1. Lecture 7: Selective Targeting of Cancer Cells
Contents:
Passive Targeting
Angiogenesis
Leaky Vasculature
Tumor Microenvironment
Local Drug Application
Incubation Time
Adding Coatings
Active Targeting
Antibodies
Over-Expression of Epidermal Growth Factor Receptors
Conjugation of Monoclonal Antibodies
Carbohydrate Directed Targeting
Receptor Directed Targeting
Antigen Directed Targeting
Advances in Drug Delivery
Conclusions

2. Cell Targeting
Current treatments of cancer are very invasive or intrusive through surgery, chemotherapy or radiation therapy.
Side effects can change drastically the life of the patient.
These changes are directly related to how effective the targeting of the tumor or cancer cells was.
One important aspect of nanomedicine is selective targeting of cancer cells.
It is very important to be able to deliver nanodrugs specifically to the cancer, and not to the healthy tissues.
There are two major types of selective targeting of cancer cells called passive and active targeting.

3. Passive Targeting Angiogenesis
In passive targeting, nanodrugs injected into the bloodstream circulate throughout the body to reach the tumor through its porous blood vessels.
A phenomenon that can potentially be taken advantage of is angiogenesis.
This is the process by which tumors signal blood vessels to supply nutrients to the tumor via vascular epidermal growth factors (VEFGs).
The blood vessels have receptors, i.e., VEGF receptors (VEGFRs) that respond to the VEGFs by growing new vessels and capillaries to the tumor.
The tumor then has a free supply of nutrients to feed from, stealing away from the host organism.

4. Passive Targeting Leaky Vasculature Method
Passive targeting is divided into three sub-methods: leaky vasculature, tumor microenvironment, and direct local application.
The leaky vasculature method uses the ability of nanoparticles to penetrate the tumor. The new blood vessels grown for the tumor are hastily made, and thus are very leaky.
Nanoparticles can enter the tumor with great ease by passing through the porous blood vessels into the tumor cells.
This phenomenon is known as enhanced permeability and retention (EPR). Since the tumor is uncontrollably growing, it is continually “hungry” and needs more and more nutrition. This “eating habit” of cancer cells can be used for the passive targeting by mixing the nanodrugs with glucose. The tumor uptakes the glucose with nanoparticles in it from the bloodstream at a much higher rate than the normal cells.

5. Passive Targeting Incubation Time
The effectiveness of passive targeting depends on the incubation time, which is the time required to collect the nanoparticles at the tumor site.
The incubation time is a function of the size of the nanoparticle and the surface chemistry.
Small particles (1-50 nm) pass through the cells rather quickly, while larger particles ( nm) take more time to accumulate.
For example, the incubation time for 1-50 nm nanoparticles is 1-5 hours; and for nm is 6-12 hours.
Using the glucose solution and surface chemistry can increase the uptake of the nanoparticles by the tumor cells and reduce the incubation time.

6. Passive Targeting Adding Coatings
Adding coatings to the nanoparticles is another way to increase the effectiveness of the EPR phenomenon.
Many types of coatings exist  organic and inorganic  that help increase the ‘stickiness’ of the nanoparticles. By adding ‘arms to the nanoparticles, they are more likely to get caught within the cell wall.
Examples of organic nanoparticles include proteins, carbohydrates, peptides and nucleotides.
The inorganic coatings consist of gold, silver, silica, polymers, and magnetic materials like iron oxide, cobalt oxide, and nickel oxide.
These coatings can provide biocompatibility and stability of the nanoparticles and reduce the toxicity of the nanodrugs to the healthy tissue.

7. Passive Targeting Adding Coatings (continued)
The body is fairly adept at recognizing foreign bodies introduced into the organism, so every nanoparticle must be biocompatible first and foremost, or risk the body attacking the nanoparticles and spoiling the treatment.
There are a few coatings that have been found to be biocompatible like silica and polyethylene glycol (PEG). Additionally, biotin and streptavidin coatings can act as masks or bonding agents for other materials.
An interesting application of these coatings is for those that are magnetic. It has been shown that external magnetic fields can have advantageous effects on nanoparticles inside the body. It is possible under certain conditions to guide the nanoparticles using external fields.

8. Passive Targeting Tumor Microenvironment and Direct Local Application
Once the nanoparticle with its chemotherapeutic drug reaches its destination, the tumor microenvironment will make the drug active and volatile. Also called tumor-activated prodrug therapy, this method takes advantage of the special tumor environment (like pH) as compared to the normal surrounding tissue.
More invasive but possibly effective is local drug application. Using the intra-tumoral administration technique, the drug can be injected directly on the tumor. For example, p53 DNA loaded nanoparticles used to treat cancer cells have shown great results.

9. Active Targeting Antibodies
Active targeting presents an interesting and innovative way to selectively target cancer cells.
Active targeting exploits the differences in receptors between the specific cancer cells and healthy cells.
One way to do this is by taking advantage of the body’s own active targeting system: antibodies.
Antibodies are proteins that recognize foreign objects in the body and send signals to the white blood cells to come ‘clean up.’
Antibodies have the unique property in that they are specific to only one antigen.
The body’s immune system contains hundreds of millions of different antibodies and is ready for just about anything.
Using the body’s own antibody for the targeting of the tumor cells is ideal because it is not recognized by the immune system and will not be rejected.

10. Active Targeting Antibodies (continued)
The body’s own antibodies can fight many diseases but not all of them.
Because of this the antibodies could be designed outside the body, grown in animals against the specific types of cancer, and then used for targeting drug delivery.
Studying cancer cells helps to find unique differences in things like protein expression, so that antibodies can be designed to stick directly to the cancer (and nothing else!)

