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|Title:||DEVELOPMENT AND EVALUATION OF NANOMATERIAL BASED TARGETED DRUG DELIVERY SYSTEM|
DRUG DELIVERY SYSTEM
|Abstract:||The advent of nanotechnology has led to exciting applications in various domains of science and technology, including rapid development in nanoscale materials towards developing targeted drug delivery system. Reduction in the sizes of the drug carriers to nanoscale dimension could overcome the biological barriers and can pass through the smallest capillary vessels which could improve in the efficacy and site specificity. As polysaccharides are conventionally used as matrices for carrying drugs, so polysaccharides based nanomaterials have attracted a lot of scientific attention in drug delivery system. In this regard, dextran, chitosan, alginate, starch, pectin etc. have been explored. These drug carriers of nanoscale dimension could be functionalized by various means, e.g., attaching ligands for receptor mediated targeted drug delivery, or by incorporating magnetic materials, e.g., superparamagnetic iron oxide nanoparticles (SPIONs) for guided drug delivery. A few studies have been reported on coating MNPs with chitosan, dextran, polylactic co-glycolic acid (PLGA) and polyvinyl alcohol (PVA) which also facilitated loading of drugs. Among various iron oxide phases, it was noted that the magnetite nanoparticles (MVPs) of about 5-20 nm in diameter were suitable- as they exhibit superparamagnetism, high saturation field, biocompatibility and non-cytotoxicity. The synthesized MNPs might require stabilizers to maintain colloidal stability as well as good aqueous dispersion. In this project, we aimed at developing a novel magnetically responsive hybrid nanomaterial for potential targeted drug delivery system. It comprised of magnetite nanoparticles coated with pectin and were characterized by an array of techniques. Another batch of nanomaterial was synthesized by coating magnetite nanoparticles with pectin reinforced with chitosan and was characterized in similar manner. Here pectin and chitosan were chosen due to its biocompatibility, biodegradability and its low cost. We have evaluated the drug loading efficiencies of diclofenac sodium, 5-fluorouracil (5-FU), oxaliplatin (OHP) in these magnetite coated with polysaccharide nanomaterials. Further we have evaluated their in vitro release in different simulated conditions, at different pH. In addition, the cytotoxicity of 5-FU and oxaliplatin loaded nanomaterials were evaluated in various cancer cell lines like HT-29 (colon), HEPG2 (liver) and MIA-PA-CA-2 (pancreas). The present study comprises six chapters and a brief discussion of each is as follows: In the first chapter we have introduced the concept of targeted drug delivery using nanotechnology. A thorough literature survey has been carried out to discuss the various types u of targeted drug delivery systems. We have mainly highlighted polysaccharide based nanomaterials for this purpose. Recent development on superparamagnetic iron oxide nanoparticles (SPIONs) incorporated in polysaccharide nanomaterials for targeted drug delivery is introduced. A detailed literature survey on methods of synthesis of SPIONs is presented. This is followed by aim and scope of the present work. In Chapter two, we have described the experimental methodologies for the fabrication of hybrid nanomaterials of magnetite and pectin (MP) where pectin was cross linked with calcium ions. The formation of these hybrid nanomaterials were characterized by array of techniques. The fabrication conditions were optimized with respect to the concentration of pectin, calcium ions and experimental conditions, namely pH, time. The optimization was based on their morphology and magnetic properties. The magnetite phase was determined from X-ray diffraction (XRD) studies and was confirmed from 57Fe Mossbauer spectroscopy recorded at room temperature, and at 5 K with and without magnetic field. The average crystallite size was determined to be in the range of 2-8 nm from Debye-Scherrer formula using the most intense peak. Fourier transform infrared (FT-IR) spectroscopy and thermal analysis techniques like thermogravimetry and differential thermal analysis (TG-DTA) were used for interpreting the coating of pectin on magnetite nanoparticles (MNPs). The surface analysis of the pectin coated MNPs by X-ray photoelectron spectroscopy (XPS) studies revealed minimal concentration of Fe on the surface, which indicated that mostly MNPs were incorporated in the calcium pectinate nanomaterials. The morphology of the fabricated nanomaterials was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) and their sizes in dry condition were measured