Chapter Literature Review The previous chapter gave a brief introduction of gene delivery and its current challenges as well as my research objective. The purpose of this chapter is to review and explore topics pertinent to the main thrust of my research. The general focus in my study is on polymer micelles and non-viral vectors, in particular, cationic polymers, for delivery of drugs or genes and codelivery of drugs and genes. I will also examine some of the difficulties involved with current gene delivery systems using non-viral vectors. 2.1 Polymeric micelles Micellization phenomena produced by amphiphilic materials have been observed long before and micellization has been an important area of research in colloid science [Wihelm M., Gao Z., et. al. 1991]. Initially the study began from low molecular amphiphiles such as ionic surfactants [Wihelm M., Gao Z., et. al. 1991]. In the last few decades, micelles fabricated by amphiphilic block copolymers began to attract much attention from both academia and industry. A great number of papers have reported mechanism of self-assembling, properties and characterization methods of polymeric micelles since 1970s’. For example, Tuzar et. al. found that block copolymers of polystyrene (PS) and poly(ethylene oxide) (PEO) could form spherical micelles in water when the length of soluble PEO is significantly longer than that of the insoluble PS portion of the molecule [Tuzar Z. et. al. 1976]. Wihelm et. al. developed a fluorescence probe technique to study the critical micelle concentration [Wihelm M. et. al. 1991]. Gao and Eisenberg established a model of micellization for block copolymers in aqueous solution [Gao Z. et. al., 1993]. Astafieva et. al. investigated the critical micellization phenomena in block polyelectrolyte solutions [Astafieva I. et. al., 1993]. Chen et. al. studied the effect of block size and sequence on the micellization of ABC triblock methacrylic polyampholytes [Chen W-Y. et. al., 1995]. This section will give a review on mechanism of micelle formation, micelle structure, methods for measuring the critical micelle concentration (CMC) of polymeric micelles, and biomedical applications of micelles. 2.1.1 Mechanism of polymeric micelle formation Formation mechanism and properties of polymeric micelles have been well studied. Micelle formation is driven by two opposing forces including an attractive force between the amphiphiles leading to aggregation and a repulsive force that prevents unlimited growth of the micelles into a distinct macroscopic phase [Astafieva I., 1991]. Thermodynamically, micelle formation is mainly due to positive standard entropy of micellization. The formation of polymeric micelles in aqueous solution is influenced by the chain length of hydrophilic and hydrophobic blocks, temperature and ions presented in the aqueous solution. 2.1.2 Structure of polymeric micelles A simple but important finding obtained by the studies in the past few decades is that the polymeric micelles usually have core-shell structure (see Figure 2.1) in aqueous solution [Jones M-C., 1999]. The core consists of hydrophobic segments of copolymer while the shell is made of hydrophilic segments including ionic species [Astafieva I., 1993; Luisi P. L. et. al., 1984; Pileni M.P., 1989]. This kind of core-shell model is not the only structure that amphiphilic copolymers may have. Under some conditions they can also form long and rod like micelles, although this structure is more common for small molecular weight amphiphiles [Astafieva I., 1993; Price C. 1983; Price C. et. al., 1986; Canham P.A. et. al., 1980]. In non-aqueous solution, the core-shell model can even inverse completely, i.e. a hydrophilic core and hydrophobic corona [Astafieva I., 1993; Price C. 1983; Price C. et. al., 1986; Canham P.A. et. al., 1980]. In fact, the core-shell concept of polymeric micelles was theoretically borrowed from those formed by small molecular weight amphiphiles. However, this core-shell structure can be evidenced by some techniques such as fluorescent probes and 1H-NMR. For example, fluorescence of bis(1-pyrenyl-methyl)ether (dipyme) [Winnik F. M., et. al. 1992) and pyrene [Wihelm M. et. al., 1991] is sensitive to the polarity change in their local environments. Therefore, by studying the fluorescent change of these compounds, one can know the polarity and hence the hydrophobicity of the core. 1H-NMR can also provide some information about the core-shell structure [Jones M-C. et. al., 1999]. The H-NMR spectrum of a copolymer in a solvent (e.g. CDCl3) where micelle formation is not expected should exhibit the characteristic peaks corresponding to the hydrophilic and hydrophobic segments of the polymer. However, in D2O, the presence of micelles with a highly inner viscous hydrophobic core results in a restricted motion of the protons as demonstrated by the weak signals associated with the hydrophobic segments of the copolymer [Jones M-C. et. al., 1999; Nakamura K., 1977; Bahadur P., et. al., 1988]. 