Chapter 5

Photoresponsive and Antimicrobial

Studies on Porphyrin and

Metalloporphyrin- Anchored Linear

and Dendritic Macromolecules

5.1 Introduction

There is much current interest in the study of polymer bound porphyrins because of

their interesting electronic properties and possible use as photosensitizers, catalyst and

complexing agents. Although the synthesis of a wide variety of polymer bound porphyrins

are well developed, relatively few soluble polymer bound porphyrins are studied. Our

interest is to prepare nature friendly and water soluble linear and dendritic polymer

bound metallated as well as metal free porphyrins. Significant changes in the electronic

spectra were seen in polymer bound metal incorporated porphyrins as well as metal free

porphyrins. This chapter gives a critical discussion on the absorption and luminescence

emission properties of porphyrins, metalloporphyrins and their polymer bound analogues.

The potential use of porphyrin bound to hyperbranched polyglycerol as artificial blood

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substitutes, antimicrobial agents and its photosensitized antimicrobial action are discussed

in detail.

5.2 Tetraphenyl Porphyrin System

The spectral properties of porphyrin complexes have attracted considerable experi-

mental curiosity and theoretical interest because of their vital role in biological processes

such as photosynthesis, respiration and their potential technological applications. In the

UV -visible absorption spectrum, the highly conjugated porphyrin macrocycle showed in-

tense absorption at 400 to 450nm (the B band or soret band) followed by several weaker

absorptions (Q bands) at higher wavelengths from 500 to 650nm (figure 5.1). Peripheral

substituents on the porphyrin ring often cause changes on the intensity and wavelength

of these absorptions. The origin of intensities of the Q and B bands were successfully

explained by Goutermans four-orbital model1,2.

Figure 5.1. UV- visible spectrum of TPP

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According to four- orbital model, the B and Q bands can be described in terms of

transitions between a pair of top filled orbitals (a1u and a2u) and lowest empty orbitals

(the doubly degenerate eg). The degeneracy of the a1u, a2u, and eg? excited levels leads

to a strong configuration interaction that results in a high-lying state corresponding to B

band and low- lying state corresponding to Q band. The configuration mixing combines

the transition dipoles of the individual one electron transition in such a way that the B

band contains nearly high intensity, while the Q band is weak. The soret band is assigned

as π - π ?type transition from the two highest occupied molecular orbitals (HOMO) a1u

(π) and a2u (π) to the lowest empty doubly degenerate antibonding molecular orbitals

eg?. The schematic representation of porphyrin HOMOs and LUMOs are shown in figure

5.2.

The presence of highly delocalized π electron system in porphyrin macrocycle provides

a variety of advantages for their applications. One of the ways to enhance the use of

porphyrins in optoelectronic devices is to further expand the existing π electron system.

Porphyrins are particularly appealing because of the richness of their properties, and

because expertise is available to modify these useful platform to build more complex

molecular architectures3.

Figure 5.2. Porphyrin HOMOs and LUMOs.

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5.3 UV-Visible Absorptions of Linear Polymeric Core

Systems Modified with Tetraphenyl Porphyrins

The linear polymeric cores selected for the modification of porphyrin were poly vinyl

alcohol, poly ethylene glycol and polyglycerol poly adipate. All of them possess primary

alcoholic terminal groups which are accessible to common reactions such as esterification,

etherification reactions etc.

5.3.1 Polyvinyl alcohol modified with TPP

Polyvinyl alcohol bearing free OH groups can be bonded to chlorosulphonated por-

phyrins very well, but the resulting polymer is insoluble in almost all the solvents, the

spectral studies become difficult4. The polymer bound TPP was dissolved in DMF and

the absorptions were studied using these solutions.

The TPP have very characteristic electronic spectra having an intense band at 416

nm (soret/B band) and three or four less intense band at 520-650 nm(Q bands).

On chlorosulphonation we have observed a red shift of 23nm for soret band and 11nm

for the Q1 band. The soret band was shifted from 416nm to 439nm and the Q1 band

was shifted from 514 nm to 525 nm. The Q2, Q3 and Q4 bands were also red shifted

from 550nm to 559 nm, 591nm to 601 nm and 648 nm to 656 nm respectively. The lone

pairs on the sulphur atom causes increased electron delocalisation with the porphyrin

macrocycle π -electron framework. The SO2Cl group enhances the π conjugation and the

HOMO-LUMO energy gap is reduced. This effect is reflected in the red shift shown by

these systems.

The polymer bound porphyrin also exhibited the characteristic absorption. The elec-

tronic spectrum was recorded in DMF. The B and Q bands of TPP bound with polyvinyl

alcohol were blue shifted to 414 nm, 513 nm, 547 nm, 590 nm and 644 nm respectively

(figure 5.3).

The planarity of the porphyrin macrocycle is highly sensitive to structural changes. On

attaching to polyvinyl alcohol core, the steric effect causes a great degree of perturbation

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on the planarity of the porphyrin macrocycle and the π electron framework is seriously

affected by this change. This causes a slight blue shift in the electronic spectral signals. A

shifting of 2nm, 1nm, 3nm, 1nm and 4nm respectively of soret, Q1, Q2, Q3 and Q4 bands

were observed on binding the TPP system on to the PVA core.The blue shift was very

prominent when compared with the absorbances of chlorosulphonated TPP (table 5.1).

The absorption bands of TPP did not show any notable red shift on binding to the PVA

core. However, the water insoluble TPP system became soluble in polar solvents such as

DMF on anchoring to PVA.

PVA is a film forming linear polymer with excellent hydrophilic character. Therefore,

the photosensitivity and photoresponsive character of the hydrophobic TPP π electron

frame work can be made hydrophilic and stable by attaching TPPSO2Cl on to PVA core.

This can be used for various industrial applications due to the excellent photochromic

character of TPP and structural properties of linear PVA core.

Figure 5.3. UV-visible spectra of TPP, TPP SO2Cl and PVA-TPP.

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Table 5.1. Electronic absorptions of TPP, TPP -SO2Cl and PVA-TPP

System B band

(nm)

Q1 band

(nm)

Q2 band

(nm)

Q3 band

(nm)

Q4 band

(nm)

TPP 416 514 550 591 648

TPP SO2Cl 439 525 559 601 656

PVA-TPP 414 513 547 590 644

5.3.2 Polyethylene glycol modified with TPP

The appending of porphyrin on to the polymers was confirmed by the conspicuous

colour change of the polymer and also by the electronic spectral measurements. Once

covalently bonded to the polymer, porphyrin was not exchangeable under ordinary con-

ditions, and the system was very stable in both polar and non-polar solvents. PEG is a

linear flexible polymer with dipolar character. Because PEG contains periodically spaced

electron rich ether functions along with terminal OH functions, it is highly compatible

with an aqueous medium and ionic species5,6.

The porphyrin bound PEG also exhibited the characteristic absorption properties.

The electronic spectrum was recorded in DMF. The B and the four Q bands were blue

shifted to 411nm, 509 nm, 541 nm, 583 nm and 63 nm respectively on binding to PEG

(figure 5.4).

The PEG end groups were functionalised with TPP. Compared to PVA-TPP system,

the loading of TPP on to the PEG system is low. However the soret and Q bands showed

blue shift due to the structural perturbation caused by the entangled polymer core on

the porphyrin framework. The soret band showed a shifting of 5 nm, from 416 nm to

411 nm on binding to PEG core. The intensity of Q bands were diminished considerably

and the shifting observed were 5nm, 9nm, 8nm and 9nm respectively for Q1, Q2, Q3

and Q4 bands (table 5.2). However, the PEG-TPP system is highly promising due to its

solubility in water and other polar solvents, its film forming properties and its potential

use in medicine and medical diagnosis.

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Figure 5.4. UV-Vis spectra of TPP, TPP-SO2Cl and PEG-TPP.

Polyethylene glycol is a nature friendly linear polymer, available in a wide range of

molar masses, soluble in water and polar solvents and widely used in medicine, cosmetics

such as moisturisers, creams, gels etc and in coating applications. A typical hydrophobic

system like tetraphenyl porphyrin can be made hydrophilic and water soluble by attach-

ing the suitably made TPP on to PEG. In the present study we could develop a novel

photoactive porphyrin supported on PEG with molar mass 6000. The newly developed

system strongly absorb in the visible region (both B and Q bands). The PEG-TPP sys-

tem is soluble in water and other polar solvents and find many applications in diagnosis,

medicine and industry.

Table 5.2. Electronic absorptions of TPP, TPP-SO2Cl and PEG-TPP

System B band

(nm)

Q1 band

(nm)

Q2 band

(nm)

Q3 band

(nm)

Q4 band

(nm)

TPP 416 514 550 591 648

TPP SO2Cl 439 525 559 601 656

PEG-TPP 411 509 541 583 639

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5.3.3 Polyglycerol polyol modified with TPP

The porphyrin bound linear polyglycerol polyol adipate is a pink coloured semisolid

soluble in water and its flexible nature made the studies easier. Porphyrin is highly soluble

in CHCl3 but all the polymer bound porphyrins are insoluble in CHCl3. Both the soret

and Q bands are blue shifted but not to a notable extent after binding with the polymer

(figure 5.5).

