The thesis entitled “Syntheses and Characterization of Novel Materials for Efficacious Anticancer Drug Delivery and Selective Sensing of Bioanalytes” encompasses the syntheses and characterization of various novel materials those are primarily used for efficacious pH-targeted chemotherapy, selective sensing and quantification of ATP inside a single living cell and also for specific sensing of female sex pheromone of certain agriculturally important pests. In recent era of cancer research, pH guided anticancer drug delivery is an emerging field by which not only the drug-sensitive, but also the drug-resistant cancer cell lines can be targeted efficiently. Scientists have paid lot of attentions to this area of research to design biocompatible, pH-responsive drug delivery vehicles, where most of the literatures are end up with complex, elaborated synthetic procedures and use of expensive chemicals. There are only a few reports in the literature on small molecule based drug delivery vehicles, which is not well explored. Herein some of the biocompatible, pH-sensitive lipid and short peptide sequences are synthesized in easy and short synthetic procedures and successfully tested for their efficacious anticancer drug delivery properties by various biophysical and biological techniques. A pH and reduction dual bio-responsive short peptide sequences are also generated in simple steps for the same cause. The formation of different nanostructures from the self-assembly of these short peptides is probed from high level of theoretical calculations and ultimately a well known chemotherapeutic drug, doxorubicin, has been delivered efficiently both to the drug-sensitive and drug-resistant cancer cell lines. In a particular case, in vivo study has also been performed to establish the drug delivery efficacy of those serum-stable vehicles that led to proficient reduction of tumour volume as compared to the free drug. On the other hand, a few of the molecules are synthesized and characterized by various analytical means for the selective sensing and quantification of adenosine 5’-triphosphate (ATP) inside a single living cell. Unique surface functionalized templates are also fabricated over MEMS devices for specific sensing of female sex pheromone of Helicoverpa armigera and Bactocera oleae pest in an agricultural field to detect the early pest infestation. Toward this end, an extensive study on the design, syntheses and characterization of different novel materials is presented below.

The thesis entitled “The Role of Liposomal Hybrids and Gold Nanoparticles in the Efficacious Transport of Nucleic Acids and Small Molecular Drugs for Cancer Nanomedicine” elucidates the preparation of various liposomal formulations of cationic monomeric and gemini lipids where hydrophobic domains were consisted of tocopherol, cholesterol and pseudoglyceryl backbone for the cellular transport of nucleic acids. The thesis continues while elucidating the role of various pH sensitive molecules and gold nanoparticles in liposomes to improve the delivery efficacy levels. This thesis also elucidates the role of gold nanoparticles stabilized with natural pH sensitive molecules for efficacious drug delivery applications. Additionally, the role of such pH sensitive gold nanoparticles in association with liposomes for the co-delivery of drug and gene has been discussed. The work has been divided into six chapters. Chapter 1A: Dimeric Lipids Derived from α-Tocopherol as Efficient Gene Transfection Agents. Mechanistic Insights into Lipoplex Internalization and Therapeutic Induction of Apoptotic Activity In this chapter, we present cationic dimeric (gemini) lipids for significant plasmid DNA (pDNA) delivery to different cell lines without any marked toxicity in the presence of serum. The six gemini lipids possess α-tocopherol as their hydrophobic backbone and differ from each other in terms of their spacer chain lengths. Each of these gemini lipids mixed with a helper lipid 1, 2-dioleoyl phosphatidyl ethanolamine (DOPE), was capable of forming stable aqueous suspensions. These co-liposomal systems were examined for their potential to transfect pEGFP-C3 plasmid DNA in to nine cell lines of different origins. The transfection efficacies noticed in terms of EGFP expression levels using flow cytometry were well corroborated using independent fluorescence microscopy studies. Significant EGFP expression levels were reported using the gemini co-liposomes which counted significantly better than one well known commercial formulation lipofectamine 2000 (L2K). Transfection efficacies were also analyzed in terms of the degree of intracellular delivery of labeled plasmid DNA (pDNA) using confocal microscopy which revealed an efficient internalization in the presence of serum. The cell viability assays performed using optimized formulations demonstrated no significant toxicity towards any of the cell lines used in the study. We also had a look at the lipoplex internalization pathway to profile the uptake characteristics. A caveolae/lipid raft route was attributed to their excellent gene transfection capabilities. The study was further advanced by using a therapeutic p53-EGFP-C3 plasmid and the apoptotic activity was observed using FACS and growth inhibition assay. Figure 1. The co-liposomes of tocopheryl gemini lipids and DOPE for efficient delivery of p53-EGFP-C3 plasmid