Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy

C H A P T E R

3 Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy Kobra Rostamizadeh1,2, Vladimir P. Torchilin2 1

Zanjan Pharmaceutical Nanotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran; 2Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA, United States

1. Introduction

pathways including defects in cell signaling pathways and induces changes in the surrounding stroma and immunoresponses. Thus, targeting a specific pathway with a single chemotherapeutic agent is less successful in eradication of cancerous cells [2]. To date, improvement of the therapeutic effect of chemotherapy has been directed particularly toward combination approaches, particularly codelivery nanoplatforms that attack cancerous cells via multiple pathways. The general principle of combination chemotherapy involves the simultaneous administration of two or more selected drugs with nonoverlapping toxicities and dissimilar mechanisms of action that inhibit multidrug resistance phenomena [3e5]. Combination therapy can overcome the toxicity problems of single-drug therapies by targeting multiple-signaling pathways. Some combination therapy regimens have been established for cancer therapy and clinical practices that have demonstrated synergistic effects (a greater effect than the sum of the separately applied individual drugs) and less systemic toxicity [6]. Besides

Cancer, after heart disease, ranks as the second-most illness-related cause of death worldwide with a growing mortality number and incidence. Cancer is one of the most challenging diseases to treat because of its great diversity and complexity. To date, chemotherapy has been used most widely as an efficient and successful method in clinical practice. However, it has not provided adequate therapeutic efficacy and lacks complete effectiveness. The major issue is that most chemotherapeutic agents have poor solubility and often exhibit other deficiencies including low bioavailability, rapid blood/renal clearance, and nonspecific targeting with significant undesirable side effects on healthy tissues [1]. Nonspecific biodistribution is another hurdle that limits the localization of drugs at the tumor site and consequently generates demands for higher doses, which in turn leads to significant nonspecific toxicity. Above all, the major problem is that cancer progresses via a wide range of different

Nanopharmaceuticals https://doi.org/10.1016/B978-0-12-817778-5.00003-8

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© 2020 Elsevier Inc. All rights reserved.

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3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy

that, combination regimens can offer improvement in the treatment results, although there are still unsuccessful outcomes and significant side effects observed due to low drug bioavailability and nonuniform biodistribution. In addition, to take advantage of possible synergy with multiple drugs, specific molar ratios at the tumor site are often needed, which are hard to obtain by conventional administration methods due to differences in injection schedules, pharmacokinetic properties, metabolism, and nonuniform biodistribution of drugs [7]. One strategy to address this issue and deliver drugs to the tumor site at the desired molar ratio is to merge nanotechnology with pharmacology and take advantage of nanoscale structures as vehicles to carry multiple drugs, tune drug release, and modify the biodistribution and pharmacokinetics of chemotherapeutic agents [8]. This approach refers to codelivery systems. Codelivery systems can not only regulate the dosages and the ratio of different chemotherapeutic agents at the tumor site but also improve the efficiency of anticancer drugs through enhanced water solubility of hydrophobic molecules, low toxicity, and high stability that prolongs circulation time in blood and enhances accumulation in tumor tissues. Moreover, further enhancement of therapeutic efficacy can be achieved by taking advantage of stimuliresponsive drug delivery systems equipped with various targeting moieties that can reduce nonspecific delivery [9,10]. An ideal carrier for codelivery should have the potential for encapsulation of both hydrophobic and hydrophilic drugs. Platforms for codelivery systems should be designed so that they carry traditional chemotherapeutics and agents such as siRNA in the same delivery vehicle. Although many efforts have been made with nanotechnology-based codelivery systems, there are several challenges, which mainly include encapsulation of drugs with various solubilities and physicochemical properties, targeting tumor tissues, regulation of the

concentrations of each drug, and manipulation of sequential drug release. To date, considerable efforts have been made to develop nanoparticulate codelivery systems for combination chemotherapy [11,12]. Various nanocarriers have been investigated, including lipid nanoparticles [13,14], liposomes [15,16], dendrimers [17,18], and polymeric nanoparticles [19,20]. Among these, increasing attention has been paid to polymeric nanoparticles due to their potential to carry both hydrophobic and hydrophilic drugs, their controlled drug release characteristics, low toxicity, high stability, and long circulation time, all of which ultimately enhance drug accumulation in the tumor target. Recently more attention has been given to polymeric micelles as drug delivery systems with respect to the unique properties for codelivery of drugs.

2. Micelles, principles and characterization Polymeric micelles, first developed by Kataoka and colleagues in the late 1980s and early 1990s, have spontaneously selfassembling nanoscale core-shell architectures consisting of a hydrophobic core domain surrounded by a hydrophilic shell (Fig. 3.1). Generally, the size of polymeric micelles is 10e200 nm depending on the length of hydrophobic and hydrophilic segments, temperature, and the concentration of polymer. Polymeric micelles are composed mainly of amphiphilic copolymers where polyethylene glycol (PEG) is the most common domain of the hydrophilic shell. Other hydrophilic polymers like poly(Nvinyl-2-pyrrolidone) [21], poly(N-) pNIPAM [22,23], and poly(acrylic acid) [24,25] are among the most widely used polymers as the hydrophilic segment. The hydrophobic core-forming segment typically consists of poly(L-aspartate) [26,27], poly(propylene oxide) (PPO) [28,29], poly(esters) such as (PDLLA) [30,31],

2. Micelles, principles and characterization

47

FIGURE 3.1 Illustration of polymeric micelles. From Stern T, Kaner I, Laser Zer N, Shoval H, Dror D, Manevitch Z, et al. Rigidity of polymer micelles affects interactions with tumor cells. J Controlled Release. 2017;257:40e50 with permission.

hydrophobic poly(ε-caprolactone) (PCL) [32,33], copolymers of lactic acid and glycolic acids (PLGA), and poly(ε-caprolactone) [34,35]. Having both hydrophilic and hydrophobic domains in one platform is perhaps the most attractive feature of polymeric micelles. This allows carrying a variety of therapeutic agents ranging from those that are practically insoluble to highly water-soluble drugs. Their remarkably low critical micelle concentration (CMC) (on the order of 10
6-10
7 M) is another major feature of polymeric micelles as drug delivery systems that enables them to maintain structural integrity even at a very low concentration when once diluted in body fluids. Polymeric micelles can protect a drug payload against degradation by various enzymes or metabolisms. The small size of polymeric micelles also allows targeting of specific pathological cancerous sites through the passive targeting phenomena known as the enhanced permeation and retention (EPR) effect. The EPR effect relies on greater capillary endothelial cell gaps within tumor tissues (in the range of 60e800 nm) compared to normal tissues, which enables the smaller polymeric micelles to

selectively penetrate cancerous tissues. Additionally, for reduction of the elimination by the reticuloendothelial system (RES) and kidney, the optimum size for polymeric micelles is about 100e150 nm [36]. The hydrophilic shell of polymeric micelles also exhibits stealth properties, particularly in cases when PEG serves as the hydrophilic part of the shell of the micelles structure, avoids opsonization of micelles by the RES, and prolongs circulation time of polymeric micelles in body fluids. These characteristics, as well as the biodegradability and functionality of polymeric micelles, have made them promising candidates for therapeutic delivery of desirable pharmaceutical formulations to tumors.

2.1 Preparation methods There are three current methods for preparation of polymeric micelles, including emulsification-solvent evaporation, solvent displacement, and salting out. In the emulsification-solvent evaporation method, an aqueous solution containing an appropriate

48

3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy

surfactant like sodium cholate, or polyvinyl alcohol is dispersed in a polymer solution prepared using a volatile water immiscible solvent like ethyl alcohol or chloroform to prepare an emulsion using shear force methods, i.e., homogenization, stirring, or probe sonication [38]. Eventually, the solvent is evaporated, and polymeric micelles are obtained as dispersed nanoparticles in water, which can be collected by centrifugation. Generally, a single emulsion (water in oil, W/O) or double emulsion (W/O/ W) is used for trapping hydrophobic and hydrophilic drugs, respectively. With solvent evaporation, or a solution-casting technique, a thin uniform film containing copolymer and drug is left on the surface of the substrate after the evaporation of solvent [39]. Next, the hydration of the film by aqueous solution forms polymeric micelles. Solvent diffusion, also referred to as the nanoprecipitation or solvent displacement method, is used extensively to encapsulate hydrophobic drugs [40]. In this method, a water miscible solvent like acetone is used to dissolve polymer and drug. Due to the miscibility of solvents, there is no need to use surfactant or high shear force to obtain nanoscale materials. Salting out is also used to load hydrophobic drugs [41]. In this method, a W/O emulsion is first prepared using water immiscible solvents, followed by addition of a salting out agent such as sodium chloride, magnesium acetate, or magnesium chloride to make the solvent insoluble by saturation of the solution and formation of polymeric micelles.