Figure 5.5. UV-Vis spectra of TPP, TPPSO2Cl and PG-TPP.

The TPP has aggregation tendency and which when attached to the linear system

like PEG and PG, this tendency may be enhanced due to the entanglements and that

results in spectral shifts and line broadening. Moreover, the planarity and consequently

the electron delocalisation of the π-frame work were perturbed by the steric hindrance

imposed by the polymer backbone on to the porphyrin macrocycle. When compared to

PVA and PEG, the polyglycerol polyadipate system is less entangled so that the structural

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perturbation caused on the polymer is less and the shifting observed is to a less extent.

The shifting observed in the Q bands were 2nm, 4 nm, 1 nm and 1 nm respectively for

Q1, Q2, Q3 and Q4 bands (table 5.3). The soret band get blue shifted by 2m from 416 of

TPP on binding with PG.

Table 5.3. The comparative study of TPP, TPP-SO2Cl and PG -TPP

System B band

(nm)

Q1 band

(nm)

Q2 band

(nm)

Q3 band

(nm)

Q4 band

(nm)

TPP 416 514 550 591 648

TPP SO2Cl 439 525 559 601 656

PG -TPP 414 512 546 590 647

The absorption phenomenon of PG-TPP are almost comparable to that of TPP. PG-

TPP strongly absorbs in the visible region of UV-visible spectrum. An intense absorption

was noted at 414 nm corresponding to the soret band. The Q bands were also of moder-

ately high intensities. The PG-TPP system is polar and water soluble though TPP is a

hydrophobic system.

5.4 UV-visible Absorptions of TPP Bound Hyper-

branched Polyglycerol

The porphyrin bound hyperbranched polyglycerol possessed wonderful photochemical

properties7,8. It is insoluble in CHCl3 but freely soluble in water and other polar solvents

though TPP shows the opposite behaviour. The UV-visible spectrum shows signals at

472 nm (soret band) and at 563 nm, 600 nm, 640 nm and 694 nm respectively for Q1, Q2,

Q3, and Q4, bands on binding to HPG (table 5.4).

The aggregation tendency of TPP is not found when it is anchored on to hyperbranched

polyglycerol due to the non-entangled structural architecture of HPG. But the aggregation

was very prominent when TPP was bound to linear polymeric cores such as PVA, PEG

and PG. A red shift of 56 nm was observed for the soret band and red shifts in all the Q

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bands in the electronic spectra of HPG systems confirmed this. The intensity of the Q 4

band was tremendously increased on attaching to HPG backbone (figure 5.6).

Figure 5.6. UV-vis spectra of TPP, TPPSO2Cl and HPG-TPP.

The phenyl ring of TPP is not coplanar with the porphyrin macrocycle. On binding

or coupling with polymers like PVA, PEG or linear polyglycerol systems, the soret band

seems to be slightly blue shifted while the Q bands nearly disappeared but they appear

clearly and red shifted while the TPP is coupled with HPG.

The peaks of the porphyrin in UV-visible region have generally interpreted in terms

of π- π* transition between bonding and antibonding molecular orbitals. The two inter

band transition, Q and B are assigned as π- π* type.

The significant broadening and red shift of 49 nm (Q1), 50 nm, (Q2), 49 nm (Q3), and

46 nm (Q4) bands in HPG-TPP system indicate increased electron delocalisation due to

increased polarity9. It is observed that after coupling with HPG there is increase in the

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intensity of absorption band as shown in the figure 5.6. This is because, coupling with

the polymer rearranges few of the electric dipoles from opposite in to parallel direction

and consequently the oscillator strengths increased.

Table 5.4. The comparative study of TPP, TPPSO2Cl and HPG -TPP

System B band

(nm)

Q1band

(nm)

Q2 band

(nm)

Q3 band

(nm)

Q4 band

(nm)

TPP 416 514 550 591 648

TPP SO2Cl 439 525 559 601 656

TPP-HPG 472 563 600 640 694

The red shift of TPP moieties after coupling with HPG may be due to the variations

in the electronic charge delocalization within the porphyrin macrocycle assisted by the

highly branched and heavily functionalised hyperbranched polyglycerol core system. It

is well known that any increase in the electron density within a molecular system would

result on an increase in the energy of HOMOs. In TPP the phenyl rings are not coplanar

with the porphyrin macrocycle. Hence such an electric flow from the phenyl rings would

not be possible for the free TPP or MTPPs.

Table 5.5. Comparative study on absorption spectra of TPP bound linear polymers

and HPG

System B band

(nm)

Q1 band

(nm)

Q2 band

(nm)

Q3 band

(nm)

Q4 band

(nm)

TPP 416 514 550 591 648

PVA -TPP 414 513 547 590 644

PEG -TPP 411 509 541 583 639

PG -TPP 414 512 546 590 647

HPG-TPP 472 563 600 640 694

5.4.1 Percentage loading of TPP and UV-visible absorptions

A series of new and novel HPG-TPP system have been developed with varying amounts

of TPP and subjected to spectral measurements. The results showed that all the systems

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exhibited red shift to almost the same extent with an increase in absorption values (figure

5.7). All of them gave well-resolved spectra indicating the absence of any dipolar interac-

tions in them and proving that porphyrin was well separated on the polymer matrix. The

intensity of both soret and Q bands were enhanced tremendously as a function of loading

of TPP on the HPG core. 100% TPP loaded HPG core shows very intense absorptions

both in the soret and Q regions. The enhanced absorption of HPG-TPP system opens

immense possibilities in photo responsive applications.

Figure 5.7.UV-Vis spectra of 20%, 40%, 60%, 80% and 100% TPP loaded HPG.

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5.5 Emission Studies on Tetraphenyl Porphyrins and

Polymer Bound Tetraphenyl Porphyrins

Porphyrins are known for their interesting emission properties. Among these, the

fluorescence properties are very unique. Many of the special applications of these systems

are based on such emission properties. When a molecule is excited to an upper electronic

state, it may return to the ground state either by radiationless cascade or by its emission

from lowest excited singlet and triplet states. The nature and quantum yield of such

emissions are determined by the relative rates of various deactivation processes. The

fluorescence yield of most porphyrins is less than 0.2.

Thus the excited state S1 is primarily deactivated by radiationless decay. It appears

fairly certain that the spin forbidden process S1→ T1 is the predominant route for ra-

diationless deactivation of S1 in porphyrins. The fluorescence spectrum of pure TPP is

shown in figure 5.8. A strong emission in the range from 600nm to 800 nm with two

maxima at about 649 nm and 716 nm was observed with an intensity of 34.3 a.u and 24.2

a.u respectively (figure 5.8).

Figure.5.8. Fluorescence spectrum of TPP.

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5.5.1 Emission spectra of TPP bound polymer systems

The fluorescence spectra were recorded under the excitation wavelength of 400 nm on

a Hitachi F-3000 fluorescence spectrometer. In the entire polymer bound TPP systems,

the positions of the bands are not much shifted, but we could observe blue shifting of

3-5 nm in both the bands in the case of linear polymers like PEG and PG. PEG-TPP

system exhibited an intense emission at 646 nm and a shoulder at 714 nm and the extent

of shifting was by 3nm and 2nm respectively(figure 5.9).

Figure 5. 9. Fluorescence spectrum of PEG-TPP.

When TPP was bound with linear polyglycerol system the intensity of fluorescence

emission was decreased to 0.39 a.u and 0.23 a.u respectively for the bands. The emission

peak of the PG-TPP system appeared blue shifted by 4nm from 649 nm to 645 nm and

from 716 nm to 712 nm compared to that of TPP (figure 5.10).

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Figure 5.10. Fluorescence spectrum of PG-TPP system.

When TPP was anchored with HPG we have noticed red shifting in the emission and

the peak appeared at 649 nm in TPP was shifted to 652 nm and the peak at 716 nm was

shifted to 718 nm and the extent of red shift was by 7 nm and 2 nm respectively (figure

5.11).

Figure 5.11. Fluorescence spectrum of HPG-TPP system.

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From the figure 5.11 it is clear that the fluorescence intensity was reduced to 0.91a.u

at 652 nm and 0.51a.u at 718 nm.The emission properties are summarized in table 5.6.

Table 5.6. Summary of emission behaviour of polymer bound TPP

System Emission Wave-

length λ1

Emission Wave-

length λ2

Intensity

I

Intensity

II

TPP 649 716 34.3 24.2

PEG-TPP 646 714 11.1 9.4

PG-TPP 645 712 3.9 2.3

HPG-TPP 652 718 0.91 0.51

On attaching the linear polymeric cores such as PEG and PG, the emission spectra

showed a remarkable decrease in intensity and the emission maxima were blue shifted.

