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Summary of the Minor Research Project
Principal Investiator: Dr. LUCY MATHEW P
Project Title:
Antimicrobial Phototherapy using Porphyrin and Metalloporphyrins
Anchored with Linear and Dendritic Macromolecules
Introduction
The various polymeric systems employed for the study are Polyethylene glycol, linear
polyglycerol adipate and hyperbranched polyglycerol systems. All the above polymers have a
common feature of free hydroxyl groups for coupling with chlorosuphonated porphyrin systems.
The hydrophobic polymeric systems achieves some hydrophilicity on binding with these linear
as well as hyperbranched polymeric systems. Th spectral results gave evidences for functional
changes and the electronic properties of porphyrin macrocycle assisted by the dendritic structure
of HPG were studied in detail.
Porphyrins are naturally occurring compounds having vital biological role in the form of
haemoglobin, myoglobin, chlorophyll, cytochromes, peroxidases, vitamin B12 etc. Porphyrins
and their metal derivatives exhibit enormously rich and novel photochemistry, which is widely
used in carrying out several special reactions and in mimicking biological systems. We paid
attention to properties of porphyrins and metalloporphyrins, which play important role in
photochemical systems and living systems.
The most useful spectroscopic technique for the study of porphyrins and
metalloporphyrinsis the electronic absorption spectroscopy. The metal free porphyrins shows an
intense band B (soret) band at ~ 420nm and four weaker band Q (Q1, Q2, Q3 and Q4) bands at
500-650 nm. The metallo porphyrins also show an intense B (soret) band at ~ 420nm but the Q3
and Q4 bands were found to be diminished. These spectral absorptions arise from the π-π*
transitions of the aromatic porphyrin ligand. The resulting spectrum shows a high-energy band B
in which the transition dipoles add (high intensity) and a low energy band Q in which the
transition dipoles nearly cancel (low intensity). The two Q bands are vibronic components of the
same transition. In metal free porphyrins these vibronic components appear as 4 bands. We paid
attention to functions of porphyrins and metallo porphyrins, which play important role in
photochemical system and living systems. We prepared polymers containing porphyrin moieties
in their side chains.
Selection of Polymer systems
Polymers can be endowed with special functions ascribable to physical and chemical
properties of functional groups bonded to polymer chain. When special behaviour of polymer
chain is added to physical and chemical properties of the functional group, new functions, which
cannot be observed in functional group in itself, appear in the polymer. Dendritic polymers,
comprising dendrimers and hyperbranched polymers with highly branched backbone structures
have several applications as film forming resins and as toughening additives etc. Hyperbranched
polymers have several applications in medicine due to their biocompatibility and water
solubility.
The polymers selected for the present study were polyglycerol polyadipate and
hyperbranched polyglycerol. We succeeded in preparing high molecular weight polymer bound
porphyrin of the above said polymeric systems. The tetra phenyl porphyrin was
chlorosulphonated using sulphonyl chloride and coupled with polymeric cores such as PG and
HPG. The polymer bound TPP was purified by column chromatography. The polymer bound
TPP systems were characterized by UV-visible, FTIR and NMR spectroscopic methods.
The main interest to select hyperbranched polyglycerol as the core material is its water
soluble and non-toxic nature. Compared to other dendrimers hyperbranched polyglycerol can be
easily synthesized using a single step process. This clear viscous liquid, which becomes free
flowing on heating up to 800C is highly soluble in water and also in polar solvents. It is non
volatile at room temperature, contains hydroxyl groups and a poly ether back bone and resembles
the well known linear systems like PVA, PEG and PG, which are also approved for a large
variety of medical and biomedical applications.
On comparing the properties of linear and hyperbranched polymer bound porphyrins we
noticed several significant differences. The linear polymers exhibited blue shifts in the soret as
well as the Q bands of UV-visible spectra indicating that there is an electronic interaction
between porphyrin moieties and the polymers. The blue shift may be caused by the structural
perturbations caused by the entanglement of the polymer core on the porphyrin framework. The
TPP has aggregation tendency and which when attached to the linear system like PG, this
tendency may be enhanced due to the entanglements and steric effect which results in spectral
shifts.