2.2 Characterization techniques The suitability of polymeric micelles as drug delivery systems is evaluated with respect to properties including particle size, surface charge, and stability [42,43]. The CMC is the concentration of copolymer in a solution above which copolymers self-assemble to form micelles. Below the CMC, due to low surface tension of

the solution, amphiphilic copolymers disperse only within the solution and show no tendency to form micelles. The CMC is one of the most important properties as it indicates the kinetic stability of polymeric micelles [44]. Spectrofluorometry by pyrene and light scattering are among the most popular techniques used to measure the CMC. Static or dynamic light scattering (DLS)/ photon correlation spectroscopy is an intensively used method for evaluation of the size (hydrodynamic diameter) and polydispersity index (PdI) of micellar structures [45]. In fact, DLS works based on the intensity distribution of relaxation times and the time dependence of the light intensity fluctuations to obtain the diffusion coefficient of nanoparticles, which is then used to calculate hydrodynamic diameter (RH) of nanoparticles using the StokeseEinstein equation. The PdI indicates the polydispersity of polymeric micelles in terms of particle size on the scale of 0e1. The PdI values of 0 and 1 indicate a monodisperse solution and the highest solution polydispersity, respectively. Various types of electron microscopy, including transmission electron microscopy, scanning electron microscopy, and atomic force microscopy, are widely used for determination of micellar morphology and size. The stability of polymeric micelles, either thermodynamic or kinetic, is another critical parameter [46]. Thermodynamic stability indicates how the system acts as micelles when in equilibrium. Thermodynamic stability of polymeric micelles depends mainly on the surface charge, the zeta potential, and the steric hindrance provided by surfactants. Kinetic stability indicates the rate at which the micelles disassemble when the copolymer concentration is lower than the CMC. Several factors affect the in vitro and in vivo stability of polymeric micelles, including the strength of hydrophobic interactions between hydrophobic segments of polymeric micelles and the size of the hydrophilic part of the copolymer. DLS is the most popular

3. Polymeric micelles for codelivery of chemotherapeutics

method to measure zeta potential. DLS uses a phase analysis approach to determine the surface charge of the nanoparticles.

2.3 Drug-loading methods One of the most desirable features of polymeric micelles is their relatively high drugloading capacity. The loading efficiency for a micellar carrier is defined as the amount of incorporated drug per micelle. Generally, different drug-loading strategies for polymeric micelles can be classified in three main categories, including: L physical entrapment L chemical conjugation L polyionic complexation methods. The physical entrapment method, the simplest, has been used widely to incorporate different drugs, particularly hydrophobic drugs, using emulsification or dialysis [47]. In this method, hydrophobic interactions like van der Waals forces and hydrogen bonding determine drug loading. In the chemical conjugation approach, a strong covalent bond is formed between the polymer backbone and the drug [48]. In this case, the amount of incorporated drug is limited to the number of functional groups in the polymer backbone, and the rate of drug release is dependent on the rate of the bond cleavage. Finally, the ionic complexation method is preferred for loading species able to form strong electrostatic interactions with charged polymers [49]. The compatibility between the drug and the copolymer play a determinant role in loading efficiency. In addition to the size and type of the core and corona forming copolymer, the stability of polymeric micelles in aqueous medium, molecular weight of the copolymer, the type and concentration of the drug and copolymer, the method of drugloading, and the ratio of organic to aqueous solution, as well as their order of addition are among

49

the factors that influence drug-loading efficiency. However, hydrophobic drugs represent the majority of those loaded into micelles. There are some reports on encapsulation of hydrophilic drugs into micelles, particularly by the doubleemulsion method [50].

3. Polymeric micelles for codelivery of chemotherapeutics Increasing attention is now being paid to the use of polymeric micelles for codelivery of therapeutic agents in cancer therapy [51,52]. Mainly, these codelivery systems are used to overcome multidrug resistance, which is perhaps the most challenging issue in chemotherapy. They also show a high potential for codelivery of chemotherapeutic agents for synergistic therapy and mitigation of side effects. They have also been employed as stimuli-responsive codelivery vehicles for codelivery of therapeutics using different therapeutic approaches. In the remainder of this chapter, different aspects of polymeric micelles as codelivery platforms with emphasis on applications are explored.

3.1 Codelivery of chemotherapeutics to overcome multidrug resistance Multidrug resistance (MDR) remains a major challenge for successful treatment of cancer in the clinic. MDR is defined as the resistance of tumor cells toward a broad range of chemotherapeutic agents by inactivation of the drug or by pumping it from tumor cells. Different mechanisms have been proposed for MDR, including increased drug efflux mediated by the overexpressed MDR-related transporters, the increased capability of DNA repair, dysfunctional apoptosis, and activation of prosurvival pathways (Fig. 3.2). However, MDR is a very complex phenomenon, and no single mechanism of resistance is likely. It usually happens as the

50

3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy

FIGURE 3.2 Cellular changes occurring in MDR cancer cells in comparison to normal cells. MDR cancer cells show: (1) Increased extracellular acidity, (2) Change in cell membrane lipid composition resulting in thicker, less fluid, and less permeable membranes, (3) Overexpression of drug efflux pumps and cell surface receptors like EGFR, (4) Increased intracellular alkalinity, (5) Increase in cytoplasmic vesicle number and volume, (6) Increased intravesicular acidity, (7) Overexpression of ion/ drug transporters on intracellular vesicles, (8) Modulated levels of detoxifying and drug activating enzymes, (9) Modulated levels of importins, (10) Overexpression of exportins, and (11) Increased mutations and altered gene expression levels. From Singh MS, Tammam SN, Shetab Boushehri MA, Lamprecht A. MDR in cancer: Addressing the underlying cellular alterations with the use of nanocarriers. Pharmacol Res. 2017;126:2e30 with permission.

result of the action of a combination of several mechanisms. Various strategies have been adapted to overcome MDR, including the use of monoclonal antibodies against P-glycoprotein (P-gp), ATP-binding cassette transporter inhibitors, and inactivation of MDR-associated gene expression using small interfering (si) RNAs. Inhibitors have only limited indications for clinical usage due mainly to their association with chemotherapeutics and high toxicities. On the other hand,

the mass ratio of chemotherapeutic agent and MDR inhibitor is a determinant parameter that has been used to achieve the highest therapeutic efficiency [51]. Polymeric micelles have been used to deliver different bioactive agents simultaneously, including chemotherapy agents and inhibitors, at an optimized ratio to the site of interest. The MTT assay has been widely used to obtain the optimal mass of drugs for the best synergistic composition. For instance, using the MTT assay of micelles composed of

3. Polymeric micelles for codelivery of chemotherapeutics

poly(ethylene oxide)-blockepoly (propylene oxide)-blockepoly(ε-caprolactone) (PEOePPOe PCL), and the payload with different mass ratios of docetaxel (DTX) and chloroquine (CQ), an autophagy inhibitor, the highest synergetic anticancer effect was obtained for the DTX/ CQ ¼ 0.8/0.2 [28]. It has been shown that the synergetic therapeutic efficacy of codelivery systems containing chemotherapeutic agents and inhibitors in a single nanoformulation can be related to the increase in the cellular uptake and prolonged drug retention in the cytoplasm. Codelivery of rapamycin with piperine as chemosensitizer through Poly(D,L-lactide-co-glycolide) (PLGA) micelles increased the uptake and bioavailability of the rapamycin by about 4.8-fold [54]. Indeed, the use of nanoformulations for codelivery of different therapeutics is a promising method to obtain synergistic effects and eliminate most of the shortcomings of chemotherapy due to MDR. Two strategies that have been used to reverse MDR with a micellar codelivery system, including codelivery of chemotherapeutics with a chemosensitizer and codelivery of chemotherapeutics with downregulating gene agents, will be discussed in the following sections. 3.1.1 Codelivery of chemotherapeutics and chemosensitizers Generally, multiple mechanisms are involved in any single MDR phenotype. To overcome MDR, based on the mechanisms involved in its development, a combination of two or more drugs are used to target multiple oncogenetic pathways. For example, combinational therapy using P-gp inhibitors, tyrosine kinase inhibitors, or proapoptotic agents enhanced the cytotoxicity of formulations [55,56]. To improve therapeutic efficiency of drugs, additional nanoformulations have emerged that target more than one MDR mechanism. The codelivery of small molecule chemotherapeutic drugs and chemotherapy sensitizers to reverse MDR has attracted growing attention [57]. Chemosensitizers are chemical

