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Material Science & Engineering International Journal

Research Article Volume 6 Issue 3

Interaction of protoporphyrinix (PPIX) and 5-amino levulinic acid (ALA) in nanoemulsion

Maurice O Iwunze

Department of Chemistry, Morgan State University, USA

Correspondence: Maurice O Iwunze, Department of Chemistry, Morgan State University, Baltimore, Maryland 21251, USA, Tel +1 443- 885-3634, Fax +1 443- 855- 8286

Received: September 19, 2022 | Published: September 23, 2022

Citation: Iwunze MO. Interaction of protoporphyrinix (PPIX) and 5-amino levulinic acid (ALA) in nanoemulsion. Material Sci & Eng. 2022;6(3):118-121 DOI: 10.15406/mseij.2022.06.00188

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Abstract

Nanoemulsion is an interesting and unique fluid system in that it is used to solubilize both ionic and non-ionic molecules.  Because of its uniqueness it has been used as a medium for drug delivery.  It is therefore used in this work to study the interaction of Protoporphyrin (PPIX) and 5-Amino levulinic acid (ALA).  While ALA is a distant precursor of PPIX, PPIX itself is not only a precursor of Heme but also a photosensitizer in the modality of Photodynamic Therapy (PDT). Both compounds are used use in PDT regimen.  A steady-state fluorescence technique is used for the study of the interaction of these very important biological compounds.  It is found that ALA quenches the fluorescence of PP IX in nanoemulsion.  This observed quenching is diffusion controlled. The bimolecular quenching constant, kq, was determined as 2.86x 1010/M-s with an interaction constant, Ka, of 4.48 x 105 with the free energy of interaction, ΔGa of -32.234kJ/mole.

Keywords: interaction, nanoemulsion, protoporphyrin ix, 5-aminolevulinic acid, fluorescence

Introduction

Nanoemulsion is a heterogeneous fluid system that is extensively used for drug delivery and solubilization of poorly soluble compounds.1-6 It is made by dispersing oil in water using surface active agent (Surfactant) and a co-surfactant, usually a short chain alcohol, in appropriate ratios. PPIX and AlA are very important in biological processes including photodynamic therapy (PDT)1-7 While ALA is a distant precursor of PP IX, PP IX itself, is a precursor of the Heme (oxygen carrier) found in mammals and plants.  The scheme of the formation of any given Heme may be represented thus:

ALA →→→  PP IX →→→ HEM

The full and complete scheme of biosynthetic route of the ALA to Heme production is given in reference.8  PPIX is poorly soluble in water9-17 but it is freely soluble in Nanoemulsion, hence its use as a medium for studying its interaction with ALA using steady-state Fluorescence spectroscopy. Figure 1 shows the chemical structure of PP IX, ALA and the SEM image of Nanoemulsion.

Figure 1 SEM Image of Nanoemulsion.

Experimental

Chemicals

All the chemicals used (tetradecane, surfactant (CTAB), 1-pentanol) were of analytical reagent grade obtained from Acros Chemicals and used as received without further purification.

Instrument

The Fluorescence spectra were obtained using Perkin Elmer’s Luminescence Spectrophotometer, model LS 50 B

All solution was prepared using triply distilled deionized water from Photronix Reagent Grade water system.

Preparation of Nanoemulsion

The chemical composition of the Nanoemulsion used is given in Table 1.

Component      

Wt., g  

   Percentage, %

   Volume, mL   

Water

174

76

174

CTAB (Surfactant)

12

5

12.63

Oil (Tetradecane)  

14

6

18.25

 Co-Surfactant (1-pentanol)               

29.9

13

31.8

Table 1 The chemical composition of the Nanoemulsion

Literature methodology14 was followed in the preparation of the Nanoemulsion used in this work.  Briefly, a measured weight of 12.0 g of CTAB as added to 174 mL of water and the mixture formed a slurry.  This slurry was mechanically stirred for about two or three minutes after which 18.25 mL of n-tetradecane were added to the slurry drop wise while the mixture is still being stirred. Thereafter 31.80 mL of n-pentanol were added, again dropwise. The stirring continued until the mixture became clear and translucent.  The translucent solution was transferred to an ultrasonic sonicator where it was sonicated for about 10 minutes.  The Nanoemulsion so prepared was found to be isotonic, clear, and translucent and was found to be stable for a considerable length of time.

Methodology

Stock solutions of 2.808 x 10-4 M PPIXS and 3.408 x 10-3 M ALA were prepared in Nanoemulsion solution.  0.40 mL aliquot of the PPIX stock solution were added to 10 5mL volumetric flasks.  A measured volume of 2.0 mL of Nanoemulsion solution were added to each volumetric flask.  The first flask contained no ALA and was diluted to the fiduciary mark with Nanoemulsion solution.  Thereafter, aliquot volumes of the ALA sock solution were added to the rest of the flasks and the resulting mixture were carefully diluted the fiduciary mark with Nanoemulsion solution.  The final concentrations were 2.246 x 10-4 M PPIX, and the concentration of ALA varied from 6.96 x 10-5 M to 6.96 x 10-4 M.