in the range of 100 - 150 nm. While in aqueous medium.they were found to be in the range of 300 --400 nm, as measured by dynamic light scattering (DLS). The increase in size in aqueous medium was attributed to swelling effect. The elemental analysis of the material was determined from energy dispersive X-ray analysis (EDAX) coupled to SEM and the concentration of MNPs was determined by estimating total Fe in the fabricated nanomaterials by instrumental neutron activation analysis (INAA). Zeta potential measurements were used for understanding the nature of interaction among MNPs, pectin, chitosan in the nanomaterials. The magnetic studies were carried out by using superparamagnetic quantum unit interference device (SQUID). The saturation magnetization of these hybrid nanomaterials was 46.21 emu/g, measured at room temperature and applied field of ±2.5 T. The field cooled (FC)-zero field cooled (ZFC) measurement studied from vi SQUID measurements confirmed superparamagnetic behavior of these materials. These results were corroborated by 57Fe Mossbauer spectroscopy studies. In the third chapter we have discussed the method of fabricating a targeted drug delivery system by loading diclofenac sodium (DS) drug in magnetite-calcium pectinate nanomaterial. The fabricated system was characterized in the similar manner as mentioned in Chapter 2. Maximum drug loading efficiency was found in the batch fabricated with 1 % pectin solution (w/v). But this composition of pectin led to formation of viscous matrix.. On the other hand, the batch prepared with 0.4 % pectin (w/v) resulted in spherical nanomaterials with 60.6 ± 1.4 % encapsulation efficiency. The concentration of the drug loaded in these nanomaterials was found to be 28.9 ± 1.6 % (w/w) on dry weight basis. These drug loaded spherical nanomaterials materials of 100 - 150 rim (in dry condition, as reflected from TEM and SEM studies) exhibited superparamagnetic property as evident from VSM (Ms =44.05 emu/g at room temperature and ±10 kOe) and SQUID (TB = 75.4 K at an applied field of 500 Oe). The in vitro release study of the MP-DS was carried out sequentially in simulated gastric, intestinal and colonic fluids by using specific enzymes and by maintaining the pH of the medium. There was negligible release of drug in gastric medium while 88 % of the drug was released rapidly in simulated intestinal fluid. The remaining drug was released in the colonic fluid. On the other hand the MP-DS showed sustained release in phosphate buffer at pH 7.4. The release profile agreed well with the Korsemeyer-Peppas model which satisfied the conditions for non-Fickian transport. This was attributed to swelling effect of calcium pectinate in the aqueous medium which resulted in diffusion based drug release. In the chapter four we reported our studies on enhanced loading of the diclofenac sodium in pectin which was reinforced with chitosan where magnetite nanoparticles were incorporated (MPCh-DS). Notably, here pectin was not cross linked with Cat+ ions but was reinforced with chitosan by electrostatic interaction. The methodology for fabrication of the MPCh-DS system was optimized by varying the chitosan concentration, where minimum concentration of chitosan was 0.025 %. The drug loading efficiency was more than 99 %. The formation of the nanomaterials MPCh-DS was confirmed from XRD, FT-IR, TG-DTA, 57Fe Mossbauer, TEM, SEM, DLS, zeta potential. The sizes of MPCh-DS were in the range of 100 — 150 rim in dry condition and in aqueous medium an average size of 350 nm was measured from SEM, TEM and DLS measurements. The magnetic measurements by VSM revealed a saturation magnetization of 34.40 emu/g (at room temperature and ±10 kOe) and the superparamagnetic property was confirmed from SQUID measurement. The in vitro vii release of the diclofenac sodium from MPCh-DS system was studied in simulated gastric, intestinal and colonic fluids at respective pH. The in vitro release study was also carried out in phosphate buffer solution at pH 7.4 to mimic the release of the drug in blood. Similar to the release pattern observed for MP-DS system in the previous chapter, the release of DS from MPCh-DS was negligible in gastric fluid while 68 % of the .drug was released rapidly in simulated intestinal fluid. This was followed by 31.5 % drug release in the colonic fluid over a period of 55 h. Overall, 99 % of the drug was released in a sustained manner. Similar to the MP-DS system, the sustained release of drug was better in phosphate buffer (pH -7.4) and the release profile agreed well the Korsemeyer-Peppas model, satisfying non-Fickian transport. Thus the release mechanism was attributed to swelling effect of pectin reinforced with chitosan