The Investigation of P7 Peptide Delivery Targeting Cdc24 in

Ras-driven Pancreatic Cancer by L-fucose-bound Liposome

Yurui Yang

1,+

, Hongbin Lan

2,+

, Huawei Zhu

3,*

and Yiran Zhu

Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario M5S 2E8, Canada

School of Pharmacy, University College London, London, WC1E 6BT, U.K.

College of life science, South China Agricultural University, Guangzhou, Guangdong, 510642, China

RDFZ Xishan High School, Beijing, 100193, China

These authors should be regarded as co-first authors because they contributed to the work equally

Keywords: Cell Division Control Protein 42, Ras-driven Pancreatic Cancer, Liposome, Reduced Cytotoxicity.

Abstract: Pancreatic adenocarcinoma is one of the most common leading causes of cancer deaths with an increasing

incidence in the developed world. Most pancreatic cancers are induced by Ras protein abnormality. Since Ras

is involved in many important cellular functions, targeting Ras alone is difficult with little progression.

Currently, the only surgical resection offers the potential cure to this disease. However, among those who

have the chance to receive surgery, most of them suffer from recurrence within a year. The previous study

has developed novel peptides targeting Cell division control protein 42 (Cdc42), which is a type of Rho family

small GTPase activated by Ras and achieved great success in inhibition of tumor cell growth. Since Cdc42 is

also expressed in normal cells, a suitable drug delivery system is required for targeted therapy. This study

investigates the cytotoxic and cell penetration effects of L-fucose-bound liposome-P7 targeting pancreatic

cancer cells, in both in vitro and in vivo conditions. The experiments will use know human pancreatic cancer

cell lines, human pancreatic epithelial cell lines, and Xenograft Murine Models. The concentration of P7 in

each L-fucose-bound liposome will be assessed through half-log dilution. Cell proliferation and cytotoxicity

are measured through colony formation assay, MTT assay, and Annexin V/ propidium iodide (PI) assay. The

cell penetration effect will be reflected by fluorescence microscopy. There are three most possible results: (1)

L-fucose-bound liposome-P7 inhibits the pancreatic cancer cell proliferation in both in vitro and in vivo cell

lines without causing significant cytotoxicity to normal cells; (2) L-fucose-bound liposome-P7 only inhibits

the cancer cell proliferation in vitro cell cultures without causing significant damage to normal cells; (3) L-

fucose-bound liposome-P7 inhibits tumor cell proliferation in both normal pancreatic epithelial cells and

cancer cells. The result of our study will provide important information for deciding whether to continue P7

peptide development in clinical trials. Future studies should focus on improving the drug delivery system and

investigating P7 effects on transformed tumor cells.

1 INTRODUCTION

Pancreatic adenocarcinoma, ranked as the fourth

leading cause of cancer deaths in the United States,

has poor outcomes and an increasing incidence in the

developed world (McGuigan, et al, 2018, Patra, et al,

2010). The incidence rates vary between countries.

Generally, the highest incidence rates are detected in

Europe and North America, while the lowest

incidence rates are measured in Africa and South-

central Asia (McGuigan, et al, 2018). This type of

cancer often has a late detection and ineffective

treatment at the advanced stage, which contributes to

a poor 5-year survival rate of 2-9% (McGuigan, et al,

2018). Nowadays, even with advancements in

detection and management, the 5-year survival rate

remains relatively unchanged with negligible

improvements. Currently, the only treatment that

offers a potential cure for pancreatic cancer is

surgical resection. However, due to the late detection

of the disease, only 20% of patients have the chance

to receive surgery, and most of them may suffer from

disease recurrence within a year (Zeng, et al, 2019).

The addition of adjuvant chemotherapy has been

shown to improve long-term prognosis in some

patients, while other patients have developed chemo-

Yang, Y., Lan, H., Zhu, H. and Zhu, Y.

The Investigation of P7 Peptide Delivery Targeting Cdc24 in Ras-driven Pancreatic Cancer by L-fucose-bound Liposome.

DOI: 10.5220/0011251700003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 651-660

ISBN: 978-989-758-595-1

651

resistance with unclear mechanisms, which

significantly limits the effect of these therapeutic

drugs. Therefore, novel drug developments are

necessary (Zeng, et al, 2019).

About 95% of Pancreatic cancer are driven by the

mutations in Ras family genes (McGuigan, et al,

2018, Patra, et al, 2010). However, targeting Ras gene

alone is a hard task since it is involved in many

essential signaling pathways promoting cell

proliferation and differentiation (Arias-Romero,

Chernoff, 2013). Alternatively, targeting the

downstream small GTPase that is activated by Ras is

favorable. Cdc24 is an important downstream

effector of Ras and plays an important role in Ras-

induced transformation, and previous studies have

developed a novel cyclic peptide, P7, targeting Cdc24

with nanomolar affinity. P7 is tested for binding to

the binding surface of Cdc24, preventing it from

interacting with its downstream effectors and thus

inhibits the transformation pathway in Ras-induced

tumorigenesis (Tetley, Murphy, Bonetto et al, 2020).

