Engineering Polymers for Biological Applications

Rao, Vijayakameswara N (2014) Engineering Polymers for Biological Applications. PhD thesis, Indian Institute of Science Education And Research Kolkata.

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Abstract

Successful administration of chemotherapeutic agents for cancer treatment requires a balance between the efficacy and the safety of the drug. This often limits physicians to a very narrow therapeutic window. To avoid the harmful side-effects, chemotherapeutic agents may be administered at a suboptimal dose. This is not only a less effective treatment, but can lead to the development of drug resistance by cancerous cells. The therapeutic window can be increased through targeted, stimuli-responsive delivery, which increases the drug concentration at the diseased site, and releases or activates the drug only when it reaches the target. Cancer is a highly variable disease occurring in many organs. There is a need for delivery systems that are easily adaptable for a number of targets in different forms of cancers, and that can accommodate various cytotoxic drugs. The motivation of this project was to develop flexible synthesis procedures for the targeted delivery of chemotherapeutic agents. In this work, we have synthesized and tested three drug delivery systems. The first system is the synthesis and complete characterization of both norbornene derived doxorubicin (mono 1) and polyethylene glycol (mono 2) monomers are clearly described. Secondly their copolymerization by ring-opening metathesis polymerization (ROMP) to get the block copolymer (COPY-DOX) is vividly elaborated. The careful design of these conjugates exhibits properties like well-shielded drug moieties and well-defined nanostructures; additionally, they show solubility in both water and biological medium, and also have the important tendency of rendering acid-triggered drugs release. Drug release profile suggests the importance of having the hydrazone linker that helps to release the drug exactly at the mild acidic conditions resembling the pH of the cancerous cells. It is also observed that the drug release from micelles of COPY-DOX is significantly accelerated at mildly acid pH of 5.5 to 6 compared to physiological pH of 7.4, suggesting the pH-responsive feature of the drug delivery systems with hydrazone linkages. Confocal laser scanning microscopy (CLSM) measurements indicate that these COPY-DOX micelles are easily internalized by living cells. MTT assay against HeLa and 4T cancer cells showing COPY-DOX micelles have a high anticancer efficacy. All of these results demonstrate that these polymeric micelles self-assembled from COPY-DOX block copolymers have great scope in the world of medicine and they also symbolize promising carriers for the pH-triggered intracellular delivery of hydrophobic anticancer drugs. For the the second system we have varied the chain length of Doxorubicin in the block copolymer, to check the drug releasing efficacy, effect on assembly, effect on cell viability In the third system, we have synthesized three different monomers with hydrazone (mono 1), amide (mono 2), ester (mono 3), linkers. Next, homopolymerization of mono 1, mono 2 and mono 3 was carried out by using second generation Grubbs’ catalyst, to evaluate the best linker among others. In the fourth system, we have prepared efficient method to prepare a stimuli responsive smart nanocarrier for controlled doxorubicin drug delivery and MRI imaging. All the three functionalities, namely, phosphonate ester (PHO), folic acid (FOL) and doxorubicin (DOX) are delicately conjugated to the norbornene backbone to produce novel monomers. ROMP of these monomers has produced well-defined block copolymerization to get the triblock copolymer (PHO-FOL-DOX) with excellent control in polydispersity index (PDI). We believe that the resulting unique nanocarrier of PHOS-FOL-DOX-Fe copolymer has several advantages as follows. First, the system demonstrates the uniqueness by conjugating the drug as well as target ligands to the polymer backbone. This is entirely in contrast to the existing literature examples that show the encapsulation technique where they encapsulate either drug or the targeting ligands such as magnetic particles and FOL. Second, the amount of drug, NPs and FOL can be precisely controlled as per the requirement. This is achieved because of the controlled polymerization of norbornene monomers that have been conjugated with drug and targeting ligands. Third, the drug delivery process could be easily guided by magnetic field due to the magnetic NPs conjugated in the nanocarriers. In addition, the presence of FOL motif will be effectively received at the surface of the cancer cell due to the over expressed folate receptor. In the fifth system, site-specific, stimuli responsive, anticancer drug cocktails nanocarrier has been synthesized by conjugating doxorubicin, indomethacin, folate to the backbone of norbornene polymer via ester linkage. Monomers namely NOR-DOX (mono 1), NOR-IND (mono 2), norbornene grafted poly (ethyleneglycol)-folate, NOR-CHO (mono 3), NOR-PEG-FOL (mono 4), are designed by ester linkers to demonstrate the smart nanorcarrier capabilities. To prepare NOR-DOX ester linker, first Fmoc is used to protect the amino group of doxorubicin. Then Fmoc-doxorubicin is conjugated through 14-hydroxy group with carboxylic acid group of 5-Norbornene-2-exo-carboxylic acid. The ester linker strategy is followed to the indomethacin cancer drug and folic acid. The synthesis and complete characterization of all three monomers are elaborately discussed. Their copolymerization is done by controlled/living ring-opening metathesis polymerization (ROMP) to get the triblock copolymer TBCP-1, TBCP-2. NMR spectroscopy and gel permeation chromatography confirms the formation of the triblock copolymers. The particles have been characterized by both dynamic light scattering and transmission electron microscopy. Drug release of active molecules was monitored by UV and fluorescence. The release profile shows the importance of having the ester linker that helps to release the drug exactly at the mild acidic conditions resembling the pH of the cancerous cells. In summary, we have synthesized targeted drug delivery systems. The drug conjugates would be retained in tumors due to the enhanced permeability and retention effect, and release the drug at the target site.

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