DNA that induces significant apoptotic response. Chapter 1B: Efficacious Gene Silencing in Serum and Significant Apoptotic Activity Induction by Survivin Downregulation Mediated by Cationic Gemini Tocopheryl Lipids Non-viral gene delivery offers cationic liposomes as promising instruments for the delivery of double-stranded RNA (ds RNA) molecules for successful sequence-specific gene silencing (RNA interference). The efficient delivery of siRNA (small interfering RNA) to cells while avoiding the unexpected side effects is an important prerequisite for the exploitation of the power of this excellent tool. We discuss in this chapter about six tocopherol based cationic gemini lipids, which induce substantial gene knockdown without any obvious cytotoxicity. All the efficient co-liposomal formulations derived from each of these geminis and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE) were well characterized using physical methods such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Zeta potential measurements were conducted to estimate the surface charge of these formulations. Flow cytometric analysis showed that the optimized co-liposomal formulations could transfect anti-GFP siRNA efficiently in three different GFP expressing cell lines, viz. HEK 293T, HeLa and Caco-2 significantly better than a potent commercial standard Lipofectamine 2000 (L2K) both in the absence and presence of serum (FBS). Notably, the knockdown activity of co-liposomes of gemini lipids was not affected even in the presence of serum (10% and 50% FBS) while it dropped down for L2K significantly. Observations under a fluorescence microscope, RT-PCR and western blot analysis substantiated the flow cytometry results. The efficient cellular entry of labeled siRNA in GFP expressing cells as evidenced from confocal microscopy put forward these gemini lipids among the potent lipidic carriers for siRNA. The efficient transfection capabilities were also profiled in a more relevant fashion while performing siRNA transfections against survivin (an anti-apoptotic protein) which induced substantial apoptosis. Furthermore, the survivin downregulation improved the therapeutic efficacy levels of an anticancer drug, doxorubicin significantly. In short, the new tocopherol based gemini lipids appear to be highly promising for achieving siRNA mediated gene knockdown in various cell lines. Figure 2. The co-liposomes of tocopheryl gemini lipids and DOPE for efficient delivery of siRNA against survivin that induces significant apoptotic response. Chapter 2: Efficacious in Vitro EGFP Expression and Silencing in Serum by Cationic Pseudoglyceryl Gemini Lipids To elicit the desirable efficacy levels in cationic liposome mediated nucleic acid therapeutics has been part of extensive scientific efforts. This chapter describes three cationic gemini lipids and application of their co-liposomes with DOPE as potent pDNA (plasmid DNA) and siRNA (small interfering RNA) cytofectins for remarkably advanced efficacy levels in numerous cell lines in the presence of serum. The hydrophobic structural lineament of cationic gemini lipids is made up of pseudoglyceryl backbone linked to the hydrocarbon chains via oligo-oxyethylene units. The stable aqueous co-liposomal suspensions of gemini lipids showed an efficient binding to pDNA or siRNA and their significant intracellular delivery in various cell lines. The transfection capabilities of different co-liposomal formulations were profiled based on EGFP expression (pEGFP-C3 pDNA transfection) and EGFP knockdown (anti-GFP siRNA transfections) in EGFP expressing cell lines. The cellular EGFP expression levels and intracellular delivery of labeled nucleic acids were thoroughly studied using flow cytometry (FACS), fluorescence and confocal microscopy. The MTT based cell viability assay revealed no loss in cell viabilities for all of the transfection optimized lipoplexes of siRNA or pDNA. The transfection profile of gemini co-liposomes was noted to be significantly much better than a commercial lipofection reagent, Lipofectamine 2000 used for pDNA and siRNA applications in each of the cell lines studied. The co-liposomes and their transfection optimized lipoplexes were physiochemically characterized extensively by means of zeta potential, dynamic light scattering (DLS) and atomic force microscopy (AFM). In brief, these new gemini co-liposomal formulations seem to offer a great opportunity for successful nucleic acid (DNA and siRNA) delivery in a practical scenario. Figure 3. Efficacious EGFP expression (pDNA transfection) and EGFP silencing (anti GFP siRNA transfection) mediated by co-liposomes of pseudoglyceryl gemini lipids and DOPE. Chapter 3: Efficient Elicitation of Liposomal Nucleic acid delivery through the Eminence of Gold Nanoparticles Stabilized with pH Responsive Short Tripeptide Derived from Tyrosine Kinase NGF Receptors The prerequisite in the area of gene therapy today is to serve transfection efficient formulations nullifying the enduring key issues. To this end, we discuss in this chapter, the role of hybrid liposomal formulations derived from structurally distinct cationic lipids, a neutral lipid (DOPE) and pH responsive short tripeptide (KFG, Lys-Phe-Gly) capped gold nanoparticles (PAuNPs). The hybrid liposomes are presented to be efficient enough to transfect pDNA leading to remarkably high gene expression