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Abstract Oxaliplatin loaded albumin nanoparticles for cancer immunotherapy Summary Cancer management strategies include surgery, radiotherapy and chemotherapy. Recently, gene therapy, hormonal therapy and immunotherapy are progressively investigated in cancer management. Chemotherapeutics as oxaliplatin, stimulate cancer cells to induce immunogenic cell death (ICD). ICD is an apoptosis process that amplifies an inflammatory immune response. Interestingly, previous reports demonstrated that the implantation of tumour cell lines previously treated with ICD inducers acts to vaccinate mice from rechallenge with the same tumour cells. In addition, tumour local treatment with ICD inducers can lead to diminution of distal tumour proving the conception of systemic immune recognition. The main physiological characteristics of ICD are cell surface expression of calreticulin, release of different Damage-associated molecular patterns (DAMPs) as heat shock proteins (HSPs) and Adenosine triphosphate (ATP) as well as high mobility group box 1 (HMGB1). Calreticulin is an “eat me signal” that combines with low-density lipoprotein receptor-related protein 1 (CD91) on phagocytes, DAMPs release provides a ‘find me’ signal and HMGB1 maturates antigen-presenting cells (APCs). Consequently, these factors lead to stimulation of host immune system. Calreticulin expression is considered the crucial factor in ICD bona fide. Oxaliplatin is a platinum-based alkylating cytotoxic agent that acts via impeding DNA replication. Oxaliplatin has a wide range of activity against colorectal, pancreatic, gastric, ovarian, bladder, breast, small- and non-small-cell lung, and head and neck cancers. Oxaliplatin suffers from various side effects as neuropathy, low blood count and dysesthesias. These harmful side effects could Summary 200 be attributed to the nonspecific biodistribution of oxaliplatin into the healthy tissues. As a consequence, the development of a suitable carrier with an enhanced tumour internalization is crucial. In addition, CD44 is a transmembrane glycoprotein with different isoforms intricate in cell-cell and cell-matrix interactions. Moreover, CD44 is highly expressed on Cancer Stem Cells and has a key role in tumor growth, metastasis and chemotherapeutic resistance. In colon cancer, CD44 isoforms are reported to trigger tumour invasion, impede apoptosis and patient survival. Downregulation of CD44 could be accomplished via different strategies as antibodies, aptamer, siRNA, miRNA and is usually associated with improved therapeutic efficacy. Zinc oxide nanoparticles (nZnO) are FDA approved biodegradable, biocompatible nanocarriers with proved anticancer activity as well as antibacterial and antiviral activity. In addition, nZnO are reported to have immunomodulatory effect. Nanotechnology is considered as the most noticeable tool in the development of new drug carrier systems, providing versatile clinical applications and scale-up for industrial production. Polymeric nanoparticles and lipid nanocarriers are the mainly investigated types of nanoparticles which had been approved by FDA for clinical applications. Commonly, drug targeting is divided into passive and active targeting strategies. Cancer passive targeting of nanoparticles relies on the discontinued fenestrations in the endothelial layer in the tumor microenvironment that ranges from 300 to 4700 nm in diameter. Moreover, poor lymphatic drainage is encountered in the tumors due to the dysfunctional lymph angiogenesis and compression of lymphatic vessels by proliferating cancer cells. This phenomena of nanomedicine accumulation in tumors is referred to as enhanced permeability and retention effect. Summary 201 Accordingly, the ultimate goal of this study was to combine oxaliplatin and nZnO into albumin NPS to improve cellular uptake, cytotoxicity and apoptosis. The effect of RBCs coating on macrophage uptake in vitro and in vivo was also assessed. A special focus was given to the effects of the proposed platform on immunological stimulation against colon cancer cells. To fulfill the above goal, the work in this thesis was divided into three chapters: I. Preparation and characterization of zinc oxide nanoparticles. II. Preparation and characterization of RBCs Coated Oxaliplatin-Zinc Oxide Loaded Albumin Nanoparticles III.Immunological and Biological Studies on RBCs Coated Oxaliplatin- Zinc Oxide Loaded Albumin Nanoparticles. Chapter I Preparation and characterization of zinc oxide nanoparticles In this chapter, we aimed to synthesize nZnO with minimum crystallite size as a possible adjuvant in cancer immunotherapy. Briefly, two studies were performed: a preliminary study to set the growth time and temperature for efficient fabrication of nZnO. The second study involved the optimization of different CPPs including precursor concentration, pH and stirring speed affecting the nZnO crystallite size to achieve the QTPP. nZnO was successfully prepared by chemical precipitation technique, where the preparation process optimized