Aptamer-based self-assembled nanomicelle enables efficient and targeted drug delivery

The study addresses the instability of amphiphilic aptamer micelles upon interaction with lipophilic cell membranes, which can lead to micelle disassembly, aptamer leakage, and loss of specific targeting. Aptamers offer advantages over traditional ligands (antibodies, peptides, small molecules) including synthetic accessibility, modifiability, stability, and low immunogenicity, enabling construction of diverse DNA-based nanomaterials. However, amphiphilic DNA micelles often destabilize in physiological environments. Prior stabilization approaches include crosslinking of micelles (e.g., by Mirkin and Tan) or using polyvalent hydrophobic chains; these can be complex or limit applicability. The research question is whether a superstable, non-crosslinked aptamer-based amphiphilic copolymer micelle can be created to retain targeting specificity and enhance drug delivery efficacy against cancer cells. The study aims to build an amphiphilic polymer comprising a polyvalent hydrophobic backbone (C18 PMH) and multiple DNA aptamers (sgc8 targeting PTK7), hypothesizing improved stability, specificity, and therapeutic delivery.

Background covers aptamer capabilities to bind diverse targets and advantages over protein ligands. Amphiphilic DNA micelles can target cancer but destabilize in lipid bilayers, leading to nonspecific interactions. Stabilization strategies include crosslinking of DNA micelles (reported by Mirkin and Tan) and use of stable polyvalent hydrophobic chains conjugated with aptamers. These methods improve stability and targeting but often require additional steps or complex chemistries, limiting broad use. The study builds on this literature by proposing a non-crosslinked, amphiphilic copolymer micelle using C18 PMH to provide multivalency and stability while maintaining aptamer-based specificity.

• Design and synthesis: Conjugate the anhydride groups of poly(maleic anhydride-alt-1-octadecene) (C18 PMH) with amino-modified aptamer sgc8 (NH2-sgc8) to form an amphiphilic polymer (C18-sgc8) bearing multiple C18 chains and DNA strands in tandem. Purify by dialysis.
• Characterization: Negative-stain TEM and DLS to assess morphology and size; EDS P-mapping to confirm DNA incorporation; agarose gel electrophoresis to verify higher molecular weight versus free sgc8; determine critical micelle concentration (CMC ~0.01143 mg/mL). Stability assessed by PDI over 48 h (PDI < 0.2).
• Drug loading (in vitro): Load hydrophobic photosensitizer chlorin e6 (Ce6) into C18-sgc8 via hydrophobic interactions to form Ce6@C18-sgc8. Characterize size shift (to ~68.06 nm), UV–vis (DNA peak 260 nm; Ce6 Soret redshift to 416 nm), and fluorescence (emission ~660 nm). Similarly load doxorubicin (DOX) and paclitaxel (PTX) to form DOX@C18-sgc8 and PTX@C18-sgc8 and characterize by DLS and UV–vis (DOX peak ~480 nm; PTX peak ~230.5 nm). Determine loading efficiencies (Ce6 89.48%; PTX 86.36%).
• Targeting and cell binding: Use sgc8 aptamer (targets PTK7) to assess specificity to HeLa (PTK7-high) vs Ramos (PTK7-negative) cells via confocal microscopy and flow cytometry at 4 °C. Compare C18-sgc8 with lipid–sgc8 and cholesterol–sgc8 constructs.
• Cellular uptake and PDT-related assays: Incubate HeLa with Ce6@C18-sgc8 vs free Ce6 at 37 °C; image by confocal to monitor Ce6 uptake kinetics. Measure singlet oxygen generation using SOSG under laser irradiation in solution; assess intracellular ROS with DCFH-DA after light irradiation. Evaluate apoptosis using Annexin V-FITC/PI by flow cytometry and confocal microscopy.
• Cytotoxicity and IC50: Assess biocompatibility of empty C18-sgc8 (0–160 µg/mL) on HeLa via CCK-8. For drug-loaded micelles (DOX@C18-sgc8, PTX@C18-sgc8, Ce6@C18-sgc8), incubate cells (4 °C 2 h), wash, incubate in fresh medium 24 h, perform CCK-8 to calculate IC50. For Ce6 groups, apply 660 nm light (1 W/cm2, 10 min).
• In vivo evaluation: For cost-effective in vivo targeting to 4T1 tumors, prepare a PEGylated block copolymer by partially modifying C18 PMH with PEG and conjugating EpCAM aptamer to create C18-PEpCAM. Load Ce6 to form Ce6@C18-PEpCAM. In Balb/c mice bearing 4T1 tumors, compare biodistribution of free Ce6 vs Ce6@C18-PEpCAM by in vivo fluorescence imaging and ex vivo organ imaging at 6 h. PDT treatment at 6 h post-injection with 660 nm NIR light (100 mW/cm2, 10 min) on days 0, 3, and 6; monitor tumor growth, body weight; perform H&E histology of tumors and major organs.

