DGIST Scholar: Functionalized magnetotactic bacteria with anti-cancer drug for improved bio-distribution in cancer therapy

Title

Functionalized magnetotactic bacteria with anti-cancer drug for improved bio-distribution in cancer therapy

DGIST Authors

Richa Chaturvedi ; CheolGi Kim ; Daeha Seo

Advisor

김철기

Co-Advisor(s)

Daeha Seo

Issued Date

2024

Awarded Date

2024-02-01

Citation

Richa Chaturvedi. (2024). Functionalized magnetotactic bacteria with anti-cancer drug for improved bio-distribution in cancer therapy. doi: 10.22677/THESIS.200000729081

Type

Thesis

Description

Magnetotactic bacteria; chemotherapeutic agent; stealth material; phagocytosis; drug delivery; lenalidomide; cytotoxicity

Abstract

The most commonly used chemotherapeutic agents for cancer treatment exhibits a poor performance due to the multidrug resistant properties of tumor cells and poor efficacy of these treatment drug. Along with all the above mentioned issues the drug delivery carriers loaded with drugs also phases an early removal from the system before reaching the affected target remains an area of interest for the researchers. To overcome this widespread issue, one effective approach based on physiological differences between normal tissue and the tumor tissue has been developed, which will help in improving the efficacy of anticancer drugs. The formation of this drug delivery system is based on a pH sensitive release of drug in the cancer cell moiety as it contains the drug which is soluble in acidic environments. Along with many other methods used to attain the above mentioned requirements, the usage of a self-propelled bacteria called as magnetotactic bacteria is proved to be a promising solution. The bacterial surface has been bio functionalized and in-turn they act as potential drug delivery carrier. In the present study, we have developed a drug carrier by three step process in which, first, the bacterial surface has been functionalized by a biotinylated PEG which allows the bacteria to escape the phagocytosis process as they become a stealth material and reach the affected area of interest with the drug load. In the next step, the anti-cancer lenalidomide drug has been PEGylated to improve its solubility and lastly, both the complexes have been attached by the streptavidin-biotin conjugation. The whole reaction process makes it a sandwiched type reaction. The bond formation, bacterial viability upon treatment with drug was investigated. The pH dependent release and the cytotoxicity of different cell lines were also examined. The development of this drug delivery carrier was based on targeted and efficient drug delivery process to the cancer cell moiety without damaging the viability of normal cell line. Also, these drug delivery carriers loaded with drugs focuses mainly on an easy, time-saving, and stable technique of drug loading and drug releasing functions that makes it a strong and reliable agent. Keywords: Magnetotactic bacteria; chemotherapeutic agent; stealth material; phagocytosis; drug delivery; lenalidomide; cytotoxicity|암 치료에 가장 일반적으로 사용되는 화학요법제는 종양세포의 다약제 내성 특성과 약물의 낮은 효능으로 인해 성능이 떨어진다. 또한, 약물을 탑재한 약물 전달체가 표적 위치에 도달하기 전에 면역 시스템에 의해 조기 제거되는 문제를 극복하는 것도 연구자들의 관심 영역으로 남아 있다. 이러한 광범위한 문제점을 극복하기 위해 정상 조직과 종양 조직의 생리학적 차이에 기반한 효과적인 접근법이 개발되었다. 이 접근법은 암세포 주변의 산성 환경에서 용해되는 약물을 활용하여 pH 조건에 민감한 약물 전달 시스템의 형성에 기반한다. 위의 요구 사항을 달성하기 위해 자기주성 박테리아의 사용이 유망한 해결책으로 입증되었다. 박테리아 표면은 생물학적 기능화를 통해 잠재적인 약물 전달체로 작용할 수있다. 본 연구에서는 세 단계 공정을 거쳐 자기주성 박테리아를 활용한 약물 운반체를 개발하였다. 첫 번째로 식작용을 피하고 약물을 표적 위치까지 전달할 수 있도록 박테리아 표면을 biotin-PEG 로 기능화한다. 두 번째는 항암제인 lenalidomide 약물을 PEG 화하여 용해성을 높이고, 마지막으로 두 복합체를 streptavidin-biotin 결합을 통해 샌드위치 형태의 구조로 연결하였다. 본 연구에서는 개발된 약물 운반체의 박테리아 생존율, 결합 구조, pH 의존적인 약물 방출 특성, 그리고 다양한 세포주의 세포 독성을 조사하였다. 본 약물 전달체는 정상 세포주의 생존력을 손상시키지 않으면서 암세포 부분을 표적하는 효율적인 약물 전달 과정을 기반으로 하였다. 또한, 약물 로딩과 약물 방출 기능을 쉽고, 빠르고, 안정적으로 수행하여 강력하고 신뢰할 수 있는 약제로 만드는 데 초점을 두고 있다.