When attached to HPG also the intensity showed a hypochromic shift but the emission

maxima was red shifted. When the fluorophore is in hydrophobic environment the fluores-

cence intensity is enhanced due to the slow radiationless decay. TPP is highly hydrophobic

in nature. So its emission spectra show maximum intensity. Introduction of TPP on to

hydrophilic polymer backbones therefore decrease its hydrophobicity and hence the inten-

sity is decreased. The blue shift in the linear systems may be attributed to the coiling in

these systems. The extent of coiling is less due to the presence of bulky fluorophore and

so the shifting is less. No such coiling is possible in HPG and hece it shows red shift10.

5.6 Electronic Spectra of Metalloporphyrins

Upon metallation the porphyrin ring system deprotonates, forming a dianionic ligand.

The metal ions behave as Lewis acids, accepting lone pairs of electrons from the dian-

ionic porphyrin ligand. Unlike most transition metal complexes, their colour is due to

absorption(s) within the porphyrin ligand involving the excitation of electrons from π to

π * porphyrin ring orbitals. The electronic absorption spectrum of a typical porphyrin

consists of a strong transition to the second excited state (S0 → S2) at about 400 nm

(the Soret or B band) and a weak transition to the first excited state (S0 → S1) at about

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550 nm (the Q band). Internal conversion from S2 to S1 is rapid so fluorescence is only

detected from S1. The B and the Q bands both arise from π to π * transitions and can

be explained by considering the four frontier orbitals (HOMO and LUMO orbitals). This

is known as the Gouterman four orbital model (figure 5.12).

Figure 5.12 Orbital diagrams showing possible transitions for porphyrins.

According to this theory, the absorption bands in porphyrin systems arise from tran-

sitions between two HOMOs and two LUMOs, and it is the identities of the metal center

and the substituents on the ring that affect the relative energies of these transitions.

Mixing splits these two states in energy, creating a higher energy 1eu state with greater

oscillator strength, giving rise to the soret band, and a lower energy 1eu state with less

oscillator strength, giving rise to the Q-bands.

Metalloporphyrins can be divided into two groups based on their UV-vis and fluores-

cence properties11. Regular metalloporphyrins contain closed-shell metal ions (d 0 or d

10)for example Zn (II) in which the d π (dxz , dyz ) metal-based orbitals are relatively

low in energy. These have very little effect on the porphyrin π to π* energy gap in por-

phyrin electronic spectra (fig. 5.13). Hypsoporphyrins are metalloporphyrins in which

the metals are of dn, n= 6 to 9, having filled d π orbitals. In hypsoporphyrins there is

significant metal d π to porphyrin π* orbital interaction (metal to ligand π- backbonding)

(figure 5.14). This results in an increased porphyrin π to π* energy separation causing

the electronic absorptions to undergo hypsochromic shifts.

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Figure 5.13. Molecular orbital diagram for metalloporphyrins. Interactions between dπ

and π* occur in hypsoporphyrins.

Figure 5.14. The dπ metal orbital overlap with the π system of the porphyrin ring.

5.7 Electronic Spectra of Metalloporphyrins Bound

to Linear and Hyperbranched Polymers

Metalloporphyrins have similar spectra as that shown by TPP. But on binding with the

polymers, ie, linear as well as hyperbranched polymers, exhibit shifts in their absorption

spectra. We have developed several metalloporphyrins of Cu (II), Zn (II) and Fe (II),

bound with linear polymers such as PVA, PEG and polyglycerol polyol and hyperbranched

polyglycerol, looking forward to carry out their possibility of applying in photoresponsive

applications, artificial blood products and in photodynamic therapy.

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5.7.1 PVA-MTPP (M=Zn,Cu,Fe) system

The electronic spectra of all the MTPPs are very characteristic, with an intense band

near 415 nm and one or two less intense bands at 520-650 nm. On comparing with

metal free TPP, tetraphenyl porphyrin metallated with Zn (II) did not exhibit shift in

wavelengh while CuTPP and FeTPP exhibited red shift The metal free TPP bound with

PVA gives absorptions at 414 nm corresponding to soret band. The PVA-ZnTPP also

showed corresponding transitions at 415 nm. The soret bands of PVA-CuTPP and PVA-

FeTPP were, however, observed at 419 nm. There were considerable shifts observed in the

Q bands. In Zn (II) the dπ(dxz, dyz) metal-based orbitals are relatively low in energy. So

it has very little effect on π to π* energy gap in porphyrin electronic spectra. The same

trend is observed when ZnTPPs are bound to linear as well as hyperbranched polymers.

The UV-visible spectra of MTPPs (M= Zn (II), Cu (II) and Fe (II)) bound to PVA is

given in figure 5.15 and a comparison of the absorption maxima are given in table 5.7.

Figure 5.15. Comparative study of UV-vis spectra of PVA-MTPP system.

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Table 5.7. Summary of UV-vis absorptions of PVA-MTPP systems

MTPP-

Polymer

System

B band

(nm)

Q1 band

(nm)

Q2 band

(nm)

PVA -TPP 414 513 547

PVA- ZnTPP 415 514 549

PVA- FeTPP 419 517 554

PVA- CuTPP 419 519 553

5.7.2 PEG-MTPP (M=Zn,Cu,Fe) system

Metallated porphyrins bound with PEG exhibited characteristic transitions in their

UV-visible spectra. Compared to PEG-ZnTPP, PEG- CuTPP and PEG-FeTPP systems

exhibited red shifts. The UV-visible spectra of MTPPs (M= Zn (II), Cu (II) and Fe (II))

bound to PEG are shown in figure 5.16 and the results are summarised in table 5.8.

Figure 5.16. Comparative study of UV-Vis spectra of PEG-MTPP system.

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Table 5.8. Summary of UV-vis absorptions of PEG-MTPP systems

MTPP-

Polymer

System

B band

(nm)

Q1 band

(nm)

Q2 band

(nm)

PEG -TPP 411 509 541

PEG- ZnTPP 412 512 545

PEG- FeTPP 414 515 554

PEG- CuTPP 416 518 555

5.7.3 PG-MTPP ((M=Zn,Cu,Fe) system

The PG-MTPP systems also exhibited characteristic absorptions from the UV-visible

region. PG-CuTPP and PEG-FeTPP exhibited prominent red shifts compared to PEG-

ZnTPP. The UV-visible spectra of the PEG-MTPP systems are shown in figure 5.17. The

spectral shifts in both soret and Q bands are given in the table 5.9.

Figure 5.17. Comparative study of UV-vis spectra of PG-MTPP system .

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Significant changes in the electronic spectra (redshifts in both B and Q bands) were seen in

polymer-incorporated MTPPS in comparison with metal free systems. This is explained

in terms of the molecular distortions and associated changes in the metalloporphyrin

orbital overlap and the charge delocalization from the peripheral substituents 12.

Table 5.9.Summary of UV-vis absorptions of PG-MTPP systems

MTPP-

Polymer

System

B band

(nm)

Q1 band

(nm)

Q2 band

(nm)

PG- TPP 414 512 546

PG- ZnTPP 416 512 544

PG- FeTPP 418 516 551

PG- CuTPP 418 518 550

5.7.4 HPG-MTPP (M=Zn,Cu,Fe) system) system

The metallated porphyrin bound to HPG also showed significant shifts in the UV-

visible spectra. The HPG- ZnTPP system showed only small transitions while FeTPP

and CuTPP bound to HPG showed significant red shifts in their Q1and Q2 bands (figure

5.18). The UV-visible spectra of the PEG-MTPP systems are shown in figure 5.18 and

the UV-visible absorption wavelengths of HPG-MTPP systems are given in table 5.10.

The absorption characteristics of the HPG-MTPP systems show superior properties

over PVA-MTPP, PEG-MTPP and PG-MTPP systems (tables 5.7-5.10). The B (soret)

band of the MTPP systems bound to PVA were found to be at 415nm, 419nm and 419nm

for M=Zn, Fe and Cu respectively. The Q1 and Q2 bands of the PVA-MTPP systems

varies from 514 nm to 519 nm and 549 nm to 553 nm respectively. PEG-MTPP systems

showed absorption maxima for soret and Q bands at 416 nm (soret band of PEG-CuTPP)

and 555 nm (Q2 of PEG-CuTPP). The metallated TPP bound to linear polyglycerol

polyadipate showed maximum B-band absorption at 418 nm for PG-CuTPP and Q1 band

maximum at 518 nm and Q2 band maximum at 550 nm. The hyperbranched polyglycerol

anchored with MTPP showed excellent absorption characteristics. The B-bands of these

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systems observed at 458 nm,475 nm and 476 nm for HPG-ZnTPP, HPG-FeTPP and

HPG-CuTPP respectively. The Q bands also showed remarkable red shift. The Q1 bands

where observed at 557 nm, 574 nm and 566 nm for HPG-ZnTPP, HPG-FeTPP and HPG-

CuTPP systems. The corresponding Q2 band positions where found to be at 587 nm,

609 nm and 603 nm respectively. The absorption intensities of HPG-MTPP systems were

of high value compared to metal analogues. The remarkable red shift and highly intense

absorptions of HPG-MTPP systems make them excellent photoresponsive materials.

Figure 5.18. Comparative study of UV-vis spectra of HPG-MTPP system

(a)HPG-TPP, (b)HPG- ZnTPP, (c)HPG-FeTPP, (d)HPG-CuTPP.

