Multifaceted approach for cancer therapeutics

Siddhartha Sankar Ghosh, Department of Biosciences & Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India for the award of the degree of Doctor of Philosophy. It is hereby declared that this thesis entitled “A Multifaceted Approach for Cancer Therapeutics” has been submitted by Bidkar Anil Parsram (Role No. to the Indian Institute of Technology Guwahati for the award of the degree of Doctor of Philosophy.

Development of novel nanocarriers (selenium-

Establishment of membrane coated

Targeted drug delivery using membrane coated

This chapter describes the characterization and functional assays of drug-loaded NPs on monolayer and hypoxic spheroids. In Chapter 5, the therapeutic efficacy of transferrin-bound red blood cell membrane-coated PLGA NPs was studied for the delivery of doxorubicin and methylene blue for chemo- and photodynamic therapy.

Selenium dioxide XRD: X-ray diffraction SDS-

Scheme 3.1 Schematic representation of the combined therapeutic module consisting of folic acid targeted selenium nanoparticles and

Schematic representation of the preparation of the RBC membrane-coated PLGA NPs for hypoxia-targeted therapy of cancer

Schematic representation of the therapeutic approach by combining chemo- and photodynamic therapy

Conventional strategies of cancer treatment

Application of cytotoxic drugs to cancer cells results in the re-establishment of cell signaling, resulting in apoptosis-mediated cell death.6. However, a high dose of radiation causes DNA strand breaks to the extent that cells cannot repair such damage.9 A laser beam can be projected directly onto the affected area.

Nanocarriers in Cancer Therapy

Targeted drug delivery

Passive targeting
Active targeting

Binding of trastuzumab to the HER2 receptors results in selective internalization of the drug conjugate. In addition to the direct conjugation of the drug to the target moiety, surface-modified nanocarriers are also used for targeting.

Types of nanocarriers for cancer therapy .1 Polymeric nanocarriers

Inorganic NPs
Protein-based nanocarriers
Carbon-based nanomaterials
Liposomes
Dendrimers

Animal or plant proteins are a great source of starting material for the preparation of nanocarriers. Examples of branched polymer dendrimers include PAMAM dendrimer, Poly(propylene imine) (PPI) and Poly-l-lysine (PLL).

Selenium

Involvement of Se in Cancer prevention
Selenium nanoparticles in cancer treatment
Selenium NPs as drug carrier

Along with these compounds, Se nanoparticles (SeNPs) have also gained the attention of researchers due to their cytotoxic properties. Similarly, green synthesis of SeNPs involves the use of microorganisms or biomolecules based on plant extracts.

Membrane-based drug delivery systems

Stem cells as a drug carrier
Tumor cell-derived particles
Exosomes
RBC membranes for drug delivery

The fluorescent labeling of these NPs by ruthenium complexes yields the fluorescent NPs that can be tracked within the cells.59 Several studies have shown that the synergistic response of the loaded drug and SeNPs represents an effective treatment strategy. Recent studies have reported the synthesis of the stimuli-responsive nanocarriers for drug delivery upon various stimuli including temperature and pH.60.

Some important anticancer drugs and signaling pathway blockers .1 Paclitaxel

MAP Kinase pathway inhibitors
Curcumin
Tirapazamine
Doxorubicin
Methylene blue

MEK proteins are phosphorylated by RAF, which causes phosphorylation of the ERKs, which is a next protein in the cascade. The growth of the tumor mass leads to the formation of an inner hypoxic core surrounded by oxygen-rich cells.

Evaluation of therapeutic potential on spheroids

MCS are used as a model system to study the targeting and penetration ability of the nanocarriers due to their complex nature. Studies conducted to compare the size-dependent penetration of the gold NPs confirmed that the permeability of the NPs is inversely proportional to size (Figure 1.13).100.

