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2.

Technical Review
2.1 Background Knowledge
Many antibiotics were once effective in killing bacteria in our bodies. However,
overtime, bacteria have developed several mechanisms of resistance towards antibiotics.
These mechanisms include:
1. Changing the permeability of its membranes
2. Reducing the number of channels available for drugs to diffuse through,
3. Changing the structure of the antibiotics target so that the antibiotic can no
longer recognize the target.
4. Neutralizing the antibiotic (destroying the antibiotic, for example, some
bacteria produce enzymes that denatures penicillin)
5. Pumping out the antibiotic quickly
6. Altering the active sites of antibiotics.

Bacteria with these resistance mechanisms survive and thereby allowing the
multiplication of antibiotic-resistant bacteria.

On the other hand, current antibiotics employ several methods to stave off the growth of
bacteria. These methods include:

1. Prevent the production of bacterial cell wall that protects the cell from the
external environment.
2. Disrupt the synthesis of proteins that bacteria requires by binding to the
machinery that builds proteins
3. Disrupt bacterias metabolic process (for example, the production of folic acid)
4. Disrupt the synthesis of DNA and RNA
5. Prevent metabolic actions
2.2 Technical Review of Existing Technologies and Research
As mentioned above, some bacteria have developed antibiotic resistant mechanisms. This
poses as a serious public health issue. Some examples of antibiotic resistance bacteria
include Staphylococcus aureus (golden staph or MRSA) and Neisseria gonorrhoeae
(the cause of gonorrhoea). However, there are current research conducted that can
provide a solution without bacteria forming a resistance towards the technology:

2.2.1 Structurally nanoengineering antimicrobial peptide polymers (SNAPPs)1


Antimicrobial peptides (AMPs) have also been found to combat multidrug-resistance
(MDR) bacteria, but were unsuccessful in clinical drug trials due to its high toxicity
towards mammalian cells. On the other hand, the development of ring-opening
polymerization of -amino acid N-carboxyanhydrides (NCAs) provided another
pathway for the synthesis of peptide polymers with antimicrobial properties. This
advancement in method facilitated the development of SNAPPs, an antimicrobial agent.
Structurally nanoengineered antimicrobial peptide polymers (SNAPPs) has shown to
exhibit sub-M activity against Gram-negative bacteria, such as ESKAPE, colistin-
resistant and MDR (CMDR) pathogens. Additionally, the polymer showed low toxicity.
A. baumannii did not show resistance towards the polymer. SNAPPs employs multimodal
mechanisms to enable bacterial cell death: 1. the destabilization of bacterias cell wall 2.
disrupt the regulation of ion movement across the plasma membrane 3. inducing
apoptotic-like death pathway (programmed cell death). Due to the tendency of SNAPPs
to aggregate, it reduces its potency. (the negative part of it making us support using the
ball) Yet, it remains to be more effective as opposed to AMPs. When tested for its
viability in mammalian cells, the therapeutic indices (TI) is slightly higher than the TI of
the last option for MDR Gram-negative pathogens. SNAPPs exist in two forms, 16-arm
and 32-arm star peptide polymer nanoparticles. It consist of a multifunctional core, also
known as the star polymer mde of poly(amino amine) PAMAM-(NH2)m, with arms

1 Lam, S. J., O'Brien-Simpson, N. M., Pantarat, N., Sulistio, A., Wong, E. H., Chen, Y. Y.,
... & Qiao, G. G. (2016). Combating multidrug-resistant Gram-negative bacteria with
structurally nanoengineered antimicrobial peptide polymers. Nature microbiology, 1,
16162.
consisting of Lys and Val amino acids. A figure of the synthesis of the SNAPPs
developed by Lam can be seen in Figure 1.

Figure 1: Synthesis of SNAPPs developed by Lam et. al.