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Table 5.10. Summary of UV-vis absorptions of HPG-MTPP systems

MTPP-

Polymer

System

B band

(nm)

Q1 band

(nm)

Q2 band

(nm)

HPG- TPP 472 563 600

HPG- ZnTPP 458 557 587

HPG- FeTPP 475 574 609

HPG- CuTPP 476 566 603

5.8 EPR Spectral Studies of Polymer Bound CuTPP

We have made an attempt to confirm the presence of Cu(II) in the polymer by record-

ing the EPR spectra of the compounds CuTPP anchored on to linear and hyperbranched

polymers. The EPR spectra indicate the presence of Cu(II) in all the compounds. The

EPR spectra of Cu (II) complexes were recorded using a Varian E-112 EPR spectropho-

tometer operating at 9.1 GHz with a microwave power of 5mw. The field set is around

3000G and the scan range used was either 2000 or 1000 G. DPPH was used as g marker.

(i) PVA- CuTPP system

The EPR spectrum of PVA- CuTPP system was recorded in DMF at room temper-

ature and the spectrum is given in figure 5.19. The spin Hamiltonian parameters of the

spectrum are A‖ (gauss) 178, A⊥ (gauss) 30, g‖ 2.235 and g⊥ 2.075, characteristic of the

copper porphyrin.

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Figure 5.19. The EPR spectrum of PVA- CuTPP system.

(ii)PEG- CuTPP system

The EPR spectrum of PEG-CuTPP system recorded in DMF at room temperature

is given in figure 5.20. The spin Hamiltonian parameters of the spectrum are A‖ (gauss)

180, A⊥ (gauss) 20, g ‖ 2.239 and g⊥ 2.080.

Figure 5.20. The EPR spectrum of PEG- CuTPP system.

(iii)PG- CuTPP system

The following figure represents the EPR spectrum of PG- CuTPP system. The spin

Hamiltonian parameters are A‖ (gauss) 173, A⊥ (gauss) 30, g‖ 2.243 and g⊥ 2.047,

indicating the presence of copper incorporated in the porphyrin macrocycle.

161

Figure 5.21. The EPR spectrum of PG- CuTPP system.

(iv) HPG- CuTPP system

The EPR spectrum of HPG- CuTPP system recorded at room temperature showed a

strong signal at high field and the representative of the spectrum is given in figure 5.22.

The spin Hamiltonian parameters of the spectrum are A‖ (gauss) 175, A⊥ (gauss) 30, g‖

2.234 and g⊥ 2.048. all these results give evidence for the incorporation of copper in the

macrocyclic frame work of porphyrin system.

Figure 5.22. The EPR spectrum of HPG- CuTPP system.

162

We could evaluate the spin-Hamiltonian parameters assuming axial symmetry for all

the four systems. The giso and G could be evaluated using the following equations,

giso = g⊥ + g‖ and

The values are given in Table 5.11. Various spin-Hamiltonian parameters were cal-

culated from the spectra using DPPH as the g marker. The trend g‖ > g⊥ > 2.0023

observed for these complexes show that the unpaired electron is localized in the dx2-y2

orbital. All the four compounds gave only broad peaks characteristic of axial symmetry in

the solid state. G> 4.0 indicate that the local tetragonal axes are only slightly misaligned

while for G >4.0 indicate that the misalignment is appreciable. Axial spectra with lowest

g > 2.04 exhibited by all compounds refer to axial symmetry with all the principal axes

aligned parallel, and would be consistent with square planar stereochemistry.

Table 5.11. Summary of Spin Hamiltonian parameters of the polymer bound

Cu(II)TPP systems

compound A ‖ A ⊥

(gauss)

g ‖ g⊥ giso G

PVA- CuTPP 178 30 2.235 2.076 2.131 3.15

PEG-CuTPP 180 20 2.239 2.080 2.133 3.05

PG-CuTPP 173 30 2.243 2.047 2.113 5.30

HPG-CuTPP 175 20 2.234 2.048 2.110 5.04

5.9 Fluorescence Studies of Polymer Bound Metallo-

porphyrins

In order to study the effect of metallation on the emission properties, the emission

spectra of the metallo porphyrin derivatives of linear and hyperbranched polymers were

subjected to luminescent emission studies. We have selected metallated PEG-TPP as a

representative of linear polymeric systems and compared it with hyperbranched polymer.

It was found that the introduction of metal ion increases the fluorescence intensity in both

the cases. This may be attributed to the increased rigidity of the molecule upon metal-

163

lation. The increase in rigidity of the molecule is expected to decrease the intersystem

crossing and hence an increase in fluorescence intensity13.

The fluorescence emission spectra of polymer bound metalloporphyrins were recorded

in DMF at an excitation wavelength of 400 nm. The metalloporphyrins were anchored

on to linear and hyperbranched polymers and the fluorescence emission properties were

investigated. The PEG-ZnTPP, PEG-FeTPP and PEG-CuTPP showed interesting emis-

sion properties. The spectra are given in figure 5.23. The λ max as well as the intensity

values of PEG- ZnTPP, PEG-Fe TPP and PEG-CuTPP are given in table 5.12. PEG-

TPP system gave emission maxima at 646 nm and 714 nm with an intensity of 11.1 a.u

and 9.4 a.u respectively. The corresponding values of PEG- ZnTPP, PEG- FeTPP and

PEG-CuTPP are 645 nm and 707 nm (33.1.u and 21.2 a.u), 648 nm and 715 nm (34.1a.u

and 24.3a.u) and 649 nm and 716 nm (36.8 a.u and 27.7 a.u ) respectively.

Figure 5.23.Emission spectra of (a) PEG- TPP, (b) PEG -ZnTPP, (c) PEG- FeTPP

and (d) PEG- CuTPP.

164

Table 5.12. Summary of emission behaviour of PEG bound MTPP

System Emission Wave-

length λ1

Emission Wave-

length λ2

Intensity

I

Intensity

II

PEG-TPP 646 714 11.1 9.4

PEG-ZnTPP 645 707 34.1 24.3

PEG-FeTPP 648 715 36.8 27.7

PEG-CuTPP 649 716 33.1 21.2

The fluorescence emission studies of HPG-MTPP systems gave promising results.

HPG-TPP system showed emission maxima at 652 nm and 718 nm with an intensity

of 0.91a.u and 0.51a.u respectively. The HPG-CuTPP system showed maximum intensity

of absorption at 649 nm and 716 nm (intensity 33.9a.u and 24.4 a.u ). the spectra are

given in figure 5.24 and the results are summarised in table 5.13.

Figure 5.24. Comparative study of emission spectra of (a) HPG-TPP, (b) HPG

-ZnTPP (c) H PG- FeTPP (d) H PG- CuTPP.

165

Table 5.13. Summary of emission behaviour of HPG bound MTPP

System Emission

Wave-

length

λ1

Emission

Wave-

length

λ2

Intensity

I

Intensity

II

HPG-TPP 652 718 0.91 0.51

HPG-ZnTPP 645 714 4.1 2.2

HPG-FeTPP 646 714 11.1 9.3

HPG-CuTPP 649 716 33.1 21.2

5.10 Solvation Studies

The unique absorption spectra of porphyrin systems have allowed their identifica-

tion and study throughout the biological realm. It is generally observed that metallic

porphyrins exhibit notable variations in spectral properties, which are dependent on the

nature of the solvents. This is attributed to the relative ligation properties of the solvents

used with the metal. Compared to the metalloporphyrins, the free-base porphyrins lack

the ability to co-ordinatively add the ligands. Consequently the metal- free porphyrins

are not expected to show such change in the electronic spectra depending on the solvent,

except due to any possible tendency towards aggregation. But remarkably enough, while

measuring the absorption spectra of TPP-polymer system under study, we found that the

absorption changes were significant enough in solvents. A series of solvents with varying

polarity is used for the solvation studies. The solvents used were DMSO, DMF, methanol,

toluene, hexane and chloroform.. The electronic spectra of the TPP-polymer system in

various solvents were investigated and the λ max values are given in table 5.14.

166

Table 5.14. The λmax(nm) values of TPP and TPP-polymer systems in various

solvents

System Bands DMSO DMF methanol chloroformtoluene

TPP B 439 416 414 414 412

Q1 522 514 512 512 510

Q2 558 550 527 526 526

Q3 603 591 556 550 550

Q4 710 648 641 640 645

PVATPP B 449 414 insoluble insoluble 412

Q1 514 513 insoluble insoluble 506

Q2 548 547 insoluble insoluble 512

Q3 601 590 insoluble insoluble 540

Q4 661 644 insoluble insoluble 582

PEG-TPP B 422 411 408 405 405

Q1 518 509 506 500 501

Q2 535 541 523 518 516

Q3 598 583 578 578 577

Q4 682 639 628 620 620

PG-TPP B 444 414 413 412 405

Q1 518 512 512 501 501

Q2 555 546 548 538 538

Q3 603 590 586 578 577

Q4 691 647 644 640 640

HPG-TPP B 488 472 463 463 460

Q1 582 563 560 558 557

Q2 610 600 597 596 596

Q3 652 640 638 637 637

Q4 710 694 692 687 685

167

The data clearly reveal that all the bands (B, and Q) are red shifted on increasing

the polarity of the solvent. This behavior indicate that the excited state of these com-

pounds are more polar than their ground state and thus, this red shift can be ascribed to

stabilization of the polar excited state as the polarity of the solvent increased i.e., lower

excitation energy is required in DMSO or DMF relative to CHCl3. This confirm the local

excitation nature, i.e. the π- π* character of the bands.

