Design of Targeted and Release Controlled Liposome for Paclitaxel

and Doxorubicin Combination in Breast Cancer Therapy

Chengpei Ouyang

1,*

, Qi Zhu

, Yifan Liu

and Xiangqi Meng

College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China

JSerra Catholic High School, San Juan Capistrano, California 92675, U.S.A.

Beijing No.101 High School, Beijing 101407, China

Beijing City International School, Beijing 100022, China

Keywords:

Combination Therapy, Paclitaxel, Doxorubicin, Bi-Layer Liposome, Co-Delivery.

Abstract: Paclitaxel and doxorubicin are commonly used in chemotherapy of breast cancer. Now we have developed a

kind of nanocarrier liposome that can deliver the combination of Dox and PTX to alleviate the pain brought

by side effects and decrease the resistance. In this paper, we expected the result data of the drug loading

capacity and the drug loading efficiency, the release of PTX and Dox in different pH environment, and the

absorption of drugs which is estimated by the amount of free drug remains in the cells. The combination of

Dox and PTX in liposome was modified with folic acid for tumor targeting, and achieved pH responsive drug

release in tumor cell by introduction of N-(4-carboxybenzyl)-N, N-dimethyl-2,3-bis (oleoyloxy) propan-1-

aminium (DOBAQ), a kind of pH sensitive lipid. The double layers were loaded with hydrophobic drug PTX,

and the hydrophilic drug DOX is loaded in the aqueous core of the vesicle. We expected the results to reduce

side effects and improve specificity when treating the cancer.

1 INTRODUCTION

Breast cancer is the disease in which breast epithelial

cells proliferate out of control under the action of a

variety of carcinogens. The early stage of the disease

often manifests as breast lumps, nipple discharge,

axillary lymphadenopathy, and other symptoms (Yin,

2010). In the late stage, cancer cells may metastasize

to a distance, and multiple organ diseases may appear,

which directly threaten the life of the patient. Breast

cancer is often called the "pink killer", and its

incidence ranks first among female malignant tumors.

Male breast cancer is relatively rare. With the

improvement of medical treatment, breast cancer has

become one of the solid tumors with the best curative

effect. Risk factors for breast cancer include genetic

factors, hormonal changes, mental and psychological

factors, and history of past breast diseases. According

to the latest data from the International Agency for

Research on Cancer (IARC) in 2018, the incidence of

breast cancer in female cancers worldwide is 24.2%,

ranking first among female cancers, of which 52.9%

occur in developing countries (Feng, 2021).

Breast cancer treatment is personalized. All

patients need to be formulated by authoritative

experts based on their own conditions. The treatment

plan depends on many factors, including tumor

subtypes, patient’s age, general health, menopause

and eating habits, stage of the tumor, hormone

receptor status (ER, PR) and HER2 status, genetic

information (such as BRCA1 or BRCA2), and genetic

testing results, such as full genetic testing and breast

cancer 21 gene testing (Oncotype DX™) (Reddy,

2011). At present, the conventional methods of

treatment of breast cancer mainly include surgery,

radiotherapy, chemotherapy, hormone therapy,

targeted therapy, immunotherapy, etc. Radiation

therapy, chemotherapy, targeted therapy and/or

hormone therapy can be used to assist the treatment

(Bodei, 2007).

1.1 Surgery

For ductal carcinoma in situ and early invasive breast

cancer, doctors usually recommend surgery to remove

the tumor (Trayes, 2021). Most patients with invasive

breast cancer will undergo sentinel lymph node

biopsy or axillary lymph node dissection. Most

important thing in the treatment of early- stage breast

cancer is to reduce the risk of recurrence by removing

Ouyang, C., Zhu, Q., Liu, Y. and Meng, X.

Design of Targeted and Release Controlled Liposome for Paclitaxel and Doxorubicin Combination in Breast Cancer Therapy.