11. Active Targeting Over-Expression of Epidermal Growth Factor Receptors
The selective targeting of cancer cells takes advantage of their property called over-expression of epidermal growth factor receptors (EGFRs).
Normal cells are smaller in size than cancerous cells, and have regular spherical shape and few EGFRs on the surface of the membrane.
The cancer cells are bigger in size and have irregular shapes. Because of their greater size, the cancer cells have a larger number of EGFRs on the surface of the cell membrane.
Thus they are more likely to encounter the epidermal growth factor signal (EGFS) to grow and divide.
This gives the cancer cells the property of rapid growth and division because of the many growing signals that they pick up.

12. Active Targeting Monoclonal Antibody Therapy
The increased number of EGFRs on the surface of cancer cells makes it easier to the target them.
The monoclonal antibody can be designed to stick to the receptors of specific cancer cells only, occupying and blocking them from receiving the EGFSs.
As a result, the tumor stops dividing and growing, and eventually shrinks.
This property has been exploited using breast cancer cells with the over-expression of the human epidermal growth factor receptor 2 (HER2). Herceptin was developed as the antibody to bind with the HER2 receptor.
Selective targeting has been shown for the first hour after combination. At the start, the odds of an antibody finding a receptor on a cancer cell is huge, but the odds decrease over time as receptors get claimed.

13. Active Targeting Monoclonal Antibody Therapy Demonstration
EGFRs
Cancer Cell
Normal Cell
Monoclonal Abs Y Y Y
Normal cells – EGFRs
Cancer cells – Lots more EGFRs, what causes them to grow and divide at such a rapid pace.
Monoclonal Abs – ‘Single clone’, engineer to block EGFRs
Ab-conjugated NPs Y
EGFs Y Y

14. Active Targeting Conjugation of Antibodies to Nanoparticles
The final step in this process is bringing in the nanomedicine component.
This is done by conjugation of monoclonal antibodies to the nanoparticles. Researchers can take the same antibodies described above to cover the nanoparticles.
This will deliver and attach the nanoparticles to the cancer cells receptors only, producing the active selective targeting delivery system.
Then, for example, the radiation can be applied to activate these nanoparticles to realize the nanodrug, or heat them to a temperature high enough for biological ablation of the tumor.

15. Active Targeting Selective Targeting Delivery System
EGFRs
Cancer Cell
Normal Cell
Monoclonal Abs Y Y Y
Normal cells – EGFRs
Cancer cells – Lots more EGFRs, what causes them to grow and divide at such a rapid pace.
Monoclonal Abs – ‘Single clone’, engineer to block EGFRs
Ab-conjugated NPs Y
EGFs Y Y

16. Antibody Conjugation
There are two available techniques for bonding the nanoparticles to the antibody: direct bonding and indirect bonding.
In direct bonding, the nanoparticles are mixed with the antibody, which sticks to the surface of the nanoparticle directly.
However, the direct bonding is not an efficient technique for attaching the antibody to the surface of the nanodrug.
In the indirect bonding technique, the nanoparticles are first covered by adaptor molecules like streptavidin, biotin, PEG, etc., which are designed to stick effectively to the surface of the nanoparticle, such as metal, and covered secondly by the antibody.
These adaptor molecules also have the ability to stick to the specific antibody used for the delivery system. The indirect bonding technique forms more stable drug delivery system.

17. Active Targeting by Nanoparticles
There are three sub-methods which have been studied and researched for active targeting by nanoparticles: carbohydrate-directed, receptor and antigen directed targeting.
It has been found that lectin-carbohydrate affects tumor survival by preventing vascularization and other processes which help tumor growth.
These binding proteins can be conjugated with nanoparticles. The lectins-nanoparticle combination can be directed to the surface of the tumor cells, thus directly targeting them.

18. Active Targeting by Nanoparticles (continued)
Endocytosis is the process in which a cell takes in materials from the outside by engulfing and fusing them with its plasma membrane.
The receptors and antigens of human cancer cells, as mentioned earlier, have great efficiency in uptake.
When endocytosis is used, the polymeric nanoparticles are taken into the microenvironment via interactions of receptors with ligands (molecules that binds to other molecules).
The choice of a ligand or antibody requires great properties such as immunogenicity, biodegradability and physiochemical properties.

19. Advantages of Antibody-Conjugated Nanoparticles
-Specificity
-Biocompatibility
-Part of the
Immune System
-Functional
-Versatile
-Controllable
Antibodies
Nanoparticles
Accurate
Selective
Targeting

20. Advances in Drug Delivery
The multistage drug delivery method has shown great promise by using mesoporous silicon particles (MSPs) which encapsulate many nanoparticles that are loaded with an anticancer drug.
Porous silicon is very safe to the human body because it is biodegradable and is approved by the Food and Drug Administration (FDA).
The geometry of the particle is important for its journey because it must be able to adhere and be internalized without being changed or altered by the outside environment.
Hemispherical particles have shown the most promise because of the highest accumulation on the tumor.
The efficacy ultimately depends on the cellular interactions between the MSPs and tumor cell surface, and adequate release of the therapeutic drug. The porosity of the MSP affects the release timing.

21. Conclusion
Specifically-designed nanoparticles can be selectively delivered to the tumor, and then can be used in many diagnostic and treatment applications, including bioseparation, sensing and imaging, immunoassays, and purification.
The general consensus of the scientific community on the best method for selectively targeting cancer cells appears to be antibody conjugated nanoparticles.
It is very accurate and has already been shown to work for in vivo studies.
Diagnosis & Imaging
Design of NPs
Selective Targeting
Activation
Assessment