The electronic spectra of polymer-porphyrin system in mixed solvents (DMF and

CHCl3) were also studied. The results also reveal that as the percentage of DMF increases,

the λ max values shifts to the longer wavelength region. This may be due to increased

πconjugation due to increased polarity and the red shift is consistent with the decrease

of HOMO-LUMO gap. This also confirms the π- π* character of the bands. The λmax

values of different TPP polymer systems in various mixed solvents are given in the table

5.15.

5.11 Light Fastening Studies on Polymer TPP Sys-

tems

From the viewpoint of photochemistry and photobiology, interactions of solar radiation

with matter are considered to occur when one photon interacts with one molecule to

produce a photochemically altered molecule or two dissociated molecules. Porphyrin is

highly photosensitive and this macrocyclic ring in chlorophyll acts as the photosensitizer

in photosynthesis.

To study the action of light on TPP and polymer bound TPP we have irradiated the

equimolar solutions of TPP and polymer bound TPP systems under visible radiant ener-

gies and measured the absorbances in definite time intervals. We have noticed significant

changes in the intensity of absorptions for the free TPP as well as the polymer bound

TPP. Compared to other photochromic systems, TPP is very fast towards light but it

also exhibits shifts in the intensity of absorptions (figure 5.25).

168

Table 5.15. The λmax(nm) values of polymer-porphyrin system in mixed solvents

(DMF and CHCl3)

System Bands 0%DMF 20%DMF 40%DMF 60%DMF 80%DMF100%DMF

TPP B 411 412 412 414 414 416

Q1 508 510 510 512 512 514

Q2 526 532 538 546 549 550

Q3 550 559 563 576 583 591

Q4 640 642 642 646 648 648

PVATPP B 406 408 411 414 414 414

Q1 499 502 507 507 507 513

Q2 511 512 512 512 514 547

Q3 532 540 541 544 544 590

Q4 571 568 572 577 578 644

PEG-TPP B 406 408 410 410 410 411

Q1 500 506 508 508 509 509

Q2 520 522 522 524 525 541

Q3 578 578 580 580 582 583

Q4 620 622 627 628 630 639

PG-TPP B 405 410 412 414 414 414

Q1 501 508 508 510 510 512

Q2 538 542 542 544 546 546

Q3 577 588 588 588 590 590

Q4 640 640 643 644 645 647

HPG-TPP B 456 458 461 461 462 472

Q1 557 557 559 560 560 563

Q2 590 591 593 597 600 600

Q3 634 635 635 638 638 640

Q4 685 685 689 691 694 694

169

Figure 5.25. Variation of absorbance of TPP on irradiation.

The curve (a) represents the absorption band of TPP at zero time and the absorbance

intensities in the visible region are B (2.2), Q1 (1.02), Q2 (0.69), Q3 (0.59) and Q4 (0.52).

On irradiation for five hours the intensities were decreased as B (1.83), Q1 (0.84), Q2

(0.36), Q3 (0.24) and Q4 (0. 17). Further irradiation to infinite time did not cause any

change in the spectra.

Table 5.16. Results of light fastening studies on TPP system

time(hrs) B band

(inten-

sity(a.u))

Q1 band

(inten-

sity(a.u))

Q2 band

(inten-

sity(a.u))

Q3 band

(inten-

sity(a.u))

Q4band

(inten-

sity(a.u))

0 2.2 1.02 0.69 0.59 0.52

1 2.04 0.86 0.36 0.24 0.21

2 1.86 0.86 0.36 0.24 0.17

3 1.83 0.84 0.36 0.24 0.17

4.5hr 1.83 0.84 0.36 0.24 0.17

24 1.83 0.84 0.36 .24 0.17

170

On irradiation, chlorosulphonated TPP was found to be more stable than TPP, to-

wards irradiation though there was small regular decrease in the intensity of absorbance

on prolonged irradiation (figure 5.26).

Figure 5.26. Variation of absorbance of TPP SO2Cl on irradiation.

Table 5.17. Results of light fastening studies on TPP-SO2Cl

time(hrs) B band

(inten-

sity(a.u))

Q1band

(inten-

sity(a.u))

Q2band

(inten-

sity(a.u))

Q3band

(inten-

sity(a.u))

Q4band

(inten-

sity(a.u))

0 1.9 0.74 0.48 0.30 0.21

0.5 1.8 0.59 0.38 0.22 0.15

1 1.79 0.54 0.35 .0.19 0.12

2 1.79 0.45 0.29 0.19 0.13

3 1.79 0.41 0.23 0.14 0.08

4 1.79 0.41 0.23 0.14 0.08

5 1.79 0.41 0.23 0.14 0.08

24 1.79 0.41 0.23 0.14 0.08

171

PVA bound TPP was found to be highly stable on irradiation with visible light. No

appreciable change in intensity was noted even after 5 hours of irradiation. The light

fastening behaviour of the TPP macrocycle binding to PVA core is evident from the

results shown in figure 5.27. The intensities observed for PVA-TPP systems were 1.98

and 0.25 for the soret and Q bands respectively.

Figure 5.27. Variation of absorbance of PVA-TPP on irradiation.

Table 5.18. Results of light fastening studies on PVA-TPP system

time(hrs) B band

(inten-

sity(a.u))

Q1band

(inten-

sity(a.u))

Q2band

(inten-

sity(a.u))

Q3band

(inten-

sity(a.u))

Q4band

(inten-

sity(a.u))

0 1.98 0.25 0.16 .12 .08

1 1.76 0.21 0.12 0.09 0.06

2 2.04 0.19 0.10 0.05 0.05

3 1.86 0.19 0.10 0.05 0.05

4 1.83 0.19 0.10 0.05 0.05

4.5 1.83 0.19 0.10 0.05 0.05

24 1.86 0.19 0.10 0.05 0.05

172

When PEG bound TPP was irradiated and measured the absorbance spectra we ob-

served a small decrease in the in the absorbance values during the first one hour. After

that the values remain constant for many hours. In figure 5.28 the band (a) represents

the absorbance at zero time and absorbances were measured for different time intervals.

The spectra do not show any appreciable change in intensity on prolonged irradiation.

This shows the light fastening property of PEG-TPP system and this may be due to the

stabilization of porphyrin macrocycle when it is bound to polymeric core such as PEG.

Figure 5.28. Variation of absorbance of PEG-TPP on irradiation

Figure 5.28. Variation of absorbance of PEG-TPP on irradiation.

173

Table 5.19. Results of light fastening studies on PEG-TPP system

time(hrs) B band

(inten-

sity(a.u))

Q1band

(inten-

sity(a.u))

Q2band

(inten-

sity(a.u))

Q3band

(inten-

sity(a.u))

Q4band

(inten-

sity(a.u))

0 1.20 0.06 0.03 0.01 0.003

1 1.20 0.06 0.03 0.01 0.003

4 1.20 0.06 0.03 0.01 0.003

4.5 1.20 0.19 0.10 0.05 0.05

24 1.20 0.06 0.03 0.01 0.003

Polyglycerol polyol bound TPP was subjected to time controlled irradiation studies

using radiant energy from the visible light. The PG-TPP system showed high stability

and light fastening on prolonged irradiation. The intensity of absorption also shows that

this new system is highly stable even after prolonged exposure to radiant energies. The

results are shown in figure 5.29.

Figure 5.29. Variation of absorbance of PG-TPP on irradiation.

174

Table 5.20. Results of light fastening studies on PG-TPP system

time(hrs) B band

(inten-

sity(a.u))

Q1band

(inten-

sity(a.u))

Q2band

(inten-

sity(a.u))

Q3band

(inten-

sity(a.u))

Q4band

(inten-

sity(a.u))

0 2.11 0.21 0.12 0.07 0.06

1 2.10 0.13 0.06 0.04 0.03

2 2.10 0.21 0.12 0.04 0.04

3 2.10 0.21 0.12 0.04 0.04

4 2.10 0.21 0.12 0.04 0.04

4.5 2.10 0.21 0.12 0.04 0.04

24 2.10 0.21 0.12 0.04 0.04

TPP bound HPG had been irradiated for 7 hours continuously in sunlight and the ab-

sorbances were measured by UV-visible spectroscopy. The absorption values were found

to remain constant and there was no change in λmax as well. This indicated that por-

phyrin macrocycle was highly stabilized when bound to HPG. The intensity of absorption

was appreciably high in this system and the intensity remains unchanged on exposure for

long time. The high loading, non-entangled configuration and optimum spacial rigidity

of the hyperbranched polyglycerol contribute to these properties (figure 5.30)

Figure 5.30. Variation of absorbances of HPG-TPP on irradiation .