DOI: 10.5220/0012032000003633

In Proceedings of the 4th International Conference on Biotechnology and Biomedicine (ICBB 2022), pages 425-434

ISBN: 978-989-758-637-8

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

425

all remaining cancer cells. These cancer cells are

undetectable, but they can cause cancer to recur

because they grow over time.

1.2 Radiation Therapy

Radiation therapy uses high-energy X-rays or proton

rays to destroy cancer cells. Radiation therapy helps

reduce the risk of breast recurrence. Through surgery

and radiotherapy, within 10 years of treatment, the

recurrence rate of breast cancer is now less than 5 %

(McGale, 2014). In the past, traditional radiotherapy

increased the long-term risk of heart disease in

women with left breast cancer. Now, proton therapy

can protect the heart from radiation damage.

Therefore, more breast cancer patients can reasonably

obtain a longer survival period and better quality of

life through modern medical methods.

1.3 Medication

Systemic therapy using medication can kill cancer

cells comprehensively. Types of systemic therapy for

breast cancer include chemotherapy, hormone

therapy, targeted therapy, and immunotherapy

(Bodei, 2007).

Chemotherapy began in the 1940s and 1950s, and

its application has greatly improved the efficiency of

anti-tumor. Although surgery can remove tumor

tissue, it cannot be completely removing all cancer

cells, especially for circulating cancer cells in the

blood. At this time, chemotherapy is needed to kill the

remaining cancer cells and reduce the risk of

recurrence and distant metastasis. Therefore,

chemotherapy is of great significance to tumor

treatment (Hassan, 2010). In the classification and

treatment of breast cancer today, chemotherapy also

plays a very important role. For early breast cancer

patients, the risk of recurrence and metastasis can be

reduced; for advanced patients, it can alleviate the

condition, prolong survival, and improve the quality

of life.

After decades of development, the commonly

used chemo-drugs for breast cancer currently include

anthracyclines (such as DOX), taxanes (such as

PTX), and antimetabolites (such as gemcitabine).

DOX is an anti-tumor drug commonly used in clinic.

Its main function is to insert the flat n-loop to the

middle of the base strand of DNA, which prevents the

transfer and replication of DNA and +RNA, thus

avoiding cells’ proliferation and metabolism. Breast

cancer is a major indication of DOX, which is often

used in combination with cyclophosphamide, in

addition, it can be combined with PTX and docetaxel,

which can increase the efficacy (Darya Alizadeh,

2014). In addition, DOX can be used to treat bladder

cancer, head and neck malignancies, testicular

malignancies, liver cancer, and stomach cancer.

PTX is a natural secondary metabolite extracted

from the bark of Taxus chinensis, which has good

antitumor effect. It is often used in the treatment of

ovarian cancer, uterine cancer, and breast cancer. PTX

is a natural secondary metabolite extracted from the

bark of Taxus chinensis; it allows tubulin and tubulin

dimers that make up microtubules out of dynamic

equilibrium, which leads to them death of cells (Sun,

2008). DOX and PTX can be administered separately,

but the therapeutic effect is not good. A single drug is

easy to cause drug resistance. Once drug resistance

occurs, the therapeutic effect of drugs will be

significantly reduced, even large amounts of doses

will not achieve the original effect. And improved

drug dosage will lead to the relatively large toxicity

and side effects. Therefore, we decided to use a Co-

administration Combination therapy, refers to the use

of two or more drugs for treatment, which can

improve the efficacy and reduce adverse reactions.

The combination of more drugs can reduce the dose

of individual drugs, thus reducing the toxicity and

side effects (Moussa, 2018). The combination of the

two drugs can reduce the side effects and resistance

significantly. PTX has the function of preventing the

cell division form beginning of cell division cycle.

