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Tumor-targeting glycol chitosan nanoparticles as a platform delivery carrier in cancer diagnosis and therapy

A natural based polymer, chitosan has received widespread attention in drug delivery systems due to its valuable physicochemical and biological characteristics. In particular, hydrophobic moiety-conjugated glycol chitosan can form amphiphilic self-assembled glycol chitosan nanoparticles (GCNPs) and simultaneously encapsulate hydrophobic drug molecules inside their hydrophobic core. This GCNP-based drug delivery systems exhibit excellent tumor-homing efficacy, attributed to the long blood circulation and the enhanced permeability and retention effect; this tumor-targeting drug delivery results in improved therapeutic efficiency. In this review, we describe the requisite properties of GCNPs for cancer therapy as well as imaging for diagnosis, such as their basic characteristics, in vitro delivery efficiency and in vivo tumor-targeting ability. Keywords:  amphiphilic nanoparticles • cancer therapy • diagnosis • EPR • glycol chitosan

Nanomedicine in cancer treatment Cancer is a devastating disease and is the leading cause of death around the world [1] . In order to overcome and prevent cancer, numerous research institutes participate in cancer clinical trials and also offer a thorough knowledge and understanding of cancer mechanisms and treatment methods. To date, there have been various cancer treatment approaches including surgery, radiation therapy, chemotherapy and many others [2] . One of the most important aspects of cancer therapy is strong and specific therapeutic efficacy without toxicity to normal healthy tissues. Unfortunately, in the case of cancer chemotherapy, large proportions of the administered drugs are distributed throughout the whole body, including normal tissues, subsequently leading to adverse side effects [3–5] . Therefore, anticancer drugs most likely have an intention of enhancing their tumorspecific delivery passively or actively to offer more effective and less-toxic treatments for patients. Over the last decade, nanotechnology has emerged as a fascinating tool in the field of nanomedicine, in particular for cancer therapy [6,7] , since nanocarriers can

10.2217/NNM.14.99 © 2014 Future Medicine Ltd

selectively deliver drugs to tumors by passive or active targeting [8–10] . Compared with normal tissues, nanocarriers can more effectively reach and accumulate in the tumor tissues through the abnormally leaky blood vessels surrounding the tumor; this phenomenon is called the enhanced permeability and retention (EPR) effect, known as a major driving force for passive drug targeting [11,12] . In addition, active targeting of drugs can occur via the introduction of various ligands (including peptides, oligosaccharides and antibodies) onto the surface of nanocarriers [13,14] . For successful clinical use of therapeutic nanocarriers, selecting the materials of the nanocarrier is an important issue, because this step will mainly determine the therapeutic efficiency and safety of anticancer drugs. The category of materials for nanocarriers is quite broad, including liposomes, natural or synthetic polymers, gold nanoparticles and other substances [15,16] . Among these, chitosan, acquired from a natural polymer, has attracted considerable attention due to its physical and biological properties including abundant availability, biocompatibility and low immunogenicity [17,18] .

Nanomedicine (2014) 9(11), 1697–1713

So Jin Lee‡,1, Hyun Su Min‡,1, Sook Hee Ku1, Sohee Son1, Ick Chan Kwon1,2, Sun Hwa Kim*,1 & Kwangmeyung Kim1 Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, Republic of Korea 2 KU-KIST School, Korea University, 1 Anam-dong, Seongbuk-gu, Seoul 136701, Republic of Korea *Author for correspondence: Tel.: +82 2 958 5916 Fax: +82 2 958 5909 kim@ kist.re.kr ‡ Authors contributed equally 1

part of

ISSN 1743-5889

1697

Review  Lee, Min Ku et al. Herein, we will limit the content on nanocarriers for cancer therapy to glycol chitosan (GC)-based delivery systems. This review particularly focuses on the distinguishing properties of GC nanoparticles (GCNPs) as an anticancer drug carrier in vitro and in vivo. In addition, we will further discuss different diagnostic and therapeutic applications of GCNPs depending on the drug type. GC self-assembled nanoparticles Chitosan, a linear aminopolysaccharide consisting of β-(1–4)-linked d-glucosamine (deacetylated unit) and N-acetyl-d-glucosamine (acetylated unit), is easily derived by N-deacetylation of chitin [19] . Chitosan has received special attention with regard to its pharmaceutical availability due to excellent biological properties, such as biocompatibility, biodegradability, nontoxicity and low immunogenicity [20–22] . For the development of efficient drug delivery systems, it is particularly important that chitosan has a positive charge, increasing adhesion to the mucosal surface, leading to improvement in drug penetration [23] . As a vector for gene therapy, the positively charged chitosan can readily form complexes with nucleic acids through ionic interactions [24] . However, the water solubility of chitosan in acidic conditions only limits its wide applications in nanomedicine. Chitosan is not soluble in neutral water and only dissolves in some acidic aqueous solutions with pH values lower than its pKa value of 6.5 [22] . To overcome this drawback, numerous chitosan derivatives have been developed by different modifications of functional groups in chitosan, such as quaternization of the primary amine. GC is obtained by grafting of a glycol group onto chitosan chains at position C6. The GC molecules exhibit complete water solubility at any pH. In particular, GC has great abilities as a drug carrier due to its enhanced water solubility and functional groups for further chemical modification. The amine groups present along the backbone of GC can be modified to improve its characteristics and in vitro/in vivo delivery efficiency. In recent years, several GC derivatives have been developed for biomedical uses because of possible nanoparticle formulations. In general, amphiphilic copolymers are used to deliver hydrophobic anticancer drugs. These amphiphilic polymers are able to self assemble in water via hydrophobic interactions to form nano-sized particles with a hydrophobic core surrounded by a hydrophilic outer shell. As a result, insoluble drugs can be easily loaded into the hydrophobic part of the polymeric nanoparticles. Thus, a hydrophobic moiety can also provide amphiphilicity to GC, resulting in the formation of self-assembled nanoparticles [25–28] . The amphiphilic GC derivatives with

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modified hydrophobic parts are able to carry poorlysoluble drugs with a better stability in physiological conditions than innate chitosan derivatives. Synthesis of GCNPs

