In functional protein. In this way, genes

In Hong Kong, lung cancer is the first leading cause of cancer death in recent years. According to the statistics published by Department of Health, 4,031 patients died due to lung cancer in 2015, which contributed to 28.2% of all types of cancer death. Among these death cases, men account for a much higher crude death rate than women. Therefore, lung cancer is the “first killer” in Hong Kong population.

There is a variety of causes leading to lung cancer. The major one is tobacco smoking and inhaling second-hand smoke. According to the data provided by Hong Kong Integrated Oncology Centre, approximately 90% of male lung cancer patients were found to be frequent smokers. It is believed that smoking sharply increases the risk of developing lung cancer.

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Currently, common treatments for lung cancer include chemotherapy, radiotherapy and surgery1. However, the therapeutic efficacy of existing treatment is quite limited. It is mainly because these therapies induce severe side effects. Despite killing the cancer cells, normal cells may also be killed at the same time. Therefore, these therapies lack specificity and they are considered to be “aggressive”. Besides, cancer cells may develop drug resistance to the cytotoxic drugs, in turn lowering the therapeutic efficacy. Due to these barriers, there is a tremendous difficulty in treating lung cancer effectively.

 

Recently, some studies have explored pulmonary delivery of RNA interference (RNAi) molecules as a new treatment for many diseases including lung cancer2. Genes that are associated with tumor cell growth and drug resistance are potential targets for gene silencing through RNAi. RNAi achieves specific post-transcriptional gene silencing, by preventing the translation of target mRNA into functional protein. In this way, genes which are associated with tumor cell growth can be suppressed. It can be used as an effective therapeutic agent to control the rapid growth of tumor. Some research found that the epidermal growth factor receptor (EGFR) is a validated therapeutic target in non-small cell lung cancer (NSCLC)3,4,5. EGFR is a transmembrane protein located on cell surface. It is a receptor responsible for binding with a group of growth factors from the extracellular environment. In patients with NSCLC, over-expression of EGFR was observed due to mutation. Activated EGFR follows a series of signaling cascade to induce cell proliferation, angiogenesis, metastasis and inhibit apoptosis. This promotes the massive growth of cancer cells. Therefore, EGFR is a potential target for gene silencing via RNAi. It is anticipated that by suppressing the activity of EGFR, the spread of NSCLC tumors can be retarded.

Nevertheless, some patients with NSCLC exhibited mutation in the intracellular kinase domain of EGFR. This mechanism is a threonine-to-methionine substitution at amino acid position 790, which is regarded as T790M. Due to this mutation, EGFR can process constitutive activation without ligand binding. As a result, the therapeutic effect of a single agent receptor targeting is barely satisfactory. Usually, EGFR tyrosine kinase inhibitors (TKIs) are used as an adjuvant to treat NSCLC6. Three generations of receptor TKIs have been reported so far3. First generation of EGFR TKIs are reversible inhibitors. They competitively bind to the tyrosine kinase domain of EGFR but not selectively bind to the mutated EGFR. Therefore, their clinical efficacy is limited because of the dose-dependent toxicity resulting from inhibition of wild-type (WT) EGFR. Second-generation of EGFR TKIs are irreversible. They covalently modify the tyrosine kinase domain of EGFR, in turn altering the downstream signaling pathway. However, they are not specific to mutated EGFR, especially the T790M mutated type. Third-generation of EGFR TKIs show the greatest efficacy. They irreversibly bind to EGFR tyrosine kinase domain. In this way, the inhibitors prevent tyrosine phosphorylation of the receptor. Then, the signaling cascade can be terminated. Most importantly, they only work on the T790M mutants, but not the WT EGFR7. Thus, they are highly selective, and they cause less inhibition of WT EGFR. As a result, third-generation of EGFR TKIs exhibit a promising potency with few adverse effects. They can be used clinically to induce a significant shrinkage on NSCLC tumors. With the combination of siRNA molecules and EGFR TKIs, the anticancer efficacy can be greatly enhanced. The below diagram summarizes the inhibitory action of the three developed generations of EGFR TKIs.

In conclusion, RNAi targeting provides a new insight for treating lung cancers more effectively. It is noteworthy to explore this area in order to investigate a novel, safe and effective therapeutic strategy against NSCLC. Eventually, it is anticipated that the research work can be translated into clinical usage to reduce the mortality rate of lung cancers.

 

Hypothesis

It is hypothesized that the combination of small interfering RNA (siRNA) against EGFR and EGFR inhibitory agents can improve the anticancer efficacy, especially NSCLC.

 

Objectives

In this project, there are several objectives:

(i)                 To formulate siRNA into inhalable spray-dried powders through spray drying technique for enhanced pulmonary delivery performance.

(ii)              To perform siRNA transfection targeting the EGFR in human NSCLC cell lines in the combination of EGFR TKIs agents.