• Formation and stability: C18-sgc8 formed spherical nanomicelles with average DLS size ~37.84 nm (Fig. 1c; caption also notes ~38.8 nm), uniform distribution, and PDI < 0.2 over 48 h. EDS P-mapping confirmed DNA incorporation. CMC ~0.01143 mg/mL, indicating stable micellization.
• Drug loading and characterization: Loading Ce6 increased micelle size to ~68.06 nm; UV–vis showed DNA 260 nm and Ce6 Soret peak redshift to 416 nm; fluorescence emission at 660 nm confirmed successful loading. DOX@C18-sgc8 and PTX@C18-sgc8 displayed increased sizes (~51.75 nm for DOX micelles in water; ~43.82–50.75 nm for PTX micelles) and characteristic absorption peaks (DOX 480 nm; PTX 230.5 nm). Maximum loading efficiencies: Ce6 89.48%, PTX 86.36%.
• Targeting specificity: C18-sgc8 bound selectively to HeLa (PTK7-high) but not Ramos cells, unlike lipid–sgc8 and cholesterol–sgc8 which bound nonspecifically to both cell types (confocal and flow cytometry).
• Cellular uptake: Ce6@C18-sgc8 rapidly entered HeLa cells showing strong intracellular fluorescence, whereas free Ce6 showed poor uptake.
• Photodynamic activity: SOSG assays showed time-dependent 1O2 generation under laser irradiation for both free Ce6 and Ce6@C18-sgc8. In cells, DCFH-DA indicated robust ROS generation after light only in the Ce6@C18-sgc8 group.
• Apoptosis induction: Annexin V-FITC/PI analysis showed massive apoptosis (>90%) in HeLa cells after light for both free Ce6 and Ce6@C18-sgc8; confocal imaging showed membrane Annexin V binding in Ce6@C18-sgc8-treated cells post-irradiation.
• Cytotoxicity and IC50: Empty C18-sgc8 exhibited negligible cytotoxicity up to 160 µg/mL. Drug-loaded micelles had significantly lower IC50 than free drugs: DOX@C18-sgc8 2.590 µg/mL vs free DOX 9.099 µg/mL; PTX@C18-sgc8 0.6317 µg/mL vs free PTX 1.607 µg/mL; Ce6@C18-sgc8 0.5104 µg/mL vs free Ce6 1.175 µg/mL under 660 nm irradiation (1 W/cm2, 10 min).
• In vivo targeting and therapy: In 4T1 tumor-bearing mice, Ce6@C18-PEpCAM showed significantly higher tumor accumulation than free Ce6 at 6 h (in vivo and ex vivo imaging). PDT (660 nm, 100 mW/cm2, 10 min on days 0, 3, 6) significantly delayed tumor growth, reduced final tumor weight, and induced extensive tumor cell necrosis (H&E), without abnormal changes in body weight or histopathology in major organs, indicating good biocompatibility.

The study demonstrates that integrating a polyvalent hydrophobic backbone (C18 PMH) with multiple aptamer strands creates superstable, non-crosslinked micelles that resist disassembly upon membrane interaction. This stability preserves aptamer presentation and multivalency, yielding high specificity toward target cells (HeLa via sgc8–PTK7), outperforming traditional lipid- or cholesterol-anchored DNA constructs that suffer nonspecific membrane insertion. Enhanced cellular uptake of payloads (e.g., Ce6) enables efficient intracellular delivery and robust photodynamic activity, as evidenced by normal singlet oxygen generation and high apoptosis rates upon irradiation. The platform’s versatility is underscored by efficient loading of multiple hydrophobic drugs (Ce6, DOX, PTX) with high loading efficiencies and significantly improved cytotoxicity relative to free drugs, likely due to receptor-mediated endocytosis and increased intracellular accumulation. In vivo, an EpCAM-targeted variant achieved superior tumor localization and PDT efficacy with favorable safety, supporting translational potential. Collectively, the findings address the central challenge of stability and specificity in aptamer micelles, offering a broadly applicable drug delivery platform for cancer therapy.

A self-assembling C18-sgc8 nanomicelle system was developed that forms stable, spherical micelles in aqueous conditions without crosslinking. The amphiphilic architecture imparts excellent stability and preserves aptamer-mediated targeting during cellular interactions, enabling efficient delivery of hydrophobic drugs to target cells. The platform successfully loads multiple therapeutics (Ce6, DOX, PTX) and enhances their in vitro cytotoxicity and in vivo PDT efficacy with good biocompatibility. These results position C18-sgc8 micelles as a promising universal nucleic acid nanoplatform for targeted delivery of hydrophobic drugs in biomedical applications.