Combination of Nanoparticles with Physical Stimuli toward Cancer Therapy

Abstract

Drug delivery systems represent an important strategy for cancer treatment. The targeted delivery of drugs is required for effective and safe cancer therapy. In cancer therapy, the target cells include cancer cells and immunocompetent cells such as antigen presenting cells. Anticancer drugs utilized include small molecular drugs, proteins and nucleic acid medicines. In order to deliver these drugs into the target cells, various nanoparticles have been developed. However, the efficacy of the nanoparticulate system itself is generally insufficient for the safe and effective treatment of cancer. For example, polyethylene glycol (PEG)-modified (PEGylated) nanoparticles accumulate in cancerous tissues; however, the PEG moiety on the surface of the nanoparticles disturbs cellular uptake, which is known as the ‘PEG dilemma.’ Thus, additional strategies such as receptor-mediated targeting are necessary to improve the delivery and cellular uptake of nanoparticles. Among additional strategies, in this review we have focused on the combination of nanoparticles with various physical stimuli, such as electric pulse and ultrasound, to improve the targeted delivery of the nanoparticles.

1. INTRODUCTION

Beating cancer is a challenging task. Approaches toward cancer therapy include several strategies such as surgical excision, drug treatment, and immunomodulation. In spite of progress in the development of regimens of chemotherapy and molecular target drugs, the outcomes from cancer therapy are not yet enough. In most cases, the reason for the insufficient outcome of cancer therapy may be problems in drug delivery. Targeted delivery of anticancer drugs is an important issue that determines not only the efficacy but also the safety of cancer therapy. To deliver anticancer drugs selectively to cancer regions, the development of nanoparticulate systems is a promising approach.

Nanoparticles can deliver various materials including small molecular drugs, proteins and nucleic acid medicines. Nanoparticulate systems can also modulate the immune system. Various platforms for delivering drugs have been developed such as liposomes, polymeric micelles, dendrimers, etc. Targeting cancer and/or immunocompetent cells is a rational strategy in cancer therapy. However, disposition of nanoparticles is dependent on their physicochemical characteristics such as size and surface charge. Thus, it is important to regulate the physicochemical characteristics of nanoparticles.¹⁾ Interaction of nanoparticles with blood components also affects the biodistribution of the nanoparticles.^2–4) Polyethylene glycol (PEG)-modification (PEGylation) of nanoparticles is useful to passively deliver the nanoparticles to cancerous tissues via an enhanced permeability and retention (EPR) effect.⁵⁾ However, the PEG moiety on the surface of the nanoparticles disturbs cellular uptake, which is known as the ‘PEG dilemma.’⁶⁾ Using receptor-mediated endocytosis, the disposition and cellular uptake of nanoparticles can be controlled.^7,8) Moreover, microenvironment-responsive systems such as matrix metalloproteinase-cleavable PEG-lipids^9,10) and acidic pH-responsive nanocarrier systems¹¹⁾ have been developed. Especially, combination of nanoparticles with various physical stimuli is a rational approach to improving the efficiency of cellular uptake of the nanoparticles. In this review, we focused on the physical stimuli-mediated delivery of nanoparticles and on applications of these systems toward cancer therapy.

2. PHYSICAL STIMULI-MEDIATED DELIVERY OF NANOPARTICLES

Various physical stimuli, such as electric pulse, ultrasound, hyperthermia and so on, have been used to improve the efficacy of nanoparticles. Table 1 summarizes the in vivo delivery of nanoparticles in combination with physical stimuli.