2.2.2 Graphene Derivatives2
Research on graphene-based materials has been increasing due to its potential in
electronics, biomedical devices and membranes (Still not scientifically mature yet, nor
easy to engineer, may be easy to produce and cost effective). However, graphene
derivatives may have harmful effects on human health and the environment. On the other
hand, graphene and its derivatives has been shown to cause bacterial death by a group of
researchers from A*STAR. It was observed that graphene oxide was able to kill
significantly more bacteria than reduced graphene oxide and graphite oxide. It is
observed that the reason for its effectiveness is due to the size of the particles. E. Coli
cells gets wrapped by layers of graphene-oxide while E. Coli gets trapped by larger
reduced-graphene oxide and graphite-based materials. The researchers propose that cell
wrapping causes membrane stress and irreversible damage. Graphene oxide and graphite
oxide can also disrupt and kill bacteria through chemical action. Glutathione, an
antioxidant necessary for bacterium survival, is oxidized when exposed to graphite and
2 Liu, S., Zeng, T. H., Hofmann, M., Burcombe, E., Wei, J. et al. Antibacterial activity of
graphite, graphite oxide, graphene oxide and reduced graphene oxide: membrane and
oxidative stress. ACS Nano 5, 69716980 (2011).
reduced graphene oxide. However, it was realized that the number of bacteria cell death is
dependent on the concentration of graphene derivatives. This suggests that graphene
derivatives active sites, places where bacteria is trapped or causes oxidative action,
becomes deactivated (limited effectiveness). This limits the action of graphene
derivatives in its antibacterial properties.

2.2.3 Chitosan/Chitin3
Chitin mainly found the main skeleton of insects and crustaceans, is a polysaccharide that
is mainly characterized by a fibrous structure. In its crude form, chitin has a highly
ordered crystalline structure that has poor solubility and low reactivity. Chitin can be
further derivatized into chitosan. Chitosan is insoluble in water but soluble in dilute
aqueous acidic solutions below its pKa of 6.3 (Our stomach is 3.5 while our blood is
slightly alkaline at a value of 7.3.) Chitosan and chitin has been found to have
antimicrobial (gram-negative, gram-positive bacteria, fungi) properties.

Research has shown that chitosan employs three different mechanisms in disrupting
bacteria growth: 1. wall cell leakage is resulted due to ionic surface interaction 2. the
penetration of chitosan into the nuclei of microorganisms results in the inhibition of
mRNA and thereby disrupting protein synthesis 3. chelates metals, suppresses spore
elements and binds to nutrients to microbial growth (good variety of mechanical bacteria
disruptions). During the above mechanism, chitosan molecules surround the bacteria and
therefore, causing cell death. Water-soluble chitosan can also be derived through the
introduction of permanent positive changes in the polymer chains. The formation of these
water-soluble chitosan can be seen in figure 2. It was also observed that the antimicrobial
activity is higher in water-soluble chitosan as opposed to insoluble chitin and chitosan. A
reason for high activity is due to the presence of a hydrophobic and hydrophilic region on
the water-soluble chitosan. This structure resembles the bacteria cell wall, resulting in
high affinity.
(unknown cost, but seems simple to synthesize even though it has not been tried yet,
unknown safety, use natural materials)
3 Goy, R. C., Britto, D. D., & Assis, O. B. (2009). A review of the antimicrobial activity
of chitosan. Polmeros, 19(3), 241-247.
2.2.4 Bacteriophage4
Interest in phage therapy and phage-derived proteins have increased in the past two
decades. However, phage-derived proteins are more advantageous over phage therapy for
these reasons: a wider host spectrum, provides no opportunity for the transmission of
antibiotic resistance genes and lack of resistance development to phage lysins. Phages,
also known as bacteriophage, are an alternative to antibiotics in the treatment of bacteria,
especially with a growing number of antibiotic resistance bacteria. The bacteriophage
contains a protein that encodes a DNA or RNA genome. This DNA or RNA genome can
be encoded with enzymes. Currently, most phages are encoded with cell wall lytic
proteins. Phages inject the genome into the bacteria where the genome gets multiplied
(genomic method, less effective, safety issues if attack human cells). The enzymes are
able to degrade the cell wall of bacteria, such as Gram-positive bacteria. Currently,
ContraFect Corporation is undergoing Phase I clinical trials on the treatment of life
threatening, drug-resistant infectious diseases using phage lytic proteins (Good scientific
maturity and engineering ease, but not sure about lowered costs yet, good environmental
friendliness).