The fluorescence measurements were made by adding 3.0 mL of each solution to a 3.5-mL of a four-clear sided cuvette.  The excitation was at 350 nm and the emission was observed at 695 nm.  The instrument slit widths (excitation and emission) were kept constant at 5 nm.

Results and discussion

We show in Figure 2 the fluorescence spectra of PP IX without and with the quencher, Q, (5-Aminolevulinic acid

-5-

As can be seen, the fluorescence intensity of PP IX is decreased as the concentration of ALA increased

The Stern-Volmer equation (equation 1) is used to analyze the data obtained in Figure 2.

Figure 2 The fluorescence spectra of PPIX without and with different concentrations of ALA.

I o I =1+ K sv Q=1+ k q τQ MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqkY=Mj0xXdbba91rFfpec8Eeeu0xXdbba9frFj0=OqFf ea0dXdd9vqaq=JfrVkFHe9pgea0dXdar=Jb9hs0dXdbPYxe9vr0=vr 0=vqpWqaaeaabiGaciaacaqabeaadaqaaqaaaOqaamaalaaabaGaam ysamaaCaaaleqabaGaam4BaaaaaOqaaiaadMeaaaGaeyypa0JaaGym aiabgUcaRiaadUeadaWgaaWcbaGaam4CaiaadAhaaeqaaOGaamyuai abg2da9iaaigdacqGHRaWkcaWGRbWaaSbaaSqaaiaadghaaeqaaOGa eqiXdqNaamyuaaaa@47B1@   ;(1)

KSV = kq/τ MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqkY=Mj0xXdbba91rFfpec8Eeeu0xXdbba9frFj0=OqFf ea0dXdd9vqaq=JfrVkFHe9pgea0dXdar=Jb9hs0dXdbPYxe9vr0=vr 0=vqpWqaaeaabiGaciaacaqabeaadaqaaqaaaOqaaabaaaaaaaaape Gaam4saiaadofacaWGwbGaaeiiaiabg2da9iaabccacaWGRbGaamyC aiaac+cacqaHepaDaaa@405B@   (2)

In this equation Io and I represent the fluorescence intensity of PP IX without and with quencher, respectively, and kq is the bimolecular quenching rate constant. KSV is the Stern-Volmer quenching constant. τ and Q are, respectively, the fluorescence lifetime of PPIX and the concentration of ALA used as the quencher, Q.  

A plot of versus Q gives a straight line as can be seen in Figure 3.

Figure 3 The KSV Plot of the PPIX-ALA Interaction.

From the slope of this plot, KSV was determined to be 314.44 and with the aid of the literature value of τ (15-18), kq was determined. A value of 2.86 x 1010/M-s was obtained.  This value is consistent with a theoretically determined diffusion-controlled bimolecular reactions.19

In other to determine the interaction constant and the ratio, n, of the molecules reacting, the equation due to Bai and his co-workers20 shown in equation 3 was used.

"log("  (I^o-I)/I)="log"(K)+"nlog"([Q])  

A linear plot was obtained as can be seen from Figure 4.

Figure 4 Plot of log(Io-I/I) versus log(Quencher).

Analysis using this equation gave Ka, the interaction constant of 4.48 x 105/mole.  This value is approximately consistent with what was observed by other workers and the n is a 1:1 ratio.21 The free energy, ΔG, of interaction was obtained using the relation of equation 3.

ΔG =  RTlnK      MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqkY=Mj0xXdbba91rFfpec8Eeeu0xXdbba9frFj0=OqFf ea0dXdd9vqaq=JfrVkFHe9pgea0dXdar=Jb9hs0dXdbPYxe9vr0=vr 0=vqpWqaaeaabiGaciaacaqabeaadaqaaqaaaOqaaabaaaaaaaaape GaeuiLdqKaam4raiaabccacqGH9aqpcaqGGaGaeyOeI0Iaaeiiaiaa dkfacaWGubGaamiBaiaad6gacaWGlbGaaiiOaiaacckacaGGGcGaai iOaiaacckaaaa@4754@   (3)

A free energy of the interaction was calculated as -32.24 kJ/mole indicating that the reaction is favorable and spontaneous as expected of all exergonic reactions we give in Table 2 the observed parameters of the reaction of PP IX and 5-aminolevulinic acid in Nanoemulsion.