(iii)            To analyse siRNA transfection efficiency by various parameters, including the expression of EGFR at mRNA and protein levels, and the proliferation and viability of NSCLC cells.

(iv)             To evaluate the therapeutic effects of the siRNA in combination with TKIs agents in animal models by applying the bio-imaging techniques.

 

 

Methodology

 

1          Formulation of siRNA

In this project, one of the aims is to utilize siRNA as the therapeutic agent targeting EGFR for the treatment of NSCLC. Therefore, the best route of administration is pulmonary delivery for the maximum delivery efficiency of therapeutics. Inhalable spray-dried powders are suitable formulation. Some related studies confirmed that spray-dried powders can improve the aerosolization and dispersion performance with low residue moisture. With these characteristics, majority of the siRNA therapeutics can be delivered to the target sites, in turn enhancing the gene silencing activity. The anticancer efficacy can be therefore improved. As a result, inhalable spray-dried powders are potential formulation for the pulmonary delivery of siRNA in this project.

 

1.1         Spray-drying

Basically, spray-drying is the major technique to formulate siRNA into spherical dry powders for pulmonary delivery purpose. Spray-drying is a method for producing dry powders from liquid or solution. It is accomplished by rapid drying with a hot gas. Spray-drying is an excellent technique to produce fine powders with consistent particle size. At the same time, siRNA can be co-sprayed along with non-viral vectors such as polymer and peptides to enhance the transfection efficiency of siRNA into NSCLC cell lines. Mannitol will be included in the formulation as a bulking agent. Besides, L-leucine can be used as a dispersion enhancer which enhance the aerosol performance of the powder. Finally, the spray-dried particles can be collected by a high performance cyclone and stored in a desiccator. Spray-dried powders can result in a higher efficacy and efficiency for the pulmonary delivery. Apart from these advantages, spray-drying technique generates spherical powders in crystalline form. It offers low residual moisture and excellent aerosol performance. Most importantly, the primitive structure of siRNA can be preserved. Thus, the gene silencing ability performed by siRNA can be retained after the formulation. Indeed, inhalable spray-dried powders exhibit many attractive properties which can eventually improve siRNA therapeutic efficacy. Consequently, this technique is becoming more popular to formulate different therapeutics, such as siRNA, polypeptides and drugs, into inhalable powders. The below diagram illustrates the schematic diagram for the production of the inhalable spray-dried powders via the spray-drying technique.

1.2         Characterization

After the production of the spray-dried powders, it is essential to characterize them by different characterization techniques. These include scanning electron microscopy (SEM), Next Generation Impactor (NGI), gel retardation assay and high performance liquid chromatography (HPLC) etc. With the aid of the first three techniques, the morphology (structure, size and shape etc.) of the spray-dried powders can be observed and evaluated. Apart from that, the integrity of the therapeutic nucleic acid can be examined by HPLC. This ensures that the functional structure of siRNA is not destroyed after the formulation and the therapeutic effects of siRNA can be retained. In fact, siRNA is very vulnerable. Its structure is sensitive to the change in pH, temperature and pressure. Since siRNA is used as the therapeutic agent, it is extremely important to keep their primitive structure. Otherwise, degraded siRNA will no longer induce their gene silencing ability. In addition, the stability of the spray-dried powders can be analyzed by dynamic vapor sorption (DVS) technique. It is used to investigate the influence of humidity on the moisture sorption of the particles. With reference of other similar research, the spray-dried powders produced are found to be porous on the surface8. This characteristic facilitates the spray-dried powders to be lighter in weight. In this way, they can be dispersed along the nasal cavity easily. Additionally, the particles will not aggregate at the bending site. This allows more portion of the spray-dried powders to get down to the lungs. The blow diagram shows some images of the inhalable spray-dried powders obtained from scanning electron microscopy (SEM), prepared by the spray-drying technique.

 

2          In vitro study

The biological effect of siRNA targeting EGFR in NSCLC cell lines will be investigated in combination with different receptor tyrosine kinase inhibitors. As previously mentioned, NSCLC cells are found to exhibit over-expression of the EGFR due to mutation. Activation of the EGFR leads to rapid development of the cancer cells. In order to inhibit this process, siRNA performs the gene silencing effect on the EGFR whereas tyrosine kinase inhibitors target the tyrosine kinase domain of the EGFR to further prevent the downstream phosphorylation signaling of the receptor. This can strengthen the therapeutic effects by regulating the growth of the cancer cells.