Kwon et al. developed amphiphilic GC derivatives, named hydrophobically modified GCNPs, by covalent conjugation of bile acid (5β-cholanic acid) to the backbone of GC (Figure 1) [29–32] . Specifically, 5β-cholanic acid was covalently conjugated to GC in the presence of ethyl(dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for 24 h at room temperature. The resulting solution was dialyzed for 3 days against the excess amount of water/methanol mixture and lyophilized to obtain GCNPs [29–32] . GCNPs have great in vitro and in vivo properties, such as high stability for long circulation and deformability for avoiding unintended accumulation and penetration in angiogenic vessels. This was proven in filtration tests; here the fluorescently-labeled GC, GCNPs and polystyrene (PS) were incubated in human serum albumin solutions for 1 day, passed through the syringe filter and fluorescence intensity observed. GCNPs demonstrated high stability and deformability by maintaining their fluorescence signal after 1-day incubation in serum conditions (Figure 2A), whereas PS with the same size of GCNPs could not pass through the filter membrane. GCNPs and PS were each administered to tumor (SCC7), macrophage (RAW264.7) and normal (human umbilical vein endothelial cell) cell lines [33] . GCNPs showed much higher uptake in tumor cells than in macrophages and vascular endothelial cells  (Figure 2B) . This result indicated that GCNPs could avoid uptake by macrophages, leading to long blood circulation times and higher accumulation in tumor tissues. In the case of PS, however, all cell types showed strong red fluorescence signals, suggesting its nonspecific uptake. Physicochemical optimization of GCNPs for in vitro & in vivo applications

In general, drugs have to pass through the cell membrane, escape from the endosome and finally reach the desired active site. In addition, the maintenance of an appropriate intracellular concentration of drugs should be considered for medicinal effect. However, some drugs have a difficulty showing their therapeutic efficacy. In particular, it is not easy to succeed in treating cancer due to its unregulated defense mechanisms that protect against cytotoxic agents such as anticancer drugs; cancer can remove drugs via multidrug resistance proteins and the acidic microenvironment causes drugs to lose activity. In the last decade, a wide variety of nanoparticles have been explored to efficiently

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Tumor-targeting glycol chitosan nanoparticles as a platform delivery carrier in cancer diagnosis & therapy  

deliver anticancer drugs. In particular, the physicochemical characteristics of nanoparticles, such as coat composition, size and fate of the internalized materials, can greatly influence the efficiency of endocytic uptake [34] . The intracellular fate according to the physicochemical characteristics of GCNPs was reported in a previous study [35] . The authors investigated the cellular uptake mechanism of GCNPs using fluorescent dye (cyanine 5.5)-labeled GC polymers. The subcellular localization of both GC polymers and GCNPs was monitored in a HeLa H2B-GFP cell line (Figure 2C) . GCNPs showed much stronger fluorescence intensity along the cell membrane and inside the cytoplasm, compared with the fluorescence signal of GC polymer. GCNPs were internalized in cells via various endocytic pathways and this was examined through the pretreatment of HeLa cells with several endocytic inhibitors (Figure 2D) . The internalized GCNPs did not strictly follow one of the classical endocytotic pathways but more than one cellular uptake mechanism. Based on the study of intracellular distribution of GCNPs using LysoTracker® (Life Technologies, CA, USA) staining and TEM analysis, GCNPs were dispersed evenly in the cytoplasm and a fraction of aggregates were found to be entrapped inside the lysosomal vesicles. To evaluate in vivo biodistribution as change in the physicochemical properties of GCNPs, three types of GC polymers with different molecular weights

(GC-20 kDa, GC-100 kDa and GC-250 kDa) were hydrophobically modified with 5β-cholanic acid (Table 1 & Figure 3A) [36] . The feed mole ratio of 5β-cholanic acid per 100 sugar residues of GC was fixed as five. The mean diameters of the three GC nanoparticles are not different greatly (approximately 231–310 nm) and also showed similar surface charge. However, in vivo tissue distribution of each GCNP was significantly different. High-molecular-weight GC-250 kDa-NPs accumulated in tumor tissue at a much higher level and circulated for a longer period of time (up to 72 h), compared with low-molecularweight GC-20 kDa-NPs and GC-100 kDa-NPs. The tissue accumulation levels of low molecular weight GC-20 and 100 kDa-NPs were monitored in the kidney at 6 h and rapidly decreased in the blood stream. GC-250 kDa-NPs remained relatively stable in the blood stream leading to prolonged circulation and tumor selectivity (Figure 3B) . To optimize tumor selectivity of GCNPs according to their amphiphilicity, degrees of hydrophobic substitution of GCNPs were studied by Kim et al. [37] . For GCNPs with 7.5, 12, 23 and 35 wt.% of 5β-cholanic acid to GC polymers, the serum stability and in vivo tumor-targeting efficiency were monitored  (Figure 3C & D) . Several factors such as size, deformability and stability determine tumor-targeting efficacy of nanoparticles. By increasing hydrophobic portion in GCNPs, the stability was increased but

CH2OCH2CH2OH CH2OCH2CH2OH CH2OCH2CH2OH O O O O O O HO HO HO NH NH2 NH O CO CH3 HC

GC

Review

H3C

3

n

5β-cholanic acid

Hydrophobically modified GC

Self-assembly in aqueous condition

Hydrophobically modified GCNP Figure 1. Hydrophobically modified glycol chitosan nanoparticles. Representatively, hydrophobic moiety, 5β-cholanic acid, was introduced into GC chain and nanoparticle structure was formed by self-assembly in aqueous conditions. GC: Glycol chitosan; GCNP: Glycol chitosan nanoparticle.

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0h

24 h

SCC7

RAW264.7

HUVEC

GCNPs

GC GCNPs PS GC GCNPs PS Control 0.8 µm 0.45 µm

PS

0.2 µm

GC

GCNPs

Intensity 4000 0 0 50 100 150 200 250 300 350 400 450 500

0 50 100 150 200 250 300 350 400 450 500

Intensity 4000 0

500 450 400 350 300 Y 250 200 150 100 50 0

X

500 450 400 350 300 Y 250 200 150 100 50 0

X

Relative ratio

1.2 1.0 0.8 0.6 0.4

0.2 0.0 GCNPs + inhibitor -

+ CPZ

+ Fil

+ + CPZ Amil + Fil

+ -

Figure 2. Stability and in vitro cellular uptake of glycol chitosan nanoparticles. (A) Stability test of GC, GCNPs and PS via filter test in human serum albumin solutions. (B) Cellular uptake images of GC and PS nanoparticles in SCC7, RAW 264.7 and HUVEC cell lines. (C) Confocal microscopic images of GCNPs and GC polymer-treated cells. HeLa cell were incubated with fluorescent compounds (25 μg/ml; 1 h).

