Novel Approaches for Targeted Delivery of Cancer Therapeutics to Nuclei of Malignant Cells
The primary goal of this project is to increase specific uptake of anti-cancer therapeutics by nuclei of cancer cells. A lot of research has been done to target therapeutics specifically across plasma membrane of malignant cells. However not much research has been done to deliver them to their target cellular compartment where the drug elicits its pharmacological response. In case of majority of anti-cancer drugs the target is usually nuclei of cancer cells. Hence nuclear delivery of therapeutics is the next frontier of pharmaceutics. In this dissertation work this issue has been investigated. Initially in the first strategy adopted, novel peptide prodrug of doxorubicin was developed which may evade over-expressed efflux pumps on breast cancer cells. This approach may lead to increased uptake and higher drug accumulation in nuclei of cancer cells. L-val-L-val doxorubicin prodrug was synthesized following standard f-moc chemistry. The prodrug was analyzed for stability, cellular and nuclear uptake and interaction with efflux and peptide transporters. Breast cancer cells (T-47D) were grown on polystyrene 12-well plates. The prodrug Val-Val-doxorubicin was found to be very stable in breast cancer cell homogenate. It was able to evade efflux pumps. The prodrug penetrated cytoplasm and nuclei of cancer cells by interacting with peptide transporters over-expressed on plasma and nuclear membrane of T-47D.Uptake of prodrug was found to be 10 fold higher than parent drug. Peptide prodrug derivatization of doxorubicin has potential to evade efflux pumps and increase availability and nuclear accumulation of doxorubcin in breast cancer cells. However due to its stability the prodrug did not biorevert to its parent drug in therapeutic concentration. Hence alternative approaches were investigated. As part of this alternate approach novel nuclear localization signal (NLS) peptide analogues have been designed that can carry therapeutic molecules specifically into nuclei of cancer cells. This strategy might be able to reduce toxicity to non-malignant cells by delivering anticancer drugs to subcellular organelle i.e. nucleus of cancer cells preferentially. Native NLS peptide-conjugated drugs can reach nucleus of any cell non-specifically. However the overarching challenge is to enhance drug uptake across plasma and nuclear membranes to enhance anticancer drug delivery in drug resistant cancer cells. Adenoviral fiber protein (AFP) that encodes for NLS has been known to penetrate both plasma and nuclear membrane nonspecifically. Therefore novel NLS peptide analogues have been synthesized by substitutions of NLS sequence specific amino acids for targeting cancer cells primarily. Specific amino acids of native NLS have been substituted based on hydrophobic interactions between the peptides with plasma membrane and nucleo pore complex (NPC) that can influence cytoplasmic and nuclear transport. These peptides can carry therapeutics selectively to nuclei of cancer cells simultaneously evading normal cells. Five NLS peptides have been synthesized. These peptides were synthesized by AAPtech automated peptide synthesizer. Following synthesis, the peptides were purified by a shimadzu Preparative HPLC which was confirmed by HPLC-MSMS. Confocal and quantitative uptake studies with various cancer and corresponding non-cancerous human cell lines were performed. It was observed that two (NLS3 and NLS5) peptides are specific for targeting cancer cell nucleus. It has led to an unequivocal conclusion that these novel peptide analogues can selectively bind to plasma membranes of cancer cells and target NPC simultaneously. Following screening of peptides which can function as targeting moieties, nanoparticle formulation was developed to deliver anti-cancer drugs. One of the important parameters for nuclear drug delivery is size. The nucleopore complex has a diameter of 30 nm. Cargo with 30-40 nm can enter the NPC passively. However particles higher than 40 nm need to be actively transported across NPC. It has been reported that particles with 60-70 nm with anti cancer drugs can be delivered into nucleus with localization signals. This is the target size which has been achieved for doxorubicin loaded nanoparticle. PLGA-PEG-NH2 was used as polymer for preparation of doxorubicin loaded nanoparticles. This is because of availability of NH2 group at the end of PEG group for conjugation of peptide moiety for targeted delivery following optimization of size. Nanoparticles were prepared by nanoprecipitation method. Parameters like effect of solvent, polymer concentration, effect of aqueous phase volume, drug concentration were optimized to yield a nanoparticle of size range 60-70 nm as this size can be taken up by nucleus if actively aided by nuclear localization signal. Following preparation of nanoparticle of optimized size, screened peptides (NLS3 and NLS5) with maximum affinity for cancer cell nuclei with minimal entry into corresponding non-malignant cells were conjugated to nanoparticles. Targeted nanoparticles were investigated for its active targeting property in a 3D model of breast cancer. It was observed that nanoparticle with NLS conjugated to its surface delivered higher concentration of doxorubicin compared to unconjugated nanoparticle or free doxorubicin.
Table of Contents
Literature review -- Rationale for investigation -- Prodrug approach for nuclear delivery of doxorubicin -- NLS peptide synthesis and purification -- Uptake of synthesized peptides in different cell lines -- Optimization of parameters with respect to size for formulation development -- Efficacy and mechanism of targeted delivery system -- Summary and Recommendations