Although the study showed a promising effect of P7

peptide as a Cdc24 inhibitor in Ras-driven cancers,

the peptide entry strategy has become another

challenge. Cell-penetrating peptide (CPP) was tagged

to the P7 and helped the delivery in the previous

study. However, the results showed a significant

cytotoxic effect with reduced target engagement

(Tetley, Murphy, Bonetto et al, 2020). Therefore, an

alternative drug delivery system with minimal

cytotoxicity and greater efficacy is necessary to be

developed.

Nanoscale drug delivery system using liposomes

is an emerging technology in cancer treatment.

Liposomes are composed of a lipid bilayer that is

enclosed as a hollow sphere with an aqueous phase

inside. Accordingly, it can encapsulate and stabilize

drugs in either aqueous compartments or lipid

bilayers, depending on the properties of drugs. Also,

the similarity of liposomes to the biological

membrane reduces their toxicity and enables the

enhanced permeability and retention (EPR) effects to

tumor tissues (Malam, Loizidou, Seifalian, 2009).

Specifically, most solid tumors have the nature of

vascular abnormalities, like hypervascularization,

aberrant vascular architecture, and a lack of

lymphatic drainage (Malam, Loizidou, Seifalian,

2009). Taking advantage of the adjustability of

nanoparticles' size, nanoscale anticancer drugs

designed ideally in a moderate size are unable to

penetrate through tight endothelial junctions of

normal blood vessels. However, they can selectively

extravasate in the tumor tissue relying on the tumor's

abnormal vascular characteristics, thereby reaching

several fold drug concentrations in the tumor than

that in the normal tissue (Malam, Loizidou, Seifalian,

2009). Therefore, liposome is a good candidate for P7

peptide delivery.

The surface of liposomes can be modified by

taking advantage of characteristics of pancreatic

adenocarcinoma. Since 80% of pancreatic cancer

cells overexpress carbohydrate antigen-19-9 (CA19-

9) and thus recruit large amounts of fucose as an

energy source, the fucose-bound liposome can be

generated for targeted delivery (Papahadjopoulos,

Heath, Bragman, Matthay, 1985, Yoshida, Takimoto,

Murase, et al, 2012). A previous study has applied a

14C-labeled L-fucose binding assay in the pancreatic

cell lines, and the result indicated the presence of

high-affinity L-fucose specific receptors (Yoshida,

Takimoto, Murase, et al, 2012). Furthermore, the

inhibition of endocytosis by chroloquine resulted in a

suppression of drug delivery (Yoshida, Takimoto,

Murase, et al, 2012). These results together supported

that L-fucose-bound liposome enters the pancreatic

cancer cells via receptor-mediated endocytosis

(Yoshida, Takimoto, Murase, et al, 2012).

In order to investigate the drug delivery system

with minimal side effects and better efficacy, a

comparative study should be designed. In the present

study, we asked whether targeting the delivery of P7

with L-fucose-bound liposome can increase the cell

penetration, enhance cytotoxicity to tumor cells and

reduce cytotoxicity to normal cells compared with

CPP-tagged P7 both in in vitro and in vivo conditions.

We chose BxPC-3 and AsPC-1 pancreatic cancer cell

lines, which secreted substantial amounts of CA-19-

9 molecules (Yoshida, Takimoto, Murase, et al,

2012). We hypothesized that treatment of P7 peptide

delivered in L-fucose liposomes to Ras-driven

pancreatic cancer can increase cell penetration,

peptide stability and reduce cytotoxicity to normal

cells. In the present study, we will treat AsPc-1,

BxPC-3, and normal pancreatic epithelial cells

(hTRET-HPNE) in culture or as a mouse xenograft

model with increasing amounts of P7-liposome

assessed by half log dilution series with various

liposome and/or P7 peptide concentrations and

measure cytotoxicity by cell counts, colony formation

assay, MTT assay, Annexin V/PI, and cell

penetration by fluorescently labeled liposomes.

2 METHOD & MATERIAL

This experiment will use two human pancreatic

cancer cell lines (AsPC-1 and BxPC-3), and one well

studied non-cancerous pancreatic cell line (hTRET-

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

652

HPNE) for in vitro studies and mouse xenograft

models for in vivo studies. Mouse xenograft models

include new subcutaneous and orthotopic models

which are generated from previous research and will

be used for the in vivo study (Yoshida, Takimoto,

Murase, et al, 2012). The subcutaneous model will

establish with mice bearing AsPC-1 cell lines, which

the orthotopic model will use mice bearing BxPC-3

cell lines. The mice will be housed under specific

pathogen-free conditions. Animal studies will be

carried out according to the Guide for the Care and

Use of Laboratory Animals of the National Institutes

of Health. All surgery will be performed under

sodium pentobarbital anesthesia, and all efforts will

be made to minimize suffering. P7 peptide and L-

fucose bound liposomes will be made according to

the method used by Tetley et al (Tetley, Murphy,

Bonetto et al, 2020). and Yoshida et al (Yoshida,

Takimoto, Murase, et al, 2012).