DOX functions in cell DNA damage. It causes the

cleavage of DNA and thus indirectly causes the

generation of hydroperoxide through the oxidative

reaction of NAD(P)H. Eventually, it leads to the

apoptosis of cells (Hideki Mizutani, 2005). When

combining the two drugs, the result suggested that it

had become more effective on tumor regression. It

can also reduce the side effects and the resistance

(Gill, 2019). In order to improve the therapeutic effect

of combined drug delivery and reduce its side effects,

we decided to load these two drugs into liposome

with. modifications of targeting and sustained release

on liposomes.

Folate acid is applied for tumor targeting in our

liposome system, although folate receptors are widely

distributed in normal tissues and tumor tissues, the

density and activity of folate receptors in most tumor

cells are much higher than those in normal cells.

Thus, it can achieve drug accumulation to the surface

of breast cancer cells and increase the concentration

of the drug in the tumor area, which can improve the

treatment effect and reduce side effects. PH

responsive drug release in tumor cell is achieved by

DOBAQ, a kind of pH sensitive Cationic lipid, which

exhibits pH dependent ionization and promote

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426

liposome disruption in pH 5.5 endosome in tumor cell

(Su, 2019). Because liposomes will dissociate and

change with each other under different pH conditions,

then control the drug release. Thus, we developed this

way of delivering PTX and DOX. Using both drugs

can limit the side effects. Additionally, in order to

ease the cardiotoxin of those two drugs, we used pH-

sensitive liposome to deliver the drug and only

targeting on the breast cancer cells that has large

amount of folate receptors (Sharma, 2006). The

structure of liposome is shown in Figure 1. We hoped

the bilayer liposome can delivering those two drugs

effectively and reach our propose of reducing the side

effects and lower the simple drug resistance.

Therefore, we will find a more efficient way that can

bring patient a better life quality and overall improve

the survival rate.

Figure 1. Schematic representation of the co-delivery of hydrophobic drug paclitaxel (purple) and hydrophilic drug

doxorubicin (red) through bilayer liposome (Liu, 2014).

2 METHODS AND MATERIALS

2.1 Materials

DSPE-PEG (2000)-Folate and Doxorubicin

hydrochloride (DOX) was purchased from Sigma-

Aldrich (Shanghai) Trading Co.Ltd. (Shanghai,

China). DOBAQ, DSPE-mPEG2000 and DSPE-

PEG2000-FITC were purchased from Avanti Polar

Lipids (Alabaster, USA). Lipoid S100 were

purchased from Lipoid GmbH (Würzburg, German).

Paclitaxel (PTX) were purchased from Macklin Inc.

(Shanghai, China). Gibco™ PBS were purchased

from Thermo Fisher Scientific (USA). Sephadex G-

50 columns were purchased from Pfizer Inc. (USA).

Amicon Ultra 0.5 mL centrifugal filters were

purchased from Sigma-Aldrich (Shanghai) Trading

Co.Ltd. (Shanghai, China). Pierce™ Protein

Concentrator PES, 3K MWCO cut-off tubes, Attune

NxT flow cytometry and Nunc™ multi-well cell

culture plates were purchased from Thermo Fisher

Scientific™. Leica SP2 CLSM Confocal laser

scanning microscopy was from Leica Microsystems

(Wetzlar, Germany).

2.2 Preparation of liposome

Liposomes were prepared by the lipid hydration

method (Schiffelers, 2003). Briefly, different masses

of lipid substances, were dissolved in round-bottomed

vials using an organic solvent mixture (chloroform:

methanol=2:1). Then, the round bottom vials are

placed on the rotary evaporator. By using the

principle of lowering the boiling point of liquids

under reduced pressure, volatile solvents are removed

by heating continuous distillation. After completion

of the rotary evaporation operation, the resulting film

in the vial was transferred to vacuum and dried for

more than 6 hours to completely remove the residual

organic solvent. The obtained films were placed in 2

mL of phosphate-buffered solution (PBS, pH 7.4) at

37°C for 30 min of hydration to obtain liposomes with

Design of Targeted and Release Controlled Liposome for Paclitaxel and Doxorubicin Combination in Breast Cancer Therapy

427

a final lipid concentration of 20 mg mL-1, followed

by 60 seconds of sonication. Then it was further

intermittently sonicated by a probe sonicator in ice-

bath at 80 W for 75 s. The free (NH4)2SO4 was

removed by passing through a Sephadex G-50

column in PBS (pH 7.4) solution. The obtained

liposomes were purified by 3 K Amicon centrifugal

filters and washed twice with fresh PBS solution (10

mM, pH 7.4).

