Novel Bio-Hybrid Nanoscale Carriers Engineered for High Therapeutic Payload and Controllable Extended Release Using Nucleic Acid Aptamers
Mohana Sundaram, Padma Priya
Type of Degreedissertation
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Controlled delivery of therapeutics to target tissues constitutes a great challenge of modern medicine, especially in the field of cancer treatment where malignant cells have to be killed without damaging the surrounding healthy cells. A drug delivery carrier capable of releasing drug in a controlled manner for an extended period of time would greatly impact the field of medicine. Such a carrier has to be small in size, non-toxic, and it should not cause any immune response. Recently, gold nanoparticles (AuNps) and nucleic acids were found to be potential tools for drug delivery. AuNps have been studied as drug delivery platforms because of their non-toxic gold core, ease of preparation of mono-dispersed particles, ease of functionalization of small molecules, and efficient cellular uptake. Nucleic acid aptamers are emerging as important drug targets and versatile therapeutic agents due to their ability to fold into complex three-dimensional structures and bind therapeutically important small molecules. A number of studies demonstrated that nucleic acids constitute attractive materials for nanotechnology, as these macromolecules can be easily programmed to carry out specific functions. Our strategy is to exploit the nucleic acid’s response to different physical and chemical triggers such as temperature and binding affinity to produce controlled and extended drug release. In this work, we have developed gold nanoparticles and DNA-based drug delivery nano-carriers which utilizes the versatile properties of nucleic acids for controlled and extended release of drugs. The drug delivery carrier consists of 15 nm gold nanoparticles (AuNps) functionalized with drug binding DNA aptamers via a single stranded (ss) anchor DNA. The presence of ssDNA makes the nano-carrier flexible to be reprogrammed for aptamers that either bind different drugs or display different affinities for the same drug. Under the optimum binding conditions (0.4 M NaCl and 4 M DNA), a maximum of 101±8 anchor DNA strands were conjugated per particle. Changes in number density influenced the mean diameter of DNA-conjugated AuNps which was determined using dynamic light scattering. The mean diameter of DNA-conjugated AuNps was also affected by conformational changes of DNA strands. Our studies revealed that short DNA strands exist in a stretched conformation while longer DNA strands adopt coiled conformation when functionalized on AuNps. For the first time, we demonstrated that neomycin could bind to DNA aptamer with very high affinity (Kd= 98.101 nM). The nano-carrier for neomycin was constructed by binding DNA-aptamer:drug complexes to the anchor DNA functionalized AuNps. Controlled and extended release of drug from the synthesized carrier was obtained by temperature and affinity modulations. The nano-carrier when modified for the anticancer drug daunomycin, demonstrated very high loading of 1157 ± 18 drug molecule per particle. The hydrodynamic size of the nano-carrier was found to be 41 ± 2 nm. This size of the nano-carrier makes it suitable for passive targeting of drug to cancer cells. The in-vitro release of daunomycin at 37oC demonstrated controlled release of drug from the nano-carrier for an extended period of time. After 6 days of release only about 75% of the bound daunomycin was released from the nano-carrier which implies that the release can be extended much further. Furthermore, the physiological relevance of the nano-carrier was demonstrated by testing the cell viability of MCF7 breast cancers cells after incubating them with the drug loaded nano-carrier. The nano-carriers displayed excellent cellular uptake, were found to be non-toxic, and invoked cell death which was observed to be proportional to the concentration of the daunomycin loaded nano-carrier. At any tested concentration, cell death by the daunomycin loaded nano-carrier was higher or equal to the cell death induced by the same concentration of free daunomycin. This nano-carrier with a very high payload and efficient drug delivery is a potentially powerful, yet flexible, tool for fighting cancer. Being versatile to be modified for any drug, it is expected to impact a number of treatment strategies.
- Padma Sundaram_ Dissertation_May 2014.pdf