Table Of Contents

List of Contents

Abstract · i
List of contents · ii
List of figuresⅴ

1. Introduction.1
2. Literature review7
2.1. Targeted cancer therapy methods8
2.1.1. Passive targeting8
2.1.2. Active targeting.8
2.1.3. Magnetic targeting9
2.2. Role of bacteria in cancer therapy.10
2.3. Magnetotactic bacteria11
2.4. Magnetospirillum magneticum (AMB-1) 12
2.5. Usage of biotinilyzed polyethylene glycol with NHS ester13
2.6. Cancer cells microenvironments and its extracellular pH15
2.7. Lenalidomide drug (LENA)17
2.8. Bioconjugation techniques18
2.8.1. Carbodiimide based chemistry.18
3. Experimental Methods21
3.1. Culture conditions of magnetotactic bacteria (AMB-1).22
3.2. Characterization of Magnetotactic bacteria and its magnetosome.22
3.3. X-ray diffraction (XRD).23
3.4. Attachment of PEG-Biotin to MTB24
3.5. Formation of MTB-Lena complex25
3.6. Characterization of MTB/PEG-biotin a drug delivery agent
loaded with drug26

3.6.1. Scanning Electron Microscopy26
3.6.2. Fluorescence assisted cell sorting (FACS)27
3.6.3. Confocal laser scanning microscopy (CLSM).28
3.7. Evaluation of bacterial viability28
3.8. Cell viability and Imaging30
3.8.1. THP-1 cell.30
3.8.2. MCF-7 cells30
3.8.3. CCK-8 based cell viability assay and Imaging31
3.9. THP-1 cell association33
3.10. pH based drug release.33
4. Results and Discussions
4.1. Preparation of drug delivery carrier and its stealth property
assessment.35
4.1.1. Characteristics of magnetotactic bacteria and magnetosomes..35
4.1.2. Formation of MTB/PEG-biotin complex38
4.1.3. Assessment of bacterial viability for MTB/PEG-biotin.41
4.1.4. Biological application of MTB/PEG-biotin complex44
4.1.5. Cell association assessment46
4.2. Loading of anti-cancer drug on the carrier and its pH
triggered release49
4.2.1. Formation of MTB-LENA complex49
4.2.2. Confirmation of drug attachment with the drug carrier50
4.2.3. MTB viability with lenalidomide drug.52
4.2.4. pH triggered drug release of lenalidomide from drug carrier55
4.2.5. Cytotoxic effect of drug carrier and drug release57
5. Conclusion and future outlook61
6. References64
List of publications75

List of figures:

Figure 1: Magnetotactic bacteria: Assests and Perspectives
Figure 2: Types of targeted drug delivery in cancer treatment [39]
Figure 3: An illustration of magnetic targeting [43]
Figure 4: Advantages of magnetotactic bacteria
Figure 5: TEM images which show the morphological structure of MTB and its magnetic
particles formed inside the bacterial body.
Figure 6: Advantages of magnetotactic bacteria with PEG-biotin for cancer treatment
Figure 7: Cancer cells extracellular microenvironments [53]
Figure 8: Chemical structure of Lenalidomide
Figure 9: Schematic for carbodiimide chemistry
Figure 10: Schematic representation of attachment of PEG-biotin with the magnetotactic
bacteria
Figure 11: Flow cytometer instrument
Figure 12: MTT reaction mechanism to turn into formazan dye
Figure 13: (a) TEM image showing the helical structure of MTB; (b) MTB with magnetosome
particles; (c) histogram showing the size distribution of magnetosomes.
Figure 14: An elemental mapping analysis to check the iron component in magnetotactic
bacteria
Figure 15: Energy dispersive X-ray (EDX) spectrum of magnetotactic bacteria
Figure 16: XRD patterns of iron oxide (Fe2O3) obtained from the magnetosomes of
magnetotactic bacteria
Figure 17: Confocal microscopy images of FITC-labeled MTB/PEG-biotin complexes.
Photomicrographs show the bright field, fluorescence, and overlay confocal microscope
images. Unlabeled bacteria are used as controls. Inset shows unlabeled bacteria (controls) that

do not exhibit any fluorescence.
Figure 18: Characterization of the MTB/PEG–biotin complex. (a) FACS histograms and (b)
density plots obtained from bare MTB, FITC-labeled PEG-biotin, and the MTB/PEG-biotin
complex show that PEG–biotin is uniformly attached to MTB, and can be distinguished from
bare MTB by fluorescence intensity.
Figure 19: Characterization of the MTB/PEG–biotin complex. (a) FE-SEM images of bare
MTB. MTB/PEG–biotin complex formation when the PEG–biotin concentration used was (b)
10 mM and (c) 20 mM.
Figure 20: Assessment of bacterial viability. (a) Quantification of the percentage of bacterial
viability where MTB was treated with different concentrations of PEG–biotin (5, 10, 15, and
20 mM) after different time intervals (6, 12, and 24 h). Results indicate that a higher
concentration of PEG–biotin (20 mM) kept the bacteria alive even after long incubation times.
Data are presented as mean ± SD, n = 3. Statistical significance (with p < 0.01 and p < 0.001)
is indicated with ** and ***, respectively, for datasets that are significantly different between
6, 12, and 24 h.
Figure 21: Assessment of bacterial viability. (a) Microscopic fluorescent live/dead images
for live bacteria stained with SYTO stain; (b) bacterial cells stained with propidium iodide; (c)
merged images for live/dead bacterial cells.
Figure 22: Cell viability assay. THP-1 cells were incubated for (a) 24 and (b) 48 h at 37 °C
with different concentrations of PEG–biotin for the MTB/PEG–biotin complex, and compared
to bare MTB and bare PEG–biotin, using the CCK-8 assay. The cell viability was found to be
concentration and time-dependent. The results indicate that cells survive long after exposure to
bare PEG–biotin and also the THP-1 cells survive long after incubation with MTB/PEG-biotin
complex as compared to bare MTB. Statistical analysis was performed using two-way ANOVA.
Data are presented as mean ± SD, n = 3. Significant difference was considered for * p < 0.05,

