Liposome-based nanovehicles for cancer therapy

Abstract

Lipid-based nanomedicine is only of the pioneer nanomaterial used as encapsulation vehicle in attempt for realizing personalized medicine. Liposomes, especially has been used widely in clinics and some others have been approved by FDA as chemotherapeutic agents in cancer therapy

as to achieve high tumor retention rate and effective cancer therapy. Chapter 2. In this chapter, I described the methods of making 9R conjugated liposome for encapsulation of ODN. The effective use of oligonucleotide therapeutics, such as antisense oli-godeoxynucleotides (ODNs) and small interfering RNAs (siRNAs), requires efficient delivery systems capable of intracellular penetration. Cell-penetrating peptides (CPPs), including arginine-rich peptides, have been extensively studied as tools for enhancing intracellular uptake efficiency of various bioactive molecules, including nanoparticles and liposomes. CPPs also have an ability to form tight complexes with nucleic acids, such as ODNs and siRNAs,making CPPs effective as packaging agents. Here, I constructed a CPP-modified liposome loaded with complexes of nona-arginine (9R) and NF-kB decoy ODNs, and evaluated intracellular uptake and anticancer activity in vitro. I found that 9R/ODN complexes were efficiently loaded into liposomes that were effectively internalized into U87MG glioblastoma cells and sensitized cells to the effects of paclitaxel. To the best of our knowledge, this is the first report describing the dual use of 9R CPP as a cell penetrating and a complexing agent within a single nanoparticle. Chapter 3. In this chapter, I described the mechanism of making aptide conjugated liposome. Aptide is a novel targeting ligand, solely produced by our lab, which has very nano-molar af-finity towards target with the advantage of small size, ease of manufacture, no patent issue and most of all, due to lack of disulfide bond in the structure, aptide remains stable in intracel-lular condition. My target is extra-domain B of fibronectin (EDB), which is over-expressed during tumor formation by alternative splicing. By conjugating $APT_{EDB}$ onto the surface of liposome, I speculate that my liposome would be efficiently targeting EDB in tumor proximi-ty, enhance the uptake rate of liposome into the tumor cells and reduce the non-specific uptake of liposome to non-cancerous area, while retaining the ability of liposome to load high amount of cargo (i.e. doxorubicin). As compared to non-targeting liposome, $APT_{EDB}$ -liposome showed significantly higher cellular uptake in U87MG human malignant glioblastoma cell line as compared to non-targeting liposome. Similar result was shown in in vivo targeting ex-periments. I observed around 55% reduction of tumor volume as compared to 26% of parental doxorubicin treatment, and 14% reduction when treated with non-targeting liposome. Taken together, our $APT_{EDB}$ -liposome encapsulating doxorubicin ($APT_{EDB}$ -LS (dox)) might be seen as an efficient drug delivery system. Chapter 4. In this chapter, I explore the possibilities of different targeting ligand density ver-sus the uptake efficacy. Since early times, research has been done by only using $PEG_{2000}$ as anti-biofouling method. Herein, I also try to find out whether the length of PEG used as anti-biofouling has any significant effect on cell uptake efficacy. I made 4 categories of liposome: (a) $APT_{EDB}$ - $PEG_{2000}$ + $PEG _{2000}$ liposome, (b) $APT_{EDB}$ - $PEG_{2000}$ + $PEG_{1000}$ liposome, (c) $APT_{EDB}$ - $PEG_{1000}$ + $PEG_{1000}$ liposome, and (d) $APT_{EDB}$ - $PEG_{1000}$ + $PEG_{550}$ liposome, Interestingly, I found out that the optimum aptide percentage on liposome is around 2.5 wt%

namely Doxil® and Taxane®. Lipid-based micelle and other lipid-coated nanoparticle are also used in nanomedical field. Lipid-based nanoparticle has been deeply studied, in-cluding its physiochemical properties, its uptake mechanism, circulation in human body, sta-bility, tumor uptake efficacy among others. To date, many research groups focused on devel-oping tumor cell specific targeting liposome in order to achieve higher tumor accumulation and better anti-tumor efficacy. In my research, I developed (i) 9R(9-arginine)-targeting liposome, that increases tumor cell uptake by the effect of cell penetrating peptide for decoy oli-godeoxynucleotide (ODN) delivery, (ii) APTEDB-liposome encapsulating doxorubicin, with aptide having high affinity towards tumor associated fibronectin and doxorubicin as anti-cancer agent, (iii) a novel hyper-cell permeable micelle (HCPMi), that has the ability to form sub-20nm micellar formation, by the combination of different lipid length, and at the same time, displays superior cancer cell targeting both in vitro and in vivo, (iv) manipulating the effect of PEG length and targeting ligand density towards the uptake of APTEDB-liposome in vitro and in vivo. It is our vision to develop a lipid-based nanoparticle that is stable, easy to manufacture, long-circulating in vivo with superior tumor specific targeting ability

with decreasing cell uptake efficiency as aptide percentage increased to 10 wt%. I also observed that with different PEG length used ($PEG_{2000}$, $PEG_{1000}$, $PEG_{550}$), reduced background PEG length showed a general increase in the cellular uptake. When anti-cancer drug doxorubicin (Dox) was loaded into each liposome, the lethal dose of dox LD50 showed the lowest (285.63nM in $APT_{EDB}$ - $PEG_{2000}$ + $PEG_{1000}$ liposome), followed by 308.93nM in $APT_{EDB}$ - $PEG_{1000}$ + $PEG_{550}$ liposome. These values are significantly lower than both the $APT_{EDB}$ - $PEG_{2000} + $PEG _{2000}$ liposome and $APT_{EDB}$ - $PEG_{1000}$ + $PEG_{1000}$ liposome, which showed 647.59nM and 559.18nM respectively. This result correlates well with the in vivo tumor uptake experiment where both $APT_{EDB}$ - $PEG_{2000}$ + $PEG_{1000}$ liposome and $APT_{EDB}$ - $PEG_{1000}$ + $PEG_{550}$ showed highest uptake in tumofollowed by $APT_{EDB}$ - $PEG_{1000}$ + $PEG_{1000}$ liposome and the lowest uptake is seen in $APT_{EDB}$ - $PEG_{2000}$ + $PEG _{2000}$ liposome. All in all, I observed a marked difference between cellular uptake, and cytotoxicity in different versions of liposome, and concluded that optimization has to be done for each type of nanoparticle to find the best combination of targeting ligand density and PEG length for maximizing therapeutic effects of the said nanoparticle.