Introduction

    Growing level of hygiene and medical care in western civilization is resulting in the change of disease types we are facing. Currently, the most dangerous, as expressed in the number of lethal cases, are cardiovascular disease (CVD) and cancer, and the last one is slowly taking the first position in the infamous statistics of death causes. Among contemporary citizens of Europe probability of developing cancer during the life time is about 42%; male 45%, female 39%. The most frequent are breast, prostate, colon and lungs. Carcinogenesis arises from a single transformed cell but it is a multistep process resulting from a combination of environmental and congenital factors. Thus, the appearance of cancer in our body is an effect of several subsequent events triggered by various factors mostly genetic predispositions, viruses, radiation or certain chemicals. They elicit many genetic and cytoplasm events including fragmentation of chromosomes, deactivation of tumor suppressor genes (protein 53, retinoblastoma protein…), functional retardation of mitochondria, what finally leads to cancer.

    Cancer, as a disease was for the first time described by ancient doctors almost 30 centuries ago but the word “carcinoma” and first effective surgical procedures were introduced by Avicenna (Ibn Sina, Al-Quanun fi al-tibb, Canon Medicae, 1020). At the beginning cancer grows without any symptoms, when it is big enough to be noticed by a patient in most of the cases it already started to spread forming new colonies in our body. First help of a doctor is a surgical removal of primary cancer. When it is already in the metastasis special anticancer drugs are administered. Contemporary cancer pharmacotherapy began in 1940s, firs by application of nitrogen mustard and folic acid antagonist (aminopterin and methotrexate), early examples of rational drug design. With the development of chemical and medical sciences new anticancer drugs appeared: taxanes, vinca alkaloids, anthracyclines, fluorouracil... Beginning of chemotherapy brought a new problem - side effects, which are frequently devastating for the patient’s organism, and drug resistance. To achieve efficient elimination of cancer cells we started using poisons at almost lethal doses, what heavily destroys also healthy cells and organs in our body.  In 1965 combinatorial - multidrug cancer treatment was introduced. This new approach was based on the hypothesis that cancer chemotherapy should follow the strategy of antibiotic therapy for tuberculosis with combinations of drugs, each with a different mechanism of action. Cancer cells could conceivably mutate to become resistant to a single agent, but by using different drugs concurrently it is more difficult for the tumor to develop resistance. Application of few drugs in specially designed “cocktails” proved to be much more efficient. With the successes of combinatorial chemotherapy and the discovery of many new agents, there was a feeling, at this time, that all cancers could be treated, if one could properly deliver to the tumor the proper combination of drugs, but it was just a feeling... Nonetheless, cancer remains a major cause of illness and death, and conventional cytotoxic chemotherapy has proved unable to cure most cancers after they have metastasized (spread). Recently a new hope on efficient and safe delivery of anticancer agents is associated with nanoparticles (NP), objects of diameter below a fraction of micrometers.

Nanotechnology

      Nanoparticles show a few very important properties, which can help to fight the beast. First, they can encapsulate cytostatic drugs which are frequently strong poisons and causes local necrosis at the place of delivery as well as systemic side effects. Nanoparticles have already been introduced in drug delivery and even in cancer therapy (liposomes with doxorubicine, taxanes-albumin coniugates). Liposomal drug carriers are not stable enough in our circulatory system. Blood vessels in tumor are found to be leaky with pores of 20-150 nm, what leads to the increased resident time of NPs in this area and are called enhanced permeability and retention (EPR) effect. NP can be equipped in surface antibodies or other special recognition moieties which increase their affinity to cancerous cells. To achieve passive (EPR) or active targeting NPs should be able to stay in our circulatory system long enough. Unfortunately our immunological system is efficiently removing any foreign objects from our bloodstream so the NP to stay there should be carefully designed and made. To escape recognition by the complement immune system and prevent coating by small plasma proteins, special coatings of NP have been developed; those are polyethylene glycol and polysaccharides. They form a sort of hydrogel which has very low surface energy and binds water around. NP can serve as a carrier for multiple drugs with various mechanisms of action; such “one package delivery” will prevent development of drug resistance of cancer cells. Additionally NP can carry molecules which are normally unable to get into the cells, like siRNA, which can trigger apoptosis. Polysaccharide NPs are especially promising due to their biocompatibility since polysaccharides are widely present in our organism. Polysaccharides are also very easy for chemical modification so various targeting molecules and multiple drugs can be covalently bound in their structure and at the surface. There is also another advantage of having glucose residues at the NP surface. Malignant rapidly-growing tumor cells typically have glycolytic rates that are up to 200 times higher than those of their normal tissues of origin. This phenomenon was first described in 1930 by Otto Warburg and is referred to as the Warburg effect. This phenomenon may simply be a consequence of the mitochondria damage in cancer, or an adaptation to low-oxygen environments within tumors, or a result of cancer genes shutting down the mitochondria because they are involved in the cell's apoptosis program which would otherwise kill cancerous cells. Despite the unclear reasons Warburg phenomenon has important medical applications; it is utilized clinically to diagnose and monitor treatment responses of cancers by imaging uptake of 2-18F-2-deoxyglucose (FDG) (a radioactive modified glucose) with positron emission tomography (PET). We plan to utilize the same mechanism by building outer surface of NP from polysaccharides. Presence of abundant glucose chains at the surface, which will be “swallowed” by glucose transporters on cancer cell wall, will cause attachment of NP to the cell surface followed by internalization. Inside, in the endosome, due to low pH and enzymatic (lizozyme) activity NP will break apart releasing drugs. 

    Presented work describes technology which allows direct formation of polysaccharide NPs in water solution at room temperature without the use of aggressive chemicals. This is very important since cleaning technologies of NPs suspensions are sometimes complex and expensive. First polysaccharides are partially oxidized to form reactive aldehyde groups along the chain. Specific oxidation agents are applied, those are periodates and hydrogen peroxides with catalyst. Then hydrophilic polysaccharide backbone is modified with hydrophobic moieties, like lipophylic amino acids or aliphatic or aromatic amines. Polysaccharides, when dissolved in water are in the form of more or less loosely coiled chain, sometimes with some branching, which are closely surrounded by water molecules. In the case of ionized polysaccharide derivatives, like hyaluronan or chitosan, since interactions with water molecules are stronger and also electrical mutual repulsion appears, the chain is less coiled – more straight. When some of the sugar rings are substituted with hydrophobic molecules side groups (hydrocarbons, fatty acids, hydrophobic amino acids), hydrophobic moieties are “trying” to be together, to minimize the internal energy of molecule. This process forces the formation of NP in water environment due to self assembly. Obtained NPs are stable and can be lyophilized and stored as dry powder, after immersion in water hydrophobic – hydrophilic interactions forms NPs again. Preliminary experiments conducted on cancer cell lines and mice are very promising. Polysaccharide NPs proved to be able to efficiently enter cancerous cells by endocytosis and to release their load in lysosomes, when pH decreases. This is a very important phenomenon, in normal body pH nanoparticles are stable for many days, in the low pH after endocytosis NP breaks down and release the load.

    Various drugs have already been covalently linked inside polysaccharide NP: Daunorubicine, Doxorubicine, Taxol, 5-F Uracile, Cytarabin, Gemcitabin… Obtained NP showed to efficiently eliminate various types of human cancer cell lines, during in vitro experiments, while polysaccharide NPs without the drug load are nontoxic for human cells and mice. Currently a series of animal experiments are in the preparation stage.

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