Table 1. Summary of in Vivo Delivery of Nanoparticles with Physical Stimuli

Physical stimuli	Cargos/drugs	Target tissues	Injection routes	Refs.
Electroporation
Electric pulse	−/pDNA	Liver	Direct injection or tail vein	33)
Electric pulse	−/pDNA	Various tissues	Direct injection or tail vein	34)
Electric pulse	Galactosylated polymers/pDNA	Liver	Tail vein	60)
Electric pulse	−/pDNA	Tumor	Direct injection	61)
Electric pulse	Liposome/pDNA	Tumor	Direct injection	62)
Electric pulse (irreversible)	−/pDNA	Liver	Hepatic artery or portal vein	14)
Sonoporation
Ultrasound+bubble liposome	−/doxorubicin	Tumor	Tail vein	17)
Ultrasound+bubble liposome	−/pDNA	Artery	Femoral artery	16)
Ultrasound+bubble liposome	Mannosylated liposome/pDNA	Liver or spleen	Tail vein	20)
Ultrasound	Mannosylated bubble liposome/pDNA	Liver or spleen	Tail vein	21)
Ultrasound	Mannosylated bubble liposome/siRNA	Liver	Tail vein	63)
Hyperthermia
Heating	Thermosensitive liposome/doxorubicin	Tumor	Tail vein	26)
High intensity focused ultrasound	Thermosensitive liposome/doxorubicin	Tumor	Tail vein	28)
Others
Tissue pressure	−/pDNA	Kidney	Tail vein	40)
Tissue suction	−/pDNA	Various tissues	Tail vein	42)
Rubbing	−/pDNA	Stomach	Organ surface instillation	43)

2.1 Electric Pulse

Electroporation uses an electric pulse to make transient pores on the cellular membrane. By electroporation, both small molecules and macromolecules can permeate the cellular membrane both in vitro and in vivo.^12,13) In general, the effect of electroporation is reversible. However, tissue damage often occurs by the electroporation. In the case of cancer treatment, such damage might be acceptable to induce tumor death. Moreover, it was reported that irreversible electroporation not only killed the tumor but also enhanced the transfection efficiency in a peripheral zone surrounding the tumor.¹⁴⁾

2.2 Ultrasound

Sonoporation creates transient pores on the cellular membrane by cavitation due to ultrasound exposure. Use of microbubbles enhances the ultrasound-mediated transfection of naked plasmid DNA (pDNA), lipoplex and polyplex in cultured cells.¹⁵⁾ As a more pharmaceutically stable system, bubble liposomes (PEGylated liposomes containing an ultrasound imaging gas such as perfluoropropane) have been developed.¹⁶⁾ The combination of bubble liposomes and ultrasound exposure with doxorubicin inhibited tumor growth in vivo.¹⁷⁾ Tumor-homing peptide AG73-modified bubble liposomes improved in vitro drug and gene delivery to tumor cells.^18,19)

As to a DNA vaccine, it is necessary to deliver pDNA to antigen-presenting cells. Since antigen-presenting cells express mannose receptors, the concomitant use of mannosylated lipoplex and bubble liposomes with ultrasound exposure can transfect the liver and spleen, in which antigen-presenting cells are abundant.²⁰⁾ As a more simple delivery system, a mannosylated PEGylated bubble lipoplex system selectively transfected antigen-presenting cells in vivo.²¹⁾ DNA vaccination by the mannosylated PEGylated bubble lipoplex with ultrasound exposure suppressed melanoma growth and metastasis in vivo.²²⁾

2.3 Hyperthermia

Tumors are generally sensitive to hyperthermia.²³⁾ The combination of hyperthermia with radiotherapy and chemotherapy improves cancer treatment.²⁴⁾ Hyperthermia targeted to the tumor region can improve the delivery of liposomes by enhancing vasculature permeability and interstitial fluid flow.²⁵⁾ Moreover, hyperthermia against solid tumors induced doxorubicin release from thermosensitive liposomes.²⁶⁾ As a hyperthermia-inducing method, high intensity focused ultrasound (HIFU) has been used for tumor ablation.²⁷⁾ The combination of HIFU with thermosensitive liposomes containing doxorubicin resulted in significant tumor regression.²⁸⁾ Magnetic nanoparticles also have been employed for hyperthermia-based therapy.²⁹⁾ In addition, the photothermic regulation of heat shock promoter-driven gene expression can be triggered by laser-induced carbon nanohorns.³⁰⁾ This system is potentially useful for hyperthermia-based therapy in the near future.

2.4 Radiation

Among other physical stimuli-based therapies, boron neutron capture therapy (BNCT) is promising for the treatment of several tumors, including gliomas. Selective delivery of the boron-10 isotope to tumor cells is important for successful BNCT. Thus, various targeting systems for boron delivery have been developed. Boronated epidermal growth factor can be selectively delivered to epidermal growth factor receptor-positive tumors by intratumoral injection.³¹⁾ Since folate receptors are often expressed on cancer cells, boron-containing folate receptor-targeted liposomes are also promising candidates for BNCT.³²⁾