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Tumor-targeting glycol chitosan nanoparticles as a platform delivery carrier in cancer diagnosis & therapy  

Review

Figure 2. Stability and in vitro cellular uptake of glycol chitosan nanoparticles (cont.). (D) Effect of endocytic inhibitors on the internalization of GCNPs. HeLa cell were either untreated or pretreated with CPZ (10 μg/ml; inhibitor of clathrin-mediated endocytosis), filipin III (1 μg/ml; inhibitor of caveolae) or amiloride (50 μM, inhibitor of macropinocytosis) in serum-free media for 1 h. Amil: Amiloride; CPZ: Chlorpromazine; Fil: Filipin III; GC: Glycol chitosan; GCNP: Glycol chitosan nanoparticle; HUVEC: Human umbilical vein endothelial cell; PS: Polystyrene. (A & B) reproduced with permission from [33] ; (C & D) reproduced with permission from [35] .

deformability was decreased, although there were no differences in size and surface charge. In the biodistribution data, h(23%)-GCNPs circulated in the blood for longer periods of time and accumulated more selectivity in tumors compared with other GCNPs. The GCNPs with low-level hydrophobic substitution were more easily dissociated because of their low stability in the blood and excreted by renal clearance, whereas the h(35%)-GCNPs with high hydrophobicity failed in tumor targeting due to the low deformability. For successful systemic drug delivery, h(23%)-GCNPs have essential characteristics, such as high stability and deformability in vivo, resulting in superior tumor-targeting efficiency. Thus, efficient tumor-targeting system using GCNPs was determined by two critical factors, stability in serum and deformability through the degree of hydrophobic substitution of nanoparticles. EPR effect of GCNPs

Nanoparticles with the size in the hundreds nanometers are known to pass through fenestrate and leaky tumor vessels via EPR effect, as mentioned earlier. The EPR effect provides efficient accumulation of nanoparticles in tumor tissues. Recently, enhanced tumor accumulation of GCNPs by the EPR effect was further investigated compared with other types of nanoparticle  [33] . Fluorescence dye labeled GCNPs and polystyrene (PS) having approximately the same size were injected systemically into tumor bearing mice and monitored in normal blood vessels (colon) and tumoral vessels inside the tumor tissues by whole-body imaging

system (Figure 4) . Interestingly, GCNPs were localized in tumor blood vessels within 5 min postinjection. After 30 min postinjection, a stronger red signal of GCNPs was detected in tumor vessels and even spread into tumor tissues through interstitial vascular leakages, whereas the diffusion of GCNPs was not shown in normal blood vessels of colon tissues. The rigid nature PSNPs exhibited poor tumor accumulation due to low deformability and low flexibility. Therefore, the improved stability and deformability of GCNPs is one of their positive attributes of tumor t­ argeting through the EPR effect. GCNPs for diagnosis of cancer As mentioned above, GCNPs possess great tumor-targeting ability by EPR effect; thus, the incorporation of imaging contrast agents into GCNPs allows noninvasive imaging of diverse cancer models (Table 2 & Figure 5) . A variety of imaging modalities, such as optical imaging, MRI, computed tomography (CT), PET and ultrasound (US) imaging, can be used to diagnose tumors by introducing appropriate contrast agents into GCNPs (Table 2) . First, fluorescence-enhanced optical imaging was achieved by GC polymers chemically conjugated with near-infrared fluorescent molecules, whose fluorescence wavelength is in the range of the therapeutic window. Near-infrared fluorescent dye, such as Cy5.5, can be covalently linked to the amine functional group of the GC chain; the resulting dye-labeled GCNPs successfully visualized several types of tumor, including brain tumor, liver tumor

Table 1. Characterization of glycol chitosan nanoparticles. Samples

Mn† 

DS

Size (nm)‡

ζ (mV)

GC-20kDa-NPs

21,180

4.7§

231

10.1

GC-100kDa-NPs

109,070

4.7

271

11.4

GC-250kDa-NPs

271,420

4.8

310

10.8

h(7.5%)-GCNPs

268,750

52 ¶

366

15.9

h(12%)-GCNPs

280,000

83

h(23%)-GCNPs h(35%)-GCNPs

§ §

349

19.5

307,500

159

¶

359

22.1

337,500

243 ¶

340

22.8

¶

Estimated from the colloidal titration result. Mean diameter, measured by dynamic light scattering. Degree of substitution of 5β-cholanic acid per 100 sugar residues of GC, determined by colloidal titration method. ¶ Degree of substitution of the number of 5β-cholanic acid per one GC polymer, determined by colloidal titration method. DS: Degree of substitution; GC: Glycol chitosan; GCNP: Glycol chitosan nanoparticle; h-GCNP: Hydrophobically modified glycol chitosan nanoparticle; Mn: Average molecular weight; NP: Nanoparticle. Adapted with permission from [36,37]. † ‡ §

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A

P -N Da k -20 GC

P a-N D 0k -10 GC

P a-N D 0k -25 GC

B

er Liv

1h

GC20kDa-NP

24 h

GC100kDa-NP

72 h

GC250kDa-NP

C

D Before filtration

Filtration (µm) 0.8

0.45

0.2

GC

Tumor

Liver

ng Lu

Lung

y ne Kid

n r lee eart umo Sp H T

spleen

Kidney

h(7.5%)

h(12%)

h(7.5%)-GCNPs h(12%)-GCNPs

h(23%)

h(23%)-GCNPs h(35%)-GCNPs

h(35%)

PSNPs

Figure 3. Effects of the molecular weight and the degree of hydrophobic substitution of glycol chitosan. (A) In vivo noninvasive near infrared fluorescence images of real-time tumor-targeting characteristics of GC20kDa-NP, GC-100kDa-NP and GC-250kDa-NP, and (B) ex vivo NIRF images of dissected organs and tumors at 72 h. (C) Deformability test of each GCNP with different degree of hydrophobic substitution.(D) Ex vivo analysis of major organs and tumors from a flank tumor-bearing mice model after intravenous injection of GCNPs. GG: Glycol chitosan; h-GCNP: Hydrophobically modified glycol chitosan nanoparticle; NP: Nanoparticle; PSNP: Polystyrene nanoparticle. (A & B) Reproduced with permission from [36] ; (C & D) reproduced with permission from [37] .