2.1.3 Critical micelle concentration (CMC) and its measurements CMC is one of the most important parameters of the polymeric micelles. It is well known that the micelles exist only above a certain concentration, i.e., the critical micelle concentration. However, the CMC characterization of amphiphilic polymer is slightly different from that of small molecular amphiphiles. The characterization of CMC is not only essential as the evidence of the formation of micelles but also very important for polymeric micelles as a drug delivery system, this will be discussed later. A number of techniques on the determination of CMC were reported. In principle, one can use any physical property that shows sudden changes at or near CMC. Most frequently, breaks or discontinuities in plots of such properties as the surface tension, electrical conductivity, osmotic pressure, interfacial tension, or light scattering as a function of polymer concentration have been used for this purpose [Wihelm M., et. al. 1991; Jones M-C., et .al. 1999]. However, since the CMC of block copolymers is normally much lower than that of low molecular weight surfactants, many techniques, which are suitable to low molecular weight amphiphiles, cannot be simply extended to amphiphilic copolymers [Wilhelm M. et. al., 1991]. For example, light scattering is known as one of the most powerful techniques for the determination of the size, shape, and aggregation numbers of micelles as well as values of the diffusion coefficient of the micelles. However, the scattering techniques are usually not sensitive enough to detect the particles at the CMC as low as that of block copolymers [Wilhem M. et. al., 1991]. Gel permeation chromatography (GPC) under aqueous conditions has been employed to study the CMC of amphiphilic copolymers since single chains and micellar chain fractions of copolymers exhibit different elution volumes [Weissig V. et. al., 1998; Yokoyama M. et. al., 1993]. It is also possible to determine molecular weight and aggregation number of micelles by GPC. It is important to note that the integrity of polymeric micelles should be maintained during their elution through the size exclusion column. However, adsorption of the polymer on the column may be a problem [Yokoyama M. et. al., 1993], especially at concentrations close to the CMC, where micelles consist of large loose aggregates. Because of the shortages of these techniques, another method was developed based on the fluorescent probe techniques [Kalyanasundaram K. et. al., 1977; Wilhelm M., et. al. 1991]. The fluorescent probe used is usually polarity-sensitive compound such as pyrene (see Figure 2.2 for its chemical structure). Pyrene is a condensed aromatic hydrocarbon, which is highly hydrophobic and sensitive to the polarity of the surrounding environment. Below the CMC, pyrene is solubilized in water, a medium of high polarity. When micelles form at a polymer concentration above the CMC, pyrene partitions preferentially into the hydrophobic domain afforded by the micellar core and thus, experiences a nonpolar environment [Kalyanasundaram K., et. al., 1977; Jone M-C., 1999]. Consequently, numerous changes such as an increase in the fluorescence intensity, a change in the vibrational fine structure of the emission spectra and a red shift of the (0,0) band in the excitation spectra, can be observed. The apparent CMC can be obtained from the plot of the fluorescence intensity ratio of pyrene such as the I1 (the first peak)/I3 (the third peak) ratio from emission spectra or the I333/I338 (peaks at 333 and 338nm) ratio from excitation spectra, against polymer concentration. A major change in the slope indicates the onset of micellization [Figure 2.3] [Jone M-C., 1999; Kalyanasundaram K. et. al., 1977]. The I1/I3 10 ratio is measured at a constant excitation wavelength (339nm) and variable emission wavelengths corresponding to I1 and I3. Many researchers have applied the fluorescent probe technique to various polymeric amphiphiles [Kalyanasundaram K. et. al., 1977; Wihelm M., Gao Z., et. al. 1991; Astafieva I. et. al., 1993]. Some claimed that CMC might be better ascertained by the I333/I338 ratio since the I1/I3 ratio is affected by the wavelength of excitation and may result in an erroneous CMC [Shin I. L., et. al., 1998; Astafieva I., 1993]. The CMC determined with fluorescence techniques needs to be carefully interpreted for two reasons. Firstly, the concentration of pyrene should be kept extremely low (10-7 M) so that a change in slope can be precisely detected as micellization occurs. Secondly, a gradual change in the fluorescence spectrum can sometimes be attributed to the presence of hydrophobic impurities or association of the probe with individual polymer chains or permicellar aggregates [Chen W. Y. et. al., 1995]. Changes in anisotropy of fluorescent probes have also been associated with the onset of micellization [Zhang X., et. al., 1996]. Figure 2.1 Schematic representations of block and random copolymer micelles. [Jones M-C. et. al., 1999] 11 Figure 2.2 Molecular structure of pyrene. 700 450 Fluorescent Intensity Fluorescent Intensity 600 I1 400 I3 350 300 250 200 500 400 300 200 150 100 100 50 360 370 380 390 400 410 300 Wavenumber (nm) 310 320 330 340 350 360 Wavenumber (nm) Figure 2.3 Emission spectra (left, excited at λex=339nm) and excitation spectra (right, monitored at λem=390nm) of pyrene. The reason of specially reviewing the two characteristics of polymeric micelles, the low critical micelle concentration and core-shell structure in aqueous phase, is that the two characteristics of polymeric micelles are especially significant for the micelles to be applied as drug carrying system. The following section will review in detail on how researchers have applied polymeric micelles’ characteristics for drug delivery. 