A Targeting Self-breakable Agent for Increased Efficacy of

Chemotherapeutic Drugs against Caco2 Cells

Ming-Hsien Tsai

, Ming-Jium Shieh

and Cheng-Liang Peng

Institute of Biomedical Engineering, National Taiwan University College of Medicine, National Taiwan University,

No1, Sec. 1, Jen - Ai Rd., Taipei, Taiwan

Isotope Application Division, Institute of Nuclear Energy Research, No.1000, Wenhua Rd., Longtan Dist., Taiwan

Keywords: SN38, Micelle, Caco2.

Abstract: Many types of nano-sized anti-cancer agents that could increase efficacy of chemotherapeutic drugs have

been created and developed in colon cancer treatment over years. Moreover, with the intention of achieving

the ideal chemotherapeutic efficacy, nano-sized anti-cancer agents were further designed to have specific

functions, efficiently killing colon cancer cells. Our research team focused on two important functions in

designing nano-sized agents, controlled drug release and targeting functions. Thus, targeting functional

micelles which entrapped chemotherapeutic drug, 7-ethyl-10-hydroxy-camptothecin (SN38) were designed

in nano-size and possessed disulfide bonds in this study. In particular, Self-Breakable SN38-loaded micelles

(SN/38 micelles), Non-Breakable micelles SN38-loaded (NB/38 micelles) and Folate-targeting Self-

Breakable SN38-loaded micelles (FSB/38 micelles) were prepared and tested to the designed agents. The

results showed that the folate-decorated functional micelles with disulfide bonds could be an effective

chemotherapeutic agent for colon cancer treatment.

1 INTRODUCTION

Chemotherapy is the most common therapy for

colorectal cancer, which is an intractable issue for

human beings due to its increased chance of death so

cancer studies are still ongoing for developing high-

efficacy chemotherapeutic drugs(Bala et al., 2013).

A good candidate for drug agents is micelle

composed of amphiphilic polymers with nanoscale

size allowing accumulation of the micelles in the

tumor through enhanced permeability and retention

(EPR) effect, thereby resulting in high tumor

uptake(Joralemon et al., 2010). However, the

efficacy of chemotherapeutic drug-loaded micelles

without specific functions is unsatisfactory.

Recently, the designed micelles with specific

functions, for instance, targeting function(Xu et al.,

2013), photodynamic function(Peng et al., 2008),

and controlled release function(Peng et al., 2010,

Peng et al., 2011b) have been created. Such

functional micelles were shown to have greater

effectiveness in cancer treatments(Sinn Aw et al.,

2014).

Folate which has exhibited outstanding ability in

increasing cellular uptake of the loaded drug was

chosen as the targeting ligands in this study (Khatik

et al., 2015, Cuong et al., 2012). In addition, unlike

many other targeting ligands used in

chemotherapeutic drugs, folate, is safe for human

consumption, and has approved by the US Food and

Drug Administration for the use in dietary

supplements. Based on these reasons, folate is a

good choice for a targeting ligand which can be used

to modify micelles, which can efficiently increase

the cellular uptake and efficacy of the

chemotherapeutic drug.

However, even if the folate-decorated functional

micelles increase cellular or tumor uptake of

functional micelles, the entrapped drug might fail to

release owing to its rigid structure, which results in

its lower efficacy(Xing et al., 2015). The folate-

decorated functional micelles in this study were

further designed for successful drug release which

plays an important role in achieving optimal efficacy

of chemotherapeutic drugs. This is attributed to the

fact that drug must reach drug action site in tumor

cell for drug action to take place(Kawato et al.,

1991). Great efforts have been made to create

controlled release micelles which are self-breakable,

particularly redox-responsive functional micelles,

216

Tsai, M-H., Shieh, M-J. and Peng, C-L.

A Targeting Self-breakable Agent for Increased Efﬁcacy of Chemotherapeutic Drugs against Caco2 Cells.

