SYNTHESIS AND CHARACTERIZATION OF ENZYME CATALYZED BIODEGRADABLE “CLICK-ENE” POLYMERS FOR TARGETED CANCER THERAPY

In this study, we report various biodegradable polymers with tunable physical properties and their possible drug delivery applications. These polymers were designed in such a way that bio-based starting materials (for example, sorbitol, hexanediol, glutaric acid) were used in order to obtain double-bond functionalized biopolymers in one-pot, and the polymerization reaction was catalyzed using an enzyme catalyst, Novozyme 435. In addition, a novel “Click-ene” chemistry was used to functionalize the resulting polymers in order to target specific cancer cells. The resulting polymers were purified using solvent precipitation method and characterized using spectroscopic techniques such as NMR, FT-IR, GPC, DSC and TGA, and the results are summarized in this thesis. In addition, to evaluate the potential biomedical applications of the DiI-encapsulating PNPs, we assessed their potential cytotoxicity by the MTT assay. Finally, these functional polymers were used to synthesize anti-tumor drug encapsulating polymeric drug delivery systems for the targeted therapy of cancer. Including synthesis and characterization results, various cell-based assays for cancer therapy will be highlighted in this work

Supramolecular anticancer drug delivery systems based on linear–dendritic copolymers

Current cancer chemotherapy often suffers severe side-effects of the administered cancer drugs on the normal tissues. In addition, poor bioavailability, due to the low water solubility of the anticancer drugs, limits their applications in chemotherapy. New delivery technologies could help overcome this challenge by improving the water solubility and achieving the targeted delivery of the anticancer drugs. Linear–dendritic hybrid nanomaterials, which combine the highly branched architectures and multifunctionality of dendrimers with the processability of traditional linear–linear block copolymers, have been introduced as ideal carriers in anticancer drug delivery applications. This review presents recent advances in the investigational aspects of linear–dendritic copolymers to be applied as anticancer drug delivery vehicles. We highlight the structures, synthesis of linear–dendritic block copolymers, interaction mechanisms between linear–dendritic copolymers and anticancer drug molecules, and findings on their drug release behavior and anticancer efficacies in vitro and in vivo

Literature Search on Using Dendrimer Nanoparticles as Drug Delivery Vehicles

The architectural design of dendrimers, multivalency, well-defined molecular weight and higher degree of branching differentiates them as unique and excellent nanocarriers in therapeutic applications such as drug delivery, gene transfection, tumor therapy, imaging and diagnostics. Nanoparticle drug-delivery systems are well known to increase the stability and selectivity of therapeutic agents. However immunogenicity, reticuloendothelial system uptake, drug leakage, cytotoxicity, hemolytic toxicity, poor aqueous solubility restrict the use of these drug delivery systems. These defects are overcame by surface engineering of the dendrimer molecule. Drug molecule can be efficiently conjugated or encapsulated into the interior of the dendrimer or physically adsorbed onto the surface of the dendrimer. And therefore, desired properties of the drug delivery system to specific needs of the medicine and its therapeutic functions such as in anticancer therapies and diagnostic imaging has highlighted the advantage of these systems as newest class of macromolecular nano-scale delivery devices. The focus of this review is mainly on the work done in the usage of dendrimer nanoparticles as drug delivery vehicles and its development in recent years