2.1.4 Polymeric micelles as drug delivery carrier Delivery systems for drugs used in the treatment of human diseases have had an impact on nearly every branch of medicine including cardiology, endocrinology, oncology, ophthalmology and immunology. Generally speaking, successful drug therapy may be 12 achieved in three ways: delivering the drug efficiently to the target, modifying the drug for increased efficiency, or finding a novel drug of inherently high efficacy. Of the three, devising an efficient means of delivery is the most cost-effective. In the search for delivery systems, Yolls and his co-worker reported the use of lactide-based copolymers for drug delivery in 1970 [Yolls S., 1975]. Such devices have become much more varied since then. Sustained-release tablets [Ng S.Y., et. al., 2000; Ng S.Y , et. al., 1997; Sintzel M.B., et. al., 1998], polymeric matrices (e.g. rods, discs and cylinders) [Hilton A.K., et. al., 1993], microparticles [Yang Y.Y., et. al., 2000; Chia H.H., et. al., 2001] hydrogels [Jeong B. et. al., 2000], lipsomes [Zalipsky S., 1995], nanoparticles [Lee J. H., 2003] and drug-polymer conjugates [Lu Z-R. et. al., 2000] are the commonly used formulations for drug delivery. The polymeric micelles were first proposed as a drug carrier by Bader et. al. in 1984 [Bader H., et. al. 1984]. Polymeric micelles exhibit a number of advantages over other forms of drug carriers because of their core-shell structure, low CMC and targeting ability. 2.1.4.1 Core of polymeric micelles as a reservoir for hydrophobic therapeutics The core of the polymeric micelles can serve as a reservoir for an insoluble drug. Incorporation of insoluble drugs into the core of micelles can be achieved by chemical conjugation or by physical entrapment through dialysis or emulsification techniques. Simple equilibration of a drug and micelles in water may not result in high levels of incorporated drug [Kwon G. S. et. al., 1995; 1997]. Chemical conjugation implies the formation of a covalent bond, such as amide bond, between specific groups on the drug and the hydrophobic core of the polymer. Such bonds are normally resistant to enzymatic 13 cleavage mainly because of steric hindrance and thus difficult to be hydrolyzed unless a spacer group is introduced [Ulbrich K. et. al., 1987]. On the other hand, the chemical conjugation of a drug to another compound may change its pharmacokinetics and pharmacodynamics. Therefore, the incorporation of a drug by a physical procedure is preferred. Physical entrapment of the drug is generally done by a membrane dialysis method or an oil-in-water emulsion procedure [Jones M-C. et. al., 1999]. For the dialysis method, a drug and a copolymer are dissolved in a solvent (e.g. ethanol or N, Ndimethylformamide) in which they are both soluble. The mixture is then dialyzed against water by using a membrane. As the solvent is replaced by water, the hydrophobic segments of the polymer and the drug molecules interact and associate to form the core of micelles while the hydrophilic segments arrange towards the aqueous phase to form the shell. In the case of the oil-in-water emulsion method, a drug and a copolymer are dissolved in a water-insoluble volatile solvent (e.g. dichloromethane) and the solution is then added to an aqueous phase with stirring to form an oil-in-water emulsion. The drugloaded micelles are formed as the solvent evaporates. The main advantage of the dialysis process over the emulsion method is that the use of toxic solvents such as chlorinated solvent can be avoided. The drug loading level of micelles could reach up to 8%-25%, depending on the fabrication techniques [Kwon G. S. et. al., 1995;1997], chemical structure of the drug and polymer, temperature and pH. For example, an increase in the length of a hydrophobic segment of polymer facilitates the entrapment of a hydrophobic drug in the core [Torchilin V. P., 2001]. Encapsulation efficiency of the drug also depends on initial drug loading [Kwon G. S. et. al., 1995; 1997] and aggregation number of copolymer. G. S. Kwon and T. Okano reported that by using oil-in-water emulsion 14 method, the encapsulation efficiency of Doxorubicin reached 65% [Kwon G. S., 1996]. In my study, the encapsulation efficiency of indomethacin reached 80% (see Chapter 4). Theoretically, the loading capacity and encapsulation efficiency of polymeric micelles depend on partition coefficiency of drug between the core phase and aqueous phase [Torchilin V. P., 2001]. It should be noted that although polymeric micelles have mostly been studied as delivery systems for drugs, they could also be used to carry plasmid DNA, anti-sense oligonucleotides or diagnostic agents [Torchilin V. P., 2001; Kataoka K. et. al., 2001]. 2.1.4.2 Nanoscale size and hydrophilic shell enabling passive targeting Another characteristic of polymeric micelles is their nanoscale size and hydrophilic nature of the shell. The size of drug-loaded polymeric micelles made from amphiphilic copolymers was reported to range from 10 to 200 nm [Kataoka K. et. al., 2001; Jones M. et. al., 1999; Trubetskoy V. S. et. al., 1996; Shin I. L. et. al., 1998; Yokoyama M. et. al., 1992]. The size and size distribution of polymeric micelles can be measured using dynamic light scattering (DLS) or observed directly under transmission electron microscopy (TEM) [Yu B. G. et. al., 1998], scanning electron microscopy (SEM) [Kim S. Y. et. al., 1998] or atomic force microscopy (AFM) [Cammas S. et. al., 1997; Kohori F. et. al., 1998]. Kwon G. S. and Kataoka K. reported the micelles made from PEO-b-BM [block copolymer micelles containing poly(ethylene oxide)] having a hydrodynamic diameter of 10-30nm [Kwon G. S. et. al., 1995]. The reversible, secondary association of a fraction of PEO-BM occurred giving an aggregate diameter of about 100nm. Another group of researchers studied a variety of drugs and tracers loaded micelles fabricated 15 example, poor specificity of most chemotherapeutic drugs may result in suppression of the bone marrow and other fast dividing tissues as well as potential genesis of secondary cancer. Another drawback of chemotherapy is the development of resistant phenotypes, which no longer respond to chemotherapy. It is also well known that