Hyperbranched Polyglycerol bound Porphyrin
When hyperbranched polyglycerol bound porphyrin is put under spectral studies, we
observed a red shift in both soret and Q bands of the porphyrin moiety. The polymer free TPP
exhibited significant absorptions at 422nm and several bands in the visible region from 500 to
650nm. When bound to linear polymers like PVA, PEG and PG we observed blue shifts in the
UV-visible spectrum while the HPG-TPP system exhibited a prominent red shift in both soret as
well as Q bands. The aggregation tendency of TPP was not found when it is anchored to HPG.
this may be due to the non-entangled structural architecture of HPG. this may be confirmed by
UV- visible spectral data of HPG-TPP system. Both B and Q bands were red shifted after
coupling with HPG. The soret band was red shifted by ~ 40nm while Q1, Q2, Q3 and Q4 bands
were red shifted by 16nm, 20nm, 41nm and 40nm respectively. The variation in electronic
charge delocalization within the porphyrin macrocycle assisted by the highly branched and
heavily functionalised HPG system caused considerable red shift. The enhanced absorption of
HPG-TPP system opens immense possibilities in photoresponsive applications. The intensity of
both soret and and Q bands were enhanced tremendously as a function of loading of TPP on the
HPG core.
Photodynamic Therapy
Photodynamic therapy is an evolving modality for the treatment of cancer. This therapy
involves the selective uptake and selection of a photosensitizer in a tumor followed by irradiation
with a light of particular wavelength, thereby initiating tumor necrosis presumably through the
formation of singlet oxygen. Porphyrins were considered as good photosensitizers but their water
insolubility make them difficult to administer systemically (via injection in to the blood stream).
Water soluble photosensitizers would therefore be expected to be the most useful since blood is a
water based system. Here comes the significance of HPG-TPP system as a photosensitizer
because of its non-toxic hydrophilic nature. It absorbs light from the longer wavelength region
(600-700nm). An ideal photosensitizer should be able to absorb light from longer wavelength
region. HPG anchored TPP retains the absorption properties of TPP and is more biocompatible.
The development PDT has provided an effective modality against antibiotic resistant bacteria
and cell free viruses. Bacterial PDT is affected by the use of various sensitizers, as a general rule,
non-charged or positively charged molecules are effective in photoinactivation of
Staphylococcus bacteria.
Bacterial Studies
We have selected two bacterial cultures, gram positive (Staphylococcus aureus) and the
other gram negative (Escherichia coli). TPP, FeTPP, HPG-TPP and HPG-FeTPP systems were
selected to test photosensitivity. The gram-negative E.coli bacterium was found to be resistant to
all the systems but the gram-positive bacteria, Staphylococcus aureus were killed by the photo
oxidation even though a complete destruction was not achieved. We have done only a
comparative study of photosensitizing property of porphyrin, its metal (Fe) incorporated system
and their hyperbranched polyglycerol bound systems. The spread plates prepared using systems
before irradiation gave uncountable colonies of microbes while after irradiation microbial killing
was achieved and have obtained countable colonies of microbes in the petri dishes. Of these
systems FeTPP exhibited a very significant toxicity and this may be due to the presence of the
metal, which is already toxic to microbes. The polymer bound TPP and FeTPP systems were also
exhibited efficiency in photokilling of the microbes. The photodynamic efficiency of TPP was
retained even on binding with HPG. The advantage is that HPG-TPP as well as the HPG-FeTPP
systems were water soluble and more biocompatible than the polymer free TPP systems.
Conclusion
Porphyrin and their derivatives are insoluble in water. porphyrin bound linear polymeric
system are also insoluble in water. But when porphyrin was bound to hyperbranched
polyglycerol, which is a biocompatible non-toxic polymer, the hydrophilicity of the system was
increased. This polymeric system of porphyrin moiety has the solubility at the extend of 100
mg/ml. From the results of the present studies, it is clear that there is provided a water soluble
hyper branched polymer comprising a porphyrin moeity which has significant photo physical
properties useful for the construction of optoelectronic devices, and in very useful in biological
systems because of their biocompatibility.