Key areas and scopes

Salient points of this thesis

Successful conjugation of transferrin molecules to RBC membrane-coated NPs resulted in high accumulation of NPs and drugs in cancer cells. Increased delivery of doxorubicin and methylene blue to cancer cells resulted in significantly improved chemo- and photodynamic therapy.

A method for coating PLGA NPs with RBC membrane was developed to prepare uniform membrane-coated NPs. Passive and active drug targeting: Drug delivery to tumors as an example; Springer, Berlin, Heidelberg, 2010; p. 3–53. Opportunities and challenges for using tumor spheroids as models for testing drug delivery and efficacy.

Introduction

Experimental Section .1 Chemicals

Cell Culture
Synthesis and characterization of selenium nanoparticles (SeNPs)
Cellular uptake of SeNPs
Loading of paclitaxel (PTX) onto SeNPs and its release
Cytotoxicity assay
Cell cycle analysis
Annexin V-PI apoptosis assay
DCF-DA assay
JC-1 staining for mitochondrial membrane potential (MMP) JC-1 is a cationic dye which enters in mitochondria and forms aggregates
Caspase-3 assay
Field emission scanning electron microscopy (FESEM)

Cells were then treated with RNase A, stained with PI and analyzed in a FACS Calibur (BD Biosciences, NJ, USA). Stained cells were incubated for 10 minutes at room temperature and analyzed with a flow cytometer (Cytoflex, Beckman Coulter). Then, cells were incubated for 30 min at room temperature, pelleted by centrifugation and resuspended in PBS for further analysis in a FACS Calibur (BD Biosciences, NJ, USA).

Results and Discussions

Synthesis and Characterization of SeNPs
Internalization of the SeNPs by cells
Drug Loading and release
Cell Viability assays
Cell Cycle analysis
Apoptosis study
Mechanism of Apoptosis

The extent of apoptosis in PTX-SeNPs treated HeLa and MCF-7 cells was quantified by Annexin V-PI assay. FESEM analysis of PTX-SeNPs treated cells (Figure 2.8) also showed characteristic membrane blebbing indicating apoptosis in treated cells. However, the appearance of green fluorescence in treated HeLa and MCF-7 cells indicated depolarization of mitochondria as a result of PTX-SeNPs treatment.

Conclusion

Selenium sensitizes MCF-7 breast cancer cells to doxorubicin-induced apoptosis through modulation of phospho-akt and its downstream substrates. Taxol-induced cell cycle arrest and apoptosis: dose-response relationship in lung cancer cells of different wild-type P53 status and under isogenic condition. In Chapter 3, a selective and targeted therapy was designed to destroy MDAMB231 (breast cancer) and A375 (melanoma) cancer cells.

Introduction

Established synthesis protocols allow the preparation of SeNPs suitable for various purposes, while targeting can be achieved by conjugation of the appropriate targeting ligand on the polymer used to stabilize the SeNPs. MDA-MB-231 (breast cancer) cells have G13D and G464V mutations in the RAS and BRAF genes, respectively, while A375 (melanoma) cells have V600E mutations in BRAF. 14, 15 These mutations result in constitutive activation of the MAPK pathway leading to uncontrolled proliferation and cancerous growth of MDA-MB-231 and A375 cells. Flow cytometric and confocal microscopy experiments were performed to study the internalization of FA-SeNPs by cancer cells, while viability assays were performed to evaluate the therapeutic potential of the present combined module.

Scheme 3.1: Schematic representation of the combined therapeutic module consisting of folic acid targeted selenium nanoparticles and PD98 for inducing

Experimental Section .1 Chemicals

Cell culture
Conjugation of folic acid to chitosan
Synthesis and characterization of FA-SeNPs
Gene expression studies
Mechanism of the internalization of the FA-SeNPs
Cell proliferation assays
Annexin V-FITC PI assay
Western Blots
Surface morphology by FESEM
Nucleus staining and comet assay
DCFDA and JC-1 staining
FA-SeNPs uptake, growth and viability of MDA-MB-231 spheroids

Similar MTT assays were performed to study the effect of DMSO (solvent for PD98), where cells were pre- or co-treated with the same volume of DMSO (0.1% v/v) along with FA-SeNPs instead of PD98. Cells were processed according to the supplier's protocol and analyzed in a Cytoflex instrument (Beckman Coulter). Then the cells were treated (PD98FA-SeNPs@20 and PD98+FA-SeNPs@20), fixed in 70% ethanol, treated with RNase and stained with PI.