chemotherapy and radiation therapies can cause serious side effects to healthy tissues or organs. With the development of the human genome project, the molecular mechanism of cancer has been substantially studied. This opens the possibility of using gene therapy for treating cancers. Several strategies have been proposed for manipulation of gene expression either on the transcriptional or translational level. A deficient gene can be replaced, and the effect of an unwanted gene can be blocked by the introduction of a counteracting gene. For instance suicide gene therapy offers the perspective to kill cancer cells selectively by using prodrug-converting enzymes and tumor specific promoters. Furthermore, antisense and ribozyme strategies offer the potential to selectively downregulate the expression of specific genes mainly on the translational level predominantly by sequencing specific interaction with messenger RNAs. In fact, most diseases have a genetic component. Hence, gene therapy holds the hope of curing, not merely treating, a broad range of ailments, including inherited diseases like cystic fibrosis and even chronic conditions like cancer and infectious diseases like AIDS. Since the first genetic treatment on September 14, 1990 [Thompson l., 2000], the hope of gene therapy has inspired a great burst of enthusiasm. However, the main challenge of gene therapy lies in the development of safe and efficient gene delivery system. This section will give a review of various types of gene delivery vectors. 23 2.2.1 Gene delivery vectors Gene delivery vector is the vector that deliver gene into the cell. The simplest and safest way to deliver gene is to transport the transgene (i.e. naked DNA) to the target site without the use of vectors. So far, skeletal muscle, liver, thyroid, heart muscle, urological organs, skin and tumor have been explored as a portal for direct gene injection [Nishikawa M. et. al., 2001; 2002]. Various physical manipulations such as electroporation, bioballistic (gene gun), ultrasound and hydrodynamics (high pressure) have been proposed to improve the efficiency of gene delivery. However, due to the rapid degradation of DNA by nuclease in the serum and clearance by the MPS, the gene expression level and the area induced by direct injection of naked DNA were generally limited. It is known that delivery of the naked gene can only be rarely applied with reasonable efficiency, as in intramuscular gene therapy [Mumper R. J. et. al., 1998]. Therefore, it is important to develop an efficient system for gene delivery and gene expression, which is applicable to both basic research and clinical settings. Basically, gene delivery systems can be classified into two categories: viral and nonviral vectors [Luo D. et. al., 2000; Merdan T. et. al., 2002; T. Niidome et. al., 2002; Thomas M. et. al., 2003]. Gene delivery by viral vectors includes employing various viruses as the vehicles to carry gene to the target cells or organs and express in the host cell or organ. This process is called infection. Viral vector appeared before non-viral vector as gene delivery system. The first major gene therapy success was the retrovirusbased treatment of infants suffering from the X chromosome linked severe combined immune deficiency (SCID-X1), displaying the potential of long-term or even permanent cure of hereditary disease [Cavazzana-Calvo M. et. al., 2000]. Because of their highly 24 evolved and specialized components, viral systems are by far the most effective means as gene delivery vehicles, which achieve high efficiencies (usually >90%) for both delivery and expression. For example, some retroviruses can integrate their genetic information into the cell nucleus very effectively. By replacing the natural genetic information of the virus with the genes desired to express in the defective cells, a genetic disease, at least in principle, can be cured [Lundstrom K., 2003]. However, viral-mediated delivery has its own limitations, including toxicity, restricted targeting of specific cell types, limited gene carrying capacity, difficulties in production and packaging, recombination and high cost [Luo D. et. al., 2000]. Furthermore, the toxicity and immunogenicity of viral systems also hamper their routine use in basic research laboratories. The random insertions of transgene may also result in mutations in the host, leading to carcinogenesis [Thomas M. et. al., 2003]. The long-term effect of the integrated transgene and the virus in the host is another serious concern [Thomas M. et. al., 2003]. Therefore, the safety issue makes the great prospects of viral vector uncertain [Lundstrom K., 2003]. Because of these limitations of viral vectors, non-viral vectors with lower cytotoxcity and immune response have been purposed. Compared with viral vectors, non-viral vectors usually not integrate the transgene into a chromosome of the target cell and only maintain as an extra-chromosomal genetic element (episome). Therefore, the nonviral vectors, unlike their viral counterparts, are safe and also amenable to large-scale production. 