An aptamer-based depot system for sustained release of small molecule therapeutics

The study addresses the challenge of delivering small, hydrophilic molecules using traditional drug delivery systems, which often suffer from poor encapsulation and burst release. Aptamers—single-stranded oligonucleotides with high affinity and specificity for small molecules—offer a potential solution due to their tunable sequences, low immunogenicity, and lack of homology to biological nucleic acids. The authors hypothesized that using drug-specific aptamer binding could create depot-type drug delivery systems that prolong local drug release and reduce systemic toxicity, even if tissue targeting by the aptamer is sacrificed. They chose local anesthesia with ultra-potent, hydrophilic site 1 sodium channel blockers (S1SCBs) TTX and STX as the testbed, where prolonged effect is desirable and rapid systemic release poses toxicity risks.

Aptamers rival antibodies in affinity and specificity and have seen wide use in diagnostics, biosensing, affinity isolation, biomarker discovery, and targeted therapeutics. Prior drug delivery efforts often used aptamers as targeting ligands on systemically delivered carriers. An aptamer-based system loading drugs via nonspecific interactions with DNA has been reported, but such approaches are limited to drugs with suitable nonspecific interactions (e.g., charge, hydrophobicity). More generalizable delivery could be achieved if specific drug–aptamer binding underpins loading and release. For local anesthetic delivery of hydrophilic S1SCBs, prior strategies include electrostatic interactions, covalent tethering to polymers, and supramolecular systems to limit burst release and toxicity, each with varying complexity and efficacy.

- Aptamer design and synthesis: A known high-affinity TTX-binding aptamer (5′-AAAAATTTCACACGGGTGCCTCGGCTGTCC-3′) was synthesized in phosphodiester (PO) and phosphorothioate (PS) backbone forms to enhance nuclease resistance. Dye-labeled variants (Cy5/Cy5.5) were prepared for imaging. All aptamers were synthesized by solid-phase phosphoramidite chemistry, purified by RP-HPLC, and confirmed by MALDI-TOF MS. - Binding affinity: Microscale thermophoresis (MST) assessed TTX binding to Cy5-labeled aptamers, comparing PO, PS, and a scrambled PS control (Scr-PS). Apparent binding constants were derived from titrations (76.3 nM–2.5 mM TTX). - Complex formation and in vitro release: Aptamer/TTX mixtures were prepared by mixing at defined molar ratios (1:1 to 40:1; typical 2:1 and 20:1) and annealed. Particle size was measured by DLS. Release kinetics were evaluated by dialysis (200 µL sample with 42 µM TTX vs 14 mL PBS, 37 °C) with ELISA quantification of TTX. - Cytotoxicity: MTS assays in C2C12 myoblasts and PC12 pheochromocytoma cells evaluated viability after 24 h exposure to free TTX, aptamers, and aptamer/TTX complexes at relevant concentrations. - In vivo efficacy and safety: Male Sprague-Dawley rats received perisciatic injections (0.3 mL) of free drug or aptamer/drug complexes. Sensory block was measured by hotplate thermal latency; motor block by weight-bearing. Contralateral deficits indicated systemic exposure. Dose–response studies varied TTX concentration and aptamer:drug ratio. Specificity controls included Scr-PS/TTX, combining TTX-specific PS aptamer with STX or with bupivacaine. Epinephrine (55 µM) co-injection tested pharmacological retention. - Tissue distribution: IVIS imaging tracked retention of Cy5.5-labeled PO and PS aptamers vs free dye at the injection site up to 72 h; confocal microscopy localized fluorescence in cryosections at 4 h. - Histology: H&E (inflammation, myotoxicity) and toluidine blue (neurotoxicity) assessed tissue response at days 4 and 14 after injection. Blinded scoring of inflammation and myotoxicity was performed. - Generality with STX: A reported STX-specific PS aptamer (PSAPSTX) was complexed with STX (2:1) and tested in the same in vitro toxicity and in vivo nerve block assays, including dose escalation and histology.