51

substances acting at small doses without cytotoxicity toward cancerous cells, which promote synergism of the effects of chemotherapeutic agents when both anticancer drugs and chemosensitizer are delivered to the same cellular location. Lower toxicity and reduced side effects are the most important differences of these formulations compared to combination chemotherapy with two anticancer drugs. P-gp inhibitors [58] and autophagy inhibitors [59] are two of the most widely used chemosensitizers. Overexpression of P-gp is likely the main mechanism of MDR, which is also referred to either as MDR1 or ATP-binding cassette subfamily B member 1 (ABCB1). P-gp is an ATPdependent efflux pump that pumps foreign substances out of cells. Most of the widely used chemotherapeutic agents such as the taxanes and anthracyclines are classified as substrates of P-gp transporters. Inhibition of the function of P-gp is one strategy used to reverse MDR and sensitize tumor cells to conventional chemotherapeutics. Currently, despite considerable efforts that have been devoted to the development of P-gp inhibitors, there is no P-gp inhibitor approved for clinic use. This is due mainly to the nonspecific action of P-gp inhibitors on other molecular targets and their nonselectivity for tumorigenic P-gp, which can result in remarkable toxicity. Recently, nanocarriers, particularly polymeric micelles, have been used to deliver P-gp inhibitors into tumors cells with high efficiency (Table 3.1) [60]. The uptake of nanocarriers by MDR cells occurs through nonspecific endocytosis, leading to high intracellular accumulation of drug. In addition, the nanocarriers can be modified with a variety of targeting moieties such as ligands or antibodies to improve their specific uptake by tumor cells. The manipulation of surface charge of micelles can also be used to facilitate nanocarrier internalization into cancerous cells. Micelles consisting of poly(ethylene glycol)-block-poly(L-lysine) (PEG-b-PLL) block copolymers with negative charge in plasma (pH 7.4) have a prolonged

52 TABLE 3.1

3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy

Polymeric codelivery micelles used to overcome MDR.

Polymer used

Drug 1

Drug 2

Study type

Refs

PHis-PLA-PEG-PLA-PHis/ Pluronic F127

Curcumin

Pluronic L61 unimers

MCF-7/ADR xenograft mice model

[9]

PEG-PLA

Cyclosporin Gefitinib A

NSCLC xenograft- BALB/c nude mouse model

[70]

BDP

Docetaxel

Silibinin

4T1 xenograft mice model

[71]

PEOePPOePCL/TPGS

Docetaxel

Chloroquine

MCF-7 and MCF -7/ADR cell lines

[28]

Drug conjugated PEG

Dox

Curcumin

HepG2 xenograft BALB/c nude mice model

[72]

(SMA)- ADH

Dox

Disulfiram

MCF-7/ADR xenograft mice model

[62]

PLGA

Dox

Chloroquine

A549 cells and A549/Taxol cells

[73]

PEO-b-PCL

Dox

MDR-1 siRNA

Athymic mice bearing MDA-MB-435/LCC6MDR1resistant tumors

[74]

NSCePLLePA

Dox

P-gp siRNA

HepG2/ADM xenograft Athymic nude mice model

[75]

PLGA

Dox

MDR1 targeting siRNA

MCF-7/ADR cell line

[76]

PDP-PDHA

Dox

shSur

MCF-7/ADR xenograft mice model

[77]

PFeDP

Dox

PTX

MCF-7/ADR xenograft mouse model

[78]

PLGA

MDR1

BCL2 siRNA

Resistant SKOV3-TR and A2780-CP20 human ovarian cancer cells

[79]

PLGA

PTX

Tetrandrine via irgd peptide

A2780/PTX cell line

[13]

PEG2k-Fmoc-NLG

PTX

NLG919, an indoleamine 2,3-dioxygenase

4T1.2 xenograft BALB/c mice model

[80]

PEOz-PLA

PTX

Honokiol

MDA-MB-231-luc-GFP xenograft nude mice model

[81]

PEG-b-PLL

PTX

Disulfram

MCF-7 cell line

[61]

PLGA

Rapamycin Piperine

Healthy SD rats

[54]

PEG-PE

PTX

DU145Xenograft BALB/C nude mice model

[82]

Curcumin

PEG-PLA (poly(ethylene glycol)-b-poly(L-lactide)); PEG (poly(ethylene glycol)), poly(D,L-lactide-co-glycolide) (PLGA) and poly(L-histidine) (PHis-PLA-PEG-PLA-PHis); PLGA (poly(D,L-lactide-co-glycolide)); BDP (polyethylene glycol-block-poly[(1,4-butanediol)-diacrylateb-N,N-diisopropylethylenediamine]); PEOePPOePCL/TPGS(poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ε-caprolactone) and D-a-tocopheryl poly(ethylene glycol)); PEOz-PLA(poly(2-ethyl-2-oxazoline)-poly(D,L-lactide)); PEG-b-PLL (poly(ethylene glycol)-blockpoly(L-lysine) block copolymer); (SMA)-ADH (poly(styrene-co-maleic anhydride) (SMA) derivative with adipic dihydrazide); Poly(lacticco-glycolic acid) and alpha-tocopherol polyethylene glycol 1000 succinate; PEO-b-PCL (poly(ethylene oxide)-block-poly(ε-caprolactone) block copolymers); NSCePLLePA (N-succinyl chitosanepoly-L-lysineepalmitic acid); PDP-PDHA (poly[(1,4-butanediol) diacrylateb-5polyethylenimine]); Polyethylene GlycolePhosphatidylethanolamine PEG-PE; Pluronic P105 and Pluronic F127 PFeDP; poly(ethylene oxide)blockepoly(propylene oxide)-blockepoly(ε-caprolactone) PEOePPOePCLPHis-PLA-PEG-PLA-Phis) poly (L-histidine)-poly (D,L-lactide)polyethyleneglycol-poly (D,L-lactide)-poly (L-histidine); PEG-PLA (poly(ethylene glycol)-b-poly(L-lactide)); BDP (polyethylene glycol-block-poly [(1,4-butanediol)-diacrylate-b-N,N-diisopropylethylenediamine]); PEOePPOePCL/TPGS(poly(ethylene oxide)-block-poly(propylene oxide)block-poly(ε-caprolactone) and D-a-tocopheryl poly(ethylene glycol)); PEG (poly(ethylene glycol)); (SMA)- ADH (poly(styrene-co-maleic anhydride) (SMA) derivative with adipic dihydrazide); PLGA (poly(D,L-lactide-co-glycolide)); PEO-b-PCL (poly(ethylene oxide)-block-poly(εcaprolactone) block copolymers); NSCePLLePA (N-succinyl chitosanepoly-L-lysineepalmitic acid); PDP-PDHA (poly[(1,4-butanediol) diacrylateb-5- polyethylenimine]); PFeDP Pluronic P105 and Pluronic F127; PEOz-PLA(poly(2-ethyl-2-oxazoline)-poly(D,L-lactide)); PEG-b-PLL (poly(ethylene glycol)-block-poly(L-lysine) block copolymer); PEG-PE (polyethylene glycolephosphatidylethanolamine).