2.1 Material

PBS (pH 7.4), Purified P7 peptide, L-fucose-bound

liposome-P7, Crystal violet 0.5% (wt/vol) in H2O,

Glutaraldehyde 6.0% (vol/vol), Trypsin-EDTA

(0.05%), Appropriate culture medium containing

serum, RPMI 1640 (Gibco) plus 10% FBS, L-

glutamine, 1% penicillin-streptomycin, and binding

buffer 10X: 0.1 M HEPES/NaOH, pH 7.4; 1.4 M

NaCl; 2.5 nM CaCl, Class 2B biocabinet, 500 mg

MTT powder, methanol, ethanol, DMS and

acidified isopropanol, Plateshaker, Pipettes 0.001–1

mL, single channel and 0.01–0.3, multichannel, Class

2B hood, Benchtop centrifuge, Microplate reader, O2

incubator, L-fucose-bound liposome-P7, L-fucose-

bound liposome without P7, liposome alone, CPP-P7,

P7 alone and PBS.

2.2 Method

a) Half log dilution

To find out the optimal drug concentration of P7

and L-fucose-P7 liposomes, half-log dilution will be

applied in both in vitro and in vivo studies. The stock

solution of pure P7 without liposome will be prepared

by dissolving P7 peptide in PBS (pH=7.4) in a

concentration of 1mg/ml. Varying concentrations of

P7 solutions will be constructed by diluting the stock

solution with PBS to seven final concentrations at

0.1mg/ml, 0.0316mg/ml, 10 ug/ml, 3.16 ug/ml, 1

ug/ml, 0.316 ug/ml, 0.1 ug/ml. These solutions will

be used to test their cytotoxicity and proliferation

inhibition effect on cell lines. Similarly, the stock

solution of L-fucose-bound liposome-P7, keep the

amount of P7 inside the liposome the same as the pure

P7 group.

Then, L-fucose-bound liposome-P7 stock

solution will be parallelly diluted with P7 group,

resulting in varying concentrations with the inside p7

concentrations of 0.1mg/ml, 0.0316mg/ml, 10 ug/ml,

3.16 ug/ml, 1 ug/ml, 0.316 ug/ml, 0.1 ug/ml, which

will be used to test cytotoxicity and proliferation

inhibition effect on cell lines. The concentration

range of P7 and L-fucose-bound liposome-P7

concentration that achieve the significant inhibitory

effect will be obtained from the in vitro MTT assay.

Subsequently, three doses will be selected to test in

vivo studies for testing treatment effects, determining

the optimal in vivo drug concentration, which will be

used in subsequent experiments.

b) Peptide synthesis and purification

Table 1: The peptide sequence of P7 (Tetley, Murphy, Bonetto et al. 2020).

Peptide Sequence

P7 P S I C H V H R P D W P C W Y R

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Table 1 shows the sequence of P7 peptide.

Synthesis and purification of the target peptide use

the method from Tetley et al (Tetley, Murphy,

Bonetto et al, 2020). Standard Fmoc solid-phase

chemistry is used to synthesize a 10μmol scale

peptide with an amidated C terminus on the

automated peptide synthesizer. After the deprotection

of Fmoc, the N terminus will be acetylated and

biotinylated as described in Tetley et al (Tetley,

Murphy, Bonetto et al, 2020). Subsequently, the resin

is washed with methanol and dichloromethane, then

dried overnight. 5% water, 5% phenol, 5%

thioanisole, 2.5% ethanedithiol in TFA is used to

deprotect and cleave the peptide from the resin for 3

h. Then, the peptide will precipitate in diethyl ether at

-20 ℃. A C18 column in reverse-phase HPLC is used

The Investigation of P7 Peptide Delivery Targeting Cdc24 in Ras-driven Pancreatic Cancer by L-fucose-bound Liposome

653

to purify the peptide with a linear gradient elution of

20 to 50% acetonitrile in 0.1% TFA. Then, the

determination of molecular masses uses a mass

spectrometry.

c) Preparation of CPP-tagged P7

The addition of CPP, which is a C-terminal Nona-

arginine motif (9R), is the same as the motif used in

Tetley et al (Tetley, Murphy, Bonetto et al. 2020).