and lung metastasis tumor [33] . GCNPs for MRI were developed by conjugation of Gd(III) complexes to primary amine group of GC, or immobilization/coating of GC on the surface of superparamagnetic iron oxide nanoparticles [38–40] . GCNPs containing Gd(III) or superparamagnetic iron oxide nanoparticles exhibited a great ability as MRI contrast agents in T1- or T2-weighted MRI, respectively [39,41] . Recently, gold nanoparticles have been considered as promising CT contrast agents instead of commercially available iodine containing agents that cause side effects [42] . Sun et al. reported that GC-coated gold nanoparticles succeeded in the imaging of colorectal metastases in liver [43] and hyperacute direct thrombus [44] . PET

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using GCNPs was also achieved by introduction of radioactive isotopes into the nanoparticles. Lee et al. developed stable 64Cu-DOTA-GCNPs using copperfree click chemistry, and the nanoparticles were used for the visualization of the tumor site [45] . Furthermore, GCNPs encapsulating bubbles or gas-generating particles enabled US imaging, a relatively low-cost and convenient imaging modality [46] . Therapeutic applications of GCNPs in cancer Nanoparticles have received great attention as smart drug carriers due to their significant tumor-targeting ability, resulting from their prolonged persistence in vivo and EPR effect. As mentioned above, the excellent

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Tumor-targeting glycol chitosan nanoparticles as a platform delivery carrier in cancer diagnosis & therapy  

tumor-targeting properties of GCNPs have enabled them to be applied in cancer therapy. Various types of drugs can be incorporated into GCNPs by physical interaction with modified GC polymers or chemical conjugation to the primary amine group in the GC backbone. For example, the conjugation of hydrophobic segments increases intermolecular hydrophobic interactions, resulting in the self-assembly of the modified GC. The hydrophobic inner core of GCNPs facilitates the incorporation of water-insoluble anticancer drugs. The conjugation of a photosensitizer to GC allows the application of GCNPs in photodynamic therapy. Furthermore, slightly positively-charged GC polymers interact with negatively-charged nucleic acids, and the resulting nanocomplexes of GC polymers and nucleic acids can be considered as valuable candidates for gene therapy. Chemotherapy

Efficient drug carriers should be designed to be nontoxic, have high drug-loading efficacy and deliver drugs into the desired tissue; thus, GCNPs, which are composed of biocompatible GCs and exhibit great tumortargeting ability, can be considered promising drug carriers. Hydrophobically modified GC self-assembles into stable nanoparticles via hydrophobic interactions of the modified group, and the resulting GCNPs provide a great opportunity of hydrophobic anticancer drug loading. The self-assembly of hydrophobic segments leads to the formation of stable nanoparticles and facilitates drug loading in the inner core of GCNPs. Moreover, the outer shell, consisting of hydrophilic GC, protects the encapsulated drugs from the reticuloendothelial system (RES) during circulation in the blood. Hydrophobic GC was obtained via conjugation of GC polymers

Review

with diverse hydrophobic groups, such as 5β-cholanic acid, hydrotropic oligomer, N-acetyl histidine and o-Nitrobenzyl succinate. These hydrophobic segments allow the incorporation of water-insoluble chemodrugs (e.g., paclitaxel [PTX], docetaxel [DTX], doxorubicin [Dox] and camptothecin [CPT]) (Table 3) . Kwon’s group developed anticancer drug delivery system using GCNPs, which were synthesized via chemical conjugation of 5β-cholanic acid to the primary amine group of GC. Hydrophobic drugs, such as PTX, CPT and DTX, were loaded in GCNPs by simple dialysis method. PTX-GCNPs containing 10 wt.% of PTX effectively suppressed tumor growth in B16F10 xenograft mice, compared with the commercialized PTX formulation in Cremophore EL. PTX-GCNPs were less toxic to both B16F10 melanoma cells in vitro and tumorbearing mice in vivo than Cremophore EL, indicating that the GCNP-based drug delivery system reduced the adverse side effects of anticancer drugs [32] . Similarly, CPT was physically loaded into GCNPs, resulting in the formation of CPT-GCNPs (280–300 nm). Sustained release of CPT from the nanoparticles led to the inhibition of tumor growth, and specific accumulation of CPT-GCNPs in the tumor sites attributed to the reduced toxic effects of CPT and the improved survival rate [31] . In particular, the physical properties of drugloaded GCNPs, such as stability and deformability, were further investigated using DTX-GCNPs. High stability of the nanoparticles in bovine serum albumin-containing solution indicated the potential of prolonged blood circulation in vivo, and distinctive deformability allowed extravasation of the nanoparticles through leaky blood vessels in the tumor site. Thus, DTX-GCNPs exhibited excellent tumor-targeting ability and antitumor effects in A549 tumor-bearing mice (Figure 6A) [30] .

Table 2. GCNP-based imaging probes in different diagnostic applications. Imaging method

Imaging site

Imaging probe

Ref.

Fluorescence 

Liver metastasis, colon, brain and lung cancers

5β cholanic-GC-Cy5.5 nanoparticle

[33]

MRI    

SCC7 cancer

GC/heparin deposited nanoparticle

[38]

T6–17 cancer

pH-responsive GC-SPIO nanoparticle

[39]

HT-29, HCT-116 colon cancer

Gd(III)-GC, Ho(III)-GC, Dy(III)-GC

[40]

SCC7 cancer

Gd(III)-5β cholanic-GC-Cy5.5

[41]

HT-29 cancer (colon cancer)

MMP-GC-gold nanoparticle

[42]

Liver metastasis cancer

GC-gold nanoparticle

[43]

CT  

Hyperacute direct thrombus

GC-gold nanoparticle

[44]

PET 

SCC7 cancer

Radiolabel GC nanoparticle

[45]

US 

SCC7 cancer

Gas-generating GC-coated nanoparticle

[46]

CT: Computed tomography; Cy5.5: Cyanine 5.5; GC: Glycol chitosan; MMP: Matrix metalloproteinase activatable peptide probes; SPIO: Superparamagnetic iron oxide; US: Ultrasound.

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Tumor/PS

30 min

5 min

Tumor/h-GCNPs Colon/h-GCNPs

Figure 4. Real-time intravascular tracking of nanoparticles in tumor tissue. GCNP-injected mice’s tumor and colon tissues and PS-injected mice’s tumor tissue were monitored by Olympus® OV100 whole mouse imaging system (Olympus, Tokyo, Japan). h-GCNP: Hydrophobically modified glycol chitosan nanoparticle; PS: Polystyrene. Reproduced with permission from [33] .

