Development Of A Multifunctional Magnetic Nanoscale Drug Carrier And Study On Its Antitumor Efficacy

Cancer is a leading cause of death world-wide and is responsible for approximately13%of all deaths, according to the World Health Organization. In Europe alone,Ferlay et al. recently estimated that in20081.7million cancer deaths occurred, and3.2million cancer cases were diagnosed. Although prognosis is better now, the largevariety of cancer types and metastases makes treatment very difficult. Surgicalresection is the treatment of choice, since this treatment is usually curative. Surgery,however, is not an option in many patients due to the tumor size, location andpresence of metastases. External beam radiotherapy is also considered a curativetreatment option. However, not all tumors are eligible for this therapy due to motionof the tumor-bearing tissue to the adjacency of radiosensitive organs. Anotherfrequently used therapy is systemic chemotherapy, but although chemotherapeuticagents are becoming more and more specific, many of the clinically usedchemotherapeutics require high tissue concentrations, which are frequently associatedwith systemic toxicity. A very promising approach to overcome systemic toxicity isthe application of drug-loaded nanosized drug carriers, such as liposomes, polymericnanoparticles, dendrimers and micelles. The incorporation of chemotherapeuticagents into nanosized drug carriers has several advantages compared to systemicchemotherapy. First, low-molecularweight drugs are mostly rapidly eliminated byliver and/or kidneys. By loading them in stealth nanoparticles, their bioavailabilitysubstantially increases. Second, due to their small size, nanosized drug carriers arepassively targeted to the tumors by the enhanced permeability and retention (EPR) effect, leading to a higher drug concentration at the tumor site and decreased toxicitycompared with systemic administration. Third, hydrophobic drugs can only beadministered intravenously (i.v.) after addition of solubilizing adjuvants like ethanolor Cremophor EL, which is often accompanied with toxic side effects. Incorporationof these drugs in micelles avoids the use of adjuvants.Similar to liposomes, polymeric micelles can deliver not only hydrophobic agentsembedded in their hydrophobic core, but also hydrophilic agents encapsulated in theiraqueous membrane. Compared to liposomes which also have a structure ofhydrophobic membrane and aqueous core, polymer micelles exhibit better stabilityand provide more possibilities of adjusted physical, chemical, and biologicalproperties by changing block lengths, chemical structure, and further functionalizingwith biomolecules. Therefore, polymeric micelles have attracted extensive attentionin the field of multifunctional nanomedicine because of their unique propertiessuperior to other types of drug delivery systems.Recently, polymer micelles have gained considerable attention as a versatilenanomedicine platform with greatly improved drug pharmacokinetics and efficaciousresponse in cancer treatment. Polymeric micelles not only can be used to incorporatetherapeutic agents, but also can be used to encapsulate magnetic or fluorescentnanoparticles for magnetic resonance imaging or optical imaging, which allowsreal-time detection of the distribution of agents in tumor tissue or tumor cells. Inaddition, magnetic nanoparticles can also be used to achieve magnetically-guideddrug delivery. Current anticancer treatments essentially target a tumor cell's ability toproliferate. Nevertheless, the major cause of death (over90%of cases) in cancerpatients is not due to their primary tumors but to the development of metastases. Cellmigration plays a crucial and primary role in tumour metastasis. Therefore, the abilityto inhibit, or at least to reduce, the migratory and invasive capacity of tumor cellsoffers a new approach to the treatment of patients with a malignant disease. Based onthe above-mentioned objectives, we developed a functionalized and magneticnanovehicle loaded with Fe₃O₄nanoparticles and hydroxycamptothecin in this paper,and evaluated its biological activity. There are mainly three parts in the dissertation:1. A double-targeted magnetic nanocarrier based with potential applications in thedelivery of hydrophobic drugs has been developed. It consists of magnetite (Fe₃O₄)nanoparticles encapsulated in self-assembled micelles of the amphiphilic copolymerMPEG–PLGA [methoxy poly (ethylene glycol)-poly (d,l-lactide-coglycolide)], andwas fabricated using the solvent-evaporation technique. The magnetic nanocarrier hasa very stable core–shell structure and is superparamagnetic. Its cytotoxicity wasevaluated using the MTT assay with three cell lines-HeLa, MCF-7, and HT1080; itexhibited no cytotoxicity against any tested line at concentrations of up to400μg/mLafter incubation for24h. Its cellular uptake was studied by Prussian blue staining andby fluorescence microscopy after encapsulating a fluorescent probe (hydrophobicquantum dots) into the nanocarrier. Finally, the magnetic targeting property of themagnetic nanocarrier was confirmed by an in vitro test. Overall, the results obtaineddemonstrate the potential of the double-targeted nanocarrier for the intracellulardelivery of hydrophobic drugs.2. A hydroxycamptothecin-encapsulated magnetic nanovehicle was fabricated byco-encapsulating Fe₃O₄nanoparticles (NPs) and10-hydroxycamptothecin (HCPT)into a micelle core self-assembled from the amphiphilic copolymer methoxypolyethylene glycol–poly(D,L-lactide-co-glycolide) via a facile dialysis method. Asatisfactory drug-loading content (9.03±0.67%) as well as a relatively highencapsulation efficiency (53.52±6.46%) was achieved. In vitro drug release wasperformed by membrane dialysis and a pH-dependent release behavior was observed.Compared to free HCPT dissolved in dimethyl sulfoxide, HCPT-encapsulatedmagnetic nanovehicle showed a greatly improved in vitro antitumor efficacy againstthree different human cancer cell lines-HeLa, A549, and HepG2-and lower IC50values were measured. The mechanism of cell death was investigated and clearlydemonstrated that the apoptosis process was triggered. An in vitro wound-healingassay and a transwell assay indicated that the HCPT-encapsulated magneticnanovehicle exerted much stronger activity in inhibiting HeLa cell migration.Magnetic targeting study indicated that HEMN could directly deliver HCPT to desired area under a magnetic guidance. These results demonstrate theHCPT-encapsulated magnetic nanovehicle might have important potential in clinicalapplications for inhibiting tumor metastasis.3. Firstly, Mal-PEG-PLGA copolymer was synthesized by ring-openingpolymerization of D,L-lactide and glycolide in toluene under a flow of nitrogen, inthe presence of Mal-PEG-OH as a macro-initiator. Secondly, Fe₃O₄nanoparticles(NPs) and10-hydroxycamptothecin (HCPT) were simultaneously loaded into amicelle core self-assembled from the amphiphilic copolymer Mal-PEG-PLGA via afacile dialysis method. Then, an RGDC-functionalized and HCPT-encapsulatedmagnetic nanocarrier (designated as RFHEMN) was fabricated by introducing atargeting peptide (RGDC) to the system. The in vitro antitumor activity of variousformulations of HCPT was investigated using human lung cancer cell line (A549),and human embryonic kidney cell line (HEK293T) was used as a control. The resultsindicated that RFHEMN exhibited the strongest antitumor efficacy to A549cells.The antimetastatic property of various formulations of HCPT was investigated andthe results demonstrated that RFHEMN exerted strongest activity in inhibiting A549cell migration. Eventually, the effect of various formulations of HCPT on cell cycledistribution was studied by flow cytometry.