by QbD by response surface methodology using BBD.The influence of different CPPs on crystallite size was statistically fitted to the quadratic model. Increasing zinc precursor concentration and/ or stirring speed decreased crystallite size. Contrarily, nZnO NPs crystallite size was directly proportional to the reaction pH .The optimum formula was selected based on the criteria of attaining minimum crystallite size. This formula Summary 202 was prepared using 500 mMZn(NO3)2.6H2O at pH 10 and 1000 rpm stirring speed. The XRD data revealed that the obtained nZnO had hexagonal wurtzite P63mc crystal structure .The optimized nZnO was also characterized by FT-IR spectroscopy where the main stretching band of ZnO at 430 cm-1 infers the successful fabrication of nZnO. The optimized nZnO was characterized in terms of UV spectroscopy with a characteristic λmax of 364 nm corresponding to the pure nZnO .The optimized nZnO showed a reversal surface charge ranged from +25.32 ±3.21 to -18.78± 1.58 depending on the dispersant pH. The morphological architecture of the nZnO demonstrated heterogeneous hexagonal and spherical nanostructure with crystallite size in good agreement with that determined by applying Scherrer’s equation on the data derived by XRD technique .The in vitro Zn2+ release from nZnO was pH dependent due to the limited solubility of Zn2+ at physiological pH. In the next chapter, attempts were made to co-load nZnO with oxaliplatin into long circulating RBCs coated albumin NPs. Chapter II Preparation and characterization of RBCs Coated Oxaliplatin-Zinc Oxide Loaded Albumin Nanoparticles. Bovine serum albumin nanoparticles (albumin NPs) have proven success in the clinical arena as a platform for cancer treatment, with an existing privation for multifunctional particulates nurturing combinatory therapies. Targeting multiple pharmacological bits are thought to improve cancer’s therapeutic outcomes. Interestingly, FDA had approved, nZnO as a multifunctional platform capable of combating various types of cancer, while achieving multiple tasks. The multi-target anticancer activity of nZnO could be attributed to its ability to provoke reactive oxygen species (ROS), macrophage polarization promoting cancer apoptosis, as well as, down regulation of CD44 expression hampering Summary 203 both cancer adhesion and migration. Currently, the use of natural membrane coat derived from RBCs would add several merits as biocompatibility, nonimmunogenicity, improved cellular interactions and targeting efficiency. In this chapter, RBCs coated albumin NPs co-loaded with oxaliplatin and nZnOwere prepared and evaluated using four studies: 1- setting the ratio of desolvent to solvent phases and stirring time for oxaliplatin albumin NPs formulation; 2- optimization of different CPPs for drug albumin NPs fabrication; 3- co-loading of different concentrations of nZnO with the optimized drug albumin NPs and 4- RBCs coating of the chosen co-loaded albumin NPs, followed by its in-depth characterization. Briefly, oxaliplatin albumin NPs were successfully prepared by desolvation technique, where the preparation process optimized by QbD by response surface methodology using BBD. The influence of different CPPs on particle size and EE % was statistically fitted to the 2FI and quadratic models respectively. Increasing BSA and oxaliplatin concentrations increased both particle size and oxaliplatin EE%, while increasing the stirring speed decreased both responses. All the obtained oxaliplatin albumin NPs had a PDI value less than 0.2 indicating the fabrication of homogenously dispersed systems. The optimum formula was selected on the criteria of attaining a minimum particle size and maximum oxaliplatin EE%. The optimum formula (Alboxa) was composed of BSA (5 mg/mL) and oxaliplatin (3 mg/mL) and prepared at 1000 rpm. Oxaliplatin albumin NPs were co-loaded with nZnO to improve the expected antitumor activity of the proposed system. The complexation between BSA and nZnO was confirmed by UV spectroscopy, fluorescence spectroscopy and FT-IR spectroscopy. The co-loading of nZnO into oxaliplatin albumin NPs significantly increased particle size. The presence of nZnO in a concentration of Summary 204 30% w/w and above respective to BSA amount significantly decreased oxaliplatin EE%. The selected nZnO concentration loaded into Alboxa was 20% w/w. Alboxaco-loaded with nZnO (Alboxa-nZnO) had a particle size of 131± 3.61nm, PDI of 0.184 ± 0.012 and oxaliplatin EE% equals 81.65± 1.01%. The presence of nZnO during the desolvation preparation of Alboxa did not interfere with the percent yield. The conformational changes in the native BSA structure during the preparation of NPs with and without oxaliplatin and nZnO were investigated by FT-IR spectroscopy. The morphological architecture of the proposed Alboxaand Alboxa-nZnO were visualized by SEM showing almost spherical and doughnut-shaped nanostructures respectively. The presence of platinum and zinc elements were confirmed by EDX analysis. RBCs coated Alboxa-nZnO NPs were successfully prepared using freshly prepared