3. Polymeric micelles for codelivery of chemotherapeutics

circulation time, while its more positive charge when exposed to the weak acid environment of tumor tissue (pH 6.5e6.8) facilitates their uptake by cancerous cells. Huo et al. [61] have shown that grafting paclitaxel (PTX) to the side chain of L-lysine and encapsulation of disulfiram, a P-gp inhibitor, in micelles significantly increases the cellular internalization and cytotoxicity of PTX against MCF-7/ADR cells. Encapsulation of disulfiram into doxorubicin (Dox)-conjugated poly(styrene-co-maleic anhydride) (SMA) derivative with adipic dihydrazide (ADH) micelles also exhibited very high-antitumor activity with almost complete inhibition of tumor growth [62]. Codelivery of verapamil and vincristine in poly(D,L-lactide-co-glycolide acid) (PLGA) micelles also demonstrated higher drug accumulation in MCF-7/ADR cells in comparison to free vincristine/verapamil combinations [63]. Tariquidar is also a P-gp inhibitor that exhibits significant tumor growth inhibition. Biotin-functionalized nanoparticles coencapsulated with both PTX and tariquidar showed high potential for inhibition of tumor growth in a mouse MDR model compared to free PTX at the same dose [64]. Drug resistance was partially abolished using polymeric micelles composed of PEG-PLA coencapsulated with a chemosensitizer, cyclosporin A (CsA), and the anticancer drug, gefitinib [65]. Cyclosporin A is a P-gp inhibitor that inactivates the STAT3/Bcl-2 signaling pathway. Coadministration of CsA and gefitinib encapsulated in polymeric micelles demonstrated the high potency of gefitinib in nongefitinib-resistant cells as well as in primarily and secondarily gefitinib-resistant cells, apparently due to increased apoptosis and impaired proliferation shown in nonsmall cell lung cancers. Together, these results confirm that codelivery of P-gp inhibitors and anticancer drugs is a promising approach to overcome MDR.

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3.1.2 Codelivery of chemotherapeutics and downregulating gene agents Inhibition of the expression of the MDR-1 gene (rather than its function) is another strategy to prevent/reduce drug efflux. Use of siRNA is a highly promising technology for downregulation of gene expression, particularly for cancer therapy [66]. Recently, several siRNA therapeutic formulations have been studied in clinical trials and successfully exhibited gene silencing of various oncogenes [67]. SiRNA has also been designed to silence MDR-1 gene expression in hopes of downregulating the expression of P-gp genes [68]. However, because of its instability and toxicity, the success of this approach depends on the development of safe and efficacious delivery systems that deliver siRNA in a safe and selective manner into cancerous cells. Micelles composed of novel poly(ethylene oxide)modified poly(beta-amino ester) (PEO-PbAE) and PEO-modified poly(ε-caprolactone) (PEOPCL) nanoparticles showed high potential for delivery of MDR-1-silencing siRNA and anticancer drugs such as PTX [69]. Generally, the high positive charge of micelles represents a favorable property that enables them to form complexes with negatively charged siRNA by electrostatic interaction. Cationic PLGA micelles (PLGA decorated with didodecyl dimethylammonium bromide (a cationic surfactant), efficiently coencapsulated siRNA and Dox at a specific high ratio of micelles-to-siRNA. Survivin is an inhibitor of apoptosis family member overexpressed in drug resistance that inhibits cell apoptosis. Survivin-targeting shRNA (shSur) colo aded with Dox into amphiphilic poly (b-amino ester), poly[(1,4-butanediol)-diacrylate-b-5-poly ethylenimine]-block-poly[(1,4-butanediol)-diacry late-b-5-hydroxy amylamine] (PDP-PDHA) micelles increased Dox accumulation, and downregulated 57.7% of the survivin expression,

54

3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy

resulting in 80.8% cell apoptosis and cell cycle changes in MCF-7/ADR cells [69]. It also increased the accumulation of Dox and shSur in the tumor tissue by 10.4- and 20.2-fold, respectively, resulting in a remarkable inhibition of tumor growth of 95.9%. Although siRNA technology has become a promising approach to chemotherapy, these reports confirm that the methods developed are based on only partial reverse MDR by silencing the expression of the genes encoding P-gp, i.e., MDR-1 and Survivin. On the other hand, siRNA makes a significant difference between the IC50 values of chemotherapeutic agents in MDR cancer cells compared to sensitive cell lines. The in vivo studies have clearly shown their capacity to slow the rate of tumor growth but not to ultimately decrease the tumor volume.

3.2 Codelivery of chemotherapeutics to achieve synergistic effects by methods other than effects on MDR Recently, combination therapy has been shown to be a promising regimen for cancer treatment of those with historically poor prognosis of cancers treated by monotherapy that are diverse, complex, and heterogeneous in nature. The coadministration of several types of therapy has resulted in remarkable synergetic effects (namely “1 þ 1 > 2”), in which the effect is more potent than any individual therapy or their theoretical combination. Given the different mechanisms involved in a cancer’s progression, it is reasonable to attack cancerous cells with combinations of drugs with different mechanism of actions in the hope of a greater effectiveness. Administration of drug combinations instead of a single drug also reduces host toxicity and adverse side effects due to the lower dosage of drugs typically used in the combinational therapy regimens. There have been several combinatorial therapeutic regimens tested in clinical

trials. Although combinational therapy provides the opportunity for an additional and synergetic therapeutic effect with low side effects, there are some issues associated with combinational therapy that must be considered, including crossresistance and toxicity of drug combinations. To avoid these problems, the choice of synergistic drug pairs according to their mechanism of action, and administration of drugs following optimization of dosing schedules, are of great importance. Until recently, clinical experiences were used to test for synergistic drug combinations, which are time-, labor-, and costintensive. Lately, high-throughput screening (HTS) has become a relatively quick and low-cost technique used to choose synergistic drug pairs with less harm to patients [83]. Recently, using machine learning techniques for the description of the large synergistic space [84], accurate predictive models have been generated by leveraging the available HTS synergy data. Table 3.2 presents different polymeric codelivery systems with synergic properties in cancer treatment. Dox and PTX are one of the most-studied paired drugs for codelivery systems. Their different mechanisms of action on cancer cells can result in high and synergistic therapeutic effects. Dox binds to DNA and prevents nucleic acid synthesis, while PTX’s usefulness is based on promotion of microtubule assembly from tubulin dimers and stabilization of microtubules through inhibition of depolymerization. Duong et al. [85] studied codelivery of Dox and PTX in micellar formulations with two different approaches. They compared the effect of codelivery of two single-drug-loaded micelles with dual-drug-loaded micelles. The results confirmed a synergistic effect for both methods. With codelivery of single-drug-loaded micelles, it would be easier to control different drug combination ratios for specific treatments. However, there is less control of a designated ratio at the site of the tumor. Using dual-drug-loaded micelles provides the opportunity to maintain

3. Polymeric micelles for codelivery of chemotherapeutics

TABLE 3.2

55

Codelivery systems by polymeric micelles to achieve synergetic effect in cancer therapy.

Micelle composition

Drug 1

Drug 2

Study type

Refs

PEG-FTS

Curcumin

FTS

4T1 xenograft syngeneic mouse model

[89]

DTX-PEG-GEM

Docetaxel

GEM

DMBA-induced breast cancer model

[86]

PEGePLLePLLeu

Docetaxel

siRNA

MCF-7 xenograft nude mice

(87)

PLFCL

Dox

Dasatinib

4T1.2 xenograft female BALB/c mice model

[88]

PLGA-PEG, PLGA-PEG-FOL and PLGAPEG-TAT

Dox

PTX

Human oral cavity carcinoma KB cell line

[85]

HA-VES

Dox

Curcumin

4T1 xenograft mice model

[90]

PCLeSSeCTS-GA

Dox

Curcumin

HepG2 and HUVEC cells

[91]

PEG-PLA

Sildenafil (Viagra)

Crizotinib

MCF-7 cells

[92]

mPEG-b-P(Glu)-b-P(Phe)

PTX

Cisplatin

A549 xenograft Balb/C nude mice model

[93]

FTS (trans-farnesylthiosalicylic acid); GEM (Gemcitabine); PEGePLLePLLeu (poly(ethylene glycol)-b-poly(L-lysine)-bpoly(L-leucine)); PLFCL (PEG5000-lysyl-(a-Fmoc-ε-Cbz-lysine)2); HA-VES (hyaluronic acid-vitamin E succinate); PCLeSSeCTS-GA (glycyrrhetinic acid-modified chitosan-cystamine-poly(ε-caprolactone)) mPEG-b-P(Glu)-b-P(Phe) (methoxy poly(ethylene glycol)-b-poly(L-glutamic acid)-b-poly(Lphenylalanine)).