SYNTHESIS OF NEW ALIPHATIC PSEUDO-BRANCHED POLYESTER CO-POLYMERS FOR BIOMEDICAL APPLICATIONS

In this study, a hyperbranched polyester co-polymer was designed using a proprietary monomer and diethylene glycol or triethylene glycol as monomers. The synthesis was carried out using standard melt polymerization technique and catalyzed by p-Tolulenesulfonic acid. The progress of the reaction was monitored with respect to time and negative pressure, with samples being subjected to standard characterization protocols. The resulting polymers were purified using the solvent precipitation method and characterized using various chromatographic and spectroscopic methods including GPC, MALDI-TOF, and NMR. We have observed polymers with a molecular weight of 29,643 kDa and 33,996 kDa, which is ideal to be used as a drug delivery system. Thus, these polymers were chosen for further modification into folate-functionalized polymeric nanoparticles for targeted drug delivery to LNCaP prostate cancer cells. We hypothesized that due to the 3D structure of the A2B monomer, we expect a pseudo-branched polymer that is globular in shape which will be ideal for drug carrying and delivery. We used a solvent diffusion method, wherein the polymer can be simultaneously converted into water-dispersible nanoparticles and therapeutic agents (doxorubicin) can be encapsulated into the polymeric nanocavities. The efficacy of this delivery system was gauged by treating LNCaP prostate cancer cells with the drug-loaded nanoparticles and assessing the results of the treatment. The results were analyzed by cytotoxicity (MTT) assays, drug release studies, and confocal and fluorescence microscopy. The experimental results collectively show a nanoparticle that was biocompatible, target-specific, and successfully initiated apoptosis in an in-vitro prostate cancer cell model

Bionanomedicine: A “Panacea” In Medicine?

Recent advances in nanotechnology, biotechnology, bioinformatics, and materials science have prompted novel developments in the field of nanomedicine. Enhancements in the theranostics, computational information, and management of diseases/disorders are desperately required. It may now be conceivable to accomplish checked improvements in both of these areas utilising nanomedicine. This scientific and concise review concentrates on the fundamentals and potential of nanomedicine, particularly nanoparticles and their advantages, nanoparticles for siRNA conveyance, nanopores, nanodots, nanotheragnostics, nanodrugs and targeting mechanisms, and aptamer nanomedicine. The combination of various scientific fields is quickening these improvements, and these interdisciplinary endeavours to have significant progressively outstretching influences on different fields of research. The capacities of nanomedicine are immense, and nanotechnology could give medicine a completely new standpoint

Polymeric Nanocarriers for Treatment of Melanoma and Genetically Modified Mesenchymal Stem Cells to Improve Outcome of Islet Transplantation