levels in various cell lines of different origins in the presence of serum (FBS). Hybrid liposomes could deliver pDNA more effectively than the native liposomes and commercial standard lipofectamine 2000 (L2K) across the entire range of N/P ratios studied under the influence of intracellular pH response and gold nanoparticles prominence. The gene transfection capabilities are profiled based on transfections performed using two different plasmids (pGL3, luciferase activity and p-EGFP-C3, green fluorescent protein expression). pDNA cellular internalization and subsequent gene expression levels are studied using flow cytometry, fluorescence microscopy and confocal microscopic studies. The extensive physiochemical characterization of hybrid liposomal formulation and their complexes with pDNA in comparison with respective native liposomes was performed using AFM, TEM, Zeta, DLS, gel retardation assay, U.V. and fluorescence emission measurements. The hybrid liposomes are shown to possess significantly higher fusion activity at lowered pH of intracellular compartments. These hybrid liposomes are fairly biocompatible across the concentration range used in transfection experiments. Precisely, introduction of these pH responsive tripeptide capped gold nanoparticles in to liposomal formulations straightforwardly must be more advantageous for a practical application in biomedical scenario to achieve therapeutic levels. Figure 4. The hybrid of liposomes and tri-peptide capped gold nanoparticles for significantly improved gene expression levels. Chapter 4: RNA Aptamer Decorated pH Sensitive Liposomes for Active Transport of Nucleic Acids in Specific Cancer Cells This chapter describes the target specific transport of pH sensitive liposomes loaded with a RNA aptamer for promising nucleic acid therapeutics. The pH sensitive liposomes are constructed from a cationic cholesteryl gemini lipid (CGL), neutral helper lipid (DOPE) and gemini analog of a pH sensitive lipid, palmitoyl homocysteine (GPHC). The liposomes are shown to be significantly fusogenic that deliver the cargoes upon lowerin the pH (6.0). The fusogenic behaviour of the liposomes was thoroughly studied by means of dynamic light scattering (DLS), zeta potential, lipid mixing, calcein dequenching and atomic force microscopy (AFM). The facile integration of cholesterol conjugated RNA aptamer in liposomes derived from cholesteryl gemini lipids was exploited for their delivery to specific cancer cells. The RNA aptamer specifically binds to epithelial cell adhesion molecule (EpCAM) with high affinity which is a cell surface marker in various solid cancers such as colorectal and breast carcinoma. These aptamer decorated pH sensitive liposomes could efficiently enter the EpCAM expressing COLO-205, Caco-2, MCF-7 and MDA-MB-231 cell lines while no such noticeable liposome transport was observed in EpCAM negative HEK 293T cells as evidenced by flow cytometry and confocal microscopy. Additionally, the liposomes are shown to be actively transported inside the cells, i.e., receptor mediated endocytosis. These liposomes could complex the nucleic acids (pDNA) in an efficient manner. The MTT based cell viability assay accounted no noticeable loss in cell viabilities for liposome treatments. Concisely, we have formulated RNA aptamer loaded pH sensitive liposomes that would certainly be promising tool in target based cancer nanomedicine. Figure 5. (A) Cellular internalization of DY-647 labeled aptamer loaded pH sensitive liposomes. (B) The liposomes were actively internalized through receptor mediated endocytosis. Each panel (A and B) represents (from left to right) bright field image, aptamer fluorescence, DAPI stained nuclei and merge of previous three impressions. Chapter 5: Natural Tri-peptide Capped Gold Nanoparticles for Efficacious Doxorubicin Delivery in Vitro and in Vivo Nanotechnology has gained ever increasing interest for the successful implementation of chemotherapy based treatment of cancer. This chapter describes the role of gold nanoparticles (AuNPs) capped with a natural pH responsive short tri-peptide (Lys-Phe–Gly or KFG) for significant intracellular delivery of an anti-cancer drug, doxorubicin (DOX). A significantly increased apoptotic response was noted for DOX treatments mediated by KFG-AuNPs in comparison with drug alone treatments in various cell lines (BT-474, HeLa, HEK 293T and U251) in vitro. Furthermore, KFG-AuNPs mediated DOX treatment significantly decreased cell proliferation and tumor growth in BT-474 cell xenograft model in nude mice. In addition, KFG-AuNPs showed efficacious drug delivery in DOX-resistant HeLa cells (HeLa-DOXR) in comparison with drug alone treatments. Figure 6. Representative images of excised tumors after doxorubicin treatment mediated by pH responsive tri-peptide capped gold nanoparticles (DOX-KFG-AuNPs) (C) in comparison with doxorubicin alone treatments (B) and untreated tumors (A). Extensive cell death as observed under Hematoxylin/eosin (H&E) (D) and TUNEL (E) staining of DOX-KFG-AuNPs treated tumor sections. Chapter 6: Significant Apoptotic Activity Induction by Efficacious Co-delivery of p53 Gene and Doxorubicin Mediated by the Combination of Co-liposomes of Cationic Gemini lipid and pH Responsive Tri-peptide Combining chemotherapy with gene therapy has appeared as an efficient tool to treat complex biological disorder like