Antibacterial Nanoplatelets via Crystallization-Driven Self-Assembly of Poly(l-lactide)-Based Block Copolymers

Membrane-active antimicrobial materials are promising substances to fight antimicrobial resistance. Herein, crystallization-driven self-assembly (CDSA) is employed for the preparation of nanoparticles with different morphologies, and their bioactivity is explored. Block copolymers (BCPs) featuring a crystallizable and antimicrobial block were synthesized using a combination of ring-opening and photoiniferter RAFT polymerizations. Subsequently formed nanostructures formed by CDSA could not be deprotected without degradation of the structures. CDSA of deprotected BCPs yielded 2D diamond-shaped nanoplatelets in MeOH, while spherical nanostructures were observed for assembly in water. Platelets exhibited improved antibacterial capabilities against two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) compared to their spherical counterparts. The absence of hemolytic activity leads to the excellent selectivity of platelets. A mechanism based on membrane permeabilization was confirmed via dye-leakage assays. This study emphasized the impact of the shape of nanostructures on their interaction with bacterial cells and how a controlled assembly can improve bioactivity.


Figure S18
. Hemolysis assay of APs.PBS was used as medium and RBCs were threatened with polymer solutions containing different concentrations between 4096 µg mL -1 to 8 µg mL -1 for diamond-shaped nanoparticles and 2048 µg mL -1 to 8 µg mL -1 for nanospheres for 1 h at 37 °C.After separation of the cells via centrifugation at 500 G the absorbance of the supernatant was measured using Absorption (544 nm) to analyze hemolysis.RBC solution containing Triton X solution (1 % in PBS) was used as positive control and pure PBS solution served as negative control.A triplicate determination was executed and results below 10 % were set as non-hemolytic.

Figure S5 .
Figure S5.DLS analysis of Boc deprotection process from the polydisperse fiber-like micelle of PLLA 50b-PBocAEAm133-co-PNiPAAm181 at a concentration of 1 mg mL -1 .(A) After treatment with 5%TFA for 1 h at RT. (B) After treatment with 5%TFA for 24 h at RT. (C) After treatment with 10%TFA for 24 h at RT. (D) After treatment with 15%TFA for 24 h at RT. (E) After treatment with 10%TFA for 24 h at 40 °C.(F) After treatment with 5%TFA for 7 days at RT. (G) After treatment with 10%TFA for 7 days at RT. (H) After treatment with 15%TFA for 7 days at RT. (I) After treatment with 20%TFA for 7 days at RT. (J) After treatment with 25%TFA for 7 days at RT.

Figure S7 .
Figure S7.(A) AFM height image of the deprotected BCPs of PLLA 50 -b-PBocAEAm133-co-PNiPAAm181 of polydisperse cationic diamond nanoplatelets obtained after dialysis against PBS at pH of 7.5 at a concentration of 0.5 mg mL -1 .(B) AFM phase image of (A).(C) AFM height image of the deprotected BCPs of PLLA 50 -b-PBocAEAm133-co-PNiPAAm181 of polydisperse cationic nanospheres obtained after dialysis against PBS at pH of 7.5 at a concentration of 0.5 mg mL -1 .(D) AFM phase image of (C).

Figure S8 .
Figure S8.(A) and (C) AFM height image of the deprotected BCPs of PLLA 50 -b-PBocAEAm133-co-PNiPAAm181 of polydisperse cationic diamond nanoplatelets measured after dialysis against PBS at pH of 7.5 at a concentration of 0.5 mg mL -1 .(B) AFM phase image of (A).(D) AFM phase image of (C).(E) Contour length histogram from (A) and (C).(F) Contour width histogram from (A) and (C).

Figure S9 .
Figure S9.(A) and (C) AFM height image of the deprotected BCPs of PLLA 50 -b-PBocAEAm133-co-PNiPAAm181 of polydisperse cationic nanospheres measured after dialysis against PBS at pH of 7.5 at a concentration of 0.5 mg mL -1 .(B) AFM phase image of (A).(D) AFM phase image of (C).(E) Contour diameter histogram from (A) and (C).

Figure S10 .
Figure S10.DLS analysis of the self-assembly morphology obtained from the deprotected BCPs of PLLA 50 -b-PBocAEAm133-co-PNiPAAm181 at a concentration of 1 mg mL -1 after aging at RT. (A) Size distribution by the cationic diamond nanoplatelets obtained after CDSA and measured in MeOH.(B) Size distribution by the cationic diamond nanoplatelets obtained after dialysis against PBS and measured in ultrapure water.(C) Size distribution by the cationic nanospheres obtained after CDSA and measured in ultrapure water.(D) Size distribution by the cationic nanospheres obtained after dialysis against PBS and measured in ultrapure water.

Figure S12 .
Figure S12.DLS analysis of the self-assembly experiment obtained from of the deprotected BCPs PLLA 50b-PBocAEAm268-co-PNiPAAm342 at a concentration of 1 mg mL -1 after aging at RT.

Figure S13 .
Figure S13.AFM cross sections of nanoplatelets indicating they height of structures.

Figure S14 .
Figure S14.AFM cross sections of nanospheres indicating they height of structures.

Figure S15 .
Figure S15.GIWAXS pattern (A) and the respective scattering curve (B) of the nearly empty silicon substrate measured at an incident angle of 0.2°.The scattering curve in (B) was obtained by integrating the intensity in (A) over all azimuthal angles.

Figure S16 .
Figure S16.Optical microscopy images (a, b) of the drop casted sample on a silicon substrate under open (a) and crossed (b) polarizers, indicated in the upper right corners of the images.AFM height (c) and deformation (d) images of the drop cast sample on the silicon substrate.The polarized light optical microscopy image in (b) clearly shows a strong birefringence of the polymer, indicating its semicrystalline nature.Remarkably, one can identify the centers of spherulitic crystal growth in Fig. a -b, suggesting that the polymer crystallization followed the classical route consisting of nucleation and growth steps.The atomic force microscopy images (b -c) show small lamellar crystals of the order of 10 -20 nm, consistent with the chemical architecture of the polymer chains.

Figure S17 .
Figure S17.AP concentration-dependent bacterial growth.Absorption was determined via optical density at 600 nm and normalized using positive control (medium with bacteria suspension; 100 %) and negative control (pure medium, 0 %).Mueller-Hinton broth medium (MHB) was used as a culture medium.Values below 50 % were considered as antimicrobial active and a calculation using the Hill1 Fit of OriginPro 2021® was used to determine MIC50.Error bars correspond to triplicates.

Table S1 .
Summary of 1 H NMR data and DLS analysis of Boc deprotection process from the polydisperse fiber-like micelle of PLLA 50 -b-PBocAEAm133-co-PNiPAAm181 through varying the concentration of TFA at RT or 40 °C within different reaction times.