Novel biodegradable cationic core shell nanoparticles for codelivery of drug and DNA 2

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

Chapter 2

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

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

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

1

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 non-

polar 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 I 1 (the first peak)/I 3 (the third peak)

ratio from emission spectra or the I 333 /I 338 (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 I 1 /I 3

ratio is measured at a constant excitation wavelength (339nm) and variable emission

wavelengths corresponding to I 1 and I 3 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 I 333 /I 338 ratio since the I 1 /I 3 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]

Figure 2.2 Molecular structure of pyrene

360 370 380 390 400 410 0

100 200 300 400 500 600 700

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,

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

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, N-

dimethylformamide) 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 drug-loaded 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

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

using PEO-b-BM block copolymer [Jones M C et al., 1999] The size of these

polymeric micelles ranged from 15 to 165nm Polydispersity of polymeric micelles is usually very low compared with the copolymers used to form the micelles For example,

the polydispersity index of acetal-PEG-b-PLA [poly (ethylene glycol-b-poly(lactic acid)] micelles was reported to be 0.03 [Nagasaki Y., et al., 1998], which is even narrower than

that of the polymer synthesized by anionic polymerization

The nanoscale size of the polymer micelles together with the hydrophilic nature of the shell not only allows easy sterilization by filtration and minimizes the risks of embolism

in capillaries, contrary to larger drug carriers [Kwon G S et al., 1996] but also makes

polymeric micelles especially advantageous to avoid fast renal clearance and other barriers such as the reticuloendothelial system (RES) and mononuclear phagocyte system (MPS), prolonging blood circulation time Another important advantage that polymeric micelles can provide is their passive targeting ability provided via enhanced permeability and retention effect at tumor and inflammatory sites (i.e EPR effect) [Matsumura Y., 1986] This is because there are increased vascular permeability and impaired lymphatic drainage at the pathological site and this cause accumulation of the drug at the pathological site Drug targeting will be discussed in detail in Section 2.1.3

2.1.4.3 Low CMC of polymeric micelles enabling long time circulation in blood stream

Amphiphilic copolymers usually possess a much lower critical micelle concentration (CMC) compared to low molecular weight surfactants The low CMC of the polymers is

a great advantage as the stability of their micelles would be unaffected under conditions

of extreme dilution, as would be the case in the physiological environment upon

administration For instance, the CMC of PEO-b-PBLA and PNIPA-b-PSt was reported

to be between 0.0005%-0.002% [La S B et al., 1996; Cammas S et al., 1997]

Therefore, stable delivery systems mean long circulation time in blood In section 2.1.5, it will be presented that long circulation time in blood is one of the key prerequisites for efficient passive targeting Torchilin V P pointed out that it is important to differentiate between thermodynamic and kinetic stability of polymer micelles [Torchilin V P., 2001] The CMC provides the information about thermodynamic stability Below the CMC, equilibrium between unimers and micelles is shifted towards unimer formation, rendering the dissociation of core-shell structure On the other hand, the kinetic stability is related

to the actual time that micelles dissociation takes Even upon dilution to a concentration below CMC, the preformed micelles can still exist long enough to perform their carrier function The kinetic stability depends on many factors including the physical state of the micelle core, the contents of a solvent inside the core, the size of the hydrophobic

segment and the ratio of the hydrophobic segment to hydrophilic segment [Tian M., et al., 1993; Wang Y et al., 1995; Creutz S et al., 1998; Yokoyama M et al., 1996]

2.1.5 Targeting ability of polymeric micelles as drug carrier

Two important issues that concern researchers for an effective drug therapy include

temporal and distribution control of drug The temporal control refers to the ability to adjust the duration of drug release or to the possibility to trigger the drug release at a specific time Temporal control can be implemented by introducing temperature-sensitive

function to the hydrophilic block of the copolymer [Cammas-Marion S et al., 1999;

Chung J E et al., 2000; Topp M D C., 1997] or by incorporating auxiliary agents such

as channel proteins [Meier V et al., 2000; Gelder P V et al., 2000] and magnet nanoparticles [Sershen S R et al., 2000; Kato N et al., 1998; Simpson C R et al.,

1998], which are sensitive to external stimuli (electrolytes, IR light or magnetic field) The distribution control is also called drug targeting, which is aimed to direct the drug to the desired site where pharmacological activities are required

Drug targeting may be classified into two categories: active and passive targeting Active targeting aims to increase delivery of drugs to the target by utilizing biologically specific interactions such as antigen-antibody and ligand-receptor binding Biological signals used in this method include antibodies and ligands On the other hand, passive targeting is defined as a way to increase the amount of drugs delivered to targeted tissues

by minimizing non-specific interactions with non-target organs, tissues, and cells Carriers included in passive targeting utilize physicochemical interactions such as hydrophobic and electrostatic interactions and physical factors of carriers such as size and mass