Melanoma is a lethal malignancy with limited treatment options for advanced metastatic stages. New targeted therapeutic options with discovery of BRAF and MEK inhibitors have shown significant survival benefit. Despite the recent progress, inefficient tumor accumulation and dose limiting systemic toxicity remains pressing challenges for treating metastatic melanoma and there is a need for drug delivery approach to improve therapeutic index of chemotherapeutics. Nanoparticle based drug delivery represents promising approach to enhance efficacy and reduce the dose limiting systemic toxicity. Nanoparticles can be formulated either by physical encapsulation of drugs or by covalent conjugation of drugs to the polymeric backbone. Nanoparticles based strategies for encapsulation and conjugation of drugs to the polymer was reviewed in Chapter 2 where we summarized non-covalent interactions between polymer backbone and drug for physical encapsulation, various polymeric backbones for drug conjugation and application of photodynamic therapy in melanoma. Phototherapy, a light activated treatment modality is a potential therapeutic option for treatment of melanoma. Excitation of photosensitizer by light of specific wavelength can be clinically utilized for fluorescence assisted tumor surgery, photoacoustic imaging, photochemical internalization and phototherapy. Indocyanine green, water soluble FDA approved anionic tricarbocyanine with excellent safety profile and absorption in the near infrared (NIR) range is an excellent photosensitizer. However, short half-life (2-4 minutes) and limited extravascular distribution restricts PT application of ICG. In chapter 3, we have described ICG based phototherapy wherein plasma circulation and tumor accumulation of ICG was improved by designing its micelles formulation. ICG micelles were formulated by covalently conjugating ICG-NH2 to the pendant carboxyl groups of poly (ethylene glycol)-block-poly(2- methyl-2-carboxyl-propylene carbonate) (PEG-PCC) copolymer using carbodiimide coupling. ICG conjugated amphiphillic polymer self-assembled into micelles with particle size of 30-50 nm and high drug loading. These ICG conjugated micelles exhibited significant in vitro photodynamic cytotoxicity. Use of sodium azide and NIR radiation at 4° C revealed photodynamic and photothermal as primary mechanism of cytotoxicity of ICG solution and ICG conjugated micelles respectively. In vivo NIR imaging demonstrated ICG conjugated micelles prolonged ICG circulation and increased its tumor accumulation through enhanced permeability and retention effect Increase in tumor accumulation improved therapeutic efficacy with complete tumor regression in NIR irradiated ICG conjugated micelles compared to free ICG and control in A375 human melanoma tumor model in athymic nude mice. These results suggest that ICG conjugated micelles can be potentially utilized for phototherapy. Clinical translation of tubulin inhibitors for treating melanoma is limited by multidrug efflux transporters, poor aqueous solubility, and dose-limiting peripheral toxicities. Tubulin inhibitors with efficacy in taxane-resistant cancers are promising drug candidates and can be used as single agent or in conjunction with other chemotherapy. In chapter 4, we describe synthesis of tubulin inhibitors with activity in taxane resistant cell lines with IC50 in nanomolar range for the treatment of metastatic melanoma. LY293, a 5 indole derivative analog, binds to colchicine binding site and does not exhibit clinically prevalent drug resistance mechanism such as multidrug resistance (MDR) protein, breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp). Since LY293 is poorly soluble in water, LY293 was formulated as polymeric nanoparticles for systemic therapy of melanoma. Methoxy polyethylene glycol-b-poly (carbonate-co-lactide) (mPEG-b-P (CB-co-LA)) random copolymer was synthesized and characterized by 1H NMR and gel permeation chromatography (GPC). Polymeric nanoparticles were formulated using o/w emulsification method with a mean particle size of 150 nm and loading efficiency of 7.40%. Treatment with LY293 loaded nanoparticles effectively inhibited the proliferation of melanoma cells in vitro and exhibited concentration dependent cell cycle arrest in G2/M phase. In vivo, LY293 loaded nanoparticles significantly inhibited the proliferation of highly aggressive metastasized melanoma in a syngeneic lung metastasis melanoma mouse model without toxicity to vital organs. Islet transplantation has been performed in many patients especially undergoing kidney transplantation to treat Type I diabetes. Proportion of recipients who achieved insulin independence is low and is limited by long-term graft rejection and by primary non-function of islets. Primary non-function is characterized as the loss of islet viability and function caused by non-immune reasons, such as the disruption of islet microvasculature and apoptosis of islets due to production of inflammatory cytokines at the transplantation sites. In chapter 5, we studied the potential of human bone marrow derived mesenchymal stem cells (hBMSCs) as gene carriers for improving the outcome of human islet transplantation. hBMSCs were transduced with Adv-hVEGF-hIL-1Ra to overexpress human vascular endothelial growth factor (hVEGF) and human interleukin-1 receptor antagonist (hIL-1Ra). Viability of human islets co-cultured with hBMSCs was determined by membrane fluorescent method and glucose stimulation test. Transduced hBMSCs and human islets were co-transplanted under the kidney capsule of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ diabetic mice and blood glucose levels were measured over time to evaluate the efficacy of genetically modified hBMSCs. Our in vitro and in vivo results showed hBMSCs can be used as gene delivery vehicles to improve the outcome of islet transplantation without affecting their stemness and differentiation potential

Development and characterisation of clindamycin hydrochloride loaded PLA/PLGA nanoparticles

Clindamycin hydrochloride drug most commonly used as antibiotics in dental and oral infection acts upon various bacterial infections also. In this experiment PLA and PLGA nanoparticle prepared and conjugated with Clindamycin hydrochloride drug. From these two nanoparticles PLGA shows good result. It is found through SEM and Zeta sizer study that these polymers after conjugation with clindamycin hydrochloride gaining mean particular size of 178.6 nm with zeta potential -17.5 mv. Due to very less zeta potential nanoparticles are remain far apart so no clumping found among the drugs. In DSC study it is shown that the Tg (glass transition temperature) of Clindamycin hydrochloride is about 1500C so it take time to disperse inside the body but after conjugation with PLGA its Tg getting reduced to about 480C.Due to this low glass transition temperature it can easily disperse inside the body. After conjugation with Clindamycin hydrochloride there also an investigation done through FTIR studies from which we got that there must be a good conjugation of clindamycin hydrochloride with PLGA nanoparticle. This is because the abundance of OH, C=O, group both are common in PLGA and Clindamycin hydrochloride drug. The stretching band in PLGA-clindamycin hydrochloride is 3644.32 cm-1 refers to the absence of hydrogen bond among the Clindamycin hydrochloride and PLGA which may stands for hydrophobic bond due to much abundance of OH group. At the end it can be tell that after conjugation there is no serious alteration of Clindamycin hydrochloride structure but due to very easily dispersible nature it can shows its bactericidal effect more rapidly in comparison to the conventional medicine which generally takes two to three days