2.2.2 Non-viral gene vectors 25 Non-viral gene delivery vectors include cationic lipids, cationic polymers and inorganic materials. Inorganic materials such as gold nanoparticles [Yang N. S. et. al., 1995] and multisegment bimetallic nanorods [Salem A. K. et. al., 2003] have been reported to deliver DNA. However, inorganic materials have attracted very limited attention in the gene therapy community, possibly due to their poor versatility and biocompatilibility problem. Cationic lipids and cationic polymers are by far the most widely used vectors in non-viral gene delivery. Although polycations have been used for the insertion of foreign DNA into cells long before liposome [Henner W. D. et. al. 1973; Ehrlich M. et. al. 1976; Fraley R. et. al. 1980], lipids used as a gene vector were more mature than polycations [Thomas M. et. al., 2003]. Lipsome-based gene delivery formulations were first reported by Fraley R. et. al. in 1980. In the 1990s, a large number of cationic lipids, such as quaternary ammonium detergents, cationic derivatives of cholesterol and diacylglycerol and lipid derivatives of polyamines, were reported [Niidome T. et. al., 2002]. Among these lipids, lipofectAMINETM exhibits high transfection efficiency. However, it is quite toxic. The development of new types of lipid molecules appears to be saturated. Most of the efforts have shifted to improve efficiency of gene delivery and expression by modification of the existing lipids or pursuing novel polymer-based carriers [Pedroso de Lima M. C. et. al., 2001; Niidome T. et. al., 2002]. It seems that cationic polymers are the most promising non-viral vector and are attracting great interest. Recent studies have shown that polycations are potentially superior to liposomal formulations in many respects. For example, the formation of the lipoplex (lipsome-DNA complexes) involves interaction among lipid molecules, in addition to that with DNA itself. A major driving force for the complex formation is the release of low-molecular weight counter-ions that 26 make a large entropic contribution to the free energy of binding [Matulis D. et. al. 2002]. The hydrophobic segments of lipids are the key determinant in the macroscopic characteristics of the ensuing liposomes, particularly their size, shape, and stability in the dispersed state, as well as interactions with other lipids, cell membranes, and DNA. This in turn affects the transfection efficiency of the resulting lipoplexes [Smisterova J. et. al., 2001; Zuhorn I.S. et. al., 2002]. Approaches for controlling these parameters are limited, leading to instability of their macroscopic properties over time [Simberg D. et. al., 2001] and thus restricting their pharmaceutical potential. Furthermore, liposomal formulations often require an adjuvant, such as di-oleylphosphatidylethanolamine, for efficient gene delivery [Hui S. W. et. al., 1996]. Compared with lipids, self-assembly of polyplexes ( polycation-DNA complexes) does not entail interaction of the polycation molecules with each other, resulting in greater control of their macroscopic properties, and is quite efficient even without any adjuvants. Furthermore, polycations can be easily tailored by chemical methods to achieve high efficiency or cell targeting. Many of the polyplexes have superior transfection efficiency and serum sensitivity compared to lipoplexes [Gebhart C. L. et. al., 2001]. These advantages make polycations a compelling target for future exploration in non-viral gene delivery. 2.2.3 Polycations Compared with lipids, the greatest advantage of cationic polymers as gene vectors are that cationic polymers can be easily tailored and synthesized to suit the special requirements encountered by gene delivery. Some natural polymers have also been 27 employed to deliver genes. In general, the most commonly used cationic polymers as gene vectors include branched and linear polyethyleneimine (PEI), copolymers of PEI, poly(L-lysine) and its copolymers, imidazole-modified poly(L-lysine) and chitosan (Figure 2.4). Figure 2.4 Cationic polymers most frequently used for nucleic acid delivery. [Merdan T. et. al., 2002] Polyethyleneimine (PEI) PEI polymers have become the gold standard of non-viral vectors. PEI with different molecular weights and degrees of branching have been synthesized and evaluated in vitro as well as in vivo. Branched PEI with a molecular weight of 25 kDa is often used as a control to evaluate gene expression efficiency of other non-viral vectors because of its high efficiency in gene transfection [Marschall P. et. al., 1999; Campeau P. et. al., 2001]. PEI polymers are able to effectively condense DNA molecules, leading to homogeneous spherical particles with a size of ~100nm. This enables efficient in vitro gene transfection 28 [Marschall P. et. al., 1999; Campeau P. et. al., 2001]. PEI offers a significantly more efficient protection against nuclease degradation than other polycations such as poly(Llysine), possibly due to its high charge density and efficient complexation with DNA. The huge amount of positive charges, however, results in a rather high toxicity, which is one of the major limiting factors especially for its in vivo use. The high density of primary, secondary and tertiary amino groups exhibiting protonation only on every third or fourth carbons at pH confers significant buffering capacity to the polymer over a wide range of pH [Godbey W. T. et. al., 1999]. This property, known as ‘proton sponge’ is the most likely contributed to the high transfection efficiency obtained using PEI-based polymers [Boussif O. et. al., 1995]. Support for this assumption is that endosomal acidification is required for efficient gene transfection [Midoux P. et. al., 1999]. Despite this recognized association, knowledge about relationships between polymer structure and important biological properties such as toxicity or gene transfection efficiency is rather limited. An increased molecular weight of PEI led to higher gene transfection as well as greater cytotoxicity [Fischer D. et. al., 1999]. The linear PEIs have also been reported [Ferrari S. et. al., 1997; Coll J. L , et. al., 1999]. It has been demonstrated that the linear PEI (Mw 21 kDa) yielded excellent gene transfection efficiency with a rather low toxicity. Linear PEI has