Carboxyfluorescein (FAM) will be linked to the N-

terminal of the P7 peptide. Named the product

without FAM and with FAM as CPP-tagged-P7 and

CPP-tagged-P7-FAM, respectively.

d) Preparation of P7 encapsulated in liposomes

Preparation of L-fucose bound liposomes has

been described previously (Tetley, Murphy, Bonetto

et al. 2020). Generally, DPPC, Chol, ganglioside,

DCP, and DPPE will be mixed at different molar

ratios, and cholic acid will be added for micelle

formation. The mixture will be dissolved in

methanol/chloroform (1:1, v/v), and the solvent will

be evaporated at 37°C to produce a lipid film, which

will be dried under vacuum. For the P7 peptide

preparation, P7 peptide containing solution will be

added to the lipid film and sonicated to obtain

uniform micelles in the buffer, which will then be

ultrafiltered. Hydrophilization treatment and L-

fucose conjugation on the surface of liposomes will

be carried out by methods modified from Yamazaki

et al (Yamazaki, Kodama, Gabius, 1994). Aminated

L-fucose will be conjugated to the liposome surface

using DTSSP, which is a type of cross-linking agent.

e) In vitro cell culture

The pancreatic cell lines AsPC-1 and BxPC-3 will

be cultured in RPMI 1640 (Gibco) plus 10% FBS, L-

glutamine, and 1% penicillin-streptomycin

(Yamazaki, Kodama, Gabius 1994). Non-cancerous

pancreatic cell line (hTRET-HPNE) will be cultured

in DMEM (Gibco) with 10% FBS, 5% L-glutamine,

and 1% penicillin-streptomycin. All cell lines will be

kept in a 5% CO2 humidified atmosphere at 37 °C.

f) Colony formation assay

In our study, we use cell colony formation assay

to measure the ability of a cell to divide and form a

colony after delivering P7 peptide delivered in l-

fucose liposomes to Ras-driven pancreatic cancer.

We will first prepare 6-well plates and harvest

exponentially growing cells. Then, we replate 50 cells

per dish, waiting for 2 h at 37 Degree Celsius.

Followed by incubation in the incubator until cells in

control dishes have formed sufficiently large clones.

Harvest cells from a donor culture using

trypsinization, which makes the cells completely

detach from the flask and float in the medium,

allowing us to see the cells more clearly. When the

cells start to detach from the culture dishes, resuspend

the cells in medium to inhibit trypsinization. Then,

neutralize the trypsin solution and count the cells.

Dilute the cell suspension and seed into flasks.

Remove the medium and rinse the cell by PBS. After

PBS wash, 2-3 ml of a mixture of 6.0%

glutaraldehyde and 0.5% crystal violet will be added.

Leave this for at least 30 min. Finally, remove the

glutaraldehyde crystal violet mixture and dry at room

temperature. After this, we count the colony

formation of cells in different plates that with the

different treatments. We will use Equal. 1 to calculate

the surviving fraction of cells.

𝐏𝐄 =

𝐍𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐜𝐨𝐥𝐨𝐧𝐢𝐞𝐬 𝐜𝐨𝐮𝐧𝐭𝐞𝐝

𝐍𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐜𝐞𝐥𝐥𝐬 𝐩𝐥𝐚𝐭𝐞𝐝

× 𝟏𝟎𝟎 Equal.

1(Franken, Rodermond, Stap, Haveman, Van-Bree

2006)

g) MTT assay

In this study, we will use MTT assay to

investigate the killing effect of targeted drugs (L-

fucose-bound liposome-P7) on pancreatic cancer

cells and drug sensitivity in established cell lines.

Subsequently, MTT assay is also used to determine

drug concentration that is required to achieve 50%

growth inhibition as compared to the growth of the

untreated control (50% inhibitory concentration,

IC50). Briefly, set up bottom plates (120 mL) to be

filled with PBS. If the drug is unstable, the drug

diluent will be freshly prepared before adding cells,

and 20 ml of total drug and 30 ml of drug solution

must be added to the plate. If testing a stable drug,

plates can be prepared with 30 mL drug

concentrations and can be stored at −20°C for later

use. Cultured pancreatic cell lines for 4 days to

determine the optimal effect for most standard drugs.

After the appropriate incubation time, add 1:10

volume of MTT solution (5 mg/mL), Shake plates for

5 min on a plate shaker by slowly increasing the

shaking speed to a maximum of 900 shakes/min, then

incubate the plate for another 4–6 h at 37°C in a CO

incubator. 150 mL of acidified isopropanol is added

to each well, mixing the rows with drugs. The cell

lines were divided into six groups and mixed with

drugs according to the table. 150 mL of acidified

isopropanol is added to each well, mixing the rows

with drugs. The cell lines were divided into six

groups and mixed with drugs according to table 1. In

terms of measurement, the optimal density (OD) is

measured at 540 and 720 nm to get a more exact

measurement. The pancreatic cell survival is

calculated by: (OD treated well [−blank])/(mean OD

control well [−blank])×100. The LC50 (the drug

concentration which results in 50% pancreatic cell

survival) can be calculated.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

654

Table 2: Different drugs put in the cell lines (Van-Meerloo, Kaspers, Cloos 2011).

AsPC-1 cell line BxPC-3 cell line

Serial numbe

1 2 3 4 5 6

L-fucose-

ound liposome - + + - + +

P7 - - + - - +

Note. “+” represents a significant decrease in cell proliferation compared with negative controls

. “-” is not

significantly different from negative control.