the initial drug ratio in vivo. However, further optimization to change the ratio of the two drugs for specific treatment would be needed. Considering their different mechanism of action and nonoverlapping toxicity, docetaxel and gemcitabine represent another candidate drug pair for codelivery systems. However, these drugs have difference in hydrophilicity/ hydrophobicity properties, and encounter a serious problem when loaded into a single nanoformulation. Docetaxel is highly hydrophobic. Gemcitabine is a highly hydrophilic drug and is quickly metabolized into an inactive metabolite, 20 -deoxy-20 ,20 -difluorouridine. To address this issue, they were conjugated to PEG to form an amphiphilic molecule with the capability for self-assembly [86]. A 4.8-fold higher AUC (0-N) for gemcitabine compared to Gemzar was the most important feature of the resultant codelivery micelle system. Lower hepato- and nephrotoxicity was another advantage of using these micelles. This was confirmed by both histopathological sections, biochemical marker level

estimation, and hemolytic toxicity. Docetaxel has also been used with siRNA for combined cancer therapy [87]. A micelle system based on the triblock copolymers poly(ethylene glycol)(PEG-PLLb-poly-L-lysine-b-poly-L-leucine PLLeu), with PLLeu as the hydrophobic core, PLL as the cationic shell, and PEG as the hydrophilic corona, was used to encapsulate the negatively charged siRNA via electrostatic interactions and the hydrophobic docetaxel in the core segment of the micelles via hydrophobic associations. The codelivery of these micelles revealed a significant effect on the tumor growth in a xenograft model, resulting in an animal survival of 40% at 27 days for the docetaxel nanoparticles plus siRNA/nanoparticles compared to 100% for the DTX-siRNA nanoparticles group. A superior antitumor effect of Dox and dasatinib (Das) in tumor suppression has also been reported using the PEGylated peptidic nanocarrier, PEG5000-lysyl-(a-Fmoc-ε-Cbz-lysine) (PLFCL) micellar system against a variety of

56

3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy

cancer cells including colon, breast, and prostate [88]. Administration of Dox and Das at a dose of 5 mg/kg produced a 95% inhibition of tumor growth in a murine breast cancer model. Regardless of drug type, the drug ratio is also important for the synergetic effect. Duong et al. [85] used Calcusyn software to find the suitable dose combination of Dox and PTX as free drug. To confirm the combined synergistic effect quantitatively, the CI values of all Dox/PTX combinations (1/0.1, 1/0.2, 1/1, 0.2/1, and 0.1/1) were simulated as a function of the cell viability from 5% to 98%. They found that a decrease in PTX in the Dox-based combinations reduced the CI values. For the PTX-based combinations, the CI values increased with the decrease in the added Dox. A higher antagonistic interaction (CI >1) and higher synergistic interaction (CI <1) of drugs was observed in the PTXbased combinations (Dox/PTX 0.2/1 and 0.1/ 1) and in the Dox-based combinations (Dox/ PTX 1/0.2). The combination of the free drugs Dox/PTX at molar ratio of 1/0.2 showed a synergistic therapeutic effect compared to the treatment of a free single drug, Dox or PTX, which was used to prepare a micellar codelivery system of two single drugs (Dox/PTX).

3.3 Codelivery of chemotherapeutics to mitigate side effects Current chemotherapy has significantly increased the survival rate of patients with cancer. However, some chemotherapeutic agents have very harsh side effects during the treatment and even after several years. Side effects may cause a disruption in the treatment schedule and/or a reduction in dosing or even lead to a treatment’s discontinuation. Simultaneous delivery of some protective agents as well as anticancer drugs may mitigate the side effects associated with chemotherapy [94]. Moreover, codelivery of chemotherapeutic drugs with some protective agents would impact not only

disease progression but survival. Dox, in spite of its well-established use for cancer treatment, has serious life-threatening side effects, particularly dose-related myocardial toxicity with increasing total lifetime doses at and above 400 mg/m2. Amongst the different mechanisms suggested for doxorubicin hydrochlorideinduced cardiomyopathy, it is clear that freeoxygen radicals formed during the redox cycling of the quinoloneesemiquinolone ring of the doxorubicin hydrochloride is a determining parameter. Thus, codelivery of Dox with an agent capable of acting against this redox cycling might help to mitigate doxorubicin hydrochloride-induced cardiotoxicity. Some natural products with remarkable free radical scavenging properties and potent chemosensitizing properties mitigate doxorubicin hydrochloridee induced cardiotoxicity while maintaining and/ or improving its potency against cancer cells. For example, codelivery of resveratrol and curcumin at a molar ratio of 5:1 in F127 micelles (mRC) coadministered with doxorubicin hydrochloride showed cardioprotective effects. This was attributed to scavenging of free radicals with a synergistic effect on SKOV-3 cells and antagonistic effect in H9C2 cells, most probably through chemosensitization [95].

4. Stimuli-responsive codelivery of polymeric micelles Conventional chemotherapy treatments suffer from the lack of specificity, which exposes healthy cells to toxic chemotherapeutic agents as much as to cancerous cells. Moreover, the fact that low concentration of drugs at the tumor site significantly decreases the therapeutic efficiency of treatment and leads to the highest frequency and dosing of drugs and harsher side effects, suggests an urgent need to generate efficient, targeted drug delivery systems for efficient chemotherapeutics. The most important feature of the newly generated drug delivery

4. Stimuli-responsive codelivery of polymeric micelles

systems, referred to as targeted drug delivery systems, is the ability to selectively deliver drugs to the tumor site. Targeted drug delivery systems are designed to selectively deliver therapeutic agents to a site of action, accumulate at the tumor, and subsequently reduce exposure of healthy cells. Since targeted drug delivery systems release the payload drug upon exposure to specific stimuli, they are also termed stimuliresponsive, intelligent, or smart drug delivery systems. Targeted drug delivery systems are also classified as either “passive targeting” or “active targeting” systems. Passive targeting systems take advantage of the differences between the environment of tumor tissue and normal tissue, including the pathophysiological characteristics of tumor vessels, pH, temperature, and external stimuli such as a magnetic field or heat as a trigger for drug release. Passive targeting has been somewhat successful in improving the efficacy of chemotherapy, but it suffers from several limitations because of the variation in the microenvironment of tumor tissue at different stages of tumor progression and within different types of tumors. To overcome these shortcomings of passive targeted drug delivery systems, an active targeting approach has been used to improve the performance of targeted drug delivery systems. In this approach, various affinity ligands like aptamers, antibodies, peptides, and small molecules are used to direct drug delivery systems toward tumors cells via specific receptors overexpressed on the cell surface by tumor cells. After binding to the cell surface, they are often internalized by receptormediated endocytosis followed by the formation of an endosome from which they release the drug payload due to a weak acidic environment or enzymes. A variety of codelivery systems, including polymerosomes, liposomes, polymeric micelles, dendrimers, and lipid-based nanostructures, have been developed that enhance drug accumulation at the tumor site using passive or active targeting strategies [96].

57

There are many reports on codelivery systems of targeted polymeric micelles that release the payload drugs in response to a stimulus in the tumor tissue, such as a reductive environment, low pH, and high temperature (Table 3.3). Considering the long circulating time of micelles, which usually comes with small size and stealth properties, it is desirable to avoid drug leakage within the circulation and have triggered drug release as much as possible. Different parameters considered to control the rate of drug release from micellar structures include micelle stability (either thermodynamic or kinetic), the rate of drug diffusion, partition coefficient, and in the case of biodegradable polymers, the rate of biodegradation. Table 3.3 summarizes some polymeric codelivery micelles used as targeted drug delivery systems.

4.1 pH-sensitive codelivery systems Among the applied stimuli, acidic pH is the most commonly used internal trigger for the selective release of anticancer drugs because of the relatively acidic pH of cancer tissues in both primary and metastasized tumors (6.5e7.2), which is lower than the extracellular pH of normal tissue and blood (pH 7.4). Moreover, micelles experience an even more acidic condition following their uptake via endocytosis and formation of endosomes and lysosomes with pH values of 5.0e6.0 and 4.0e5.0, respectively. These differences of pH between healthy and cancerous cells make the pH value a suitable stimulus for targeted drug release. For instance, Dox has been conjugated via acid-sensitive linkage to a Pluronic F127- chitosan (F127-CS) polymer to form a self-assembling and pH-sensitive polymeric micelle system for codelivery of Dox and PTX [97]. The hydrophobic nature of PTX allows efficient entrapment via hydrophobic interactions into the core segment of the micelles. The pH sensitivity of micelles was confirmed by increase in the release rate of Dox and PTX

58 TABLE 3.3

3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy

Polymeric codelivery micelles used as stimuli-responsive drug delivery systems.