2.5 Other Physical Stimuli

The in vivo transfection efficiency of naked pDNA is generally low due to rapid degradation and poor cellular uptake. Here, the combination of naked pDNA with several physical stimuli has been investigated. As mentioned above, physical stimuli such as electroporation^33–35) and sonoporation^36,37) have been used to enhance the transfection efficiency of naked pDNA. Hydrodynamics-based transfection, i.e., rapid intravenous large volume injection of naked pDNA, is an efficient method to deliver naked pDNA to the liver.^38,39) As more mild physical stimuli, pressure-mediated transfections of intravenously delivered naked pDNA to the kidney, liver and spleen have been reported.^40,41) Moreover, tissue suction using a device enables site-specific transfection by naked pDNA to the kidney, liver, spleen, and heart.⁴²⁾ On the other hand, rubbing stimuli against the gastric serosal surface greatly enhanced naked pDNA transfer.⁴³⁾ In addition, the concomitant use of an abrasive compound calcium carbonate with naked pDNA was effective to a similar extent as the rubbing stimuli.⁴⁴⁾ These physical stimuli would improve the efficacy of the nanoparticulate systems.

3. PERSPECTIVES

In the physical stimuli-mediated delivery of nanoparticles, the development of specialized devices is important to successfully increase the cellular uptake of the nanoparticles in clinical use. In the case of electroporation, the shape of the electrodes is an important factor, not only for efficacy but also for safety.⁴⁵⁾ As another example of a device, an implantable pneumatically actuated microsystem was developed to achieve renal pressure-mediated transfection.⁴⁶⁾

As mechanisms of improved efficacy of the combination of nanoparticles with physical stimuli, both cellular uptake and intracellular disposition of the nanoparticles are important issues (Fig. 1). Especially, localization of the nanoparticles in endosomes/lysosomes can result in the degradation of contents in the nanoparticles. In the case of electroporation, nanoparticles directly enter cytosol through transient pores on the cellular membrane. Also, ultrasound exposure of bubble lipoplex increased cytosolic pDNA.⁴⁷⁾ It was reported that the concomitant use of TAT-PEG liposomes containing pDNA and bubble liposomes with ultrasound exposure promoted the endosomal escape of pDNA.⁴⁸⁾ Thus, in the case of bubble liposomes with ultrasound exposure, both transient pores (Fig. 1 (i)) and endosomal escape (Fig. 1 (ii)) might be involved in the transfer of pDNA into cytosol. On the other hand, in many cases the physical stimuli induce cellular actions such as changes in gene expression. This affects the efficacy of the nanoparticles. Especially, transcriptional activation (Fig. 1 (iii)) was involved in enhanced transgene expression by hydrodynamics-based transfection,^49,50) ultrasound-mediated transfection,⁵¹⁾ and pressure-mediated transfection.⁵²⁾ Thus, it is necessary to consider the effect of the physical stimuli on cellular functions in view of future clinical use.

Fig. 1. Schematic Illustration of the Mechanism of Enhanced Transfection Using Bubble Liposomes with Ultrasound Exposure

Three proposed mechanisms of the enhanced transfection are numbered (i–iii).

The combination of active targeting, such as utilization of receptor-mediated endocytosis, with physical stimuli would greatly improve target specificity. Moreover, the combination of several physical stimuli such as electric pulse with ultrasound^53,54) could further enhance the efficacy of the nanoparticles. On the other hand, in the tissue suction-mediated transfection, the transgene expression was not enhanced in the case of the stomach.⁴²⁾ This is probably due to the poor biodistribution of pDNA in the stomach after intravenous injection. In contrast, naked pDNA instillation onto the stomach without the physical stimuli resulted in efficient transgene expression.^55–57) As mentioned above, physical stimuli such as rubbing the stomach surface could enhance the transgene expression.⁴³⁾ Thus, appropriate selection of the administration routes would also be an important issue in the physical stimuli-mediated delivery of the nanoparticles.⁵⁸⁾ In general, endocytosis is the primary cellular uptake mechanism of nanoparticles. For example, in the case of naked pDNA instillation onto the stomach, the cellular uptake mechanism was macropinocytosis.⁵⁹⁾ Since endocytosed nanoparticles are generally degraded in lysosomes, the uptake mechanism greatly governs the intracellular fate of the nanoparticles. Therefore, appropriate selection of physical stimuli which change the uptake mechanism would be a useful strategy for developing effective cancer therapy using nanoparticles.

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