Table 2 shows the possible results of cell

proliferation of L-fucose-liposome and P7 peptide in

two different cell lines (AsPC-1 and BxPC-3) in MTT

assay.

h) Fluorescence polarization

Cell penetration effect will be detected by

confocal laser microscopy and fluorescence

microscopy. As discussed in previous work (Tetley,

Murphy, Bonetto et al, 2020), cells will be plated in

Lab-Tek chambered coverglasses at 1 X 10

cells/chamber (liposome citation). L-fucose-

liposome-P7 or liposome-P7 will be added to cells at

an optimal P7 concentration determined by half log

dilution (predicted around 0.7μg/ml). There will be

six different groups. The experimental group includes

FAM-L-fucose-P7 liposome. The negative control

group includes FAM-P7, FAM-liposome, FAM-L-

fucose liposome, and FAM-L-fucose. Positive

control group consists of FAM-CPP-P7 alone. Each

treatment will be mixed with cancer cell lines and

normal cells. Then each group will receive a beam of

Fluorescence microscope which will be used to detect

the intensity of excited fluorescent probes (Moerke

2009).

If L-fucose-p7 liposome is not bound to the Cdc42

protein, when they are hit by polarized light, the light

is going to be deflected. Since the complex is small,

the angle of rotation will be big, and the light it emits

is unpolarized, which is more diffuse and less intense,

so the degree of deflection will not be strong (Moerke

2009). However, when the L-fucose-P7 liposome

binds to the Cdc42 protein, the complex becomes

more massive. The Cdc42 protein is too large to rotate

freely, so the large complex keeps the light in the

polarized state, hence producing very fluorescent

light. By comparing the fluorescence intensity,

whether the Cdc42 protein binds to the drug can be

known. If the drug binds to Cdc42 protein, very

strong fluorescence can be obtained. On the contrary,

if they are not combined, the fluorescence intensity

will be weak. By comparing the content of

fluorescent agents in cells, the cell penetration of

drugs can be known. For example, the higher the

intensity of fluorescent light in the cell, the higher the

amount of drug entering the cell, so the cell is more

penetrating. However, if the content of fluorescent

agents in cells is relatively low, it indicates that the

drug content of forbidden cells is relatively low, and

the cell penetration is poor.

i) Annexin V/PI assay

In order to assess the cytotoxicity of newly

synthesized L-fucose-liposome-P7 compared with

previously developed CPP-P7, we apply annexin

V/PI assay to determine the fraction of apoptotic and

necrotic cells (Kabakov, Gabai, 2018). We will first

harvest the AsPC-1, BxPC-3 and hTRET-HPNE cell

lines and wash twice in PBS at 4 degree celsius and

resuspend in 1 X buffer. Then aliquot 100 μl cells into

fluorescence-activated cell sorter (FACS) tubes and

add 5 μl FITC-Annexin V and/or 10 μl PI (50 ug/ml

Propidium Iodide 10x). The mixture will incubate for

15 min at room temperature in the dark. Finally, 400

μl binding buffer (Binding buffer 10x : 0.1 M

HEPES/NaOH, pH 7.4; 1.4 M NaCl; 2.5 nM CaCl)

will be added to each tube and the result will be

analyzed by flow cytometry on FACS.

j) Fluorescence microscopy

Cell penetration effect will be detected by

fluorescence microscopy. As discussed in previous

work, cells will be plated in Lab-Tek chambered

cover glasses at 1 X 10

cells/chamber (Tetley,

Murphy, Bonetto et al. 2020). L-fucose-liposome-P7-

GFP or liposome-P7-GFP will be added to cells at an

optimal P7 concentration determined by half log

dilution (predicted around 0.7μg/ml). According to

Tetley et al. (Tetley, Murphy, Bonetto et al, 2020),

cells will be cultured in complete medium for 30 min

and then replaced with fresh medium. Cells will be

washed twice with PBS and fixed with 4%

paraformaldehyde 30 min and 2 hours post-treatment

at room temperature for 15 min. Then, the cells will

be washed 3 times using PBS and exposed to DAPI

staining the nuclei. The distribution of P7 peptide can

The Investigation of P7 Peptide Delivery Targeting Cdc24 in Ras-driven Pancreatic Cancer by L-fucose-bound Liposome

655

be assessed by fluorescence microscopy by

comparing the fluorescent intensity.

k) Peptide stability study

Inject P7 and L-fucose-bound liposome-P7 into

mice from the subcutaneous model group and detect

the drug plasma concentration after injection of 1, 2,

4, 8, 12 and 48 hours. Make profiles of drug plasma

concentration with time by HPLC. Compare the drug

plasma concentration curve, the stability of peptide to

the serum protease can be analyzed.

l) Animal model development

The subcutaneous model and orthotopic model

have been previously described (Tetley, Murphy,

Bonetto et al. 2020). Briefly, in the subcutaneous

model, mice aged 4 to 6 weeks will be modified with

AsPC-1 cell line to allow the growth of tumor to 5

mm in diameter. AsPC-1-bearing mice will be treated

with CPP-tagged P7 (2 mg/kg), L-fucose-P7-

Liposome (2 mg/kg), via the tail vein twice a week.

