Alginate-Based Amphiphilic Block Copolymers as a Drug Codelivery Platform

Abstract

Structured nanoassemblies are biomimetic structures that are enabling applications from nanomedicine to catalysis. One approach to achieve these spatially organized architectures is utilizing amphiphilic diblock copolymers with one or two macromolecular backbones that self-assemble in solution. To date, the impact of alternating backbone architectures on self-assembly and drug delivery is still an area of active research limited by the strategies used to synthesize these multiblock polymers. Here, we report self-assembling ABC-type alginate-based triblock copolymers with the backbones of three distinct biomaterials utilizing a facile conjugation approach. This "polymer mosaic" was synthesized by the covalent attachment of alginate with a PLA/PEG diblock copolymer. The combination of alginate, PEG, and PLA domains resulted in an amphiphilic copolymer that self-assembles into nanoparticles with a unique morphology of alginate domain compartmentalization. These particles serve as a versatile platform for co-encapsulation of hydrophilic and hydrophobic small molecules, their spatiotemporal release, and show potential as a drug delivery system for combination therapy.

Keywords: Alginate; Block Copolymer; Click Chemistry; Codelivery; Self-Assembly.

Figure 1.

Synthesis and characterizations of the triblock alginate-b-PEG₁₁₂-b-PLA 6: (A) Synthetic scheme of alginate-b-PEG₁₁₂-b-PLA. (B) ¹H-NMR characterization of alginate-b-PEG₁₁₂-b-PLA and full consumption of tetrazine and TCO was observed (D₂O, 500 MHz). The alginate and PEG backbones are marked in red and purple boxes. Peaks marked with * are tetrabutylammonium peaks. (C) ¹H-NMR characterization of alginate-b-PEG₁₁₂-b-PLA and full consumption of tetrazine and TCO was observed (DMSO-d6, 500 MHz) Peaks marked with * are tetrabutylammonium peaks. The designated peaks for PEG and PLA domains were marked and integrations were marked in red.

Figure 2.

Size and morphology characterizations of mosaic triblock nanoparticles. (A) The size distribution of Alginate-b-PEG₁₁₂-b-PLA₅₀ aggregates measured by DLS (Ba²⁺: 1 mM). (B) The size distribution of Alginate-b-PEG₁₁₂-b-PLA₇₅ NPs measured by DLS (Ba²⁺: 1 mM). (C) The size distribution of Alginate-b-PEG₁₁₂-b-PLA₁₅₀ NPs measured by DLS (Ba²⁺: 1 mM). (D) The morphology of Alginate-b-PEG₁₁₂-b-PLA₅₀ aggregates observed by Cryo-TEM (Ba²⁺: 1 mM) and no discernible nanoparticles were observed. (E) The morphology of Alginate-b-PEG₁₁₂-b-PLA₇₅ NPs observed by Cryo-TEM (Ba²⁺: 1 mM, scale bar: 200 nm) and discrete alginate domains were identified on the surface of nanoparticles. (F) The morphology of Alginate-b-PEG₁₁₂-b-PLA₁₅₀ NPs observed by Cryo-TEM (Ba²⁺: 1 mM, scale bar: 40 nm) and discrete alginate domains were identified on the surface of nanoparticles. Supporting Information for negative staining TEM characterizations of Alginate-b-PEG₁₁₂-b-PLA₅₀ aggregates, Alginate-b-PEG₁₁₂-b-PLA₇₅, and Alginate-b-PEG₁₁₂-b-PLA₁₅₀ NPs (Figure S17).

Figure 3.

Encapsulation efficiency of different payloads in PLA₁₅₀-domain mosaic nanoparticles and control PEG-b-PLA and alginate-b-PLA diblock copolymer nanoparticles. A 1.9–5.3 fold increase in encapsulation was observed for mosaic nanoparticles compared to corresponding PLA-b-PEG nanoparticles among payloads. In all cases, data are represented as mean ± SD (n=3) and **: p < 0.01. Supporting Information for encapsulation efficiency of different payloads in PLA₇₅-based mosaic and diblock copolymer nanoparticles (Figure S20). (LogP values of selected payloads are from PubChem database)

Figure 4.