** p < 0.01, *** p < 0.001, and non-significant (ns) for p > 0.05.
Figure 23: Bright-field and fluorescence microscopic images of live/dead assay: live/dead cell
viability assay of THP-1 cells treated with bare MTB and the MTB/PEG–biotin complex. The
cells were incubated for 24 h and then stained with a double-staining kit (calcein-AM and
ethidium dimer-1). The live and dead cells exhibited green and red fluorescence, respectively.
THP-1 cells treated with bare MTB showed 50% live cells, whereas cells treated with the
MTB/PEG–biotin complex displayed 80–90% live cells.
Figure 24: THP-1 cell association with different concentrations of PEG-biotin combined with
MTB and bare MTB after 24 h of incubation at 37 °C. Results indicate that THP-1 cells
incubated with bare MTB showed a very high cell association (12%), whereas cells treated
with the MTB/PEG–biotin complex displayed relatively less cell association. Data were
normalized to untreated cells. Error bars, mean ± SD, n = 4.
Figure 25: Schematic representation of formation and working mechanism of MTB-LENA
Figure 26: Characterization of MTB-LENA complex. Confocal microscopy images show the
bright field and fluorescence images of the FITC labeled MTB-Lena complex. Unlabeled MTB
are used as control.
Figure 27: Characterization of MTB-LENA complex. FACS histogram acquired from
MTB/PEG-biotin complex and MTB-Lena complex show that drug lenalidomide (LENA) is
uniformly attached to the MTB/PEG-biotin complex in a sandwiched manner.
Figure 28: FT-IR spectrum of PEG-Biotin and PEG-Biotin-LENA drug.
Figure 29: Bacterial viability. (a) Analysis for the percentage of bacterial viability where MTB
treated with different concentrations of Lenalidomide (LENA) drug (10, 20, 30, 40, and 50
µg/mL) for different time intervals (6, 12, and 24 hours). Results indicate that there was a
minimal amount of bacterial cell damage even after an incubation of 24 hours and most bacteria
were alive with even higher concentrations of the drug. Data are represented as mean ± SD,
viii

n=3. Statistical significance (with P < 0.01 and P < 0.001) are indicated with * and *** marks
respectively for data sets that are significantly different between 6, 12, and 24 hours.
Figure 30: Bacterial viability. (a) Fluorescent microscopic images of live bacterial cells stained
with SYTO stain, (b) Microscopic images of dead bacterial cells stained with propidium iodide.
(c) Merged image of Live/Dead bacteria cells.
Figure 31: Schematic for pH triggered drug release from the drug delivery carrier
Figure 32: Lenalidomide release over time from MTB-LENA complex at different pH (4.5,
5.5, 6.5, and 7.4). Result indicates that most amount of drug (0.05 µg/mL) was released at the
pH 4.5 and the least amount of drug released at a basic pH 7.4. This show that the lenalidomide
drug is soluble in acidic pH. Also, the maximum drug released at 3 hours of incubation.
Figure 33: MCF-7 and THP-1 cell viability upon drug release. (a) THP-1 cells were incubated with
MTB-LENA complex and only MTB/PEG-biotin complex for 12 and 24 hours. Results indicate that
almost 80-90% of THP-1 cells were alive even after the incubation of 24 hours. (b) MCF-7 cells upon
treatment with MTB-LENA complexes show that almost 60% of cells were killed after 24 hours of
incubation whereas, MCF-7 cells treated with MTB/PEG-biotin complex indicate that only 20-30% of
cells were killed. Error bars, mean ± SD, n =3.
Figure 34: Fluorescence microscpic images of Live/Dead cell viability of MCF-7 and THP-1
cells incubated with MTB/PEG-biotin complex for 24 hours. Results indicates that both the ce
lls show high viability (80% - 90%) with only drug carrier as there was no drug attached to
it. Scale bar=100 µm.
Figure 35: Fluorescence microscopic images of Live/Dead cell viability of MCF-7 and THP-1 cells
treated with MTB-LENA complex. The cells were incubated for 24 hours. The live and dead cells
exhibited green and red fluorescence respectively. MCF-& cells treated with MTB-LENA complex
show almost 60% dead cells due to drug release whereas, THP-1 cells treated with the complex did not
show much changes in viability. Scale bar = 100µm.

URI

http://hdl.handle.net/20.500.11750/48038
http://dgist.dcollection.net/common/orgView/200000729081

DOI

10.22677/THESIS.200000729081

Degree

Doctor

Department

Department of Physics and Chemistry

Publisher

DGIST

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