At 4, 8, 11, 15, 18, and 22 days after transplantation,

tumor volumes will be measured by IVIS imaging.

Representative image of mice treated with P7. For D-

mannose pre-treatment in the in vivo experiment for

L-fucose bound liposome, 5 mg of D-mannose will

be injected through the tail vein 5 minutes before

administration of agents (Tetley, Murphy, Bonetto et

al, 2020).

In the orthotopic model, BxPC-3-Luc cells in 100

µl PBS will be orthotopically injected into the

pancreas of nude mice (ages 4 to 6 weeks).

Bioluminescence will be measured on day 0-4 post-

injection, and the mice will be randomly assigned into

different groups (placebo groups, CPP-tagged P7

group and L-fucose bound liposome-P7 group)

before the initiation of treatment. The mice received

injection twice in the first week, and then received

injection once in week 2 and once in week 3. All mice

will be sacrificed on the day after the last injection

but before a final bioluminescence measurement.

m) Statistical Analysis

Results will be presented as means (± SD) for

each sample. All statistical significance of all

numerical data will be analyzed using the student’s

T-Test on GraphPad Prism® at (p <0.05).

3 POSSIBLE RESULTS

Table 3: In Vitro Cell proliferation comparison of L-fucose-liposome-P7 with negative controls on cancer cells and normal

cells.

Result 1 Result 2 Result 3 Result 4 Result 5

Colony

formation assay

+ - + _ ++ + + + - -

MTT assay

+ - + - ++ + + + - -

Annexin /PI

assay

A L A L D D A A L L

Note. In table 3, “C” represents tests in cancer cells group and “N” means in normal cells. “+” represents a

significant decrease in cell proliferation compared with negative controls*. “-” is not significantly different from

negative controls. Negative controls include L-fucose-liposome, Liposome without L-fucose and L-fucose. “A”

means apoptotic cell population is significantly larger than live and necrotic cells, while “L” represents live cells

population is larger than apoptotic and necrotic cells and “D” represents population of necrosis dead cells is

higher than both apoptotic and necrotic cells.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

656

Table 4: Cell proliferation inhibition comparison of L-fucose-liposome-P7 with P7 and CPP-P7.

Result 1 Result 2 Result 3 Result 4 Result 5

P7 Cpp-P7 P7 Cpp-P7 P7 Cpp-P7 P7 Cpp-P7 P7 Cpp-P7

Colony

formation

assay

+ ++ - + ++ + - + - -

MTT assay + ++ - + ++ + - + - -

Note. In table 4, “+” represents a significant higher cell proliferation inhibition effect of L-fucose-liposome-P7

in cancer cell lines compared with that of p7 and cpp-p7, respectively. “-” indicates that no significant greater

proliferation inhibition effect of L-fucose-liposome-P7 compared with p7 or cpp-p7, respectively.

Table 5: Possible Results on Cell penetration of L-fucose-liposome-P7 in vitro.

Cell penetration by L-

fucose-liposome-P7 in vitro

Result 1 Result 2 Result 3 Result 4 Result 5

Fluorescence microscopy + + + - +

Note. In table 5, “+" represents the fluorescence intensity inside the cancer cells is significantly higher than that

outside. “-” indicates that fluorescence intensity inside cancer cells is not significantly higher than outside.

3.1 Possible Results

Possible result 1: As shown in table 3 and table 4, L-

fucose-bound liposome-P7 has significantly greater

cell anti-proliferative effect to pancreatic cancer cells

compared with all negative controls, P7 peptide and

CPP-P7. Also, fluorescence intensity indicates that L-

fucose-bound liposome-P7 has good penetrability to

cancer cells (Table 5). Moreover, the sizes of tumors

in animal models have significantly decreased by

treating with L-fucose-bound liposome-P7.

Additionally, L-fucose-bound liposome-P7 shows

significant higher inhibitory effects on the cell growth

of in vitro cancer cell samples and in vivo xenograft

mice models without affecting normal pancreatic

epithelial cells.

Possible Result 2: L-fucose-bound liposome-P7

exerts significant greater inhibitory effect on

proliferation of the pancreatic cancer cells in the

determined human pancreatic cancer cell lines,

compared with CPP-P7 and negative controls but not

P7 peptide. Also, it shows cytotoxicity to pancreatic

cancer cells but not non-cancerous cells, and it

controls/reduces mean tumor diameter in vivo animal

models without affecting normal pancreatic cells.

Specially, L-fucose-bound liposome-P7 show

greater cytotoxicity and inhibitory effect on

proliferation of pancreatic cancer cell lines but no

significant inhibitory effect and cytotoxicity on

normal pancreatic cell lines compared with all

negative control groups. Also, L-fucose-liposome,

Liposome without L-fucose and L-fucose alone do

not show inhibitory effect on growth of both

pancreatic cancer cell lines and normal pancreatic cell

lines, while P7 alone shows comparable cell

proliferation inhibition to L-fucose-bound liposome-

P7 on pancreatic cancer cell lines.