Micelle composition

Drug 1

Drug 2

Animal model or cell line

Stimuli

Refs

PEG-PLGAeSSeDTX Docetaxel

Verapamil

Healthy male Wistar rats

Redox-sensitive

[99]

SHRss

Dox

microRNA-34a

DU145 Xenograft BALB/C nude mice model

Redox-sensitive

(82)

DTDAP

Dox

PTX

B16 xenograft mice model

Redox-sensitive

[107]

PEG-PAsp(AED)PDPA

Dox

siRNA

HepG2 and HUVEC cells

Redox-sensitive

[91]

mPEG-PAsp-NI

Dox

Chlorin e6 (Ce6)

4T1 xenograft mice model

Hypoxia-and singlet oxygen responsive

[108]

ACeCSePpIX

Dox

Apatinib

MCF-7/ADR xenograft mice model

Light-sensitive

[109]

F127-CS

Dox

PTX

Healthy male Wistar rats

pH-sensitive

[97]

mPEGeSSeDOX

Dox

PTX

B16 xenograft melanoma mice model

Redox-sensitive

[110]

pHPMA

Dox

Axitinib

A549 xenograft mice model

Dual-pH responsive

[111]

PAP

MDR-1

Survivin-targeting RNA

MCF-7/ADR xenograft Balb/c nude mice model

Redox-sensitive

[112]

PEG-pp-PEI-PE

PTX

siRNA

A549 xenograft nude mice model

Metalloproteinase 2 (MMP2)-sensitive

[113]

mPEGeSSeC18

PTX

Dasatinib

MCF-7 and MCF-7/ADR cell line

Redox-sensitive

(98)

LDLeNSCeSSeUA

PTX

Lipoprotein and siRNA

MCF-7 xenograft mice model

Dual redox and pH sensitive

[114]

PEOz-PLA

PTX

Honokiol

MDA-MB-231-luc-GFP xenograft nude mice model

pH-sensitive

[81]

(PEG-PLGAeSSeDTX) poly (ethylene glycol)-poly (D,L-lactide-co-glycolide) -docetaxel; (SHRss) poly(L-arginine)-poly(L-histidine)-stearoyl; (DTDAP) D-A-tocopherol polyethylene glycol 1000 succinate; (HA-VES) hyaluronic acid-vitamin E succinate; (PEG-PAsp(AED)-PDPA) poly(2(diisopropyl amino)ethyl methacrylate) and poly(N-(2,20 -dithiobis(ethylamine)) aspartamide) and poly(ethylene glycol); (mPEG-PAsp-NI) nitroimidazole-bearing methoxy poly(ethylene glycol)-co-poly(aspartic acid); (ACeCSePpIX) acetylated-chondroitin sulfate-protoporphyrin; (F127-CS) pluronic f127-chitosan polymer; (HPMA) N-(2-hydroxypropyl) methacrylamide; (PAP) poly[bis(2-hydroxylethyl)-disulfidediacrylate-b-tetraethylenepentamine]; (PEG-pp-PEI-PE) polyethylene glycol -peptide -polyethylenimine - 1,2-dioleoyl-sn-glycero-3phosphoethanolamine; (PEG-PE) polyethylene glycol-phosphatidyl ethanolamine; (LDLeNSCeSSeUA) N-succinyl chitosanecystamineeurocanic acid; (PEOZ-PLA) poly(2-ethyl-2-oxazoline)-poly(D,L-lactide).

during in vitro drug release studies. The pH decrease facilitated the cleavage of an acidliable cis-aconityl bond. In an in vivo pharmacokinetic study of PTX-loaded F127-CS-Dox micelles in rats, the area under the plasma concentration time curve (AUC0eN) values of PTX and Dox were 3.97- and 4.38- fold higher in comparison to those for a PTX plus Dox solution [97].

4.2 Redox-sensitive codelivery systems The higher reducibility potential of tumor cells compared to healthy cells has become a popular stimulus for targeted intracellular drug delivery systems. The reducing environment of tumor cells is controlled mainly by the reduction/oxidation states of NADPH/NADPþ and glutathione (GSH, GSH/GSSG). In tumor cells,

4. Stimuli-responsive codelivery of polymeric micelles

a high concentration of GSH compared to NADPH provides a reductive microenvironment. In fact, GSH, through the formation and cleavage of disulfide bonds and the reaction with excess reactive oxygen species (ROS) plays a determinant role with respect to the intracellular-reducing potential. The concentration of GSH determines the extent of the cellular reducing environment. The intracellular concentration of GSH in normal cells is 10 mM, whereas the concentration in tumor tissues is increased at least four times and is even higher in some multidrug-resistant tumors. The intercellularreducing potential of tumors can serve as a trigger in drug delivery systems known as redox-sensitive nanocarriers. Since redoxsensitive drug delivery systems tend to degrade and release their payload only in tumor cells due to the reducing environment, they show little toxicity toward normal cells, leading to low associated side effects of such chemotherapeutics. In addition, a high concentration of GSH in tumor cells allows rapidly release of drug payload upon exposure to the reducing-intracellular environment. PEGSS-C18 copolymer has been used to form redox-sensitive polymeric micelles for codelivery of PTX and dasatinib (SS-PDNPs) [98]. The disulfide bonds present in the backbone of the SS-PDNPs are reduced when internalized by tumor cells with a high-reducing environment. Consequently, degradation of the amphiphilic polymer structure results in quick disassembly of the micelles and fast drug release. The anticancer effect of various reduction-sensitive micelles, including blank redox-sensitive micelles (referred to as SS-NPs) and redoxnonsensitive micelles (referred to as CC-NPs), PTX-loaded redox-sensitive micelles (referred to as SS-PNP), PTX and dasatinib coloaded redox-sensitive micelles (referred to as SSPDNP), and redox-nonsensitive micelles (referred to as CC-PDNP) showed a 5.6-fold higher apoptosis incidence for MCF-7/ADR cells treated with SS-PDNPs, compared to

59

Taxol, and SS-PNPs and CC-PDNPs. A codelivery system composed of a redox-sensitive mPEG-PLGAeSSeDTX conjugate possessed the ability to carry the P-gp inhibitor verapamil (VRP) and Docetaxel (mPEG-PLGAeSSeDTX/ VRP (PP-SS-DTX/VRP)) and overcome MDR [99]. Fig. 3.3 illustrates schematically the mechanism of action of the redox-sensitive micelles of PP-SS-DTX/VRP. These findings indicate that PP-SS-DTX/VRP micelles inhibit P-gp in MCF-7/ADR cells because of verapamil’s high efficiency in lowering the efflux activity of P-gp.

FIGURE 3.3 Schematic illustration of the combination of DTX and VRP by using a redox-responsive micelle to overcome MDR in cancer cells. From Guo Y, He W, Yang S, Zhao D, Li Z, Luan Y. Co-delivery of docetaxel and verapamil by reduction-sensitive PEG-PLGA-SS-DTX conjugate micelles to reverse the multi-drug resistance of breast cancer. Colloids Surf B Biointerfaces. 2017; 151:119e27. with permission.