175

Table 5.21. Results of light fastening studies on HPG-TPP system

time(hrs) B band

(inten-

sity(a.u))

Q1band

(inten-

sity(a.u))

Q2band

(inten-

sity(a.u))

Q3band

(inten-

sity(a.u))

Q4band

(inten-

sity(a.u))

0 2.12 0.21 0.11 0.07 0.07

1 2.10 0.13 0.06 0.04 0.03

2 2.10 0.13 0.06 0.04 0.03

3 2.10 0.13 0.06 0.04 0.03

4 2.10 0.13 0.06 0.04 0.03

4.5 2.10 0.13 0.06 0.04 0.03

24 2.10 0.13 0.06 0.04 0.03

5.12 Antimicrobial Phototherapy

Phototherapy is the term used to describe treatments, which use light to achieve

their effects14. For example blue light exposure, which is used to treat newborn babies

with neonatal jaundice. In this case blue light absorbed by bilirubin, the yellow pigment

responsible for producing the skin discoloration, and turns it in to a more soluble form,

which is easier to excrete from the body.

Another application of phototherapy, called phototodynamic therapy (PDT), uses a

combination of electromagnetic radiation (usually laser light) and a drug to selectively

target and destroy cancers. Photodynamic therapy matured as a feasible medical technol-

ogy in 19806s at several institutions through out the world for the treatment of carcinomas

and sarcomas15. The German physician, Mayer-Betz performed the first study with pho-

todynamic therapy with porphyrins in humans in 1913. It is also being investigated for

treatment of psoriasis and acne, and is approved for the treatment of muscular degenera-

tion. Mayer-Betz tested the effects of haemato porphyrin-PDT on his own skin. Modern

day versions of it were tested at the Mayo Clinic at Rosewell Park Cancer Center, but

really did not become widespread until Thomas Dougherty16 initiated clinical trials and

formed the International Photodynamic Association in 1986.

176

The mechanism of photodynamic therapy involves the hopefully selective uptake and

retention of a photosensitizer in a tumor, followed by irradiation with light of particu-

lar wavelength, thereby initiating tumor necrosis presumably through the formation of

singlet oxygen17. It is a ternary treatment for cancer involving three key components:

photosensitizer, light and tissue oxygen18.

5.12.1 Photosensitizer

Photosensitizer is a chemical compound that can be excited by light of a specific

wavelength. This excitation uses visible or near infra red light. It is important that the

sensitizer should be easy to administer systemically via injection in to the blood stream.

Water-soluble sensitizer would therefore be expected to be the most useful since the blood

is a water-based system. But the sensitizer must also be able to get in to the cells by

traversing lipid membranes, thus it should ideally also be hydrophobic.

All photosensitizing agents used in PDT have very similar structures and are often

based on naturally occurring molecules including hemoglobin (the substance that makes

blood look red), vitamin B12, and chlorophyll (the chemical used by the plants for pho-

tosynthesis and which gives them green colour). These compounds are all known as

macrocycles and contain nitrogen, oxygen or sulphur atoms locked inside a large hollow

ring. In some cases the ring also contains a metal such as iron or magnesium. The pho-

tosensitizing agent used as the drug in PDT is given to the patient, which accumulates

in the diseased tissue. It is harmless in its inactive form, but when it is excited by light

of the correct wavelength (usually in the form of laser) it produces singlet oxygen, which

is highly toxic and kills the cell (figure 5.31).

Photodynamic therapy currently makes use of a range of agents from plant extracts

to complex synthetic macrocycles, but characteristically, they are all able to accumulate

selectively in the diseased tissue.

177

Figure 5.31. The process of photodynamic therapy. [A drug is given to the patient (1)

which accumulates in the diseased tissue (2) In its inactive form the agent is harmless

(3) but when it is excited by light of the correct wavelength (usually in the form of a

laser) it produces singlet oxygen which is highly toxic (4) and kills the cell]

5.12.2 Porphyrins as photosensitizers

Human tissue transmits light most effectively in red region of the visible spectrum

and hence photosensitizers with strong absorption band in this region (650-800nm) can

be activated to penetrate deeper in to the tissues. Porphyrins are 22π electron systems

whose main aromatic conjugation pathway contains 18π electrons, which explains long

wavelength absorptions and the intense colour associated with them.

Porphyrinoid photosensitizers have more than one absorption band that can be utilized

for tissue depth controlled penetration. Provided that the porphyrin posses an absorption

maximum at a wavelength corresponding to that of the incident laser light, shining light of

highly coloured porphyrin causes excitation to the singlet state (1P*). The singlet-excited

porphyrin can decay back to the ground with release of energy in the form of fluorescence-

enabling identification of tumor tissue. If the singlet excited state lifetime is suitable (and

this is true for many porphyrins) it is possible for the singlet excited state to be converted

in to the triplet-excited state (3P*), which is able to transfer energy to another triplet-

178

excited state. One of the very few molecules with a triplet ground state is dioxygen, which

is found in most cells. Energy transfer therefore takes place to afford highly toxic oxygen

(1O2) from ground state dioxygen (3O2), provided the energy of the molecules are higher

than that of the product (1O2)19,20 . The reason that singlet oxygen is liberated in the

cells is because of simple photophysics. Figure 5.32 shows a simplified Jablonski diagram

showing the photophysics of the sensitization process used in photodynamic therapy.

Figure 5.32. Photophysics of PDT sensitization.

5.13 Phorphyrin Bound Hyperbranched Polyglycerol

for Antimicrobial Phototherapy

5.13.1 Biocompatibility of hyperbranched polyglycerol

Many biologically active compounds are not suitable for therapeutic purposes because

of their poor solubility, limited bioavailability and rapid elimination. More than that,

while the beneficial effects of many drugs arise through their interactions with specific

tissues, their exposure to other cell types frequently lead to other side effects and toxic-

ity. In recent years there has been increasing interest in photoactive dendritic systems.

End group modified dendrimers are under investigation in a variety of applications such

as carriers of drug and other guest molecules. But the major drawback is their mul-

tistep synthesis and therefore hyperbranched polymers selected as an alternative. The

179

hyperbranched polyglycerol synthesized by the anionic ring-opening multibranching poly-

merization (ROMBP) of glycidol consists of an inert polyether backbone with functional

hydroxyl groups at every branch end. This structural feature resembles the well known

poly (ethylene glycol) (PEG) that is accepted for various biomedical applications. Hy-

droxyl terminated dendrimers based on polyether scaffold have been shown to be of low

toxicity. The non-toxic properties make these new polymers very promising candidates

for drug delivery devices.

The hydroxyl groups of HPG are derivatized with chlorosulphonated porphyrin groups.

The porphyrin which is also biocompatible but hydrophobic become water-soluble by

connecting with HPG.

Before considering the HPG-porphyrin system as a photosensitizer we have considered

a number of issues on PDT. It is important that the sensitizer must be easy to administer

systemically (via injection in to the blood stream). Water-soluble sensitizers would there-

fore be expected to be the most useful since the blood is a water-based system. But the

sensitizer must also be able to get in to by traversing lipid membranes. Thus it should

also be hydrophobic. The porphyrin-polyglycerol system is such an amphiphilic molecule.

Another factor is that the sensitizer must also absorb at long wavelength (i.e., at low

energy). This is because the low energy light travels further through tissue than does high-

energy light (which gets scattered). If we want to kill big tumors by generating singlet

oxygen inside them we need to have sensitizer which likewise absorbs low energy light -

one which has absorption peak in the low energy (long wavelength) area of the electronic

absorption spectrum. The absorptions of TPP in the Q band region were strongly red

shifted after coupling it with HPG (figure 5.34).

180

Figure 5.33. Structure of HPG-TPP system

The porphyrinated hyperbranched polyglycerol exhibited intense absorptions in the

visible region of the spectrum (table 5.22).

We have studied the UV-visible absorptions of linear polymer bound porphyrins like

PVA-TPP, PEG-TPP and PG-TPP and compared them with the absorbance of HPG-

TPP system. The HPG-TPP system exhibited significant red shiftin its absorptions for

the soret and all the four Q bands. of 46 nm to a longer wavelength of 694 nm, which is

a primary requirement for an ideal photosensitizer.

181

Figure 5.34. UV-Vis spectra of (a) TPP (b) HPG-TPP (c) PVA- TPP (d) PEG-TPP

and (e) PG-TPP .

Table 5.22. Light absorption by TPP and TPP bound HPG

Bands λ (nm)

(TPP)

εmax

(TPP)

λ (nm)

(HPG-

TPP)

εmax

(HPG-

TPP)

Q1 514 0.35 563 2.4

Q2 550 0.168 600 1.73

Q3 591 0.11 640 1.13

Q4 648 0.103 694 1.008

The linear systems such as PVA-TPP, PEG-TPP and PG- TPP and their metallated

systems do not have absorptions at longer wavelength region and not enough to meet

the benchmark requirements of PDT agents. However HPG-TPP system meets all these

182

requirements and can be used as an efficient system for PDT. The soret band of HPG-

TPP shows signal at 472 nm with an intensity of 2.1. The Q1, Q2, Q3 and Q4 bands were

observed at 563 nm, 600 nm, 640 nm and 694 nm with intensities 2.4, 1.73, 1.13 and 1.008

respectively.