Payload release from mosaic alginate-b-PEG-b-PLA triblock copolymer NPs (37 °C in 1· PBS): (A) 48-hr release profile of rhodamine B and coumarin 6 from PLA₇₅-domain mosaic NPs. 29.3% of coumarin 6 and 83.1% of rhodamine B were released at 48 hrs. (B) 48-hr release profile rhodamine B and coumarin 6 from PLA₁₅₀-domain mosaic NPs. 24.5% of coumarin 6 and 64.5% of rhodamine B were released at 48 hrs. (C) 48-hr release profile of therapeutic small molecules from PLA₇₅-domain mosaic NPs. 40.6%−70.5% of release was observed among payloads at 48 hrs. (D) 48-hr release profile of therapeutic small molecules from PLA₁₅₀-domain mosaic NPs. 29.2%−58.5% release was observed among payloads at 48 hrs. (E) 168-hr co-release profile of rhodamine B and coumarin 6 from PLA₇₅-domain mosaic NPs. 29.3% of coumarin 6 and 83.1% of rhodamine B were released at 168 hrs. (F) 168-hr co-release profile of rhodamine B and coumarin 6 from PLA₁₅₀-domain mosaic NPs. 33.1% of coumarin 6 and 81.1% of rhodamine B were released at 168 hrs. (G) 168-hr co-release profile of azathioprine and irinotecan from PLA₇₅-domain mosaic NPs. 47.1% of irinotecan and 76.8% of azathioprine were released at 168 hrs. (H) 168-hr co-release profile of azathioprine and irinotecan from PLA₁₅₀-domain mosaic NPs. 39.0% of irinotecan and 67.0% of azathioprine were released at 168 hrs. (I) 168-hr co-release profile of doxorubicin and erlotinib from PLA₇₅-domain mosaic NPs. 57.9% of erlotinib and 67.3% of doxorubicin were released at 168 hrs. (J) 168-hr co-release profile of doxorubicin and erlotinib from PLA₁₅₀-domain mosaic NPs. 45.8% of erlotinib and 58.8% of doxorubicin were released at 168 hrs. In all cases, data are represented as mean ± SD (n=3).

Figure 5.

Cellular uptake of rhodamine and rhodamine encapsulated in mosaic triblock nanoparticles in HeLa cells and anti-proliferation study of doxorubicin/erlotinib polymeric mosaic nanoparticles against A549: (A) Untreated cells. (B) Free rhodamine treated cells. (C) Alginate-b-PEG₁₁₂-b-PLA₇₅ NPs treated cells. (D) Alginate-b-PEG₁₁₂-b-PLA₁₅₀ NPs treated cells. Dark blue is the cell membrane, cyan is the nucleus, and red is rhodamine. (E) Flow cytometry analysis of cells treated with free rhodamine or rhodamine encapsulated nanoparticles. Compared to free rhodamine B treated cells, almost 100% of cells were labeled for both PLA₇₅ and PLA₁₅₀-based mosaic NPs. (F) 24-hour cell viability study treated with both mosaic NPs and triblock copolymer and quantified by CellTiter Glo 2.0. Mosaic NPs were biocompatible with 91.2%−100% cell viability from 25–100 μg/ml and there was no statistical significance between 100 μg/ml of each treatment group and untreated cells (p > 0.05). (G) 36-hour anti-proliferation study of doxorubicin (10 μM), erlotinib (10 μM) and doxorubicin (10 μM )/erlotinib (10 μM) dual loaded PLA₁₅₀ mosaic nanoparticles. The co-delivery system results in 13.6% cell viability in 36 hours, which is statistically significant compared to small molecules. In all cases, data are represented as mean ± SD (n=3). *: p<0.05 and **: p < 0.01.