DOI: 10.5220/0005766002160221

In Proceedings of the 9th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2016) - Volume 1: BIODEVICES, pages 216-221

ISBN: 978-989-758-170-0

for the enhancement of the drug efficacy. Disulfide

bonds created in such functional micelles quickly

react with glutathione (GSH) which renders the

micelles unstable, thereby enabling them to release

the drug spontaneously (Huo et al., 2014, Lai et al.,

2014). Therefore, the folate-decorated functional

micelles created in this study were designed to be

redox-responsive.

We attempted to enhance efficacy of the

chemotherapeutic drug, 7-Ethyl-10-hydroxy-

camptothecin (SN38), an active metabolite of the

clinical drug, irinotecan (CPT-11) which is used in

the treatment of colorectal cancer. For this purpose,

Folate-targeting Self-Breakable micelles (FSB

micelles) consisting of self-degradable copolymers,

and targeting copolymer, were created to setup an

active drug delivery system using a colorectal cancer

cell line, Caco2. FSB micelles could facilitate Caco2

in acidic microenvironment to take up the loaded

SN38, resulting in enhanced drug efficacy. In

addition, to confirm that FSB micelle can be the best

anti-cancer agent, Self-Breakable micelles (SB

micelles) without targeting function and Non-

Breakable micelles (NB micelles) which have no

specific function were also created to evaluate the

designed functions of FSB micelles.

2 EXPERIMENTAL SECTION

2.1 Synthesis of the Self-degradable,

Non-self-degradable Copolymers

and Targeting Copolymers

The non-self-degradable (ND) copolymers,

methoxyPolyEthylene Glycol-PolyCaproLactone,

(mPEG-PCL) was synthesized as described in our

earlier research(Chen et al., 2015). The self-

degradable (SD) copolymers, methoxyPolyEthylene

Glycol-S-S-PolyCaproLactone (mPEG-S-S-PCL)

was primarily obtained via two chemical reactions.

MPEG-SH was reacted with excess 2-

mercaptoethanol in deionized water to obtain

mPEG-S-S-C

OH. Then, MPEG-S-S-PCL was

obtained via the ring-opening polymerization.

To prepare the targeting copolymer, Folate-

Poly(Ethylene Glycol)-Poly(CaproLactone) (F-PEG-

PCL), FMOC-NH-PEG-PCL was used to synthesize

as described (Peng et al., 2011a). F-PEG-PCL was

obtained by conjugating the de-protected polymer,

-PEG-PCL, with folate via an amide bond. The

designed copolymers were characterized by

HNMR, FT-IR, and Gel Permeation

Chromatography (GPC) was used to determine the

molecular weight (MW) of copolymers.

2.2 Characteristics of Self-breakable,

Non-self-breakable, and Targeting

Self-breakable Micelles

The NB micelles prepared from ND copolymers and

SB micelles prepared from SD copolymers in this

study were prepared to evaluate the function of SB

micelles in triggering the release of loaded drug in

the presence of GSH in cancer cells. The FSB

micelles prepared using a mixture containing 80%

(w/w) SD and 20% (w/w) targeting copolymers were

designed to have targeting and self-breakable

function for achieving the best chemotherapeutic

efficacy in cancer treatment.

SB micelles, NB/38 micelles, SB/38 micelles,

and FSB/38 micelles were prepared using a

lyophilization-hydration method. The SN38-loaded

micelle formulations containing 10mg/mL of

polymer and 1mg/mL of SN38 in PBS were filtered

using 0.22µm filter to remove non-loaded SN38.

Then, the size of micelles was determined by

Transmission Electron Microscopy (TEM), and

Dynamic Light Scattering (DLS). Loading

Efficiency (LE) and Drug Content (DC) were

determined using the calibration curve based on

maximum absorption values of SN38 in DMSO.

Critical Micelle Concentration (CMC) of the

micelles was determined using pyrene as described

elsewhere.

2.3 Physical and Chemical Stability of

the Micelles

To access whether SB/38 micelles, and NB/38

micelles were self-breakable agents or not, the

micelles were incubated with or without 10mM DTT

in phosphate buffered saline (PBS) which was used

to simulate glutathione (GSH) in cells. Incubation

with or without DTT was conducted at 37°C over

specific time periods. Then, the micelle stability was

determined by their size and polydispersity (PdI). At

select time points, the size and PdI of the micelles

were determined by DLS.