Cytotoxicity of dendrimers

Drug delivery systems are molecular platforms in which an active compound is packed into or loaded on a biocompatible nanoparticle. Such a solution improves the activity of the applied drug or decreases its side effects. Dendrimers are promising molecular platforms for drug delivery due to their unique properties. These macromolecules are known for their defined size, shape, and molecular weight, as well as their monodispersity, the presence of the void space, tailorable structure, internalization by cells, selectivity toward cells and intracellular components, protection of guest molecules, and controllable release of the cargo. Dendrimers were tested as carriers of various molecules and, simultaneously, their toxicity was examined using different cell lines. It was discovered that, in general, dendrimer cytotoxicity depended on the generation, the number of surface groups, and the nature of terminal moieties (anionic, neutral, or cationic). Higher cytotoxicity occurred for higher-generation dendrimers and for dendrimers with positive charges on the surface. In order to decrease the cytotoxicity of dendrimers, scientists started to introduce different chemical modifications on the periphery of the nanomolecule. Dendrimers grafted with polyethylene glycol (PEG), acetyl groups, carbohydrates, and other moieties did not affect cell viability, or did so only slightly, while still maintaining other advantageous properties. Dendrimers clearly have great potential for wide utilization as drug and gene carriers. Moreover, some dendrimers have biological properties per se, being anti-fungal, anti-bacterial, or toxic to cancer cells without affecting normal cells. Therefore, intrinsic cytotoxicity is a comprehensive problem and should be considered individually depending on the potential destination of the nanoparticle. © 2019 by the authors. Licensee MDPI, Basel, Switzerland

Drug Delivery Strategies For The Treatment Of Advanced Lung Cancer And Various Lung Metastases

Lung cancer remains the leading cause of cancer-related deaths in the United States. Secondary lung tumors metastasized from other cancer sites also remains highly prevalent, in which most metastatic tumors cannot be cured with existing therapies. Chemoresistance (multi drug resistance – MDR) that develops intrinsically or acquired is one of the key factors leading to fatality in these patients. MDR develops form a variety of resistance mechanisms that can occur consecutively or concurrently, therefore, making most current treatments unsuccessful. Current therapies have known to slow tumor growth, but rarely provide a cure. Immunotherapy has seen some promise, including the use of checkpoint inhibitors, when other therapies have not proven beneficial. However, only a small fraction of tumor types and patients have benefited from this type of treatment, and some toxicity has been stated. Therefore, establishing new types of treatments, as a single therapy or combination therapy, that can i) increase the rate of survival in patients suffering at early or late stages (when MDR and metastasis have developed) of lung cancer, and ii) reduce toxicity and adverse side effects of treatments is of great importance in pulmonary oncology. In this work we describe alternative treatment modalities that suggest their potential to address MDR and prolong the rate of survival in patients suffering from primary and secondary lung tumors. A combination of local lung targeting (high payload to target side and reduced systemic toxicity), nanocarriers (to modulate interactions with physiological environment) intracellular organelle targeting (to repurpose cytoreductive therapies), siRNA as therapeutic agent (to target apoptotic pathways), and macrophage repolarization immunotherapy (to modulate the tumor microenvironment) are reported. We describe the development mitochondrial-targeted dendrimer nanocarriers (DNCs) as a platform for the repurposing of chemotherapeutics, the development of siRNA/TPP-DNC complexes (TPP-dendriplexes) as a platform for pulmonary delivery of siRNAs, the development of asymmetric dendrimers with a chemotherapeutic and varying surface functionalities to enhance tumor targeting and penetration, and address the impact of local pulmonary administration of tumor associated macrophage (TAM)-targeting immunotherapy. Overall, we conclude that all these strategies described above have the potential capability to address issues resulting from MDR and for the treatment of primary and secondary lung tumors