For the preparation of liposomes containing DOX

(D-LPs), we set up four concentration gradient groups

(10 µg/ml, 1 µg/ml, 0.1 µg/ml, 0.01 µg/ml), and

added gradient concentrations of DOX, stirred, and

then incubated at 45℃ for 20 min. The PTX-loaded

liposomes (P-LPs) were made at the same

concentration and by the same method. By using gel

filtration, the large molecule liposomes are eluted

first due to their weak retention ability, while the

small molecule compounds are retained strongly and

exit the column last, thereby removing the free

PTX/DOX. The liposomes were stored at 4℃ for

later use.

For the preparation of DOX and PTX-loaded

liposomes (DP-LPs), as shown in the Figure 1, the

PBS was replaced by 1 ml 300 Mm (pH 4)

(NH4)2SO4 solution. As mentioned above, the PBS

solution (pH, 7.4) was added to the SephadexG-50 gel

filtration column to equilibrate the pH, and then PTX-

LPs were used to replace the outer phase consisting

of (NH4)2SO4 solution. Doxorubicin was then

remotely loaded using the (NH4)2SO4 gradient

method. (Bolotin E M, 1994). Similarly, we set up

four concentration gradient groups (10µg/ml, 1µg/ml,

0.1µg/ml, 0.01µg/ml) and the molar ratio of PTX to

DOX was 1:1. Briefly, PTX-LPs were preheated at

50°C, and the appropriate amount of DOX solution

was added and incubated with liposomes at 50°C for

20 min with gentle stirring to load DOX into the

liposomal internal phase. Free DOX and PTX were

then removed by 3 K Amicon centrifugal filters.

Liposomes were stored at 4℃ and used within 24 h

of preparation (Qiu, 2016). The Tables 1-3 list the

lipid ratios of the liposomes used in the following

experiments (Walsh, 2012; Moghimipour E, 2018;

Campbell R B, 2001; Bernsdorff C, 1999; Sampedro

F, 1994; Sharma A, 1994).

Table 1: Liposome formulation of FA-LPs.

Corresponding compositions (%)

DSPE-PEG2000-Folate DSPE-mPEG2000 DOBAQ lipoid S100

Molar Ratio (%) 1 4 50 45

Table 2: Liposome formulation of FA-FITC-LPs for cell uptake study

Corresponding compositions (%)

DSPE-

PEG2000-Folate

DSPE-

mPEG2000

DOBAQ lipoid S100

DSPE-

PEG2000-FITC

Molar

Ratio (%)

1 4 49 45 1

Table 3: Liposome formulation of FITC-LPs for cell uptake study

Corresponding compositions (%)

DSPE-PEG2000-FITC DSPE-mPEG2000 DOBAQ lipoid S100

Molar Ratio (%) 1 4 50 45

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428

2.3 Physicochemical Characterization

of Liposomes

For encapsulation efficiency measurement (Shew R

L, 1985), 0.1 ml of the liposome suspension was

passed through a Sephadex G-50 gel filtration column

and the PTX and DOX contents were measured by

high performance liquid chromatography (HPLC) at

227 nm and at Ex of 470 nm, Em of 590 nm,

respectively. Drug entrapment efficiency (DEE, wt%)

and drug loading capacity (DLC, wt%) were

calculated according to the following formulas (Tang,

2014):

DEE = (amount of loaded drug/amount of drug

added) × 100%

DLC = (amount of loaded drug/amount of loaded

drug + amount of drug carrier) ×100%

The column used for high performance liquid

chromatography (HPLC) is Acclaim™ 300 C18

column, flow rate is 1 mL/min, the liquid phase is of

paclitaxel was methanol-water (65:35, v/v) with the

detection wavelength of 227 nm and the liquid phase

of adriamycin was methanol-water (70:30).