Results and Discussions

Preparation and characterization of FA-conjugated SeNPs
FA-SeNPs uptake by cancer cells
Cell viability assay
PD98 and FA-SeNPs combinedly induce apoptosis
Inhibition of spheroid growth by combined treatment of PD98 and FA-SeNPs

Because PDI of FA-SeNPs prepared at all FA-CS concentrations in the . Furthermore, Z-stacked images of the treated cells (Figure 3.7A, B) confirmed the successful internalization of RhB-loaded FA-SeNPs. The reason behind the synergistic behavior of PD98 and FA-SeNPs was the inhibition of MAPK pathway in MDA-MB-231 and A375 cells.

Conclusion

On the other hand, increased PI fluorescence (red) accompanied by decreased Calcein-AM green fluorescence in PD98FA-SeNPS@40 and PD98+FA-SeNPs@40 treated samples suggest the presence of dead cell population . Surprisingly, synergistic action was observed in the combined treatment at a very low concentration of both FA-SeNPs and PD98. Mechanistic insights revealed that FA-SeNPs functioned through ROS production and mitochondrial depolarization, while PD98 inhibited MAPK signaling required for uncontrolled cell division in cancer cells.

Simple preparation of doxorubicin- and folic acid-conjugated carbon nanotubes@Poly(N-vinylpyrrole) for targeted synergistic chemo-photothermal cancer therapy. Folic acid-conjugated chitosan for targeted delivery of SiRNA to activated macrophages in vitro and in vivo. Enhancement of paclitaxel-induced apoptosis by inhibiting the mitogen-activated protein kinase pathway in colon cancer cells.

Introduction

Clinical studies on patients with hypoxic tumors have shown an increased risk of metastasis and resistance to chemotherapy and radiotherapy.1,2 During tumor development, the supply of oxygen and nutrients to the tumor core decreases causing a hypoxic environment, which leads to the release of angiogenic activators. to activate the growth of new blood vessels around the tumor site.3 Another noteworthy point is that in most cases it is not the primary tumor that causes mortality; but the metastasis to a secondary site becomes the key deciding factor. However, distribution of the drug molecules in normal body tissues leads to systemic toxicity causing serious side effects and the possibility of chemo-resistance by various cancer cells to multiple anticancer drugs. The influence of RBC membrane coating on the internalization of the drug-loaded nanoparticles was assessed in.

Experimental Section .1 Materials

Cell Culture
Formation of RBC membrane-coated PLGA NPs
Characterization of the Nanoparticles
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE)
Biocompatibility and Hemocompatibility analysis
Drug Loading, release and nanoparticle uptake studies
Cell viability assays
Apoptosis & Cell death analysis
Western blotting
Spheroid formation and Viability assays
Microscopy of the Multi-Cellular Spheroids
Flow Cytometry of the MCSs
Comet assay
Realtime PCR for gene expression
Wound healing assay

For the release study, 5 mL of drug-loaded NPs (Cur+TPZ@RB) stored in a dialysis bag were deliberately mixed in the presence of PBS (100 mL). To confirm the NP internalization pathway, cells were stained with Lysotracker red. The uptake of Cur+TPZ@PL and Cur+TPZ@RB into MCF-7 as well as A375 MCSs was studied by microscopy.