recently been reported to mediate a cell cycle independent nuclear entry of plasmid DNA [Brunner S. et. al., 2002]. This finding is of particular importance in the therapy of slow dividing tissues. Poly(L-lysine) 29 Poly(L-lysine) was one of the first polymers used in non-viral gene delivery. Due to the peptide structure of poly(L-lysine) it is biodegradable, making it suitable for in vivo use. However, the polymer exhibits moderate to high toxicity. With poly(L-lysine) of suitable molecular weights and optimized N/P ratios, complexes with plasmid DNA displayed a size of ~100nm [Wolfert M. A. et. al., 1999] and were taken up into cells as efficiently as PEI complexes [Merdan T. et. al., 2002]. However, the transfection efficiency was several orders of magnitude lower. A possible reason is the lack of amino groups with pKa between and 7, resulting in no endosomolysis and low levels of transgene expression [Merdan T. et. al., 2002]. Imidazole-containing polymers The heterocycle imidazole displays a pKa of about 6, possessing a buffering capacity in the endolysosomal pH range, and thus possibly mediating vesicular escape by a ‘proton sponge’ mechanism. Therefore, polymers containing the heterocycle imidazole have shown promising transfection capability. In several approaches, modification of ε-amino groups of poly(L-lysine) using histidine or other imidazole-containing structures showed a significant enhancement of reporter gene expression compared to poly(L-lysine) [Fajac I., Allo J.C. et. al., 2000; Benns J. M. et. al., 2000; Midoux P. et. al., 1999]. Chitosans Chitosan is a biodegradable and linear aminopolysaccharide with randomly distributed beta linked N-acetyl-D-glucosamine and D-glucosamine, derived from the common biopolymer chitin. Chitosan displays a significantly better biocompatibility than PEI. 30 Depending on the molecular weight and the degree of deacetylation, chitosans are capable of forming small (50 fold) compared to the intact structures. However, Thomas Merdan proposed that an increased flexibility of the fractured structures with a better ability to complex DNA might play a crucial role [Tang M. X., 1996]. 32 Other cationic polymers In addition to these commonly used cationic polymers as described above, a variety of other cationic polymers such as polybrene [Mumper R. J., 1996], gelatin [Leong K. W., 1998], tetraminofullerene [Isobe H., 2001], poly(l-histidine)-graft-poly(L-lysine) [Benns J. M., 2000] have also been employed to deliver DNA. Furthermore, Leong K. W.’s group has synthesized polyphosphoramidate (PPA) bearing different side chains. PPA was synthesized from poly(1,2-propylene H-phosphonate) by the Atherton-Todd reaction. Under the optimized conditions, the polyphosphoramidate bearing spermidine side chain/plasmid DNA complexes yielded a luciferase expression level close to PEI/DNA complexes [Wang J. et. al., 2002]. M. E. Davis’ group synthesized families of linear βcyclodextrin (β-CD)-containing polycations (βCDPs), demonstrating that these polymers were efficient gene carriers [Popielarski S. R. et. al., 2003; Gonzalez H. et. al., 1999; Hwang S. J. et. al., 2001]. Cyclodextrins (CDs) are cup-shaped molecules formed of cylic oligomers of glucose. Cyclodextrins comprised of 6, and glucopyranose units are called α-, β- γ-CD, respectively. Studies of structure-dependent functions with βCDPs demonstrated the importance of intercharge spacing to transfection efficiency and toxicity [Hwang S. J. et. al., 2001]. A significant effect on transfection efficiency was observed when the interamidine distance was reduced by just 2Å. Study of linear, cyclodextrincontaining polycations conducted by Popielarski S. R. et. al. showed that cellular toxicity was related to the distance of the charge center from the carbohydrate unit, and increasing polycation hydrophilicity provided decreasing toxicity [Reineke T. M. et. al., 2003]. Azzam T. et. al. reported polysaccharide-ologoamine based conjugates for gene delivery [Azzam T. et. al., 2002]. Besides, a great number of other cationic polymers have also 33 been synthesized to deliver gene. Although these polymers may have their unique characteristics, few cationic polymers have either significantly higher gene expression efficiency or lower cytotoxicity compared to the commonly used polymeric vectors as described above. Stability of the cationic polymer/DNA complexes, successful delivery of the gene to the target site and in vivo gene transfection efficiency still remain to be great challenges. Therefore, significant efforts have been made to modify the cationic polymers. Modification of current cationic polymers The modification was mainly focused on increasing the stability of cationic polymer/DNA complexes and the targeting ability to diseased tissues. The main factor causing the instability of polyplexes is adsorption of the proteins existing in the blood onto the surface of the complexes. The adsorption of proteins results in the aggregation of the complexes, rendering rapid blood clearance following intravenous administration [Toncheva V., 1998]. One approach to overcome this issue is to introduce a hydrophilic segment into the cationic polymer, which serves as a hydrophilic shell to prevent the complexes from interacting with the serum proteins. PEG, which has been used as the excellent hydrophilic segment of amphiphilic polymers to form drug-loaded micelles, became the first choice. For example, Itaka K. et. al. have incorporated PEG with a molecular weight of 12 kDa to poly(L-lysine). Their results showed that the incorporation of PEG into poly(L-lysine) enhanced the stability of the complexes, making them serum tolerable. PEI has also