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

Traditional drug delivery systems face challenges in delivering hydrophilic small molecule therapeutics effectively. These challenges often result in short durations of action and increased systemic toxicity due to rapid drug release. This research explores a novel approach leveraging the unique properties of DNA aptamers to overcome these limitations. Aptamers, single-stranded oligonucleotides, possess high affinity and specificity for their target molecules, rivaling antibodies, without the associated immunogenicity or toxicity. Their synthetic nature ensures no sequence homology to known genetic sequences. These characteristics make aptamers ideal candidates for drug delivery applications. Previous studies have used aptamers as targeting ligands in systemic drug delivery or exploited non-specific interactions between drugs and aptamers for drug loading. However, a system relying on specific drug-aptamer binding for controlled release has not been thoroughly investigated. This study hypothesizes that aptamers' specific binding ability can create effective depot drug delivery systems (DDS), prolonging drug release and minimizing systemic toxicity. The researchers chose to test this hypothesis using local anesthesia with site 1 sodium channel blockers (S1SCBs), specifically tetrodotoxin (TTX) and saxitoxin (STX), as a model. S1SCBs are potent local anesthetics, but their short duration of effect and significant systemic toxicity limit their clinical use. The hydrophilic nature of S1SCBs makes encapsulation challenging. The researchers believed that non-covalent aptamer/S1SCB complexes could offer a solution by prolonging the duration of local anesthesia while mitigating systemic toxicity.

The existing literature extensively documents the use of aptamers in various applications, including diagnostics, biosensors, affinity isolation, biomarker discovery, and targeted therapeutics. Their high affinity and specificity for target molecules rival that of antibodies, and they exhibit limited immunogenicity and toxicity. Aptamers have been employed as targeting ligands in systemic drug delivery systems. A previous study described an aptamer-based systemic drug delivery system utilizing the non-specific interaction of drugs with DNA; however, this approach is limited to drugs capable of non-specific interactions with aptamers. The current research aims to address this limitation by focusing on specific drug-aptamer binding for sustained release, potentially expanding the range of applicable therapeutics.

The study employed a TTX-binding aptamer (5′-AAAAATTTCACACGGGTGCCTCGGCTGTCC-3′) modified with phosphorothioate (PS) to enhance nuclease resistance. The binding affinity of the PS-modified aptamer to TTX was assessed using microscale thermophoresis (MST), revealing a significant improvement in binding affinity compared to the unmodified aptamer. A scrambled sequence control aptamer showed minimal binding, confirming sequence-specific interaction. To evaluate sustained release, TTX was complexed with PO and PS aptamers at different molar ratios (2:1 and 20:1). Dynamic light scattering (DLS) confirmed that complexation did not induce aptamer aggregation. In vitro release kinetics were studied using dialysis, with TTX concentration quantified using ELISA. Cytotoxicity of aptamer/TTX complexes was assessed in C2C12 (myoblast) and PC12 (pheochromocytoma) cell lines using the MTS assay. In vivo studies examined the efficacy of aptamer/TTX complexes in rat sciatic nerve blockade. Rats were injected with free TTX, or aptamer/TTX complexes, and the duration of sensory and motor nerve block was measured using a modified hotplate test and weight-bearing test. The specificity of the aptamer was assessed by comparing the duration of nerve block with TTX, saxitoxin (STX), and bupivacaine. The optimal aptamer:TTX ratio was determined by testing different molar ratios. Dose-response studies explored the effects of varying TTX concentrations. The combination of PS/TTX with epinephrine was investigated to further enhance the duration of nerve block. In vivo imaging (IVIS) and confocal microscopy tracked tissue retention of Cy5.5-labeled aptamers. Histology (H&E and toluidine blue staining) assessed tissue reactions to free TTX, PS aptamer, and PS/TTX complexes. Similar in vivo and histological studies were conducted using STX and a PS aptamer specific to STX.

Phosphorothioate modification enhanced the binding affinity of the TTX-binding aptamer by a factor of 2. Complexation of TTX with aptamers significantly prolonged TTX release in vitro, with PS-modified aptamers showing slower release compared to PO-modified aptamers. In vivo, aptamer/TTX complexes significantly prolonged the duration of sciatic nerve block in rats compared to free TTX. The PS/TTX complex increased the duration of nerve block by 7.7-fold compared to free TTX. The effect was specific to TTX, with minimal impact on the duration of nerve block induced by STX or bupivacaine. The optimal aptamer:TTX molar ratio was determined to be 2:1. Dose-response studies showed that aptamer/TTX complexes significantly prolonged nerve block and reduced systemic toxicity at all doses tested, even at doses where free TTX was lethal. Co-administration of epinephrine further prolonged the duration of nerve block with PS/TTX, enabling the use of higher TTX concentrations without causing mortality. In vivo imaging demonstrated significantly longer tissue retention of PS aptamers compared to PO aptamers. Histology showed minimal inflammation and no myotoxicity or neurotoxicity associated with PS/TTX complexes. Similar results showing prolonged nerve block and reduced toxicity were observed using a PS aptamer specific to STX.

This research successfully demonstrated the use of drug-specific DNA aptamers as effective depot-type drug delivery systems for small hydrophilic molecules. The aptamer/drug complexes provided prolonged drug release, significantly extending the duration of local anesthesia and reducing systemic toxicity. The approach offers several advantages: simplicity of synthesis and modification of aptamers, ease of drug loading (simple mixing), and specific drug-aptamer interaction ensuring controlled release. This method could be broadly applicable to various therapeutics, provided specific aptamers can be developed. The study primarily focused on local anesthesia, but the technology has potential for other applications requiring depot drug delivery. The phosphorothioate (PS) modification played a key role in improving aptamer stability, binding affinity, and tissue retention. The unexpected decrease in nerve block duration at a high aptamer:TTX ratio (40:1) requires further investigation. However, the overall benign tissue reaction of the PS aptamers suggests good biocompatibility.

This study provides compelling proof-of-concept for using drug-specific DNA aptamers as drug delivery systems. The method is simple, safe, and effective, particularly for small-molecule drugs. Future research should explore the optimization of aptamer design, the range of applicable drugs, and the potential of this technology for systemic administration.

The study primarily focused on a rat model for local anesthesia. Extrapolation to humans requires further investigation. The unexpected reduction in efficacy at the highest aptamer:TTX ratio needs further study to elucidate the underlying mechanism. While the study demonstrated excellent biocompatibility in the rat model, further exploration is needed to confirm the long-term safety profile in various tissues and species.