- Binding and release: - PS modification improved TTX aptamer binding approximately 2-fold: apparent affinity improved from 29.5 ± 2.1 µM (PO) to 14.3 ± 1.7 µM (PS). Scrambled PS showed weak binding (3.84 mM). - DLS indicated ~3 nm complexes without aggregation. - Dialysis release: Both PO/TTX and PS/TTX (2:1 or 20:1) slowed TTX release vs free drug (p < 0.0001 at 24 h). PS/TTX released significantly slower than PO/TTX at 12 h (p < 0.0001) and 24 h (p = 0.026). Scr-PS/TTX released TTX rapidly, similar to free TTX (92.8 ± 3.6% and 94.4 ± 3.8% released by 12 h for 2:1 and 20:1, respectively). - Cytotoxicity: >90% cell viability in C2C12 and PC12 for all formulations tested. - In vivo sciatic nerve block with TTX: - At 42 µM TTX: free TTX produced 0.9 ± 0.6 h block; PO/TTX (20:1) 1.9 ± 0.8 h; PS/TTX (20:1) 7.1 ± 2.4 h (p = 0.0001 vs TTX; p = 0.0033 vs PO/TTX). Aptamers alone caused no block. - Specificity: Scr-PS/TTX (20:1) yielded only 2.1 ± 0.8 h (p = 0.0207 vs TTX; far less than PS/TTX). TTX-specific PS aptamer did not prolong block from STX (2.2 ± 0.4 h vs 2.0 ± 0.4 h; p = 0.49) or from bupivacaine (3.9 ± 0.4 h vs 3.4 ± 0.4 h; p = 0.0611). - Ratio optimization (constant 42 µM TTX): 1:1 gave 0.3 ± 0.3 h (similar to free); 2:1–20:1 yielded prolonged blocks (5.4–7.1 h) without significant differences; 40:1 reduced duration to 2.6 ± 0.4 h. - Dose–response (PS/TTX 2:1): Blocks were longer at all doses vs free TTX. Notably, 31 µM free TTX caused no block, whereas PS/TTX gave 1.9 ± 0.9 h (100% success). Free TTX at 63 µM was uniformly fatal; no deaths occurred at any PS/TTX dose. Contralateral deficits (systemic toxicity) were markedly reduced with PS/TTX. Motor block lasted ~1.3× longer than sensory block (p < 0.05). - Epinephrine co-injection (55 µM) with PS/TTX (2:1) allowed safe delivery of up to 104 µM TTX with 22.0 ± 2.8 h block and no mortality; contralateral latency markedly reduced vs without epinephrine. - Tissue retention and localization: - IVIS: Free Cy5.5 cleared rapidly (<20% at 4 h). ~37% of PO aptamer remained at 4 h; ~50% of PS aptamer remained at 24 h, significantly greater retention for PS at all time points. - Confocal imaging: PS aptamer signal ~4-fold higher than PO in tissues surrounding the nerve at 4 h; localization in connective tissue near nerve, not within the nerve. - Histology and safety: - TTX alone caused very mild inflammation; PS and PS/TTX caused mixed inflammation at day 4 (scores 2–3), diminishing by day 14 (scores 1–2); no myotoxicity or neurotoxicity observed in any group. - Generality with STX using PSAPSTX: - Free STX (33 µM): 2.6 ± 0.5 h block with contralateral deficits (2.3 ± 0.9 h). - PSAPSTX/STX (2:1, 33 µM): 6.9 ± 0.8 h block (2.6-fold increase; p < 0.0001), no contralateral deficits. At 45 µM, 11.3 ± 0.7 h block without mortality, whereas free STX at the same dose was uniformly fatal. No cytotoxicity; histology similar to PS/TTX with no myotoxicity or neurotoxicity.

Forming non-covalent complexes between drugs and their specific DNA aptamers created depot-type delivery systems that controlled release of small, hydrophilic molecules, addressing a key limitation of traditional carriers. Phosphorothioate modification improved aptamer binding to TTX, slowed in vitro release, enhanced in vivo efficacy (longer block), decreased systemic exposure, and permitted administration of doses that would otherwise be lethal. Co-administration of epinephrine further enhanced safety and duration by pharmacologically reducing vascular clearance from the injection site. Specificity studies showed that benefits are restricted to the matched drug–aptamer pair, underscoring that controlled release arises from specific molecular recognition rather than nonspecific interactions or viscosity effects. Improved tissue retention of PS aptamers likely reflects nuclease resistance and nonspecific protein interactions, which may aid depot function. The approach generalized to STX using an STX-specific PS aptamer, supporting broader applicability to other small molecules for which aptamers can be developed.

This work provides proof-of-concept that drug-specific DNA aptamers, especially with phosphorothioate backbones, can function as simple, scalable depot delivery systems for small, hydrophilic therapeutics. For TTX and STX, aptamer complexes prolonged local anesthetic duration, reduced systemic toxicity, and enabled higher, otherwise lethal doses to be administered safely; epinephrine co-administration further extended duration. The platform is highly specific: each aptamer-based DDS is effective only for its cognate drug. Given the ease of synthesis and modification, this strategy could be extended to other therapeutics where depot delivery is desired, including local or potentially systemic applications. Future work could optimize aptamer sequences and chemistries to further tune binding/release, evaluate long-term safety and immunogenicity, and explore additional drug classes and clinical indications such as postoperative pain control.

- Specificity constraint: Each aptamer DDS benefits only the matched ligand; this limits broad applicability without bespoke aptamer development for each drug. - High aptamer ratio effect: An unexpected reduction in efficacy at a 40:1 PS:TTX ratio suggests possible steric hindrance or inter-aptamer interactions; underlying mechanisms remain unclear. - Targeting trade-off: Using specific binding for drug loading may preclude using the same aptamer for tissue targeting in systemic delivery. - Model scope: Efficacy and safety were demonstrated in a rat sciatic nerve block model; translation to other species, tissues, and systemic administration requires further validation. - Inflammation: Transient mixed inflammatory responses were observed with PS aptamer and PS/drug complexes, though they diminished by day 14; the clinical significance warrants further study. - Pharmacokinetics: While tissue retention of PS aptamers was prolonged, the relationship between aptamer residence time and pharmacodynamic effect duration was not fully resolved.