Design and Synthesis of New Delivery Vehicles for Anti- Cancer Therapeutics

Abstract

The thesis entitled ‘‘Design and Synthesis of New Delivery Vehicles for Anti-Cancer Therapeutics’’ encloses the syntheses, characterization and bioactivity determination of the novel systems designed for achieving high efficacy and targeted therapeutic effects in cancer cells. Cancer is the most serious disease that causes of death of millions of people every year across the world. Reasonably good progress has been observed in the basic understanding of cancer biology, leading to improved treatment and diagnostic methods. The latest approach of treatment of cancer is the targeted delivery of drug/siRNA via nanocarriers to the tumor site effectively and efficiently. In spite of such developments there is continuing need for newer design of efficacious agents towards this end. The following chapters of this thesis present self assembled natural peptide having the ability to deliver the anticancer drug DOX to colon cancer environment. Peptide-based AuNPs (gold nanoparticles) have been designed to deliver the anticancer drug DOX selectivity in CD44 expressive cancer cell lines. A set of unique non-viral vectors of natural origin has been synthesized to deliver the reporter gene in hepatic cancer cell line selectively followed by RNAi therapy has incorporated to achieve the successful antitumor activity in hepatic cells by knocking down the anti-apoptotic protein survivin. Chapter 1: Current Strategies towards Targeted Delivery of Cancer Therapeutics A worldwide rapidly growing cancer incidence and its mortality attract the researchers to develop effective cancer therapy. However, conventional cancer therapy is unsatisfactory because of serious side effects on normal cells of the healthy organ along with the tumor. Considering these drawback, researchers have developed effective approach involving nanobiotechnology, to carry therapeutic agents to the tumor site by secure encapsulation without leakage. Development of new methodologies for therapeutic agents’ delivery and the consequent cancer therapy is at the forefront of nanobiotechnology research. Over the past few decades, a steep rise in cancer therapeutics has observed in targeted delivery approaches. Therapeutic agents’ delivery systems popularly known as nano-carriers are basic tools of targeted delivery approaches. The well documented therapeutic approach is targeted delivery of anticancer drugs by nano-carriers that are sensitive towards the tumor microenvironment (TME) and thus improving the safety, pharmacokinetic profiles, and bioavailability of locally or systemically administered drugs, leading to improved therapeutic efficacy. Another rapidly growing personalized field of cancer treatment is gene silencing by RNA interference iii (RNAi). The technique is to switch off the specific cancer genes by the use of small interfering RNAs (siRNAs) and hence stop the synthesis of corresponding diseased protein. SiRNA based therapies are limited in clinical practice due to its nuclease degradation and hence a targeted delivery system is required that protects RNA from degradation and effective release in the cytoplasm of target cancer cells without any adverse effects. Here we provide insight into various nano-carrier formulations that are in research and in clinical trials keeping the focus on chemotherapeutic drug delivery systems and targeted siRNA delivery systems. The use of various nanoparticles for siRNA/drug delivery, including cationic liposomes, gold nanoparticles and self-assembled peptides for targeted delivery in cancer are discussed. Chapter 2: Efficacious delivery of chemotherapeutics targeted for colon cancer mediated by dual stimuli responsive self-assembly of a natural tetrapeptide Efficient delivery of chemotherapeutics, specifically for colorectal cancer, draws a significant attention of the pharmacologists in the current days, particularly due to the unique physiological parameters pertaining to the colon those include factors such as pH, microflora activation etc. Scientists are therefore interested in designing delivery vehicles with different structural backbones those respond selectively at the colon specific environment. We have herein developed a new nanovesicular system generated from the naturally conserved tetra-peptide sequence, Lys-Tyr-Trp-Cys (KYWC) and its oxidized derivative, KYWCCWYK. These nanovesicles were found to be quite sensitive towards the pH of the media, where they were stable at lower pH but ruptures rapidly at physiological pH ~7.4. The nanovesicles were then loaded with the anticancer drug, doxorubicin (DOX) which showed a sustained drug release profile with response to the pH ~7.4 and reducing environment of the media. It was observed that the cellular internalization of DOX was further enhanced leading to decreased cellular viability of the colon carcinoma (CT-26) cells when they were treated with the DOX loaded octapeptide, KYWCCWYK, system compared to the drug alone. The observations were supported by the in vitro data obtained from the stomach gastric carcinoma (AGS) cell lines. Overall the present study detailed the efficacious delivery of DOX specifically for the treatment of colorectal cancer arbitrated by the multi-stimuli responsive self-assembly of a biologically conserved short peptide sequence.