To enhance the drug loading capacity, hydrotropic oligomer was introduced into GC (Figure 6B), and the PTX loading efficacy was increased up to 24.2 wt.%. PTX-HO-GCNPs were stable for 50 days in physiological conditions, and exhibited less cytotoxicity and improved anticancer effects, compare with Abraxane® (Celgene, NJ, USA; commercialized PTX-formulation)  [47] . High drug loading can also be achieved via chemical conjugation of drug to GC. Son et al. developed the Dox-conjugated GCNPs, containing 38 wt.% of Dox in GCNPs, and the resulting nanoparticles showed pH-sensitive drug release behavior due to the cis-aconityl spacer between Dox and GC. After systemic delivery of Dox-GCNPs, tumor growth was successfully suppressed over 10 days [48] . Stimuli-responsive nanoparticles have great potential for smart drug delivery. Lee et al. developed pHsensitive GCNPs through conjugation of N-acetyl histidine (NAcHis) residues to GC. The NAcHis moiety is hydrophobic at neutral pH; however, under slightly acidic conditions, the imidazole group of NAcHis is protonated, resulting in the disassembly of nanoparticles. In the weakly-acidic environment of tumor tissue and the cytoplasmic endosome, Dox-NAcHis-GCNPs can more effectively deliver the anticancer drug to tumor sites and successfully suppress the tumor growth [49] . Meng et al. developed a dual pH/light-sensitive GCNPs by conjugation of light-responsive o-nitrobenzyl succinate into GC and by crosslinking with glutaraldehyde, which resulted in the formation of acid-labile imine

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bonds. The GC–o-nitrobenzyl succinate showed fast drug-release behavior at low pH with light irradiation and good cytotoxicity in MCF7 cancer cells under UV irradiation [50] . In cancer therapy, angiogenesis is one of promising therapeutic targets; thus, antiangiogenic peptide drugs, which aim to suppress the blood vessel formation in tumor tissue, have received much attention. According to the previous literature, antiangiogenic peptide RGD (Arg-Gly-Asp), which binds to αvβ3 integrin on the endotherial cells, can be integrated in GCNPs by a solvent evaporation method [51,60] . RGD-GCNPs sustained released the RGD peptide drug for 1 week in vitro, and inhibited the adhesion/migration of HUVEC. In tumorbearing mice, the treatment of RGD-GCNPs reduced both tumor volume and microvessel density, compared with native RGD peptide injection (Figure 6C). Considering its drug loading capability for both hydrophobic anticancer drugs and peptide drugs, tumor-targeting ability and potentials for stimuli sensitivity, GCNPs would be very useful in cancer chemotherapy. Photodynamic therapy

To maximize therapeutic efficacy with reduced side effects, external stimuli-responsive chemotherapies, such as photodynamic therapy (PDT), have been developed. PDT involves the treatment of chemical photosensitizers and the irradiation of certain wavelengths onto the target tumor sites. The light irradiation of the photosensitizer causes the generation of reactive singlet oxygen through the photochemical reactions between the photosensitizer and the surrounding molecules, resulting in damage to tumor tissue [61] . However, clinical applications of PDT have been limited due to the insolubility and nonspecificity of photosensitizers. To overcome these drawbacks, efficient photosensitizer carrier systems into tumor sites have been critically required. Similar to hydrophobic chemodrugs, photosensitizers, such as chlorin e6 (Ce6), protophorphyrin IX (PpIX), pheophorbide A (PheoA) and fullerene (C60), can be loaded into GCNPs physically or chemically (Table 3) . Lee et al. investigated the differences of Ce6-loaded GCNPs and Ce6-conjugated GCNPs in Ce6 release behavior, tumor-targeting ability and antitumor effects [52] . Ce6-loaded GCNPs were prepared by incorporation of Ce6 in the hydrophobic inner core of GCNPs, and showed the initial burst release of Ce6. In contrast, Ce6-conjugated GC self-assembled into stable nanoparticles via hydrophobic interactions, and the nanoparticles were accumulated in the tumor site with higher Ce6 contents, compared with Ce6-loaded GCNPs. The successful inhibition of tumor growth was observed after systemic delivery of Ce6-conjugated GCNPs, followed by light irradiation (Figure 7A) .

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To exhibit the photosensitizer’s activity specifically in target tumor tissue, many researchers have tried to use the quenching/dequenching system of photosensitizers Lee et al. demonstrated the cellular on/off system of PpIXGCNPs, which is useful in tumor-specific PDT [53] . GC conjugated with PpIX formed a stable nanoparticle via self-assembly of PpIX molecules, and self-quenching effects of PpIX in the nanoparticles were observed due to the compact crystallized PpIX molecules. In this state, PpIX showed no fluorescence signal and phototoxicity under light exposure. In the cytoplasmic environment, however, the nanoparticles disassembled and generated singlet oxygen upon light irradiation. The improvement in anti-tumor effects of PpIX-GCNPs was attributed to this cellular on/off photoresponsibility of

PpIX-GCNPs and tumor-targeting ability of GCNPs in vivo. Similarly, PheoA was conjugated to GC with cleavable disulfide bonds, and self-assembled into nanoparticles (PheoA-ss-GCNPs) [54] . Photoactivity of PheoA in nanoparticles was suppressed by the self-quenching effects, but it was recovered after cellular uptake due to the dissociation of disulfide bonds. Considering the specific accumulation in tumor sites and the switchable photoactivity mechanism of PheoA-ss-CNPs, tumortargeted PDT was achieved with the reduced side effects  (Figure 7B). In addition, pH-responsive nanogel was developed by chemical grafting of 2,3-dimethylmaleic acid (DMA) and fullerene (C60) to GC [55,56] . GC conjugated with DMA and C60 formed multinanogel aggregates via the charge–charge interactions between

NIRF

MR

Pre

Inner part h-GCNP-23%

Hydrophobic 5 β-cholanic acid Cy5.5

Gd(III)

Cross

Pre

US Glycol chitosan surface

24 h

Glycol chitosan polymer Hydrophobic 5bcholanic acids NIRF Cy5.5

Hydrophilic polymer shell

CT

Review

CO2 generating polymer

Post 3D

Au -GCNP

5mm Figure 5. Diagnostic cancer imaging using various clinical imaging methods (near-infrared fluorescence, magnetic resonance, computed tomography and ultrasound) after injection of hydrophobically modified glycol chitosan nanoparticle-based imaging probes. CT: Computed tomography; Cy5.5: Cyanine 5.5; GC: Glycol chitosan; GCNP: Glycol chitosan nanoparticle; h-GCNP: Hydrophobically modified glycol chitosan nanoparticle; MR: Magnetic resonance; NIRF: Near-infrared fluorescence; US: Ultrasound. Reproduced with permission from [37,41,43,46] . 