mice blood. The RBCs derived membrane obtained by hypotonic treatment of erythrocytes was fused with Alboxa-nZnO. The coating of Alboxa-nZnO significantly increased particle size with a neglected effect on oxaliplatin and nZnO EE%. All the prepared formulae showed a negative zeta potential ranged from -17.62± 2.34 to -29.38± 1.95 mV depending on the presence of nZnO and RBCs coat. The presence of nZnO with and without RBCs coat decreased oxaliplatin LE% from 7.99 ± 1.15% to 4.85± 0.26%. The proper orientation of RBCs coat on the Alboxa-nZnO was confirmed by quantification of %glycoprotein, sialic acid content and the extent of surface CD47 protein expression. The successful coating of Alboxa-nZnO with RBCs was also confirmed with TEM, where a non-aggregated spherical core-shell nanostructures were observed. Incubation of RBCs coated Alboxa-nZnO with FBS (10% and 50% v/v) for 48 h had an insignificant effect on particle size, PDI and zeta potential indicating the colloidal stability of the proposed system. Summary 205 The in vitro oxaliplatin release from RBCs coated Alboxa-nZnO had a more prolonged oxaliplatin released than Alboxaand Alboxa-nZnO in PBS pH 7.4 (in presence of 50% FBS) and acetate buffer pH 5.5 (in presence of glutathione 10 mM). The in vitro Zn+2 release from Alboxa-nZnO with and without RBCs coat showed a pH dependent behavior due to the inherent solubility of Zn in acidic conditions. Stability studies of RBCs coated Alboxa-nZnO after storage for 21days at 4 °C revealed nonsignificant changes in the measured physicochemical characteristics. The aforementioned results gave a rational for immunological and biological assessment of the prepared formulation. Chapter III Immunological and Biological Studies on RBCs Coated Oxaliplatin- Zinc Oxide Loaded Albumin Nanoparticles. In this chapter, the efficacy of the developed C-AlbOxa-nZnO was evaluated by testing its cytotoxicity and cellular uptake on CT26 colon cancer cells. The effect of RBCs coating on the macrophage uptake was also assessed. The consequent in vitro immune stimulation of the proposed system was tested by assessing calreticulin CT26 cell surface expression, ATP and HMGB1 secretion, as well as, the expression of CD80, CD86 and MHC II on macrophage cells. The in vitro phagocytosis efficiency of macrophage against pre-treated cancer cells with the combinatory systems was investigated. The effect of C-AlbOxa-nZnOon CD44 expression and consequently cell adhesion and migration ability was investigated on CT26 cells. The in vivo biodistribution of fluorescence labelled C-AlbOxa-nZnOin tumour bearing mice was also assessed. The prepared C-AlbOxa-nZnO (composed of 5 mg BSA, 3 mg oxaliplatin and 1 mg nZnO and coated with RBCs) showed ≈ 2 fold increase in cytotoxicity compared to that of free drug solution. C-AlbOxa-nZnO significantly induced CT26 Summary 206 apoptosis markers namely; caspase 3 and 7 over untreated cells or those treated with either C-AlbOxaor C-AlbnZnO. The macrophage uptake of both coated and uncoated AlbOxa-nZnO was assessed qualitatively and quantitatively by confocal laser microscope and flow cytometry respectively. RBcs coating was able to diminish macrophage internalization of the proposed system. The uptake of the fluorescence labelled C-AlbOxa-nZnO by CT26 cells was investigated also qualitatively and quantitatively by confocal laser microscope and flow cytometry respectively. The confocal microscope graphs revealed the uptake of C-AlbOxa-nZnO intracellularly adjacent to the nucleus.CT26 cellular capturing expressed as MFI, was improved in time- and concentration dependent manners. Tracking different endocytic pathway indicated that clathrinmediated endocytosis is the endocytic pathway involved in the uptake of the prepared C-AlbOxa-nZnO. The incorporation of nZnO into the proposed platform was responsible for downregulation of surface CD44 and consequently hampering both cell adhesion and migration. C-AlbOxa-nZnO was able to induce macrophage polarization towards pro-inflammatory M1phenotype in terms of upregulation of IL-6, TNF-α, CD80, CD86 and MHC II expressions on J774 macrophage cells. C-AlbOxa-nZnOwas able to induce ICD hall marks on CT26 cells namely; surface calreticulin expression, ATP and HMGB1 release over untreated cells or those treated with either monotherapy. C-AlbOxa-nZnO was able to stimulate macrophage recognition and phagocytosis of the treated CT26 cells over untreated cells or those treated with either monotherapy. Lower RES deposition, yet higher tumour preferential accumulation were noticed following IV administration of C-AlbOxa-nZnO compared to the uncoated counterpart. Accordingly, C-AlbOxa-nZnO can be considered a potentially promising nanosystem triggering the anti-tumour immune responses and thus, Summary 207 could be utilized as an injectable delivery system with long-standing action, controlled release of oxaliplatin and preferential tumour deposition. To support the outcomes of this study, it seems to be essential to carry out further antitumor efficacy and re-challenge studies. |