 

2.1         siRNA transfection

siRNA transfection is carried out to deliver the gene silencing molecules to the NSCLC cell lines to inhibit the EGFR activity9. Firstly, siRNA is mixed with peptides at an optimal ratio for transfection. Then, the peptide/siRNA complexes are incubated at 37°C for 30 minutes. On the other hand, the NSCLC cells are seeded in a 24-well plate. Next, the peptide/siRNA complexes are prepared in serum-free medium and added in each well for siRNA transfection. The plates are incubated for 3 to 4 hours at 37°C. After that, the transfection medium is replaced with serum-supplemented growth medium. At the same time, EGFR tyrosine kinase inhibitors are added to the plate to inhibit the downstream phosphorylation activity. Eventually, the plate is incubated for 24 hours. After siRNA transfection, the NSCLC cells are suitable for various quantitative assays, such as qRT-PCR, Western blotting and morphological assays. These are common techniques that can be applied to investigate the biological effect of siRNA in combination with different TKIs targeting EGFR in NSCLC cell lines. Here shows the schematic diagram for siRNA transfection procedures.

 

2.2         Biological analysis

After 24-hour incubation, sufficient time should be provided for the performance of the gene silencing effects by siRNA molecules. Together with the tyrosine kinase inhibitors, the activity of the EGFR in NSCLC cells can be altered effectively. This can prevent the rapid growth of the cancer cells. Theoretically, the NSCLC cells will show a significant regression. By performing qRT-PCR and Western blotting, the level of the EGFR mRNA expression can be obtained. In addition, the NSCLC cell growth and cell viability can be assessed using a colorimetric tetrazolium (MTT) assay and fluorimetric detection of resorufin respectively. In addition, the effects of EGFR siRNA and EGFR inhibitory agents on apoptosis and nuclear morphology in the cells can be observed under fluorescent microscopy.

3          In vivo study

To further study the biological action of siRNA and EGFR inhibitory agents, animal model will be used for experiment. It is a more advanced study because not only a single type of cell is being studied. Instead, the entire body system is considered to observe the therapeutic effects. Mice are chosen as the animal model. It can mimic human biological condition and in turn predict the therapeutic effects induced in human body.

 

3.1         Mice lung cancer model development

Mice lung cancer model needs to be developed for experimental treatment. To achieve this, intrabronchial tumor cell implantation can be performed10. Firstly, the mice are anesthetized intraperitoneally. Then, cervical tracheotomy is performed, beginning with a skin incision just above the manubrium sterni and dissecting the muscle layers until uncovering the trachea. At the lung periphery location, 70-100 µL of tumor cell suspension is inoculated. After that, the trachea and skin are closed when the thread. Finally, the tumor cells will be developed in the mice model. High-resolution computed tomography can be used to observe the growth of the tumors in the lungs.

 

3.2         siRNA pulmonary delivery

With the developed inhalable siRNA delivery system, the spray-dried powder can be inhaled into the mice model via an inhaler. Aerolizer can be used as a model capsule-based device because it is a commercially available inhaler with low resistance. Meanwhile, the EGFR TKIs can be injected intravenously into the mice to induce the pharmacological effects. This can inhibit the activity of the over-expressed EGFR. The signaling cascade of EGFR can be terminated. The cancer cells can no longer proliferate and spread rapidly. Eventually, it is aimed to control the tumor growth in the mice.

 

3.3         Bio-imaging of tumors

In order to further study the effects of siRNA and EGFR inhibitory agents, bio-imaging technique can be applied to observe the progression of the tumors in the mice model11. The mice can be treated with siRNA together with EGFR tyrosine kinase inhibitory drugs and monitored for several weeks. It is hypothesized that a reduction in the tumor size can be visibly observed after the administration of the siRNA molecules and EGFR inhibitory drugs. In case an obvious shrinkage of tumor is observed, bio-imaging can provide an evidence for the potential therapeutic efficacy of the siRNA therapy in combination with the EGFR TKIs in treating NSCLC.

The below diagram shows an example of the bioluminescence imaging on lung tumors in mice.

Expected Outcomes

In this project, it is expected that the combination of small interfering RNA (siRNA) against EGFR and EGFR inhibitory agents can improve the anticancer efficacy, especially NSCLC. Besides, it is anticipated that inhalable spray-dried powder formulation can improve the pulmonary delivery efficiency of siRNA molecules to the NSCLC cells. With the technology of the bio-imaging, it is expected that a reduction in tumor size can be visibly observed in the mice model. Finally, it is expected that the research data can be contributed to clinical usage to target EGFR in providing a novel and effective treatment for lung cancer.

 

Summary

Lung cancer is a leading cause of death in Hong Kong. Despite various treatment options, current prognosis is poor. This project aims to investigate the biological effect of siRNA targeting EGFR in NSCLC cell lines in combination with different receptor tyrosine kinase inhibitors, using spray-dried powder as the formulation for enhanced pulmonary delivery. Through in vitro and in vivo study, it is targeted to design a new, safe and effective therapeutic strategy against NSCLC. Lastly, it is aimed to provide a new insight for the treatment of lung cancer in the medical sector.

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