can be attributed to the low surface area of the Boc-protected group, which delayed hydrolysis. In contrast, the unblocked group could easily release DOX due to the localization of DOX on the surface of the microsphere, resulting in an initial burst release. To evaluate the cytotoxicity of the DOX-loaded stereocomplexed microspheres, A549 lung cancer cells were incubated with medium extracts of the two types of microspheres. Figure 13b shows a very slow reduction in the cell viability of cancer cells cultured with Boc-protected microspheres after 24 h, whereas the cell viability of the cancer cells cultured with unblocked microspheres was dramatically decreased after incubation for 2 h and decreased to below 25% after 24 h. This

can be attributed to the difference in drug release rate between the blocked and unblocked microspheres. In particular, the microspheres prepared in tetrahydrofuran (THF) showed a significantly high anti-proliferative effect.

**Figure 13.** (**a**) Synthesis process of Boc-L-proline functionalized PLLA and PDLA, unblocking of L-proline end groups (top). Schematic illustration for fabrication of sc-PLA microspheres using spontaneous precipitation from mixture of enantiomeric PLAs (bottom). (**b**) In vitro release profiles of DOX from sc-PLA microspheres functionalized with (**i**) Boc-L-proline and (**ii**) L-proline. In vitro cytotoxicity test on A459 cells incubated with medium extracts of the sc-PLA microspheres functionalized with (**iii**) Boc-L-proline and (**iv**) L-proline at incubation times of 2, 6, and 24 h [63], Copyright 2019. Reproduced with permission from Elsevier B.V.

In addition, Brzezi ´nski et al. successfully fabricated stereocomplexed micelles with β-cyclodextrin (β-CD) core as an intracellular drug carrier [64]. Hence, micelles stability can be improved, and in vitro release rate of DOX from supramolecular nanocarriers can be controlled. The stereocomplexed micelles with DOX efficiently inhibited the proliferation of HeLa (cervical cancer) and K562 (chronic myelogenous leukemia). This study suggested that the supramolecular interactions facilitate effective establishment of drug delivery system as an anti-cancer therapy.