Chapter 3: Hyaluronic Acid coated pH-Responsive Natural Tetrapeptide Capped Gold Nanoparticles for the Targeted Delivery of Doxorubicin to the CD44 Expressing Cell Lines Targeted drug-loaded nanoparticles have attracted a lot of attention because of their selective, controlled and effective delivery of therapeutics to the specific organs or cells, leading to the enhancement of its antitumor efficacy. Herein we reported the hyaluronic acid (HA)-coated natural tetrapeptide, Lys-Tyr-Trp-Cys (KYWC), capped gold nanoparticles for selective delivery of doxorubicin (DOX) to the CD44 expressing tumor cells, e.g. B16F10 and A549 cells. Our observations prompted that HA-coated tetrapeptide KYWC-AuNPs (HA-KYWCAuNPs) improved the drug localization effectively to the tumor cells having CD44- overexpression. HA-KYWC-AuNPs displayed sturdy inhibition rates and persuade evident apoptosis in CD44-overexpressed A549 and B16F10 cells. However, a fall of its efficacy was observed in less expressed CD44 cell line, HepG2. Thus a new biodegradable HA-KYWCAuNPs were developed that carried a chemotherapeutic drug (DOX) and improved its potential via CD44 receptors of cancer cells. Chapter 4: Assessment of Delivery of nucleic acids using Ferrocene-tocopherol lipid conjugates Chapter 4A: (±)α-Tocopherol–Ferrocene Conjugated Lipids for the Specific Delivery of Transgene in Liver Cancer Cells at High Serum Conditions Delivery of therapeutic genes to a specific organ has drawn major attention to researchers in the last two decades. Various types of non-viral delivery vectors including cationic liposomes are potential candidates to deliver therapeutic genes due to their low immunogenic response. Here, we have developed ferrocene conjugated cationic tocopheryl liposome as a non-viral vector which can deliver a reporter gene (pGL3; encoded for luciferase protein) specifically to liver cancer cells (HepG2 and Huh7), compared to non-hepatic cells such as CaCO2 (Human epithelial cells) and HeLa (cervical cancer cells). The transfection efficiency of these liposomal formulations are greater than that of commercially available L2K (Lipofectamine KYWC-AuNPs-DOX KYWC-AuNPs HA-KYWC-AuNPs-DOX DOX loading at pH7.4 Hyaluronic Acid =HA Drug release at pH ~ 5 B16F10 A549 HepG2 0 50 100 DOX AuNP-DOX HA-AuNP-DOX %DOX Positive Cells In vitro study in CD44 more expressive and less expressive cell lines vi 2000) and also, the efficiency are retained in high serum conditions (up to 50% FBS). The combination of both ferrocene and tocopherol moieties are supposed to be the main contributing factors for their particular cells-specific delivery. Chapter 4B: Silencing of a specific gene by means of RNAi (RNA interference) recently emerges as promising cancer therapeutic tool. Small double-stranded RNA (ds-RNA) has been exploited for successful sequence specific gene silencing. Cationic liposome frequently used in nonviral delivery vehicle, an attractive agent for safe and efficient delivery of siRNA. Herein we have established in vitro delivery of siRNA against survivin (an anti-apoptotic protein, which induced substantial apoptosis) by three new tocopherol-ferrocene based cationic lipids in HepG2 cells. Gene knockdown efficiency was investigated by the co-liposomes derived from cationic lipids and a naturally occurring helper lipid Lα-dioleoyl phosphatidyl ethanolamine (DOPE). All optimised coliposomal formulations were well characterized by various physical methods like dynamic light scattering (DLS), zeta potential measurements and atomic force microscopy etc. The efficiency of transfection was checked by flow cytometry on analysis of CY5 labelled siRNA. The gene knockdown activity was checked by cell viability, RT PCR and western blot analysis. Entry of CY5 labelled siRNA into cells was also confirmed by confocal microscopy. In short we present here a set of biocompatible Ferrocene-Tocopherol based lipids for successful siRNA delivery in HepG2 cells leading to an efficient gene knockdown in the presence of serum. Some of the findings of the above have been communicated for publication and manuscript is in preparation for other parts included in this synopsis.