It is extremely important to be able to synthesize pure molecules for the use as pho-

tosensitizer. The porphyrin bound HPG, however can be easily prepared, functionally

modified and purified to equip them with intense light responsive and novel therapeutic

applications.

Simpler and economical therapies that can effectively treat cancer with minimum side

effects are the dire straits of this new millennium. Last century witnessed a renewed surge

in reinventing the medicinal aspects of light in presence of photosensitizers namely, Pho-

todynamic therapy, was found to be exceptionally useful as anticancer treatment protocol.

HPG-porphyrin based PDT can offer a promising treatment protocol for cancers and va-

riety of other diseases for which remedial measures existing are minimal. The realization

that any chromophore that can induce phototoxicity upon illumination leading to selec-

tive destruction of diseased (premalignant, malignant and benign) tissues lead to incessant

possibilities and any step taken in the design and development of such chromophores are

very much appreciated. HPG-TPP system can make wonders in this field.

5.14 Antimicrobial Activity of HPG-TPP System

The science of photodynamic antimicrobial therapy follows similar principles of PDT. It

is argued that the widespread systemic use of antibiotics is a cause of multidrug resistance

and local therapy using photodynamic agents would lessen the risk of such collateral ef-

fects. Localized infections need not be treated with systemic medication if an efficient

alternative is available. In the past decade photosensitized porphyrin derivatives in con-

junction with laser light were adopted for treating several microbial infections on skin.

There were studies on photodynamic action against microorganisms such as Salmonella

typhimurium, or the yeast, Saccharomyces cerevisiae21. Photosensitizers being readily

available and inexpensive should be attractive in the area of low cost topical health

183

care regimens. The natural product porphyrins are effective against a range of anaer-

obic bacteria. Porphyrin sensitizers are more effective against gram-positive bacteria but

gram-negative organisms are more refractory to photodyanamic activity due to their more

complex cell wall22,23. Although several light sources proposed as lasers and xenon arc

lamp etc, antimicrobial therapy uses low power density light rather than lasers. Microbial

photokilling is attained with milliwatts rather than tens or hundreds of watts19. The

power density of a light source is normally given in mW/cm2 where as the light source

describes the energy received (by a wound or a petri dish) and as such can be calculated

as the power density multiplied by the illumination time (in seconds). The use of directed

light against microbial pathogens in situ also causes the problems of the possibility of col-

lateral damage. Such effects can be minimized by keeping a minimum distance between

the light source and petri dish concerned so as to keep the temperature 370C-390C. In

the present studies we have chosen 500W tungsten filament lamp (wave length >600nm)

having a power density 0.28 mW/cm2.

The mechanism of photodynamic microbial killing is same as that of tumor cell necrosis

in PDT. On irradiation the photosensitizer gets excited to the triplet state which trans-

fers its energy to molecular oxygen, and the singlet oxygen formed in situ then reacts

rapidly with its environment-cell wall, nucleic acids, peptides etc. The short half-life of

singlet oxygen again ensures a localized response.18 Haematoporphyrin derivative is the

first preparation used in clinical PDT and has some activity against both bacteria and

viruses24,25. Many porphyrins are benign in the dark but are transferred by sunlight and

produce singlet oxygen, which is toxic to cells. In the present work, the study of photo

toxicity after 4-hr incubation with TPP, MTPPs, HPG-TPP and HPG-MTPPs on both

Staphylococcus aureus and Escherichia coli were carried out in vitro. The different steps

by which the experiment was done is as follows:

1. 18 number of 10ml nutrient broth tubes were prepared and sterilized at 121 0C and

under 15 lbs pressure. Then they are appropriately labeled.

2. A loopful inoculam of Staphylococcus aureus were asceptically transferred in to 9

tubes of the medium kept at room temperature. Similarly a loopful of inoculam

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of Escherichia coli was also asceptically transferred in to the other 9 tubes of the

medium kept at room temperature.

3. Incubated all the tubes at 37 0C for 4 hours.

4. After incubation, 5 mg of each of the samples to be tested were added in appropri-

ately labeled tubes.

5. Reincubated the culture tubes at 37 0C for further 12 hours.

6. After the incubation period, 100µl aliquot from each broth was transferred to

petridishes and spread plates were prepared. The plates were incubated at 37 0C

for 24 hours.

7. Then the tubes were irradiated using 500W tungsten filament bulb for 2minutes.

8. After irradiation, the tubes were left in atmospheric condition for 30 minutes. After

that 100µl aliquot from each tube is transferred to petridishes and spread plates

were prepared.

9. The tubes were further irradiated for another 3 minutes and spread plates were

again prepared.

10. All the plates were incubated at 370C for 24 hours.

11. After the incubation period, the plates were examined for bacterial growth.

In the present studies we have selected two bacterial cultures, gram positive Staphy-

lococcus aureus and gram negative Escherichia coli. The systems selected to test photo-

senzitivity were TPP, FeTPP, HPG-TPP and HPG-FeTPP. The observations were differ-

ent for different systems and were interesting. The gram-negative E.coli bacterium was

found to be refractory to all the systems but the gram-positive bacteria, Staphylococcus

aureuswere killed by the photo oxidation even though a complete destruction was not

achieved. We have done a comparative study of photosensitizing property of porphyrin,

its metal (Fe) incorporated system and their hyperbranched polyglycerol bound systems.

185

The spread plates prepared using systems before irradiation gave uncountable colonies of

microbes while after irradiation microbial killing was achieved and have obtained count-

able colonies of microbes in the petri dishes. Of these systems FeTPP exhibited a very

significant toxicity. The polymer bound TPP and FeTPP systems were also exhibited

efficient photokilling of the microbes. The photodynamic efficiency of TPP was retained

on binding with HPG. The advantage is that the HPG-TPP as well as the HPG-FeTPP

systems were water soluble and more biocompatible than the polymer free TPP systems.

The comparative study of photodynamic antimicrobial activity of the systems under study

were possible by examining the photographs given below. TPP strongly acted on Staphy-

lococcus aureus and the colonies became countable on irradiation for short period of two

minutes. The colony was reduced to almost 75 percent on irradiation for a time span of

5 minutes (figure 5.35).

Figure 5.35. Staphylococcus aureus colony inoculated with TPP: (a) before irradiation-

uncountable (b) after irradiation for 2 minutes- countable (c) after irradiation for 5

minutes- countable.

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The action on bacteria, Escherichia coli was not evident and the colony remains

uncountable even after irradiating for 10 minutes. This may be due to its more complex

cell wall compared to gram-positive bacteria23(figure 5.36).

Figure 5.36. Escherichia coli colony inoculated with TPP: (a) before irradiation-

uncountable (b) after irradiation for 10 minutes- Uncountable .

HPG bound metal free TPP also had photodynamic antimicrobial property against

Staphylococcus aureus and this system became superior to TPP because of its hydrophilic-

ity and biocompatibility. The antimicrobial property of HPG-TPP system before and

after irradiation was given in figure 5.37. the entire bacterial colony remain intact before

irradiation of the system with visible radiant energy. On irradiation for two minutes, the

bacterial colony was severely attacked by the newly developed photodynamic antimicro-

bial agent HPG-TPP. This is evident from figure 5.37 (b). On completing irradiation for

5 minutes, more than 75 percent of the bacterial colony was destroyed by the HPG-TPP

photodynamic antimicrobial agent (figure 5.37 (c)).

187

Figure 5.37. Staphylococcus aureus colony inoculated with HPG-TPP : (a) before

irradiation- uncountable (b) after irradiation for 2 minutes- uncountable (c) after

irradiation for 5 minutes- countable) .

When metallated systems were used, against Staphylococcus aureus, the property of

microbial killing was found to be enhanced due to the presence of the heavy metal which

itself inhibits microbial growth. Therefore FeTPP exhibited surprisingly good result when

irradiated for 5 minutes (figure 5.38). The colony becomes countable after 2 minutes of

irradiation and almost 80% of the colony was destroyed on irradation for three minutes

more. It was ensured that the distance between tubes and the bulb were arranged in a

manner that, the temperature within the tubes were not exceed as 37-390C.

188

Figure 5.38. Staphylococcus aureus colony inoculated with FeTPP : (a) before

irradiation- uncountable (b) after irradiation for 2 minutes- countable (c) after

irradiation for 5 minutes- countable.

When FeTPP bound HPG system was used in the antimicrobial phototherapy against

gram positive Staphylococcus aureus, we could notice its significant effect on microbial

killing and the effect was more enhanced than metal free HPG-TPP or simple TPP or

FeTPP system. The bacterial colony almost half on irradiation for two minutes using

visible radiant energy. On further irradiation, the effective photokilling was observed and

further after 5 minutes of irradiation almost 90% of the bacterial colony was photokilled

(figure 5.39).