To prove that disulfide bonds designed in the SD

copolymers could be broken up by GSH in the cells,

the MW of copolymers self-assembling into SB

micelles were analysed via GPC after the incubation

with 10mM DTT. In brief, SB/38 micelles were

lyophilized after incubation at 37°C for 24 h. The

lyophilized SB/38 micelles were dissolved in THF,

A Targeting Self-breakable Agent for Increased Efﬁcacy of Chemotherapeutic Drugs against Caco2 Cells

217

and then the MW of the polymers of SB/38 micelles

was determined via GPC.

2.4 Drug Release Profile

In vitro SN38 release profiles of SB/38 and NB/38

micelles, dispersed in PBS with or without DTT,

were analysed using a modified dialysis-bag

diffusion technique at 37°C. The dialysis tube

containing 0.4 mL of the micelle formulation was

suspended in 100 ml PBS in a closed bottle. A

magnetic mixer was introduced into the bottle and

incubated at 37°C. Every 1ml of aliquot was

withdrawn from the external media and refilled with

1ml of fresh PBS at select time intervals. The SN38

concentration was determined by fluorescence

intensity at 427nm (excitation at 390nm). All

experiments were conducted in triplicate.

2.5 In Vitro Cytotoxicity

The human colon cancer cell line, Caco2, was

cultured in a humidified 5% CO

incubator at 37 °C

in Minimum Essential Media, MEM (GIBCO BRL,

Gaithersburg, MD, USA) supplemented with 20%

heat-activated fetal bovine serum (FBS), 1% non-

essential amino acids, 2 mM L-glutamine, 1 mM

sodium pyruvate, 1500 mg/L sodium bicarbonate,

and 1% (v/v) Penicillin-Streptomycin Amphotericin

B Solution (GIBCOBRL). Initially, the Caco2 cells

were seeded onto 96-well plates at a density of

10,000 cells per well and cultured. After 24 h, cells

were incubated in media containing different

concentrations of SN38 for 6h. Then, the cells were

washed three times with PBS to remove the

suspended SN38 and cultured with fresh medium for

another 48 h. Cell viability was assessed using MTT

assay with a scanning multi-well ELISA reader

(Microplate Autoreader EL311, Bio-Tek Instruments

Inc., Winooski, VT, USA). The cytotoxicities of SB

micelles, NB micelles, FSB/38 micelles, SB/38

micelles and NB/38 micelles were also evaluated by

the same method.

Scheme 1: Structure of a targeting self-breakable drug-

loaded micelle.

3 RESULTS AND DISCUSSION

3.1 Synthesis of the Self-degradable,

Non-self-degradable Copolymers

and Targeting Copolymer

The FSB micelles composed of SD copolymers and

targeting copolymers were used as a targeting self-

breakable agent for the enhancement of drug

efficacy and were loaded with the chemotherapeutic

drug, SN38, used to treat colon cancer in this study

(scheme 1). The

H-NMR results revealed that SD

copolymer was successfully synthesized and had a

MW of 8,530 g/mol (data not shown). GPC analysis

indicated SD copolymer had a molecular weight,

11,253 g/mol, and a narrow PolyDispersity (PD) of

1.15 (Table 1). The FT-IR spectra showed the

linkage of NH

PEG-PCL with folate via an amide

bond which indicated the successfully synthesis of

F-PEG-PCL (Figure 1).

3.2 Characteristics of Self-breakable,

Non-self-breakable, and Targeting

Self-breakable Micelles

The characteristics of the SB/38, NB/38 and FSB/38

are shown in Table 2. In terms of the size, the size of

NB/38 micelles, NB/38 micelles, and FSB/38

micelles were determined to be all about 130nm at

10:1 ratio of polymer/drug in PBS. The TEM images

further supported this finding as the results show

that the actual sizes FSB/38 micelles used as a

targeting self-breakable agent were the same as that

determined via DLS (Figure 3). Comparison of the

in vitro and in vivo test results involving the use of

each of these three SN38-loaded micelles ruled out

the possible issues associated with the difference in

size, perhaps due to their similarity in size. In

addition, owing to their uniform nano-size, these

SN38-loaded functional micelles could successfully

accumulate in the tumor via the EPR effect and thus

the enhance efficacy of drug, SN38.