been conjugated with PEG to enhance transfection efficiency in the presence of serum proteins [Lee H. et. al., 2001]. However, the gene transfection did 34 not seem to increase but decreased due to the presence of PEG chains on the surface [Lee H. et. al., 2002; Itaka K. et. al., 2003]. Mishra S. et. al. conjugated PEG onto branched PEI and linear beta-cyclodextrin containing polymer, and studied the cellular uptake, intracellular trafficking and luciferase expression [Mishra S. et. al., 2004]. The study by using transmission electron microscopy showed that the unPEGylated particles entered cells by large aggregates while the PEGylated complexes were internalized as small and discrete particles. Mao H. Q. et. al. found that the clearance of the PEGylated chitosan nanoparticles in mice following intravenous administration was slower than unmodified chitosan nanoparticles. Peptide and protein have also been PEGylated to increase the stability of complexes [Morpurgo M. et.al., 2004]. In addition to PEG, Toncheva V. et. al. conjugated other hydrophilic polymers such as dextran and poly[N-(2- hydroxypropyl)methacrylamide] (PHPMA) onto poly(L-lysine (PLL) [Toncheva V. et. al.,1998]. The complexes formed discrete nanoparticles with a size of 100nm, displaying decreased cytotoxicity compared to PLL/DNA complexes. These studies showed that the conjugation of PEG helped to maintain the stability of complexes and delay their clearance. However, the introduction of PEG rendered the decrease in gene transfection efficiency possibly due to the lower endolysosomal escaping capacity [Mao H-Q., 2001; Lee H. et. al., 2002; Itaka K. et. al., 2003; Toncheva V., 1998]. In addition, receptor-mediated gene delivery systems have been studied to enhance gene transfection efficiency and cell-type specificity [Templeton N. S. et. al., 2000]. Many receptor-specific ligands, antibodies, vitamins have been covalently conjugated to various cationic polymers. For example, fibroblast growth factor (FGF), RGD, anti-CD3, 35 anti-CD5, transferrin (Tf), mono- or oligosaccharides, folic acid and other ligands have been exploited for targeting to various cells [Sosnowski B. A., 1996; Hart S. L., 1995; Buschle M., 1995; Merwin J. R., 1995; Wagner E., 1990; Haensler J., 1993]. Suh W. et. al. conjugated monoclonal antibody against leukemia-specific JL-1 antigen (anti-JL-1 antibody) as a targeting moiety to poly(L-lysine) (PLL) for targeted gene delivery to leukemia T cells. Antibody-PLL/DNA complexes showed significantly higher in vitro transfection efficiency than PLL/DNA complexes [Suh W. et. al., 2001]. I. J. Hildebrandt et. al. studied the targeting ability of Tf-conjugated PEI by using a noninvasive optical bioluminescence imaging system. The Tf-modified PEI showed significantly higher luciferase activity in vivo and ex vivo at the tumor as compared to other organs, including the lungs (a site of high expression with PEI) [Hildebrandt I. J., 2003]. Bennis J. M. et. al. conjugated folic acid onto PEG-PEI polymer, which was further used as the gene vector. The folate-PEG-PEI showed higher gene expression level in cancer cells compared with PEI/DNA complexes while smooth muscle cells showed no specificity for folate tethered complexes, where PEI/DNA complexes yielded higher gene expression efficiency [Benns J. M., 2001]. These studies indicated that incorporation of biological signals onto cationic polymers provided an efficient way to improve gene transfection efficiency in target cells. 2.2.4 Polymeric micelles as gene vectors Basically, the PEGylated cationic polymers can self-assemble into micellar-like coreshell structure with PEG as the shell to enhance the stability of the polymer/DNA complexes. In addition to this kind of polyion complex micelles, the nonionic polymeric micelles have also been used as a vehicle to entrap DNA directly. For example, Liu Y. 36 and Deng X. used poly(D,L-lactic acid)-poly(ethylene glycol) block copolymer to entrap DNA [Liu Y. et. al., 2002]. Jeong J. H. and Park T. G. grafted PLGA onto poly(L-lysine) and prepared a kind of surface charged micelles to bind DNA. This system exhibited much higher gene transfection efficiency with lower cytotoxcity than PLL [Jeong J. H. et. al., 2002]. 2.2.5 Improvement of gene expression efficiency by codelivery of drugs A number of codelivery formulations have been reported to improve gene expression efficiency. For instance, the endolysosomal escaping reagent such as chloroquine has been added into the cell culture medium to increase the gene expression [Choi Y. H. et. al., 1998; Mao H-Q. et. al., 2001; Zhang X. et. al., 2001]. Miyake H. et. al. studied the combined treatment with antisense (AS) clusterin oligodexoxynucleotide (ODN) and cisplatin, a coordination complex of platinum and commonly used to treat bladder cancer, in the inhibition of KoTCC-1 tumor growth and metastasis in a human bladder cancer KoTCC-1 model. Characteristic apoptotic DNA ladder formation and cleavage of poly(ADP-ribose) polymerase protein were detected after combined treatment with AS clusterin ODN and cisplatin but not either agent alone. In vivo systemic administration of AS clusterin and cisplatin significantly decreased the S.C. KoTCC-1 tumor volume compared with mismatch control ODN plus cisplatin. Furthermore, after the orthotopic implantation of KoTCC-1 cells, combined treatment with AS clusterin and cisplatin significantly inhibited the growth of primary KoTCC-1 tumors [Miyake H. et. al., 2001]. The expression of E-selectin can also be induced in in vitro models of activated endothelium such as in human umbilical vein endothelial cells (HUVEC) by exposing 37 them to TNFα or other inflammatory