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6h

12 h

24 h

48 h

2100

72 h Tumor volume (mm3)

1h

Tumor

Whole body

A

1500 1200 900 600 300 0

B

1h

6h

Saline h-GCNP Free DTX 30 mg/kg DTX h-GCNP 10 mg/kg DTX h-GCNP 30 mg/kg

1800

5

0

12h

10

24h

15 20 25 Time (day)

30

35

48h

72h

PTX-HO-h-GCNP 3500

= HO

3000

Feed ratio of PTX (wt. %)

Loading amount of PTX (wt. %)

Loading efficiency (%)

Size (nm)

–

–

–

302 + 22

PTX (10 wt. %)HO-h-GCNP

10

9.6 + 0.2

96.8 + 2.5

328 + 18

PTX (20 wt. %)HO-h-GCNP

20

19.2 + 0.6

95.1 + 3.3

343 + 12

PTX (30 wt. %)HO-h-GCNP

30

Sample

HO-h-GCNP

Tumor volume (mm3)

= PTX

Saline PTX formulation (20 mg/kg) Abraxane (20 mg/kg) PTX-HO_h-GCNP

2500 2000 1500 1000 500 0

2.42 + 0.3

78.4 + 0.9

0

358 + 21

5

10

15

20

25

30

35

Day Normal saline GCNP 100 mg/kg/over 2days, i.t.

C

Tumoral injection NP formulation

RGD 10 mg/kg/over 2 days, i.v.

cRGD

10000

RGD 10 mg/kg/over 2 days, i.t.

RGD loaded GC nanoparticle (RGD-h-GCNP) Angiogenic vessels at tumor tissue

Tumor volume (mm3)

9000

RGD-g-GCNP 10 mg/kg/over 2 days, i.t.

8000 7000 6000 5000 4000 3000 2000 1000 0 0

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2

4

6 8 Time (day)

10

12

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Tumor-targeting glycol chitosan nanoparticles as a platform delivery carrier in cancer diagnosis & therapy  

Review

Figure 6. Therapeutic behavior of chemo-drug loaded glycol chitosan nanoparticles (see facing page). (A) Near-infrared fluorescence images of tumor accumulation of DTX-GCNPs and their therapeutic behavior in A549 cancer-bearing mice. DTX-GCNPs showed tumor-targeting behavior, and tumor volume of mice was threetimes lower than nontreated mice after DTX-GCNP injection [30] . (B) Near-infrared fluorescence images of tumor accumulated Cy5.5 labeled PTX-HO-GCNPs, their loading efficiency and in vivo therapeutic results. PTX-HO-GCNPs exhibited tumor accumulated behavior and high loading amount of PTX (20% ecapsulation) due to hydrotropic properties. Therapeutic results showed significant efficacy compared with commercialized PTX formulations [47] . (C) Therapeutic mechanism of RGD-GCNPs and their therapeutic behavior. When RGD-GCNPs were directly injected in tumor site, released RGD bound to endotherial cells, and suppressed tumor growth [51] . DTX: Docetaxel; GCNP: Glycol chitosan nanoparticle; h-GCNP: Hydrophobically modified glycol chitosan nanoparticles; HO: Hydrotropic oligomer; NP: Nanoparticle; PTX: Paclitaxel; RGD: Arg-Gly-Asp. (A) Reproduced with permission from [30] ; (B) reproduced with permission from [47] ; (C) reproduced with permission from [51] .

carboxylic acid groups in DMA and amine groups in GC. At pH 5.0, DMA blocks were detached from GC backbones, leading to the dissociation of nanogels due to reduced electrostatic interactions. The disintegration of the nanogel at low pH resulted in the disassembly of close-packed C60 molecules, followed by recovery of photoresponsive properties. Taken together, GC grafted with hydrophobic photosensitizer could be considered as a valuable candidate for tumor-specific PDT agents.

Gene therapy

In the field of cancer therapy, both viral and nonviral vectors have been extensively studied as gene delivery vehicles. However, regarding the toxic and immunogenic properties of viral vectors, biocompatible nonviral vectors have been considered as favorable nucleic acid carriers [62] . In nonviral gene delivery, generally cationic materials are required to form a stable complex with nucleic acids. Despite quite efficient in

Table 3. Therapeutic applications of glycol chitosan nanoparticles in chemodrug, photosensitizer and nucleic acid delivery. Therapeutic method

Drug

Chemotherapy       Paclitaxel  

Polymer

Cancer/cell

5β cholanic-GC

B16F10 melanoma cells

Ref. [31]

HO-5β cholanic-GC MDA-MB231 human breast cancer cells

[47]

Decetaxel

5β cholanic-GC

A549 human lung cancers

[29]

Doxorubicin  

DOX/GC-DOX

II45 mesothelioma cells

[48]

N-acetyl histidineGC

HT29 tumor

[49]

5β cholanic-GC

MDA-MB231 human breast cancer

[30]

GC−o-Nitrobenzyl Succinate

MCF-7 human breast cancer cell

[50]

RGD-5β cholanicGC

B16F10 melanoma tumors

[51]

5β cholanic-GCCe6, GC-Ce6

HT-29 colon cancer

[52]

PpIX

PpIX-GC

HT-29 colon cancer

[53]

PheoA

PheoA-ss-GC

KB cells/HT-29 cells

[54]

Fullerene (C60)

GC-g-DMA-g-C60

Human nasopharyngeal epidermal carcinoma KB cells

[55]

Fullerene (C60)

Camptothecin

Small peptide drugs Photodynamic Ce6 therapy

GC-g-DMA-g-C60

KB cells

[56]

Gene therapy     DNA (Luciferase)

5β cholanic-GC

COS 1 cells

[57]

siRNA (RFP)

5β cholanic-GC 5β cholanic-PEI

RFP-B16F10 melanoma tumors

[58]

siRNA (VEGF)

Thiolated GC

PC-3 prostate cancer

[59]

Ce6: Chlorin e6; DOX: Doxorubicin; GC: Glycol chitosan; GC-g-DMA-g-C60: Glycol chitosan grafted with 2,3-dimethylmaleic acid and fullerene; PpIX: Protophorphyrin IX; PheoA: Pheophorbide A; RFP: Red fluorescent protein; RGD: Arg-Gly-Asp; VEGF: Vascular endothelial growth factor.