Hydrodynamic diameter, polydispersity index

(PDI) and zeta potential of liposomes were measured

by NS-90Z Nanoparticle size and potential analyzer

from Omec (Zhuhai, China), using dynamic light

scattering (DLS) and electrophoretic light scattering

technique. The liposome was diluted with Milli Q

water before measurement.

Transmission electron microscopy (TEM)

samples were prepared by diluting the liposome

solution to a concentration of 0.1 mg/mL and adding

dropwise 5 μL of liposome dissolved into a 200-mesh

formvar-coated copper grid (TABB Laboratories

Equipment, UK). After five minutes, the solution was

aspirated through filter paper and 2 μL of uranyl

acetate solution (2%, w/v) was added. After five

minutes, the solution was blotted off with filter paper.

The TEM sample was air-dried and then assayed.

2.4 In Vitro Release Kinetic of

Liposomes

Here, we used the dialysis method to investigate the

release kinetics of DOX and PTX from liposomes in

phosphate-buffered saline (PBS), a method that has

been used several times in previous liposome delivery

studies (Campbell R B, 2001; Lv, 2014; Wang, 2016)

The pH of the PBS release medium was set to 7.4 and

5.5, respectively. Briefly, the liposomes loaded with

PTX and Dox were suspended in 5 ml of PBS release

medium and transfer to a dialysis bag (MWCO 3500

Da). The release experiments were started by placing

the dialysis bag into 45 mL of release medium with

continuous shaking at 100 rpm at 37℃. At the

scheduled point in time (1,2,4,612,24,48,72,108 h), 4

mL of the incubation solution was extracted and

replaced with an equal volume of fresh PBS. DOX

release was determined using the HPLC method

mentioned above. The concentration of paclitaxel in

1 mL of solution will be detected at 227 nm (flow rate

1 mL/min) on a C18 column. At the end of 108 h, the

dialysis bag was opened and 2.0 ml of 10% TritonX-

100 was mixed completely with the release medium.

The concentrations of paclitaxel and Adriamycin

were then determined by HPLC and UV-Vis

spectrometer as mentioned above, and the maximum

drug release was calculated.

After calculating the free drug concentration, the

percentage of drug release can then be calculated with

the following formula: percentage of drug release =

amount of drug released / amount of drug contained

in the package × 100%. The drug co-delivery release

profiles of Dox and PTX have demonstrated that

liposomes have slow linear sustained release kinetics

and efficient drug loading yields. Drug release

profiles at pH = 7.4 and 5.5 have also been clearly

studied (Lv, 2014; Zhu, 2015). Based on previous

studies, we predicted the release profiles of bilayer

liposomes loaded with Dox and/PTX drugs

administered in combination or alone at 1, 2, 4, 6, 12,

24, 48, 72, and 108 hours and compared them.

2.5 Stability Studies in Human Plasma

At 37°C, 10 mg of liposomes were added to 1 mL of

human plasma and stirred at low speed (200 rpm)

with a magnetic stirrer. After every period (1, 2, 4, 6,

12, 24h), the particle size of the liposomes was

determined using dynamic light scattering. The

liposomes were observed for the appearance of

agglomerated precipitation.

2.6 Breast Cancer Cell and Liposome

Interaction Studies

2.6.1 Cell Culture

Two types of human breast cancer lines, including

MDA-MB-231 and MCF-7 were cultured in

Dulbecco’s modified Eagle’s medium (DMEM) with

high glucose containing 10% fetal bovine serum

(FBS), supplemented with 100 mg/ml streptomycin

and 100 U/ml penicillin. The cells were maintained at

37℃ in a humidified incubator under 5% CO2 (Qiu,

2016).