Figure 1: FT-IR spectra of folate-PEG-PCL and folate.

mPEG-SS-PCL

F-PEG-PCL

SN38

Wavenumber(cm

-1

)

800 1000 1200 1400 1600 1800 2000 2200

Absorbance

Folate

F-PEG-PCL

1550

1651

1605

1740

1114

BIODEVICES 2016 - 9th International Conference on Biomedical Electronics and Devices

218

Table 1: Molecular characteristics of mPEG-S-S-PCL,

mPEG-5,000, and SB/38 micelles incubated with DTT

for 24 h.

Time (min)

16 18 20 22 24 26

mPEG-S-S-PCL

mPEG-5000

mPEG-S-S-PCL + DTT

Figure 2: GPC elugram of mPEG-S-S-PCL, mPEG-5,000,

and SB/38 micelles incubated with DTT for 24 h.

3.3 Physical and Chemical Stability of

the Micelles

To prove that SB micelles can be a self-breakable

agent, the SB/38 micelles, and NB/38 micelles were

incubated with DTT which was used to simulate

GSH with thiol groups in cells. SB/38 micelles with

DTT became larger than those without DTT over

time (Figure 4A). The PdI data shown in Figure 4B

indicated that SB/38 micelles with DTT had a wide

range of particle distribution (PdI : over 0.3) 3h after

the start of the test. As expected, these results were

due to the thiol groups in DTT reacting with

disulfide bonds in SB/38 micelles, causing SB/38

micelles to be relatively unstable and aggregate. In

contrast, the presence of DTT did not affect the

stabilities of NB/38 micelles during the course of the

whole experiment. Compared with SB/38 micelles,

NB/38 micelles remained stable.

Figure 3: TEM image of FSB/38 micelle.

Figure 4: Stability of SB/38 micelles or NB/38

micelles incubated with or without DTT for 24h was

determined by DLS in terms of size (A) and PdI (B).

Moreover, the GPC analysis (Table 1 and Figure

2) was performed to confirm that SB micelles

composed of mPEG-S-S-PCL could be a self-

breakable agent indicated that the disulfide bonds

designed in SB copolymers were broken up by DTT

after incubation of the SB/38 micelles with DTT,

resulting in a significant decrease in molecular

weight.

3.4 Drug Release Profile

Drug release profiles of SB/38 and NB/38 micelles

with or without DTT were conducted to determine

the micelle’s ability to release drug. As shown in

Figure 5, only SB/38 micelles successfully released

SN38 with DTT over time. DTT reacted with the

disulfide bonds in SB/38 micelles, which resulted in

a significant drug release. In contrast, the other

micelles released little amount of SN38 with or

without DTT (i.e., < 5% of SN38 released) over 96h.

This implies NB/38 micelles were relatively stable

regardless of the presence or absence of DTT. These

results are in accord with those obtained via DLS

and GPC informed us that unstable SB/38 micelles

will release drug. This proved again the efficacy of

SB/38 micelles to be used a potent drug for colon

cancer treatment.

Table 2: Characteristics of NB/38 micelle, SB/38

micelle, and FSB/38 micelle.

3.5 In Vitro Cytotoxicity

To evaluate the cytotoxicity of the free drug, the

designed nano-sized agents, free SN38, SB micelles,

NB micelles, SB/38 micelles, NB/38 micelles and

FSB/38 micelles were incubated with Caco2 cell

Copolymer/micelle Mn Mw Mp P.D.

mPEG-S-S-PCL 9,742 11,253 13,699 1.15

mPEG-5000 6,679 7,015 7,606 1.05

SB/38 micelles

(mPEG-S-S-PCL)

+10mM DTT

7,115 7,792 7,569 1.08

Time (h)

0 5 10 15 20 25 30

Size (nm)

1000

2000

3000

4000

5000

6000

7000

NB/38 micelle

NB/SN micelle + DTT 10mM

SB/SN micelle

SB/SN micelle + DTT 10mM

Time (h)