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Review  Lee, Min Ku et al. Figure 6. Therapeutic behavior of chemo-drug loaded glycol chitosan nanoparticles.(A) Near-infrared fluorescence images of tumor accumulation of DTX-GCNPs and their therapeutic behavior in A549 cancerA bearing mice. DTX-GCNPs showed tumor-targeting behavior, and tumor volume of mice was 3 times lower than Before nontreated mice after DTX-GCNP injection [30] . (B) Near-infrared fluorescence images of tumor accumulated irradiation 2 days 7exhibited days Cy5.5 labeled PTX-HO-GCNPs, their loading efficiency and in vivo therapeutic results. PTX-HO-GCNPs tumor accumulated behavior and high loading amount of PTX (20% ecapsulation) due to hydrotropic properties. Therapeutic results showed significant efficacy compared with commercialized PTX formulations [47] . (C) Ce6-h-GCNP Therapeutic mechanism of RGD-GCNPs and their therapeutic behavior. SalineWhen RGD-GCNPs were directly injected in (Ce6 loaded NP) tumor site, released RGD bound to endotherial cells, and suppressed tumor growth [51] . DTX: Docetaxel; GCNP: Glycol chitosan nanoparticle; h-GCNP: Hydrophobically modified glycol chitosan = GC nanoparticles; HO: Hydrotropic oligomer; NP: Nanoparticle; PTX: Paclitaxel; RGD: Arg-Gly-Asp. 5β-cholanicwith acid permission from [47] ; (C) reproduced with (A) reproduced with permission from [30] ; (B) =reproduced permission from [51] . Ce6-GCNP = Ce6 Free Ce6 (Ce6 conjugated NP) 1000

Saline Ce6 Ce6-GCNP Ce6-h-GCNP

Tumor size (mm3)

800

Ce6-h-GCNP

600 400 Ce6-GCNP

200 0

0

5

10 15 Time (day)

20

B

120

PheoA Aqueous media

Cancer cell

Cellular 1 2 uptake Reducible condition

hv PheoA-ss-GCNPs Switchable photoin self-quenched activity of PheoAstate ss-GCNPs

PheoA-ss-GC conjugate

0 day

3 day

100

Cell viability (%)

GC Quenched PheoA

80 60 40

GC PheoA PheoA-GCNPs PheoA-ss-GCNPs

20 0

2 4 6 8 10 Laser treatment time (min)

6 day

Tumor volume (mm3)

200 Saline Free PheoA PheoA-ss-GCNPs

150

#

100 *

50

0

2

4 6 Time (days)

8

Figure 7. Therapeutic effects of photosensitizer-conjugated glycol chitosan nanoparticles. (A) Schematic illustration of Ce6-loaded or Ce6-conjugated GCNPs and their therapeutic results. As a hydrophobic site, Ce6conjugated GCNPs showed significant tumor suppression after photodynamic therapy due to higher tumor accumulation [52] . (B) Bioreducible mechanism for photodynamic therapy using PheoA–GCNPs and their photoactivity in cells and tumor.

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Tumor-targeting glycol chitosan nanoparticles as a platform delivery carrier in cancer diagnosis & therapy  

Review

Figure 7. Therapeutic effects of photosensitizer-conjugated glycol chitosan nanoparticles (cont.). Disulfide linked PheoA showed photoactivity in normal state, but the photoactivity was restored after cellular uptake in cancer cell. Significant decreased tumor volumes were observed after photodynamic therapy [54] . Ce6: Chlorin e6; GC: Glycol chitosan; GCNP: Glycol chitosan nanoparticle; NP: Nanoparticle; PheoA: Pheophorbide A. (A) Reproduced with permission from [52] ; (B) reproduced with permission from [54] .

vitro and in vivo gene delivery, this strong cationic charge is one of the main drawbacks of the conventional cationic carriers, having issues with potential in vivo toxicity, which have not yet been resolved. Chitosan, a biocompatible polymer with slightly positive charge, has been suggested as a potential candidate for gene delivery. Many researchers have investigated the nanocomplexation of chitosan or its derivatives with nucleic acids [24,63–64] . However, the resulting nanocomplexes are less compact due to the relatively low charge density of chitosan. To develop more stable and compact nanocomplexes of chitosan and nucleic acids, Lee et al. used hydrophobically modified GC and hydrophobized DNA (Figure 8A) [57] . 5β-cholanic acid-conjugated chitosan self-assembled into stable nanoparticles by hydrophobic interactions between the 5β-cholanic acid in GC and hydrophobized DNA. The contents of hydrophobic GC influenced not only the physochemical properties of the resulting DNA/GCNPs, such as size, surface charge and DNA loading efficiency, but also on the intracellular translocation efficacy. The increased contents of the hydrophobic GC facilitated DNA loading into the nanoparticles and enhanced cellular uptake. Compared with naked DNA and a commercialized transfection agent, DNA/GCNPs had superior transfection efficiencies in vitro and in vivo. As potential therapeutic agents, siRNA has been of interest in cancer therapy. This small dsRNA, consisting of 21–25 nucleotides, is incorporated with the RNA-induced silencing complex, resulting in cleavage of the complementary target mRNA [65] . Despite its therapeutic potential in various diseases, many challenges still remain; in particular, enzyme-susceptibility of siRNA causes rapid degradation in physiological fluids, and its negative charge hinders cell membrane penetration. To achieve efficient siRNA delivery systems, the carriers for siRNA are enabled to protect siRNA molecules from nuclease attack during blood circulation, deliver specifically into the target tissue, and facilitate intracellular translocation [66] . GCNPs, having superior tumor-targeting properties, can be considered good candidates for siRNA delivery. However, the slightly positive charge in GC is not sufficient to form condensed and stable nanoparticles with siRNA via electrostatic interactions. To integrate siRNA in nanoparticles successfully, strongly positively-charged polymers, such as polyethylenimine (PEI), are required to add as an additional cationic reagent. Huh et al. developed a nano-sized siRNA carrier, consisting of