With advances in the chemistry of amphiphilic polymers, polymeric micelles can be designed to have reactive functional groups on their surfaces for conjugation of pilot molecules Thus, the stage is set for promising research in active drug targeting However, over the past decade, passive targeting has shown greater success than active targeting

Passive targeting achieved much higher in vivo selectivity due to the EPR effect

[Torchlin V P., 2001] The evidence showed that drug-loaded polymeric micelles preferably accumulated to a greater extent than free drug into tumors and displayed a

reduced distribution in non-targeted sites [Weissig V et al., 1998] That is because

malignant or inflamed tissues have impaired lymphatic drainage, increasing vascular permeability The tumor vessels are also leakier and less permeation selective than normal vessels This phenomenon is called the EPR effect However, implementation of

in vivo selective drug delivery with an active targeting system is not as easy as a simple extension of in vitro specificity to selective cells Several difficulties obstruct this simple

extension, including loss of antibody specificity caused by chemical conjugation and/or enzyme degradation in the blood, non-specific uptake of drug-antibody conjugates, and increased antigenicity of antibody or antibody-drug conjugates stemming from multiple doses

Moreover, the passive component of drug targeting is important in active targeting systems for the following three reasons: (1) the majority of a living body comprises non-target sites Non-specific target sites may capture the major dose fraction Therefore, minimization of non-specific capture at non-target sites may be important to maximize amounts of drugs delivered by active targeting systems; (2) passive transfer phenomena precede biologically specific interactions even in most active targeting systems Exceptions are cases for intravascular targets such as lymphocytes and vascular endothelial cells Most targets are located in the extravascular space To reach these targets via the bloodstream, the first step must be translocation through the vascular endothelia, followed by permeation through the interstitial space to the extravascular targets Even for active targeting systems based on biologically specific receptors on cells such as tumor-specific antigens, the passive transendothelium step is a necessary pre-requisite; (3) the passive aspects may become very important as drugs are introduced to active targeting carriers For antibody-drug conjugates, the adverse physicochemical

influence of conjugated drugs on the specificity of the antibody is expected to increase with an increase in the amount of conjugated drugs Therefore, minimization of non-specific interactions is important in both active and passive targeting More precisely, inhibition of non-specific distribution/excretion at non-target sites is a critical issue in active and passive targeting In living bodies, two major routes for this non-specific distribution/excretion are present: renal excretion and uptake by RES Evasion of the renal excretion can be achieved relatively easily by conjugation of drugs to polymeric carriers or encapsulation of drugs in polymeric carriers or liposomes However, evasion

of the RES uptake has not been attained as easily as that of renal excretion One way to

resolve this problem is to use polymeric micelle structures [Yokoyama M et al., 1996; Karaoka K et al., 1993; Okano T et al., 1994] Polymeric micelles possess discretely

separated two phases The functions 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 In vivo delivery of drugs may therefore be controlled by the outer shell segment

independently of the micelle inner core that expresses pharmacological activities Polymeric micelles can suppress unfavorable (mainly hydrophobic) interactions between

drugs and the RES systems to evade RES uptake because of the presence of the

hydrophilic shell Some researchers have also reported that the size is a key factor to evade the uptake by RES Mononuclear phagocyte system (MPS) namely, fixed macrophages of liver and spleen, is another barrier that may shorten the blood circulation

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 achieving passive targeting In addition, the shell of polymeric micelles can be designed to possess functional groups for conjugation of biological signals to achieve active targeting

2.1.6 Amphiphilic copolymers used for fabrication of micelles

Both random and block amphiphilic copolymers can self-assemble into spherical micelles in aqueous solution A-B diblock copolymers have been the main focus for the fabrication of micelles Multiblock copolymers such as poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) were also able to self

organize into micelles [Malmsten M et al., 1992; Prasad K N., 1979]

The hydrophobic core generally consisted of a biodegradable block such as benzyl-L-aspartate) (PBLA), poly(DL-lactic acid) (PDLLA) or poly(ε-caprolactone) (PCL) The core was also made from a water-soluble polymer [e.g poly(aspartic acid)] P(Asp)), which was rendered hydrophobic by the chemical conjugation of a hydrophobic

poly(β-drug [Yokoyama M et al., 1990; 1992; 1996], or formed through the association of two oppositely charged polyions (polyion complex micelles) [Harada A et al., 1995; 1998;

Kataoka K., 1996] Some non or poorly biodegradable polymers such as polystyrene (PS)

[Zhao C.L et al., 1990; Zhang L et al., 1995] or poly(methyl methacrylate) (PMMA) [Inone T et al., 1998) were also used as an core-forming block In addition, a highly