in the aqueous medium. This system could be assumed to be suitable for delivering the drug at inflammatory regions, e.g. knee joints if administered intravenously where the drug can be release in a sustained manned over a longer period of time. In the chapter five we have discussed the method of fabricating targeted drug delivery system with anticancer drug 5-FU loaded in magnetite calcium pectinate nanomaterials (MP-5FU). The drug loading efficiency was found to be 29.8 f 3.3 %. Similar to the previous chapters the fabrication of MP-5FU was confirmed from XRD, FT-IR, TEM, SEM, DLS and zeta potential studies. In aqueous medium, DLS measurement reflected an average size of 300 urn which was attributable to swelling effect of the polymer. The saturation magnetization of these materials was 43.15 emu/g recorded by VSM at room temperature and ±10 kOe. The superparamagnetism was confirmed from the FC-ZFC profile recorded from SQUID measurements. The in vitro release studies in simulated gastric condition showed 11.8 % release by weight. The release of 5-FU was — 40 % in simulated intestinal condition. It was followed by --- 46 % release of drug in simulated colonic fluid. It accounted for a total release of - 98 % over a period of 48 h which exhibited sustained release of the drug in gastrointestinal conditions. Similar to MP-DS system, the in vitro release of 5-FU from MP- 5FU showed better sustained release in phosphate buffer (pH —7.4) over a period of 48 h and was in good agreement with swelling controlled drug release mechanisms as discussed in previous chapters. Further the cytotoxicity of the fabricated nanomaterials MP-5FU was studied on HT-29 (colon), HEPG2 (liver), MIA-PA-CA-2 (pancreas) cancer cell lines by SRB assay after 48 h. The % cell viability decreased with increase in the MP-5FU concentration (1-5 mg/mL). The cell viability of MP-5FU at 5 mg/mL was found to be 60.9 ± 3.3 % for HT-29 and 68.2 ± 16.1 % for HEPG2 cancer cell lines. Strikingly, the cell viability for 5 mg/mL viii of MP-5FU in MIA-PA-CA-2 pancreatic cancer cell line was 23.9 ± 5.1 % and the corresponding GI50 was 3.7 mg/mL. On the other hand, the GI50 of MP-5FU for HT and HEPG2 was more than 5 mg/mL. The magnetite-pectin system without 5-FU, did not indicate any antiproliferative effect. Therefore decrease in cell viability for MP-5FU was only due to release of the anticancer drug 5-FU from the nanomaterials. These results indicated the successful fabrication of magnetic nanomaterials of pectin for potential delivery of 5-FU. In chapter six we have reported the fabrication of anticancer drug, oxaliplatin (OHP), loaded in the hybrid nanomaterials of magnetite-pectin and in magnetite-pectin reinforced with chitosan. The sizes of these nanomaterials were between 100 - 150 nm in dry condition, confirmed by SEM and TEM. In aqueous medium, DLS measurement reflected an average size of —330 nm which was attributable to swelling effect of the polymer. The VSM studies revealed high saturation magnetization (45.65 emu/g) at room temperature and ±10 kOe. The superparamagnetic property was characterized by SQUID magnetometry. The drug loading efficiency was calculated with respect to platinum (Pt) content by ICPMS, after calibrating the Pt contents in the known concentrations of the drug. The loading efficiencies were 50.2 ± 1.5 % in MPCh-OHP and 55.2 f 1.2 % in MP-OHP. The in vitro release profile of oxaliplatin from MP-OHP in the phosphate buffer pH 5.5 and 7.4 indicated sustained release. On the other hand the release of OHP from MPCh-OHP was rapid. From this we concluded that MP-OHP was a better drug delivery system for sustained release of oxaliplatin and was further studied for its cytotoxicity in MIA-PA-CA-2 (pancreas) and HT-29 (colon) cancer cell line. The % cell viability decreased as the concentration of MP-OHP was increased from 1 mg/mL to 5 mg/mL. However, the GI50 value for MP-OHP in MIA-PA-CA-2 (pancreas) and HT-29 (colon) cancer cell lines was found to be above 5 mg/mL. Finally we have summarized our work by highlighting the successful fabrication of a novel drug delivery system of nanoscale dimension with sustained release capability of different types of drugs namely DS, 5-FU and oxaliplatin. The additional magnetic property induced due to the presence of superparamagnetic magnetite nanoparticles has enhanced its functionality as a potential magnetically guided targeting ability. It may be interesting to study the in vivo applicability of these magnetite-pectin-drug nanomaterials materials for assessing their capacity for magnetically targeted drug delivery system.|
|Appears in Collections:||DOCTORAL THESES (chemistry)|
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