189

Figure 5.39. Staphylococcus aureus colony inoculated with HPG- FeTPP : (a) before

irradiation- uncountable (b) after irradiation for 2 minutes- countable (c) after

irradiation for 5 minutes- countable.

From the above results it is clear that both porphyrin and HPG bound porphyrin

systems are effective photosensitizers which can be used against gram -positive bacteria

and by increasing the duration of irradiation cidal effect can be achieved.

5.15 Porphyrin Bound Hyperbranched Polyglycerol

as Potential Artificial Blood Product

One of the principal tasks of blood is to transport oxygen throughout the body and

then to release the oxygen to tissues. This is accomplished by the oxygen-carrying pro-

tein haemoglobin, which possess an Fe(II) porphyrin unit at its active site. It is this

Fe(II) porphyrin that is responsible for (reversibly) binding oxygen. The protein serves

to protect and isolate the oxygen binding porphyrin units, as well as helping prevent its

deactivation. Traditional attempts to mimic the reversible oxygen binding properties of

190

heme containing proteins concentrated on constructing simple Fe(II)porphyrins appended

with large groups26.

There are reports on porphyrin bound hyperbranched polyester, which was obtained

by using just one synthetic step, could mimic hemoglobin. The porphyrin cored hy-

perbranched polyester was therefore synthesized by reacting as excess of the branching

monomer, 3-5-diacetoxy benzoic acid with a small amount of tetrakis (4-acetoxy phenyl)

porphyrin under reversible trans esterification. Fe (II) is also inserted using standard con-

ditions. UV-visible spectrophotometric techniques were used to assess the oxygen binding

potential for porphyrin bound hyperbranched polyester.

In the present work the tetrakis Fe (II) porphyrinato sulphonyl chloride was coupled

with hyperbranched polyglycerol and the UV absorption was measured. It exhibited an

absorption maximum at 475 nm in DMF. Oxygen was bubbled through the solution for

1 minute and the absorption maximum was noted and it was found to be get shifted to

486 nm. Nitrogen is bubbled through the solution for 5 minutes, the UV spectrum was

found to be get restored to 475 nm (figure 5.40). By removing oxygen from the solution

the uncomplexation of the Fe (II) species was observed. The experiment was conducted

with the solutions of varying concentrations and the shift observed was consistent.

Figure 5.40. The UV-visible absorptions of HPGFeTPP system. (a) O2 free

HPGFeTPP system and (b)Fe(II)-O2 complex.

191

The oxygen binding capability of the HPG-TPP system was tested in aqueous solutions

of variable pH values. For this, solutions of HPG-TPP system in three different pH were

prepared. The different pH values of the solutions prepared were 4.2, 7.0, and 8.3. All

the solutions are of same concentrations (2x 10−4 millimoles). Through these solutions

N2 was bubbled for five minutes to remove any dissolved O2 and the UV spectra were

taken. At pH 4.2 the soret band was observed at 453 nm (figure 5.41) and at pH 7 and

pH 8.3 the soret band was observed at 430 nm (figure 5.42).

Figure 5.41. The UV-visible spectrum of HPG-Fe TPP system at pH 4.2.

192

Figure 5.42. The UV-visible spectrum of HPG-FeTPP system at pH 7.0 / 8.3.

Oxygen was bubbled through these solutions for half an hour and UV spectra were

taken. No significant shift was observed at pH 4 (figure 5.44) but at pH 7 and at pH 8.3 a

characteristic shift in the soret band was observed by 5nm and 7 nm respectively (figure

5.45 and 5.46). Then N2 was bubbled for 5 minutes and the UV spectra were taken again.

No change was observed at pH 4 but the soret band came to the original position for

solutions at pH 7 and at pH 8.3, that is at 430 nm.

193

Figure 5.43. The UV-visible spectrum of HPG-FeTPP system after O2 binding at pH

4.0.

Figure 5.44. The UV-visible spectrum of HPG-FeTPP system after O2 binding at pH

7.0.

194

From the results it is evident that oxygen-binding capability of HPG-FeTPP system

is appreciably high only at pH 7 or above. When the pH is below 7 it doesnt bind oxygen.

These results approved the efficacy of porphyrin bound HPG as artificial blood product

to reversibly bind oxygen. We came to the conclusion that the solutions at pH 7 and above

are efficient for binding oxygen than solutions at pH below 7. Since the porphyrin bound

HPG was water-soluble and biocompatible it can be used as safe artificial blood product

for human use.

Figure 5.45. The UV-visible spectrum of HPG-FeTPP system after O2 binding at pH

8.3.

In the present work we have synthesized different polymeric derivatives of tetraphenyl

porphyrin and metalloporphyrins. We used both linear and hyperbranched polymeric

systems. The linear systems used were polyvinyl alcohol (PVA), polyethylene glycol

(PEG) and polyglycerol polyadipate (PG). The hyperbranched polymeric system used

was hyperbranched polyglycerol (HPG). All these polymeric systems are water soluble,

nature friendly and having free hydroxyl functional groups in common which can be used

to bound with porphyrin by condensation reaction.

195

Porphyrin on binding with linear polymers like PVA, PEG and polyglycerol polyadi-

pate, exhibited blue shift in the absorption spectra. This is due to the structural per-

turbations caused by the entangled linear polymer core on the porphyrin frame work.

The planarity of the porphyrin macrocycle is highly sensitive to structural changes. On

attaching to linear polymers, the steric effect causes a great degree of perturbation of the

planarity of the porphyrin macrocycle and the p electron framework is seriously affected

by this change. This causes a notable blue shift in the electronic spectral signals. The

TPP has aggregation tendency and which when attached to the linear system like PVA,

PEG and PG, this tendency may be enhanced due to the entanglements and that results

in spectral shifts and line broadening. On attaching to hyperbranched polyglycerol no-

table red shift was observed in the absorption spectrum of porphyrin due to the variations

in the electronic charge delocalization within the porphyrin macrocycle assisted by highly

branched and heavily functionalized HPG core system. Metallation of TPP caused further

red shift in the absorption spectra.

The emission studies of metalloporphyrins bound with linear and HPG polymers re-

vealed several significant and established facts about metallated porphyrins. We noticed

an increased fluorescence yield for FeTPP, ZnTPP and CuTPP bound to linear and hyper-

branched systems. This is due to increased rigidity of TPP structure on metal insertion,

which is expected to decrease the intersystem crossing yields.

Light fastening studies conducted on the various polymer systems proved that the sta-

bility of the TPP was very much increased when anchored to a polymer system. To study

the action of light on TPP and polymer bound TPP we have irradiated the equimolar

solutions of TPP and polymer bound TPP systems under visible radiant energies and

measured the absorbances of definite time intervals. We have noticed significant changes

in the intensity of absorption for the free TPP as well as the polymer bound TPP. Com-

pared to other photochromic systems HPG-TPP is very fast towards light, and it also

exhibits shifts in the intensity of absorption. The red shift and stability towards conti-

nous light exposure make HPG-TPP and its metallo derivatives excellent photoresponsive

systems for photoprobing in medicine and medical diagnosis and in various industrial and

laboratory applications.

196

TPP and MTPP are insoluble in water but when modified with HPG it became sol-

uble. HPG is non toxic and biocompatible and hence the system can find applications

as photosensitizer in photodynamic therapy. A good sensitizer should be able to absorb

light from longer wavelength and should be water soluble. We have studied the UV-visible

absorptions on linear polymer bound porphyrins and compared them with the absorbance

of HPG-TPP system. The HPG-TPP system exhibited significant red shift which is a

primary requirement for an ideal photosensitizer. Since the HPG-TPP system is such an

ambhiphilic molecule it can be used as a photosensitizer.

Photodynamic antimicrobial therapy was done by using this compound and found to be

successful. The antimicrobial studies where carried out against a gram positive bacterium

Stayphylococcus aureus and gram negative bacterium Escherichia coli. The photoinduced

anti microbial activity was studied with TPP, FeTPP, HPG-TPP and HPG-FeTPP. The

gram negative E.coli bacterium was found to be refractory to all the systems but the gram

positive bacterium Stayphylococcus aureus where killed by the photo-oxidation process.

UV-visible spectrometric techniques were used to assess the oxygen binding potential for

porphyrin bound hyperbranched polyester. The oxygen binding capability of HPG-TPP

system was tested in aqueous solutions of variable pH values. From the results it is evident

that oxygen-binding capability of HPG-FeTPP system is appreciable at pH 7 or above.

When the pH is below seven it doesnt bind oxygen. These results approved the efficacy

of porphyrin bound HPG as artificial blood product to reversibly bind oxygen. Since

the porphyrin bound HPG was water soluble and biocompatible it can be used as safe

artificial blood products for human use.

The HPG-TPP system developed in the present work and its metalloderivatives are

excellent systems, which could find many biomedical applications. The very intense ab-

sorptions at longer wavelength region make the system unique in this field. The water

soluble environmental friendly HPG-TPP and its metalloderivatives offer a wide spectrum

of applications in medicine, medical diagnosis, photodynamic therapy and so on.

197

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