cancer. Herein, we show efficient co-delivery of DNA and an anti-cancer drug, doxorubicin (DOX) by means of gemini cationic liposome (GCL) based lipoplex nanoaggregates that are coated with DOX encapsulated pH responsive tripeptide nanovesicles. The lipoplex, tripeptide vesicles and their association was thoroughly studied using dynamic light scattering (DLS), zeta potential, atomic force microscopy (AFM). Flow cytometry, fluorescence and confocal microscopic analysis revealed that the GCL-tripeptide association could significantly co-deliver the p53 expression plasmid (p53-EGFP-C3) and DOX in HeLa and HEK 293T cells in the presence of serum. A synergistic increase in gene expression level and DOX internalization was observed in co-delivery which was even substantially higher than individual lipoplex transfection and DOX treatment. The apoptosis induced due to p53 expression and DOX was profiled with the help of annexin-V positivity analysis under flow cytometry and nuclear damage analysis by DAPI nuclei counterstaining under confocal microscopy which noted to be significantly higher in cells during co-delivery. The MTT based cell viability assay revealed a significantly increased loss in cell viability counts for co-delivery treatments. Such a system delivering synergistically increased significant efficacy levels in combinatorial drug and nucleic acid therapeutics would be certainly advantageous for practical biomedical applications. Figure 7. The co-delivery of pDNA and drug (doxorubicin) mediated by GCL-tripeptide association as observed under (A) confocal microscopy (pDNA; green and doxorubicin; red) and (B) flow cytometry.

The biggest hurdle in success of gene and drug therapy is designing and preparation of suitable bio-nanomaterials to carry the desired nucleic acid and drug to the targeted site. The work described in the present thesis encompasses two different approaches for the delivery of bioactive oligo/polynucleotides and drugs in vitro and in vivo using either cationic lipids or their nanocomposites with different carbon nanomaterials. The idea of using carriers for oligo/polynucleotides and drugs came into existence because of numerous physiological barriers in pathway of delivery of naked oligo/polynucleotides or drugs which reduces the overall activity of these bioactives in biological systems. These barriers trigger scientific research toward the preparation of appropriate biomaterials which can overcome the physiological barriers and improve the activity of bioactive oligo/polynucleotides and drugs in cellular systems. Toward this end, the design and synthesis of different cationic lipids and carbon nanomaterials were undertaken as described in seven chapters of the thesis. A series of novel cationic lipids with structural variability was prepared and used for gene delivery in vitro. They were further tuned chemically to sustain delivery efficiency in high serum percentage during in vitro transfection. These serum compatible lipids were used to perform transfection of reporter gene plasmid and found to be more efficient compared to the some well known commercial products for the same purpose. Another series of novel lipids were synthesized for the targeted gene delivery in vitro. These tryptophan based cholesteryl lipids were used to prepare mixed liposomes. These mixed liposomes were highly efficient in targeting sigma receptor rich HEK293T over sigma receptor negative HeLa cells. Mixed liposomes were also prepared for selective targeting of αvβ3 and αvβ5 integrins in gene transfection protocol using a palmitoyl-RAFT-RGD4 template. A mixed liposomal formulation was developed to carry out anti-sense siRNA mediated knockdown of Smad-2 protein with better efficiency compared to some of the best known commercial products for the same purpose. These mixed liposomes were also highly efficient for regression via induction of p53 mediated apoptosis in xenograft tumors developed in nude mice. Carbon nanomaterials have been extensively explored as nanoscale gene/drug carriers for potential applications. But the challenge is to solubilize these highly hydrophobic materials in aqueous medium for use in biological systems. Although there are reports for covalent modifications of such nanomaterials but it could be done only with the loss of some beneficial features of these materials. Herein a non-covalent technique has been efficiently used to suspend single walled carbon nanotubes in water using biocompatible cationic lipids. These nanosuspensions were used to complex plasmid DNA and transfect them in vitro. They proved to be highly serum compatible DNA carriers which did not drop the efficiency even in very high percentage of serum. Similarly exfoliated graphene was modified with cationic lipid and serum components to improve IC50 of Tamoxifen citrate and Methotrexate to a considerable extent in vitro. The improved Methotrexate formulations were highly efficient for regression in size of xenograft tumors developed in nude mice. Thus, the present thesis entails generation of cationic lipids and carbon nanomaterials based nanocomposites which were not only highly biocompatible themselves but their efficiency was found many fold better compare to some of the best commercial delivery agents. These were useful for the delivery of various bioactive oligo/polynucleotides and drugs in vitro and in vivo.