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hydrophobically modified GC and PEI (Figure 8B) [58] . Both GC and PEI were conjugated with 5β-cholanic acid, and the modified polymers self-assembled into compact nanoparticles via hydrophobic interaction of the pendant groups. Cationic PEI facilitated siRNA loading in the nanoparticles through charge–charge interactions. The resulting siRNA-PEI-GCNPs, which were approximately 250 nm in diameter and had slightly positive surface charge of 9.95 mV, protected siRNA against nuclease attack for up to 6 h, whereas naked siRNA was degraded within 30 min in the presence of serum. The in vitro gene silencing efficacy of siRNA-PEI-GCNPs was comparable to the commercialized transfection agent, Lipofectamine2000 (Life Technologies™, CA, USA). siRNA-PEI-GCNPs exhibited negligible cytotoxicity, compared with cytotoxic PEI complexes. Further, siRNA-PEI-GCNPs exhibited the excellent tumor-targeting properties, and abolished the expression of the target gene (red fluorescent protein) in RFP/B16F10 tumor-bearing mice, after systemic delivery of the nanoparticles. It is generally known that siRNA is hard to form into condensed nanoparticles with cationic lipids or polymers due to its relatively low charge density and short and rigid structure [67] . Furthermore, the use of strong cationic polymers for nanocomplex formation with siRNA may induce undesired in vivo toxicity. To overcome these weaknesses, poly-siRNA with higher molecular weight and strong negative charges was synthesized through disulfide bond formation between thiol groups at the 5´ end of each strand [68] . Lee et al. developed poly-siRNA delivery system using thiolated GC (tGC) (Figure 8C) [59] . Poly-siRNA and tGC were loosely bound to each other via weak electrostatic interactions, and thiol groups in tGC induced self-crosslinking of polymers, resulting in the formation of more condensed poly-siRNA/tGC nanoparticles (psi-GCNPs). The stable psi-GCNPs successfully protected siRNA from physiological anionic proteins/carbohydrates or nucleases. Since crosslinking in psi-GCNPs occurrs via cleavable disulfide bonds, intact siRNA could be released from the nanoparticles in cytosolic reductive environments, followed by participating in the RNA interference mechanism. As a result, psi-GCNPs exhibited high gene silencing efficiency in vitro, similar to that of siRNA/Lipofectamine complexes. To investigate tumor-targeting ability, in vivo biodistribution of psi-GCNPs was observed after intravenous injection; psi-GCNPs were specifically accumulated in tumor

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Review  Lee, Min Ku et al. sites, whereas most psi-PEI complexes were found in the liver. When psi-GCNPs containing siRNA targeted against VEGF were systemically delivered, tumor growth and microvessel formation were remarkably suppressed in PC3 tumor-bearing mice. Considering the negligible immune responses, psi–GCNPs have great potential for systemic delivery of therapeutic siRNA, particularly in treating cancers. Conclusion & future perspective Over the last decade, nanoparticles have attracted much attention as a smart delivery system in cancer therapy, because they can be accumulated in tumor tissue via EPR effects and have potentials for controlled drug release. Among various nanomaterials, GCNPs have been considered as a promising delivery vehicle for cancer therapy due to their superior properties, such as biocompatibility, high stability in physiological fluids and tumor-targeting ability.

According to previous reports, GCNPs can incorporate: contrast agents for optical imaging, MR imaging, CT, PET, or US imaging; and anticancer drugs (e.g., chemodrugs, photosensitizers and therapeutic genes) through physical loading or chemical conjugation. Accordingly, the targeted delivery of imaging agents and anticancer drugs to tumor site can be achieved by using GCNPs, with the reduced side effects. Recently, combination therapy has received much attention as a potentially effective cancer treatment, since there are numerous factors that are associated with cancer. As described above, GCNPs have been used as a delivery carrier for chemical drugs and therapeutic genes; this property enables the combinatorial treatment of both therapeutic agents. Among various characteristics of cancer, multidrug resistance is an important issue in the failure of many forms of chemotherapy. Blockade of the expression of multidrug

A

+

DNA

GC-CA

B

+

+

+

+

+

+

PEI-CA

GC-CA

siRNA

C

+

Thiolated GC

Poly-siRNA (psi)

Figure 8. Glycol chitosan nanoparticle-based gene delivery systems. (A) 5β-cholanic acid-modified glycol chitosan nanoparticles for DNA delivery. (B) 5β-cholanic acid-modified glycol chitosan and PEI nanocarriers for siRNA. (C) Thiol residue-modified glycol chitosan nanoparticles for poly-siRNA. CA: 5β-cholanic acid; GC: Glycol chitosan; PEI: Polyethylenimine.

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Tumor-targeting glycol chitosan nanoparticles as a platform delivery carrier in cancer diagnosis & therapy  

resistance protein such as p-glycoprotein (Pgp) is considered as an effective strategy to overcome multidrug resistance, and the treatment of siRNA targeting Pgp remarkably reduce the Pgp expression and drug resistance. GCNPs have a superior potential for the codelivery or sequential delivery of chemical drugs and siRNA targeting Pgp, leading to the enhancement of anticancer efficacy. In addition, GCNPs incorporating both imaging contrast agents and therapeutic agents enable the simultaneous diagnosis and treatment of cancer. Real-time monitoring of the distribution of therapeutic agents allows rapid feedback and optimization of the regimen for individual patients. Taken together, biomedical applications of GCNPs

Review

are expected to greatly contribute to cancer diagnosis and therapy. Financial & competing interests disclosure This study was funded by GiRC and GRL program (2012K1A1A2A01056095, 2013K1A1A2A02050115) through the National Research Foundation of Korea (NRF) and the Intramural Research Program of KIST. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary Development of glycol chitosan nanoparticles (GCNPs) • Hydrophobically modified glycol chitosan (GC) is self-assembled into stable nanoparticles via hydrophobic interactions between pendant groups. • Molecular weight and degree of substitution are critical factors determining stability in physiological conditions and the deformability of GCNPs. The best tumor-targeting ability of GCNPs is obtained when GCs with a molecular weight of 250 kDa and 23% 5β-cholanic acid are used.

GCNPs for cancer therapy • GCNPs integrate diverse imaging contrast agents for optical imaging, MRI, computed tomography, positron emission tomography, ultrasound imaging, and the resulting nanoparticles successfully visualize the tumor site. • Chemical or peptide anticancer drugs are loaded in GCNPs and selectively delivered to tumor tissue, resulting in reduced side effects. • Photosensitizer-conjugated GCNPs, have cellular on/off photoactivity, which can be used in photodynamic cancer therapy. • The cationic properties of GCs allow formation of nanocomplexes with nucleic acids. Further, chemically modified GC is useful to develop more stable and condensed nanoparticles for gene delivery.

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