2.3. Drug Delivery Technologies


2.3.1 Molecular Machines5 6 7
1. Molecular Machines: The 2016 Nobel Prize for Chemistry was awarded to a group of
scientist for the development of synthetic molecular machines. A type of molecular
machine that shows potential for further application is known as nanorobots. These
naonrobots can made up of molecular machines which can be categorized based on its

4 Rodrguez-Rubio, L., Gutirrez, D., Donovan, D. M., Martnez, B., Rodrguez, A., &
Garca, P. (2016). Phage lytic proteins: biotechnological applications beyond clinical
antimicrobials. Critical reviews in biotechnology, 36(3), 542-552.
5 Abendroth, J. M., Bushuyev, O. S., Weiss, P. S., & Barrett, C. J. (2015). Controlling
motion at the nanoscale: rise of the molecular machines. ACS nano, 9(8), 7746-7768.
6 Tam, E. S., Parks, J. J., Shum, W. W., Zhong, Y. W., Santiago-Berros, M. E. B., Zheng,
X., ... & Ralph, D. C. (2011). Single-molecule conductance of pyridine-terminated
dithienylethene switch molecules. Acs Nano, 5(6), 5115-5123.
7 Orozco, J., Corts, A., Cheng, G., Sattayasamitsathit, S., Gao, W., Feng, X., ... & Wang,
J. (2013). Molecularly imprinted polymer-based catalytic micromotors for selective
protein transport. Journal of the American Chemical Society, 135(14), 5336-5339.
functions: 1. Molecular switch: a molecule that reverses between two or more stable
conformations in response to factors such as pH, temperature, light etc. 2. Molecular
motors: molecules that enable converts external energy input into unidirectional rotation
motion. 3. Molecular propeller: consists of several molecular-scale blades attached at an
angle. These components can be combined together to produce a collective motion.
Therefore, theoretically, nanomachines can be synthesized to transport things at a
macroscopic level. Therefore, nanomachines can provide application in drug delivery
methods. However, these nanorobots are comparatively poor efficiency, speed and
capabilities (lower effectiveness, higher cost, and lower engineering ease, and higher risk,
unknown safety)

2.3.2 Bis-MPA Dendrimers8


In the field of nanomedicine, research on dendrimers and dendrons have been increasing
due to its potential in drug delivery and gene therapy applications. A dendrimer is high
branched polymeric molecule that radiate from a central core. Dendrimers poses
significant advantages over linear polymers: 1. Synthesis can be controlled such that
precise distribution of functional groups can be achieved. 2. This allows for consistency
in the replicating the polymeric structure (high precision and effectiveness) 3.
Biodegradability property, low toxicity and water solubility (safe, but uncertain about
environmental impacts of manufacture) 4. Loading capacity is adjustable 5. Drug release
can be controlled and tuned based on its environment.

A group of researchers from the Royal Institute of Technology has developed a promising
family of dendritic macromolecule, bis-MPA as the building block (in large scale
production alr, so good maturity and even better engineering ease, thus low cost too).
Some of the more relevant applications for bis-MPA dendrimers consist of:
1. Passive encapsulators: The dendrimers do not form covalent bonds with the drug. This
application is especially useful to delivery drugs that are insoluble in the body fluids.

8 Dendritic Polyester Scaffolds: Functional and Biocompatible Precision Polymers for


drug delivery application. Sandra Garcia-Gallego, Michael Malkoch. Polymeric Drug
Delivery Techniques. Translating Polymer Science for Drug Delivery, Aldrich Material
Science
Based on the environment at which the drugs are to be released, the dendrimer can be
tailored to release the drug according to pH, temperature or enzymes.
2. Unimolecular Carriers: The dendrimers and the drug make up one molecule. This
involves covalently bonding the drug into the central core of the dendrimer. Similar to the
above, the dendrimer can be tailored to release the drug based on its surroundings.
3. Grafting-to strategy is an application developed by Carlmark et. al. The surface of
Bis-MPA can be used to amplify specific functional groups and surface area. The grafting
strategy involves covalent attachment of dendrons on the surface of the dendrimer. This
covalent attachment is usually a normal condensation reaction. 9 (we know the method to
graft the peptides, but not tried yet)

9 Carlmark, A., Malmstrm, E., & Malkoch, M. (2013). Dendritic architectures based on
bis-MPA: functional polymeric scaffolds for application-driven research. Chemical
Society Reviews, 42(13), 5858-5879.