Design of Targeted and Release Controlled Liposome for Paclitaxel and Doxorubicin Combination in Breast Cancer Therapy

429

2.6.2 Confocal Laser Scanning Microscopy

(CLSM) Observation of Liposome

Uptake by Cells

The uptake of liposomes by both MDA-MB-231 and

MCF-7 cells was determined by observing the

fluorescence characteristics of DOX released from

FITC-labeled liposomes (FITC-LPs) by confocal

laser scanning microscopy (CLSM) at an emission

wave of 480 nm and an excitation wave of 590 nm.

Firstly, the cells were seeded on the coverslips in 24-

well cell culture plates at a density of 1 ×105

cells/well in 2 mL of DMEM. Then the cells were

incubated for 24 h to 50% confluence and the original

medium was replaced with 200 ml of 3 mmol/ml

DOX-PTX-FA-FITC-LPs (DP-FA-FITC-LPs) and

DOX-PTX-FITC-LPs (DP-FITC-LPs). After 3 or 6 h

incubation, then the supernatant was removed by

centrifugation to obtain a cell mass. The cell masses

were washed three times with cold PBS, fixed in 4%

paraformaldehyde at room temperature for 30 min,

followed by cell nuclei staining with DAPI for 5 min

before washed three times with PBS for confocal

microscopy analysis. Later, the coverslips were

detected on CLSM. Fluorescence images of cells

were analyzed using ImageJ or FIJI software (Lv,

2014).

2.6.3 Qualitative Analysis of Cellular

Uptake Using Flow Cytometry

1.0 ml of MDA-MB-231 and MCF-7 cells (1 × 10

cells/well) were inoculated into 24-well tissue culture

plates and cultured at 37°C, 5% CO

for 24 h until the

cells grew almost confluently. The medium was then

replaced with 1.0 ml of 0.5 mg/ml DP-FITC-LPs and

DP-FA-FITC-LPs diluted with DMEM medium,

respectively, and the plates were incubated at 37°C

and 5% CO

.After 4 h, the medium was removed, and

cell monolayer was suspended by brief treatment with

trypsin and then washed three times with cold PBS.

Then the cell samples were examined by flow

cytometry using a Attune NxT (Thermo Fisher). the

level of cells that have taken up liposomes (positive

event, %) and the level of liposomes that have been

taken up (mean fluorescence) are measured. 10,000

sensor events collected and analyzed using FCS

Express software (Qiu, 2016).

2.7 In Vitro Cytotoxic Studies

The cells were cultured as shown above. MDA-MB-

231 and MCF-7 cells were plated at a density of 5 ×

cells/well in 10% FBS-containing medium in 96-

well plates and grown for 24 h. The cells were then

exposed to liposomes (single drug/drug

combinations; FA/without FA) at different

concentrations of combined drugs or free drugs

(10µg/ml, 1µg/ml, 0.1µg/ml, 0.01µg/ml), for 48 h. To

determine the cytotoxicity of empty liposomes, 100

ml of 6 mmol/ml liposomes were added to each well

of a 96-well plate and incubated with the cells

described above. The cell viability was assessed using

the CellTiter from Promega according to the

manufacturer’s instructions. The principle of

detecting cell viability is that the fluorometric signal

of CellTiter is proportional to the ATP content of the

cells, which is proportional to the number of cells.

Toxicity of each drug concentration was subsequently

determined for each well. The data was analyzed by

nonlinear regression to get the IC50 value. The

combination index (CI) values were calculated by the

equation:

CI =



,



,



,



,

(1)

CA,x and CB,x are the concentrations of drug A and

drug B used in combination to achieve x% drug

effect. ICx,A and ICx,B are the concentrations for

single agents to achieve the same effect. A CI of less

than, equal to, and more than 1 indicates synergy,

additivity, and antagonism, respectively (Zhao,

2004). Using this analysis method, a CI = 0.9−1.1

reflects additive activity, and a CI >1.1 indicates

antagonism, while a CI < 0.9 suggests synergy (Liu,

2014).