Possible Result 3: L-fucose-bound liposome-P7

shows cytotoxicity to cancer cells to a large extent

and revels cytotoxicity to normal cells (Table 3).

Also, L-fucose-bound liposome-P7 reduces mean

tumor diameter in vivo mice models but causes side

effects to mice.

L-fucose-bound liposome-P7 exerts greater

cytotoxicity to both pancreatic cells and normal

pancreatic epithelial cells in vitro and in vivo. Also,

all negative control groups do not show significant

cell cytotoxicity to the pancreatic cancer cells in vitro

and in vivo studies. As shown in table 3, results of

Annexin V/PI assay indicates that L-fucose-bound

liposome-P7 causes cell deaths by necrosis to

pancreatic cells and normal pancreatic epithelial

cells. In table 4, L-fucose-bound liposome-P7 shows

a significant higher cell proliferation inhibition effect

in cancer cell lines compared with P7 and CPP-P7.

Moreover, fluorescence microscopy results show that

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L-fucose-bound liposome-P7 has poor penetration to

cancer cells (Table 5).

Possible Result 4: L-fucose-bound liposome-P7

inhibits cell growth of both pancreatic cancer and

normal cells (Table 3). Fluorescence imaging results

reflect poor cell penetration of L-fucose-bound

liposome-P7 to pancreatic cancer cells.

L-fucose-bound liposome-P7 exerts great

inhibitory effects on cell growth of cancer cells and

normal cells mainly by apoptosis (Table 3). In vivo

animal study, the sizes of tumors are decreased by L-

fucose-bound liposome-P7. As shown in table 4, L-

fucose-bound liposome-P7 shows a significant higher

cytotoxicity to cancer cell lines compared with CPP-

P7 but not P7. FAM-L-fucose-liposome and FAM-L-

fucose-bound liposome-P7 are observed to locate and

accumulate significantly in tumor sites, while FAM-

P7, FAM-liposome, FAM-L-fucose and FAM-CPP-

P7 do not show significant target delivery to the

pancreatic tumor site.

Possible Result 5: L-fucose-bound liposome-P7

does not exert any significant effect to either

pancreatic cancer cells, or normal pancreatic

epithelial cells (Table 3). Also, all negative control

groups show not significant effect on the growth of

both pancreatic cancer cells and normal pancreatic

cells. Colony formation assay, MTT assay and

Annexin assay all demonstrate insignificant effect on

pancreatic cancer cells and normal cell lines (Table

3). Although L-fucose-bound liposome-P7 shows

good cell penetration to cancer cells (Table 5), it has

no comparable cytotoxicity to P7 and CPP-P7 on in

vitro pancreatic cancer cells (Table 4). Also,

fluorescence imaging experiments of FAM-L-fucose-

bound liposome-P7 and all negative control groups

including FAM-P7, FAM-liposome, FAM-L-fucose

liposome, and FAM-L-fucose and positive control

(FAM-CPP-P7) do not show significant distribution

and accumulation in the targeted pancreatic tumor

site.

4 DISCUSSION

Previous studies report that P7 peptide binds with

Cdc42 protein and inhibits the downstream functions

exerted by Ras-mediated signaling, leading to

blockage of cancer cell transformation in Ras-driven

cancers (Malam, Loizidou, Seifalian, 2009).

However, the lack of a proper delivery system slows

down the drug development processes. To determine

a better delivery system that would enhance

therapeutic effects of P7 peptide and reduce OFF-

target cytotoxicity, this study uses L-fucose-modified

liposome in comparison with CPP-tagged P7 peptide

used in previous studies to deliver P7 peptide to two

well studied pancreatic cancer cell lines from humans

and to in vivo pancreatic cancer animal models.

Possible result 1 indicates a great cytotoxic effect and

proliferation inhibitory effect on pancreatic cancer

cells without affecting normal pancreatic epithelial

cells, indicating target delivery of L-fucose-bound

liposome-P7 to pancreatic cancer cells. Fluorescence

imaging indicates that L-fucose-bound liposome-P7

has good penetrability to cancer cells, which may

explain its higher cytotoxicity than P7. According to

Tetley et al. (Tetley, Murphy, Bonetto et al. 2020),

binding with CPP reduces the efficacy of P7, which

explain the much higher cytotoxicity of L-fucose-

bound liposome-P7 than CPP-P7 (Table 3). Overall,

possible result 1 fully supports our hypothesis that L-

fucose-modified liposome has a significant treatment

effect with enhanced cell penetration and targeted

delivery to pancreatic cancer cells. Further studies

assessing the pancreatic cancer cell transformation

should be done for a thorough understanding of the

blockage of Ras-driven transformation. More

complex and representative animal models should

also be done in preclinical trials before entering

clinical trials to prevent potential damages that could

not be found in mouse models.