cytokines [Harari O. A. et. al., 1999; Walton T. et. al., 1998; Murakami T. et. al., 2000]. Dexamethasone has also been employed to enhance the gene expression after hepatic DNA injection [Malone R. W. et. al., 1994]. Dexamethasone was originally used to treat inflammation. Acute hepatic inflammation likely occurred at the site of direct injection and possibly reduced expression from transfected genes. Malone R. W. et. al. used dexamethasone to treat the inflamed site and resulted in enhanced and prolonged gene expression. Similarly, bleomycin has also been combined with interleukin-12 gene to treat subcutaneous and metastatic melanomas in mice, resulting in marked suppression of the treated tumors as well as bystander metastatic lesions by using electrochem-gene therapy [Kishida T. et. al., 2003]. Additionally, cyclosporin A [Parkar M. H. et. al., 2004], a kind of immunosuppressive agent, and verapamil [Li D., 2003], the inhibitor of P-glycoprotein have also been found to affect the gene expression. 38 [...]... block copolymer to entrap DNA [Liu Y et al., 20 02] Jeong J H and Park T G grafted PLGA onto poly(L-lysine) and prepared a kind of surface charged micelles to bind DNA This system exhibited much higher gene transfection efficiency with lower cytotoxcity than PLL [Jeong J H et al., 20 02] 2. 2.5 Improvement of gene expression efficiency by codelivery of drugs A number of codelivery formulations have been... macrophages of liver and spleen, is another barrier that may shorten the blood circulation 20 time of the drug Nanoscopic drug carriers can evade recognition and uptake by MPS, and circulate for a more prolonged period of time In summary, the size and relatively high stability of polymeric micelles enable them to evade the renal clearance and uptake by the RES and MPS, prolonging their blood circulation and. .. derivatives of cholesterol and diacylglycerol and lipid derivatives of polyamines, were reported [Niidome T et al., 20 02] Among these lipids, lipofectAMINETM exhibits high transfection efficiency However, it is quite toxic The development of new types of lipid molecules appears to be saturated Most of the efforts have shifted to improve efficiency of gene delivery and expression by modification of the existing... generally required by drug carriers can be distinctly shared by structural separated dual phases of the micelles The inner core plays the role of a drug loading/release depot for pharmacological activities, while the outer shell is responsible for interactions with biocomponents such as cells and proteins These interactions determine biodistribution and pharmacokinetic behavior of this drug carrier system... addition to that with DNA itself A major driving force for the complex formation is the release of low-molecular weight counter-ions that 26 make a large entropic contribution to the free energy of binding [Matulis D et al 20 02] The hydrophobic segments of lipids are the key determinant in the macroscopic characteristics of the ensuing liposomes, particularly their size, shape, and stability in the... cell membranes, and DNA This in turn affects the transfection efficiency of the resulting lipoplexes [Smisterova J et al., 20 01; Zuhorn I.S et al., 20 02] Approaches for controlling these parameters are limited, leading to instability of their macroscopic properties over time [Simberg D et al., 20 01] and thus restricting their pharmaceutical potential Furthermore, liposomal formulations often require... polymers have also been 27 employed to deliver genes In general, the most commonly used cationic polymers as gene vectors include branched and linear polyethyleneimine (PEI), copolymers of PEI, poly(L-lysine) and its copolymers, imidazole-modified poly(L-lysine) and chitosan (Figure 2. 4) Figure 2. 4 Cationic polymers most frequently used for nucleic acid delivery [Merdan T et al., 20 02] Polyethyleneimine... al., 20 01], and provide good protection for the complexed DNA against DNase degradation comparable to PEI [Koping-hoggard M et al., 20 01] Relationships between structure and property of chitosans show that the percentage of positively charged monomer units must be greater than 65% in order to obtain stable complexes capable of transfecting cells in vitro [Koping-Hoggard M et al., 20 01] Small and very... 1998] The buffering capacity of PEI may result in rapid endosomal escape However, in the case of chitosans, degradation of the polymers may be crucial [Merdan T., 20 02] Lysosomal enzymes may break down chitosans into small molecules, leading 31 to increased osmolarity and eventually to release of DNA by rupture of lysosomes [Koping-Hoggard M et al., 20 01; Merdan T et al., 20 02] Dendrimers Dendrimers are... the cationic polymers Modification of current cationic polymers The modification was mainly focused on increasing the stability of cationic polymer /DNA complexes and the targeting ability to diseased tissues The main factor causing the instability of polyplexes is adsorption of the proteins existing in the blood onto the surface of the complexes The adsorption of proteins results in the aggregation of . a number of advantages over other forms of drug carriers because of their core- shell structure, low CMC and targeting ability. 2. 1.4.1 Core of polymeric micelles as a reservoir for hydrophobic. macrophages of liver and spleen, is another barrier that may shorten the blood circulation 21 time of the drug. Nanoscopic drug carriers can evade recognition and uptake by MPS, and circulate for. the onset of micellization [Zhang X., et. al., 1996]. Figure 2. 1 Schematic representations of block and random copolymer micelles. [Jones M-C. et. al., 1999] 12 Figure 2. 2 Molecular