3 EXPECTED RESULTS AND

CONCLUSIONS

3.1 Characteristics of Co-Delivery via

pH Release FA Targeted Liposomes

Firstly, we characterized the physical properties of the

dual- versus single-drug-loaded liposomes to

determine whether the drug combination could alter

the physical properties of the liposomal formulation.

Dynamic light scattering (DLS) measurements

showed that the resulting dual-loaded liposomes had

similar mean hydrodynamic diameters as the single-

loaded liposomes. Therefore, the effect of drugs on

liposome particle formation is negligible (Qiu, 2016).

Next, we determined the encapsulation efficiency

or loading yield of the liposomes. Particle size and

PDI are important characteristics of drug-laden

liposomes that determine the release kinetics of the

drug, as shown in the table 4. Single- and dual-loaded

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430

liposomes were dissolved in the release medium to

gradually release the encapsulated drug (Dox and/or

PTX). DOX and PTX concentrations were quantified

by fluorophotometer and/or HPLC, respectively. The

results of in vitro drug release assays showed that this

liposome has slow and linear slow-release kinetics for

DOX and PTX, similar to that of single drug

liposomes. These results confirm the ability of the

method to load different hydrophobic drugs into the

same nanoparticles with efficient drug loading rates

and sustained drug release profiles (Wang, 2016; Liu,

2014; Kurbacher C M,1996)

Table 4. Physico-chemical characterization of different liposomes formulations (Wang, 2016).

Sample

Particle size

(nm)

PDI

Zeta

potential

(

)

DLC of

PTX (%)

DLC of

DOX (%)

DEE of

PTX (%)

DEE of

DOX (%)

LP 126.8±3.3 0.14±0.02 +45.3±2.8 NA NA NA NA

PTX LP 125.6±3.5 0.15±0.03 +37.6±2.6 18.6±2.0 NA 82.5±4.3 NA

DOX LP 128.3±3.4 0.12±0.03 +34.0±2.4 NA 10.3±1.6 NA 85.5±4.0

PTX-DOX

129.5±4.4 0.19±0.06 +26.8±3.3 12.5±1.8 9.6±1.2 81.7±4.6 83.6±4.3

* NA means not available.

3.2 In Vitro Release Kinetic of

Liposomes

The DOX and PTX release behavior of liposomes

was evaluated by phosphate-buffered saline (PBS)

dialysis at different pH values (7.4 and 5.5). Among

them, the release of DOX was strongly influenced by

the ambient acidity. At pH 5.5, about 79% of DOX

was released; at pH 7.0, less than 23% was released,

as illustrated in Figures 2 and 3. To summarize, we

present a robust combination chemotherapy approach

that encapsulates two different types of antitumor

therapeutics into a specific liposome formulation

through a controlled 1:1 molar ratio. We

demonstrated that liposomes could release Dox and

PTX in vivo at predefined ratios of loaded drugs and

induce synergistic effects in tumor cells. Such

targeted reactive release liposomes have the superior

ability to act as drug carriers, providing a controlled

and sustainable spectrum of Adriamycin drug release

with improved antitumor activity.

Figure 2: In vitro release of DOX in DOX loaded liposome and DOX/PTX co-loaded liposome at pH 7.4 and 5.5 buffer (Lv,

2014; Wang, 2014).

Design of Targeted and Release Controlled Liposome for Paclitaxel and Doxorubicin Combination in Breast Cancer Therapy

431

Figure 3: In vitro release of PTX in PTX loaded liposome and DOX/PTX co-loaded liposome at pH 7.4 and 5.5 buffer (Lv,

2014; Wang, 2014).