60

3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy

5. Targeted codelivery of polymeric micelles The use of polymeric micelles for targeted drug delivery is a promising strategy to improve the efficacy of pharmaceuticals in chemotherapy. The principle of targeted drug delivery is based on nanoparticles acting as carriers for pharmaceutical agents, with a targeting molecule such as antibody or ligands attached to the surface of the nanoparticles, which then directs them to the receptors/antigens on the surface of tumor cells. To achieve the maximum therapeutic efficiency, it is very important to find a suitable target and potent drug. In addition, however, the type of carrier plays an important role in the success of targeted drug delivery systems. This high drug-loading capacity and the possibility of surface modification of micelles make them promising candidates for targeted drug delivery systems. Polymeric micelles have also been used as codelivery agents in development of targeted drug delivery systems for chemotherapy. A variety of targeting moieties have been used to design micellar-based targeted drug delivery systems [100e105]. The nucleus has been a very important site to target and improve the therapeutic efficiency of cancer treatment. To date, drug delivery systems have been designed mainly to enable drug delivery at endo/lysosomal sites of cancer cells. To reach the nucleus, in these systems, drugs need to escape from endo/lysosomal compartments and overcome a variety of intracellular resistance mechanisms, including drug metabolism and detoxification, overexpression of drug efflux pumps (e.g., P-gp), drug sequestering to acidic compartments, and drug deactivation. Only a small portion of any drug is likely to reach the cytosol with eventual delivery to the nucleus, particularly in drug-resistant cells. For instance, it was found that most of the cisplatin delivered to the cytoplasm bound to protein and only a small portion (less than

10%) of the total covalently bound cellassociated cisplatin reached the DNA fraction [106]. These barriers markedly decrease the efficacy of some chemotherapeutic drugs, such as Dox, which acts via DNA injury. To solve this problem, nuclear-targeted vehicles that facilitate drug translocation into the nucleus have been explored. Generally, nuclear-targeted molecules like transactivator of transcription (TAT) have been used to modify the surface of drug delivery systems. TAT is a cell-penetrating peptide and has the ability to quickly enter almost all living cells. It actively delivers proteins, DNA, and nanoparticles into the nucleus. Micelles consisting of grafted poly-(N-3-carbobenzyloxy-lysine) (CPCL) with TAT-chitosan decorated on the surface have been used as a codelivery system for p53 and Dox into the nucleus of cancer cells [106]. Using confocal laser scanning microscopy, the vector delivered more TAT-CPCL into the nucleus than did CPCL alone. The TATmodified vector served as a highly efficient gene and drug codelivery system with high gene transfection efficiency and a high anticancer effect leading to low viability in HeLa cells [106]. An MTT cell viability assay clearly confirmed that TAT-CPCL/p53/Dox micelles possessed a higher cytotoxicity compared to the blank TAT-CPCL’s negligible cytotoxicity.

6. Application of polymeric micelles in codelivery for multiple therapies Due to the diversity, and complexity of cancer, the integration of two or more therapies with different therapeutic mechanisms of action in one nanoplatform (e.g., chemotherapy and photothermal therapy) seems a promising strategy for significant enhancement of the therapeutic efficacy and a better long-term prognosis. Codelivery nanoplatforms provide the opportunity to integrate chemotherapy with multiple therapeutic approaches. Treatment of tumors

61

6. Application of polymeric micelles in codelivery for multiple therapies

with multiple approaches has attracted a great deal of attention because of their success in overcoming the challenge of tumor heterogeneity, reversing MDR, and achieving additive or synergistic anticancer effects (Table 3.4).

6.1 Chemo-immunotherapy The function of the immune system is a determinant parameter in cancer’s initiation and growth. Cancer cells have the potential to escape, inactivate, or overpower the immune system. Immunotherapy is now a wellestablished clinical regimen that boosts the TABLE 3.4

immune system’s activity against cancerous cells. One strategy is to prohibit cancer cells from expressing inhibitory receptors referred to as immune checkpoints, or to inhibit cancerous cell expression of immune inhibitory ligands. Despite the high potential for immunotherapy in cancer treatment, the overall clinical success of this regimen is still far from an acceptable situation. This is probably because the efficacy of this treatment is closely associated with preexisting antitumor immune responses. By initiating an antitumor immune response, chemotherapy agents could improve the efficiency of immunotherapy. Chemo-immunotherapy combines immunotherapy and chemotherapy. Recently,

Polymeric micelles in codelivery for multiple therapies.

Micelle composition

Drug 1

Drug 2

Animal model or cell line

Therapeutic approach

Refs

Heparin-D-a-tocopheryl Dox succinate

Toll-like receptor 7 agonist imiquimod (IMQ)

4T1 xenograft BALB/c mice

Chemoimmunotherapy

[115]

POEG-b-PMBC

Dox

Immune checkpoint inhibitor NLG919

4T1.2 xenograft mice model

Chemo-immunotherapy

[115]

F127/PDPP

Dox

poly(dithienyldiketopyrrolopyrrole)

HeLa cells

Chemo-phototherapy

[116]

PCL-ss-PEG-ss-PCL and Dox PCL-acetal-PEG

Indocyanine green

BEL-7404 xenograft nude mice model

Chemo-photothermal therapy

[117]

mPEG-S-S-C16

Dox

Semiconducting polymer dots PCPDTBT dots (Pdots)

HepG2 xenograft nude mice model

Chemo-photothermal therapy

[118]

Porphyrin-based telodendrimer

SN-38

-

HT-29 xenograft mice model

Chemo-phototherapy

[119]

PEG-b-PHEA

GW627368X Cayman chemical

Gold nanorod

Sarcoma S180 xenograft mice model

Photothermal therapy

[120]

PEG-PLA

PTX

CaWO4 (CWO)

HN31 xenograft mice model

Chemo-radio therapy

[121]

POEG-b-PMBC (poly(oligo(ethylene glycol) methacrylate) -b-poly N,N0 -(t-butyoxycarbonyl)cystamine); F127/PDPP (PluronicF127/ poly(dithienyl-diketopyrrolopyrrole)); PCL-ss-PEG-ss-PCL and PCL-acetal-PEG (poly(ε-caprolactone)-ss-poly(ethylene glycol)-ss-poly(εcaprolactone) and poly(ε-caprolactone)-acetal-poly(ethylene glycol)); mPEG-S-S-C16 (monomethoxy-poly(ethylene glycol)-S-S-hexadecyl); SN38 (7-thyl-10-hydroxycamptothecin), PEG-b-PHEA (Poly(ethylene glycol) monomethy ether, poly 2-hydroxyethyl acrylate); PEG-PLA(poly (ethylene glycol)-poly(lactic acid)).

62

3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy

there have been a few reports on the application of polymeric micelles for concurrent codelivery of chemotherapeutic and immunotheraputic agents. Chen et al. [80,122] used a dualfunctional, immunostimulatory nanomicellar carrier composed of a NLG919-conjugated PEG with a Fmoc linker (PEG2k-Fmoc-NLG) as an indoleamine 2,3-dioxygenase (IDO) inhibitor for delivery of PTX. They showed that the codelivery immune-chemotherapy they developed led to a significantly improved antitumor response in both breast cancer and mouse melanoma models. Polymeric micelles have also been used for simultaneous delivery of Dox and NLG919 for the treatment of leukemia [123]. The Dox-loaded micelles self-assembled from a PEG-Fmoc-NLG conjugate showed cytotoxicity comparable to that of free Dox. In vivo studies showed significant improvement in antitumor activity for the Dox/PEG-Fmoc-NLG group compared to Doxil or the free Dox group in an A20 lymphoma mouse model.

6.2 Chemo-angiogenic therapy Angiogenesis, defined as the growth of new blood vessels from a preexisting vasculature, plays a crucial role in the progression of cancer because the proliferation and metastatic spread of cancer cells strongly depends on an abundant supply of oxygen and nutrients, and the removal of metabolites. Thus, the inhibition of angiogenesis represents a promising approach to limitation of access to oxygen and nutrients and sensitization of tumor cells to chemotherapy. The combination of antibodies against endothelial growth factor (anti-VEGF) and cytotoxic therapeutic agents has been used intensively in the clinic for cancer treatment. The synergy of treatment might be explored by the high apoptosis rate of the cancer cells, which increases blood flow in tumor sites after tumor vascular normalization consequently promotes the penetration of nano-vehicles into the deepest part of

tumor tissues through the tumor interstitial matrix [124]. On the other hand, the lack of selectivity of anti-VEGF agents and free cytotoxic drugs can also result in significant side effects (e.g., hypertension, and thrombotic events). Thus, there is an apparent need for targeted codelivery of anti-VEGF agents with common chemotherapeutic drugs. For instance, a dualpH responsive cross-linked micelle for two-step release of the small molecule receptor tyrosine kinase inhibitor, axitinib (AXI), which acts by an antiangiogenesis effect and a cytotoxic agent, Dox, has been reported [111]. A pH-sensitive hydrazone linkage was used to conjugate Dox to amphiphilic N-(2-hydroxypropyl) methacrylamide (HPMA) to take advantage of the EPR of micelles in tumors and accumulate selectively at the tumor site. In a related study, axitinib was also encapsulated into the core segment of selfassembling micelles [111]. In addition, to prolong the micelles, circulation time, and increase their stability, the micelles were cross-linked through benzoiceimine bonds, which are triggered to break at lower extracellular pH (pH 6.5) and enable delivery of the axitinib and Dox selectively to the tumor site. Axitinib interacts with tyrosine kinase receptor located on the cell membrane for vasculature modulation (Fig. 3.4). After nanoparticle uptake by cancer cells, exposure of micelles to the more acidic environment of the lysosome (pH 5.0) results in the hydrolysis of hydrazone linkages, which in turn releases the conjugated Dox and induces cell death. The in vivo antitumor effect of dualdrug-loaded cross-linked micelles (DA-CM) showed the highest tumor growth inhibition (88.38%) with no apparent body weight loss. Such a high growth inhibition might be explained by the synergistic effect of axitinib and Dox delivered by the cross-linked micelles. Free-Dox and single-Dox-loaded cross-linked micelles (D-CM) resulted in 49.16% and 67.23% suppression of tumor growth, indicating the significance of the EPR effect for tumor-targeted delivery.