0 5 10 15 20 25 30

PdI

0.0

0.2

0.4

0.6

0.8

1.0

1.2

NB/38 micelle

NB/38 micelle + DTT 10mM

SB/38 micelle

SB/38 micelle + DTT 10mM

Micelle Size (nm)

PdI

LE(%)

DC(%)

CMC

(wt%)

NB/38

130.0±2.1 0.14±0.03 94±4.3 8.6±0.21

0.0021

SB/38

132.4±1.2 0.10±0.04 93±4.1 8.5±0.13

0.0025

FSB/38

131.5±2.3 0.13±0.02 92±3.9 8.4±0.24

0.0023

A Targeting Self-breakable Agent for Increased Efﬁcacy of Chemotherapeutic Drugs against Caco2 Cells

219

under conditions mimicking in vivo tumor

environment at a low pH(Vaupel et al., 1989,

Estrella et al., 2013). No toxicity was observed over

24 h in NB micelles and SB micelles (data not

shown), which confirmed that SB micelles could be

nontoxic owing to their biocompatibility. Free SN38

achieved the highest efficiency in killing cancer cells,

which was expected in this study, as it is known to

be the most toxic in vitro in cellular experiments

(Figure 6B). However, it is not clinically used.

Among the designed anti-cancer drugs without a

targeting function, the toxicity of SB/38 micelles

was significantly higher than that of NB/38 micelles

which was due to their successful self-controlled

drug release. Regarding to FSB/38 micelles, it was

found that they were able to achieve the highest

level of effectiveness in killing cancer cells among

the anti-cancer drugs studied. This can be attributed

to the fact that FSB/38 micelles had decorated-folate

on their surface which caused the Caco2 cells to take

up more FSB/38 micelles, resulting in the much

higher efficacy of SN38. In addition, to evaluate the

effect of medium pH on the cytotoxicity of FSB/38

micelles, a comparison of the cytotoxicity of FSB/38

micelles at a medium pH of 7.4 with that of FSB/38

micelles at a medium pH of 6.7 or 6 was conducted.

The highest cytotoxicity of FSB/38 micelles was

observed at pH 6 (Figure 6A). This could be

Time

(

)

0 20 40 60 80 100 120

Drug release (%)

-10

NB/38 micelle

NB/38 micelle + DTT 10mM

SB/38 micelle

SB/38 micelle + DTT 10mM

Figure 5: SN38 release profile.

Figure 6: Cell viability of FSB/38 micelles was evaluated

in media with different pH values, pH = 6, 6.7, and 7.4 (A).

Cell viability of SN38, NB/38 micelles, SB/38 micelles,

and FSB/38 micelles (B).

attributed to the fact that Caco2 cells quickly took up

folate in the medium at low pH, resulting in more

uptake of the folate-decorated FSB/38 micelles.

These results show that FSB/38 micelles could be an

effective drug for colon cancer treatment.

4 CONCLUSIONS

In this study, the nano-micelles were designed to be

self-breakable micelles, called SB/38 micelles. The

intention of creating SB/38 micelles was to improve

the drug release and enhance drug efficacy. The

results of DLS and GPC prove that SB/38 micelles

disassembled with DTT which was used to simulate

GSH with thiol groups in the cells, resulting in drug

release. Furthermore, the release profiles showed

that not only SB/38 micelles successfully released

SN38, but also a great amount of SN38 was released

with DTT. To effectively kill cancer cells and thus

ensure better results of cancer treatments, a targeting

smart anti-cancer agent, FSB/38 micelles, which

consisted of 80% SD and 20% targeting copolymers

were successfully designed and produced. It was

then confirmed that FSB/38 micelles are an ideal

anti-cancer drug through cytotoxicity experiments

and cellular uptake experiments. The in vitro

cytotoxicity results showed that FSB/38 micelles

achieved the best effectiveness in killing colon

Caco2 cells among the designed anti-cancer drugs.

Hence, these findings confirm the efficacy of

FSB/38 micelles as an effective chemotherapeutic

drug for colon cancer treatment.

ACKNOWLEDGEMENTS

This study was supported by the National Science

Council of the Republic of China (NSC 102–2320-

B-002–038-MY3). We appreciate the help from

National Taiwan University College of Medicine.

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