3.3 Stability Studies in Human Plasma

The particle size of liposomes was determined by

dynamic light scattering at 37℃ for 24h to

characterize liposome stability. No significant

precipitation of liposomes with plasma proteins was

observed, demonstrating good stability of liposomes.

3.4 Cellular Uptake Behavior of the

Dual Drug Loaded Liposomes

The cellular uptake behavior of DP-FA-FITC-LPs in

MDA-MB-231 and MCF-7 cells was studied using

confocal laser scanning microscopy (CLSM). Nuclei

were stained with DAPI (blue) and FITC (green)

labeled liposomes were used for subcellular

observation. After 3 h incubation, green fluorescence

was observed in the cells. When the incubation period

was increased to 6 h, the uptake of liposomes by the

cells was enhanced and the green fluorescence was

widely distributed in the cytoplasm, indicating that it

could be successfully internalized by the tumor cells

through endocytosis (Marinello P C, 2019)

3.5 In Vitro Cytotoxic Studies

The semi-inhibitory concentration values (IC50

values) for D-LPs and P-LPs were higher than those

for free DOX and free PTX, respectively, as

demonstrated in Tables 5 and 6. The reasons may be

related to the different cellular uptake pathways of

free drug and drug-loaded nanoparticles, as well as

the different modes of controlled release of drug-

loaded nanoparticles. In cell culture medium, most of

the free drugs can show their effects rapidly after

being transported into the cells by passive diffusion.

In contrast, drug-loaded nanoparticles are mainly

taken up by cells through the endocytic pathway and

exert antitumor activity after the drug molecules are

released from the nanoparticles.CI values below,

equal to or above 1 indicate synergistic, additive or

antagonistic effects, respectively. The calculated

CI50 of free PTX or DOX were greater than one,

indicating that they had no synergistic effect.

Nanoparticles had a significant synergistic effect

with CI50 values less than one, indicating that DOX

and PTX coadministration was significantly better

than free drug coadministration.

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Table 5: IC50 of free drug and drug loaded liposomes on MCF-7 cell (Lv, 2014).

Sam

les IC50 of PTX IC50 of DOX IC50 of combination

Free PTX 0.151±0.060 NA NA

Free DOX NA 0.132±0.055 NA

PTX LP 0.054±0.010 NA NA

DOX LP NA 0.049±0.012 NA

PTX-DOX LP NA NA 0.017±0.010

* NA means not available

Table 6: IC50 of free drug and drug loaded liposomes on MDA-MB-231 cell (Lv, 2014).

Sam

les IC50 of PTX IC50 of DOX IC50 of combination

Free PTX 0.193±0.067 NA NA

Free DOX NA 0.176±0.069 NA

PTX LP 0.062±0.012 NA NA

DOX LP NA 0.059±0.009 NA

PTX-DOX LP NA NA 0.024±0.006

* NA means not available

4 CONCLUSIONS

In brief, we developed a liposome with folate-

targeting and pH-responsive release capabilities for

DOX and PTX co-delivery. It has sufficient structural

stability, efficient delivery capacity, and good

biocompatibility to show its potential to deliver

antitumor drugs by intravenous injection. FITC-

labeled this liposome can be absorb by MDA-MB-

231 and MCF-7 tumor cells and has a synergistic

inhibitory effect on tumor cells. It has high tumor

accumulation, significant tumor suppression

efficiency, and reduced systemic toxicity in vivo.

Thus, our co-delivered liposomes are likely to

achieve excellent results in the treatment of human

breast cancer and also to provide additional design

and value or combination therapy in other diseases. In

the future, when the final dose-dependent response of

the two anticancer drugs, the optimal concentration of

anticancer effects, and the application of this strategy

in the treatment of different tumors s are clearly

investigated, clinics will be able to use lower

concentrations of conventional drugs to provide

better therapeutics and reduce patient suffering.

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