Parameter

Value

Unit(s)

Stern-Volmer Quenching constant (KSV)

314.44

M-1

Bimolecular Quenching Constant (kq)

2.86 x 1010

M-1s-1

Interaction Constant (Ka)

4.48 x 105

mol-1

Free Energy of Interaction (ΔGa)

-32.24

kJ/mol

Interaction Ratio (n)

1:01

-

Table 2 The Observed/Calculated parameters of the reaction Between PP IX and ALA in nano emulsion

Conclusion

We have shown in this work that PPIX which is poorly soluble in water is freely soluble in Nanoemulsion.  Steady-state fluorescence spectroscopy is used in this medium to study and to obtain relevant data regarding the interaction between two important biomolecules in PDT regimen, PPIX and ALA.  It is further observed that the interaction is diffusion-controlled and quite exergonic.  Consistent with the finds of other workers, the binding of ALA to PPIX is observed to be in the ration of 1:1 with a binding interaction constant, Ka, of 4.48 x 105/mol and free energy is interaction, Ga, of -32.24 kJ/mol

Acknowledgments

The author is very grateful to the Chemistry Department of Morgan State University for her support of this work and to Prof. Michael McCaffery who prepared the SEM image of the Nanoemulsion.

Conflicts of interest

The author hereby declares of having not conflict of interest in this article.

Funding

None.

References

  1. Sonnville-Aubrun O, Simonnet JT, Alloret FL. Nanoemulsion: a new vehicle for skincare products. Adv Colloid and Interface Sci. 2004;108-109:145–149.
  2. Manjit Jaiswal, Rupesh Dudhe, Sharma PK. Nanoemulsion: an advanced mode of drug delivery system 3 Biotech. 2015;5(2):123–127.
  3. Devarajan V, Ravichandran V. Nanoemulsions: As Modified Drug Delivery Tool.  Pharmacie Globale (IJCP). 2011;2(4):1–6.
  4. Zhang Y, Shang S, Gao C, et al. Nanoemulsion for solubilization, stabilization, and in vitro release of pterostilbene for oral delivery. AAPS PharmSciTech. 2014;15(4):1000–1008.
  5. Lovelyn C, Attama AV. Current state of nanoemulsion in drug delivery. Journal of Biomaterials and Nanobiotechnology. 2011;2(5):626–639.
  6. Khushwinder K. Nanoemulsion as an effective medium for encapsulation and stabilization of cholesterol/ꞵ-Cyclodextrin inclusion complex. J Sci of Food Agric. 2014;95(4):2718–2728.
  7. Mahmoudi K, Garvey KL, Bouras A, et al. 5-aminolevulinic acid photodynamic therapy for the treatment of high-grade gliomas. J. Neurooncol. 2019;141(3):595–607.
  8. Valdes PA, Milesi M, Widhalm G, et al. 5-Aminolevulinic acid induced protoporphyrin IX (ALA-PpIX) fluorescence guidance in meningioma surgery. J Neuroocol. 2019;141(3):555–565.
  9. Protoporphyrin IX. 2022;1–5.
  10. Desganges S, Juzenas P, Vasovic V, et al. Amphiphilic protoporphyrin ix derivatives as new photosensitizing agents for the improvement of photodynmic therapy. Biomedicines. 2022;10(2):1–23.
  11. Sachar M, Anderson KE, Ma X. Protoporphyrin IX: the Good, the bad, and the Ugly. J Pharmacol Exp Ther. 2016;356(2):267–275.
  12. Protoporphyrin IX. NIH. 2022.
  13. Maurice O Iwunze. Electrooxidation of ferrocene in nano-emulsion. Journal of Chem. Chem Eng. 2020;14, 37–40.
  14. Myrzakhmetov B, Arnoux P, Mordon S, et al. Photophysical properties of protoporphyrin ix, pyropheophorbide-a and photofrin in different conditions. Pharmaceuticals.2021;14(2):138.
  15. Postnikova GB, Yumakova EM. Fluorescence study of the conformational properties of myoglobin structure 3.  ph-dependent changes in porphy and tryptophan fluorescence of the complex of sperm whale apo myoglobin with protoporphyrin ix; the role of the porphyrin macrocycle and iron in formation of native myoglobin structure. Eur J Biochem. 1991;198:241–246.
  16. Sicchieri LB, Da Silva MN, Samad RE, et al. Can measurement of the fluorescence lifetime of extracted blood PPIX predict atherosclerosis?  Journal of Luminescence. 2018;195:176–180.
  17. Hungerford G, Cumberbatch N, Holland A, et al. Rapid (Flash-Flim) imaging of protoporphyrin IX I a real time using a CMOS based widefield fluorescence lifetime imaging camera. Mmc 2021 Congress.  2021;5–9.
  18. Erkkila MT, Reichert D, Gesperger J, et al. Macroscopic fluorescence-lifetime imaging of nadh and protoporphyrin ix improves the detection and grading of 5-aminolevulinic acid-stained brain tumors. Scientific Reports. 2020.
  19. Lewkowicz JR.  Principles of fluorescence spectroscopy. Springer. 1983;284.
  20. Feng XZ, Lin Z, Yang LJ, et al. Investigation of the interaction between acridine orange and bovine serum albumin. Talanta. 1998;47(5):1223–1229.
  21. Sulkowskiu L, Pawelczak B, Chudzik M, et al. Characteristics of the protopophyrin ix binding on human serum albumin using molecular docking. Molecules. 2016;21(11):1519.
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