Results & Discussions

Preparation, Characterization and Biological Response of RBC- NPs
Preparation of Drug-Loaded RBC-NPs
Improved Internalization of Cur+TPZ@RB
Endocytosis pathway study
Macrophage escape of the RBC-NPs
Synergistic Antiproliferative Action of Cur+TPZ@RB in Monolayer Culture
Apoptosis and cell death analysis study
Enhanced uptake and cell death in hypoxic 3D multicellular spheroids

As shown in Figure 4.8B, long-term storage (2 weeks) at 4 °C did not affect the hydrodynamic diameter of Cur+TPZ@RB. Furthermore, fluorescence imaging by confocal laser scanning microscope (CLSM) (Figure 4.10A, B) revealed that Cur+TPZ@RB NPs were successfully internalized and localized mostly in the cytoplasm of the MCF-7 and A375 cells. The results confirmed the presence of the Cur fluorescence in the lysosome, suggesting that Cur+TPZ@RB followed the endocytosis pathway for internalization.

Cur+TPZ@RB induced cell death observation by Calcein-AM PI staining
ROS mediated DNA damage in hypoxic conditions
Inhibition of Epithelial to Mesenchymal Transition

The comet assay result (Figure 4.22C) showed that Cur+TPZ@RB treatment of MCF-7 spheroids resulted in DNA strand breaks in treated cells. After this, the expression of vimentin and fibronectin was studied in MCS treated with Cur+TPZ, Cur+TPZ@PL and Cur+TPZ@RB. Surprisingly, only 22% wound repair was observed in treated cells due to reduced cell migration due to treatment with Cur+TPZ@RB.

Conclusions

The reduced migration and downregulation of mesenchymal markers of the treated cells make the Cur+TPZ@RB system an effective candidate for drug delivery to solid tumors. Uptake studies in A375 and MCF-7 monolayer cells indicated that the internalization of the Cur+TPZ@RB occurred via endocytosis with a specific preference for the caveolin-mediated pathway. The generation of the reactive oxygen species, DNA damage and activation of caspases were found to be the major drivers of apoptotic cell death in Cur+TPZ@RB-treated cancer cells.

Further investigations revealed that Cur+TPZ@RB exhibited a synergistic cell death response in A375 and MCF-7 cell monolayers and spheroids. In addition, Cur+TPZ@RB was able to reduce the elevated levels of mesenchymal markers including vimentin and fibronectin in MCF cell spheroids, suggesting a reduction in EMT with improved therapeutic efficacy of anticancer drugs. Curcumin-induced rapid generation of reactive oxygen species (ROS) leads to caspase-dependent and -independent apoptosis in L929 cells.

Chapter 5 demonstrates the therapeutic efficiency of the transferrin bound RBC membrane-coated PLGA NPs to deliver doxorubicin and methylene blue

Introduction

Therefore, most cancer treatment includes drug combinations as well as the use of radiotherapy.6,7 In this domain,. Along with specific action of Dox, red laser irradiation of the treated cells resulted in the production of ROS due to MB inside the cells. Anti-proliferative effects of the laser-irradiated TF-DoxMB (TF-DoxMB+L) were investigated on a monolayer (2D) as well as the 3D spheroids.

Experimental Section .1 Materials

Preparation of PLGA NPs and RBC membrane coated PLGA NPs (RB-NPs)
Preparation of the TF-conjugated RBC membrane coated PLGA NPs (TF-RB-NPs)
Characterization of the PLGA-NPs, RB-NPs, and TF-RB-NPs All the prepared NPs were characterized for their hydrodynamic diameter
Western blot analysis
Drug loading and release study
Uptake study of the NPs

The amount of TF bound on the RBC membrane surface was determined by Bradford assay. The prepared TF-conjugated membrane-coated NPs (TF-Dox-NPs) were named as TF-0.02- to TF0.4-Dox-NPs based on the membrane used for coating (Table 5.1). RBC membrane proteins from intact RBC, RBC membrane pellet and RBC membrane coated NPs were isolated in RIPA buffer.