6. Application of polymeric micelles in codelivery for multiple therapies

pH6.5

63

pH5.0

Tumor microenvironment

Endo-Iysosome

nucleus Iysosome

HPMA

Axitinib

Doxorubicin

β -sitosterol

FIGURE 3.4 Illustration of dual-pH responsive micellar platform for codelivery of axitinib (AXI) and doxorubicin (Dox) based on HPMA copolymers. From Xu X, Li L, Zhou Z, Sun W, Huang Y. Dual-pH responsive micelle platform for codelivery of axitinib and doxorubicin. Int J Pharm. 2016;507(1):50e60 with permission.

6.3 Chemo-photothermal therapy Photothermal therapy uses electromagnetic radiation, including visible light, NIR, radio frequency, and microwaves, to stimulate a photosensitizer that converts this energy into heat. Minimal invasiveness and high specificity with minimal toxicity are the most important features of photothermal therapy. Recently, integration of photothermal therapy with chemotherapy has been considered a useful technique

for a targeted synergistic chemo-photothermal strategy [125,126]. Aptamer (Apt)-polydopamine (pD) and its derivatives are well known for their effective photothermal properties. pDfunctionalized CA-(PCL-ran-PLA) micelles have been developed for effective delivery of DTX and improved therapeutic efficacy [127]. The results demonstrated that DTX-loaded Apt-pD-CA-(PCL-ran-PLA) micelles had high-therapeutic efficacy that was due to its synergistic chemo-photothermal properties.

64

3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy

6.4 Chemo-radiotherapy

6.5 Chemo-enzyme prodrug therapy

A radiosensitizer, or a radiosensitizing agent, is a pharmacologic agent that potentiates the toxicity of radiation therapy. Radiation therapy uses various external energy sources such as X-rays and protons to shrink tumors by disrupting their DNA. Some radiosensitizers are directly toxic by themselves, while others show toxicity only on exposure to radiation. A different mechanism has been proposed for radiosensitizing agents. For instance, the mechanism of action of PTX as a radiosensitizer involves the inhibition of cell cycle progression at a radiosensitive phase (G2/M). The 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) augments radiosensitization by inhibiting the function of heat shock protein 90 (Hsp-90) [128]. Rapamycin (RAP) radiosensitizes cancer cells by inhibiting mTOR, which is downstream of the PI3K-Akt survival pathway [129]. A sensitivity enhancement ratio is used to compare the potency of radiosensitizing agents in chemoradiotherapy [130]. It is defined as the ratio of radiosensitivity of radiation-treated cells to radiosensitivity of drug combined with radiation-treated cells. Tomoda et al. [131] studied a codelivery micellar system, referred to as Triolimus, containing a variety of radiosensitizing species including PTX, 17-allylamino-17demethoxygeldanamycin (17-AAG), and rapamycin used to investigate the corresponding potential of different radiosensitization agents on A549 cells and the suppression of tumor growth with their combinations in an A549 xenograft mouse model. The radiosensitizing effects were as follows: PTX alone > (PTX and RAP) >Triolimus> (PTX and 17-AAG) > (17-AAG and RAP). However, Triolimus showed less acute toxicity compared to PTX alone or radiation alone, indicating the high potency of polymeric micelles as carriers for codelivery of radiosensitizing and chemotherapeutic agents.

Chemo-enzyme prodrug therapy (EPT) has recently been introduced as a therapeutic approach to improve specificity and anticancer efficiency of chemotherapeutics [132]. EPT consisting of a foreign prodrug-activating enzyme and a nontoxic prodrug are delivered to the tumor cells using various targeting approaches. The prodrug is then activated and converted into a potent pharmacologic agent through enzymatic activity. The time interval between enzyme and prodrug treatment is the most challenging step. Too short a time interval means the prodrug will be activated by the enzyme while in the circulation with decreased drug concentration at tumor sites and enhanced side effects. On the other hand, too long a time interval means the prodrug-activating enzymes are eliminated by the immune cells of the body and consequently cannot activate prodrug into a potent cytotoxin. Therefore, delivery of prodrugs and enzymes simultaneously to tumor sites with a single nanoplatform is important. The combination of 3-indoleacetic acid (IAA) and horseradish peroxidase (HRP), which is highly cytotoxic to mammalian cells, is one of the most studied enzymeeprodrug for anticancer therapy. IAA stimulates cell division and promotes cell differentiation. HRP is a widely studied heme containing an enzyme with the ability to convert the auxin IAA into cytotoxins via catalysis. Neither IAA nor HRP is cytotoxic at clinically used concentrations. But when combined, HRP can activate IAA to produce a series of free radicals, which can initiate the tumor cell killing process and apoptosis. Among the different chemical bonds used to prepare prodrug, esters are the most common linkages. They are readily hydrolyzed by esterases widely distributed in the body to facilitate drug release. Ethyl 3-indoleacetate (EIA) is a hydrophobic prodrug that is quickly

65

References

hydrolyzed by the double action of esterases and the low-pH conditions of tumors to produce IAA. Nanomicelles formed by self-assembly of the amphiphilic copolymer PEG-PAsp(-AED)CA have been used for codelivery of HRP and EIA [133]. To enhance the targeted delivery of nanomicelles, a reductive-sensitive disulfide bond has also been added to the polymeric micelle structure to initiate the drug and enzyme release once the micelle has been taken up by tumor cells [133]. As a result, IAA activated by HRP produces an abundance of ROS, offering great potential for cancer treatment.

7. Conclusions The codelivery of chemotherapeutic drugs represents a promising strategy to achieve high efficacy in cancer therapy. To date, a variety of drug delivery systems have had success as codelivery systems. Micelles as self-assembled structures of amphiphilic copolymers have unique properties as drug carriers, including small size, long circulation time, and high drug-loading capacity, which have attracted extensive attention compared to other systems. To date, micelle-based codelivery systems have been developed with synergistic anticancer effects and suppress multidrug resistance. In addition, multiple-drug-loaded micelles have been widely used to design targeted drug delivery systems by attachment of different targeting agents along with chemotherapeutics that are pH-sensitive, redoxsensitive, and nuclear-targeted drug delivery systems. Micelles, by concurrently delivering multiple types of agents, also provide the opportunity to combine chemotherapy with other therapeutic approaches like radiotherapy, immunotherapy, photothermal therapy, and hyperthermal therapy. Thus, development of these micellar codelivery systems affords the opportunity to take advantage of micelle properties as efficient and novel drug delivery

systems for combinational therapy. Despite the high potential and variety of codelivery systems developed, there are still issues to be addressed. Optimization of the dose ratio of different therapeutic agents is still a big challenge for generation of synergetic effects. There remains a need to control the level of drug payload to maintain dose ratios. To minimize toxicity, the design of new methods to find the best pairs of drugs for concurrent delivery would be of more than a little interest. The possibility of payload release in a controlled manner and the need for optimum dosing must be taken into consideration in order to have a highly efficient codelivery system. Development of such codelivery systems will ultimately lead toward more effective therapies for cancer treatment.

Acknowledgments The authors would like to acknowledge William Hartner for helpful comments on the manuscript.

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