Similar with possible result 1, possible result 2

indicates L-fucose-modified liposome P7 peptides

achieve the great efficacy with minimal side effects

and toxicity, which is consistent with previous

investigations in targeted delivery of L-fucose

liposome to pancreatic cancer cells. Differently, in

possible result 2, L-fucose-modified liposome P7

shows significant greater proliferation inhibition

effect than cpp-p7 but not p7, which indicates that the

efficacy of P7 is not enhanced by L-fucose liposome.

This could be also possibly because binding with L-

fucose liposome reduce the efficacy of P7 when

increasing drug distribution of L-fucose-bound

liposome-P7 to cancer cells. This result partially

supports our hypothesis that L-fucose-modified

liposome has targeted drug delivery to the tumor site

and increased cell penetration to cancer cells.

Compared with possible result 1 and 2, possible

result 3 shows that L-fucose-modified liposome P7

peptides has strong cytotoxicity and side effects at the

same time. It may be due to the existence of other

unknown molecular mechanisms induced by L-

fucose-modified liposome P7 peptides in the cell,

resulting in the apoptosis of normal cells and

pancreatic cancer cells induced. Simultaneously，in

result 3 both L-fucose-modified liposome P7 and

cpp-p7 shows significant proliferation inhibition and

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

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L-fucose-modified liposome P7 is stronger, which

may because the 9R motif binds non-specifically to

other moieties or causes aggregation in the

lysate(Tetley, Murphy, Bonetto et al, 2020).This

result partially supports our hypothesis that L-fucose-

modified liposome has targeted drug delivery to the

tumor site but also has strong cytotoxicity to the

normal cell.

Possible result 4 shows that L-fucose-modified

liposome P7 peptide has cytotoxicity to both cancer

cells and normal cells by apoptosis (Table 4), while

L-fucose-modified liposome P7 has poor penetration

to the cancer cells, indicating the possibility that L-

fucose-modified liposome P7 induce an unknown

path on the cell surface which cause apoptosis of

cells. compared with cell. by cell apoptosis, cell

intracellular content is not released while by cell

necrosis, cell intracellular content is released which is

prone to cause inflammatory reaction (Helewski,

Kowalczyk-Ziomek, Konecki, 2006). Hence, drug

cytotoxicity induced by cell apoptosis is more

desirable than by cell necrosis. However, L-fucose-

modified liposome P7 causes significant harm to

normal pancreatic cells which would potentially

increase the suffering of patients. Hence, future work

should modify liposome carriers or find a new

strategy to increase the cell penetration of P7, which

can eventually achieve the efficacy of P7. Overall,

possible result 4 does not support our hypothesis.

There could be many reasons for the failure of

possible result 5. However, we have set up several

negative controls to eliminate the problem. If it does

not show any alteration compared with negative

controls, which include P7 alone, liposome alone, L-

fucose-liposome alone, it is likely that P7 is unable to

exert its inhibitory effect in pancreatic cancer cells. If

P7 alone shows a significant inhibitory effect in

pancreatic cancer cells compared with L-fucose-

liposome-P7, then probably the delivery system is the

problem that prevents P7 from exerting its effect.

Other failed results in animal models could be due to

enzymatic activity in the animal body, clearing

liposomes before they reach the pancreas. Therefore,

further study should investigate liposome

modifications that make them last longer in the

animal body is necessary.

5 CONCLUSION

In conclusion, the objective of our experiment was to

determine whether targeting P7 delivery with L-

focused binding liposomes can increase cell

penetration, enhance cytotoxicity to tumor cells, and

reduce cytotoxicity to normal cells compared to CPP-

tagged P7 under in vitro and in vivo conditions. This

study explores the therapeutic effect of L-fucose-P7

liposome in CA19-9 overexpressing pancreatic

cancer cell lines and Xenograft Murine Models. The

results of this study will examine whether L-fucose-

P7 liposome has a better therapeutic effect and a

weaker OFF-target effect compared with CPP-

tagged-P7 used in previous studies and preparing it

for entering clinical trials. These possible results and

the experiment itself provide the potential for future

improvement in the delivery system including

adjustment of liposome size to increase half-life and

adding monoclonal antibodies specific for pancreatic

cancer cells overexpressing EGFRs. The

modifications on liposome surface proteins will alter

pharmacokinetics of the P7 peptide to make it more

efficacious and the enhanced targeting skill of the

liposome will potentially minimize the OFF-tumor

effects.

ACKNOWLEDGEMENT

We would like to thank Hongbin Lan, Yurui Yang,

Huawei Zhu, and Yiran Zhu who

cooperatively contribute to this paper, especially

Hongbin Lan and Yurui Yang who did wonderful

work to this paper equally and they two should be

regarded as co-first authors. Moreover, we sincerely

thank Professor Arthur Salomon and teachers who

give useful guidance and helps.

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