Engineered nanomaterials for biomedical applications

Engineered nanomaterials (ENM) have emerged as attractive and promising candidates for a wide range of advanced applications including in particular in medicine. However, the increased development of ENM raises the need to carefully assess their potential impact on human health and environment. For that, detailed evaluation of the intrinsic and biological identity of ENM is required for the safe design and use of these materials. To this effect, the present thesis focuses on the synthesis and biocompatibility assessment of two different classes of nanomaterials, dendrimers and superparamagnetic iron oxide nanoparticles (SPIONs), promising future nanomedicines for drug delivery and imaging agents in magnetic resonance imaging (MRI). Assessment was performed on primary human monocyte derived macrophages (HMDM), primary human bronchial epithelial cells (PBEC), and cell lines. Hereby an insight on the impact of these materials on the immune system and on their promising and potential use as nanomedicines has been obtained. Furthermore, we attempted to use systems biology approaches as a novel tool to identify possible hazard of ENM by using next generation sequencing RNA-Seq and computational tools. Finally, we assessed the bio-nano-interactions by evaluating the effect of the protein corona on the targeting capabilities of ENM and their behaviour. Importantly, the ENM were extensively characterized, using different techniques prior to the toxicity studies. In Paper I, we evaluated the biocompatibility of a library of polyester dendrimers based on 2,2-bis(methylol)propionic acid (bis-MPA) including dendrimers with two different surface functionalization, hydroxyl and carboxylic end groups, and two commercial polyamidoamine dendrimers (PAMAM) with amine and hydroxyl end groups. We found excellent biocompatibility for the entire hydroxyl functional bis-MPA dendrimer library, whereas the cationic, but not the neutral PAMAM exerted dose and time dependent cytotoxicity in the cell models tested. In paper II, using system biology approaches and bioinformatics tools, we were able to identify and validate the toxicity mechanism of PBEC exposed to PAMAMs dendrimers at low doses. Our studies showed that PAMAM-NH2, but not PAMAM-OH, caused down-regulation of cell cycle-related genes and cell cycle arrest in Sphase. Our findings provide evidence of the beneficial use of these new toxicology tools for the future risk assessment of nanomaterials. SPIONs have emerged as promising nanomaterials for biomedical applications, due to their excellent magnetic properties, chemical stability and biocompatibility. In paper III, ultrasmall superparamagnetic iron oxide nanoparticles (USIRONs) were prepared by a one-pot aqueous approach by using Fe(OH)3 as iron precursor, vitamin C as reducing agent, and dehydroascorbic acid (DHAA) as capping agent. We showed that USIRONs present high crystallinity, long-term colloidal stability, enhanced saturation magnetization, and exhibit excellent biocompatibility as demonstrated in the toxicity evaluation using primary HMDM. When nanoparticles are in contact with physiological fluids, adsorption of proteins on the surface of the nanomaterial will occur, resulting in the establishment of aprotein corona. Whether the protein corona will affect the targeting capabilities of the ENM was investigated. In paper IV, folic acid (FA)-conjugated iron oxide nanoparticles with poly(ethylene glycol) (PEG) or SiO2 surface coatings were synthetized. We evaluated their biocompatibility and specific targeting effects on HMDM and on ovarian cancer cells, that over express the folic acid receptor. Notably, we demonstrated the nanoparticles (NPs) were nontoxic to cells and that FA specific uptake was observed only for the FA iron oxide SiO2 coated NPs in the presence of serum proteins. Our studies contribute to the development of new nanomaterials and their applications, which may facilitate the clinical translation of the nanomedicines