radiological: pre-clinical comparative study of...

268Current Trends in Biotechnology and PharmacyVol. 11 (3) 268-277 July 2017, ISSN 0973-8916 (Print), 2230-7303 (Online)

AbstractPresent study was to do comparative study

of microbial produced surfactant with FDAapproved drug survanta by using Near IR and FarIR spectroscopy this is useful for pre clinical testfor RDS treatment. More recently IR spectroscopycomes as novel diagnostic tests to assess fetallung maturity. Two spectral diagnostic windowsfor deep tissue imaging in Near IR spectrum (300-1100nm) and for Far IR spectrum (1200nm-2200nm) were used for comparable study of drugsurvanta with microbial produced Surfactin andLipopeptide (Biosurfactant) from B.subtilisMTCC2423 and C.tropicalis MTCC230respectively. Near IR spectrum (300-1100nm)shows the absorbance peak for surfactin andlipopeptide (Biosurfactant) was fall in samewavelength as survanta shows i.e, 396nm and967nm. Far IR spectrum (1200nm-2200nm) showsthat the least absorbance frequency was obtainedat 1249nm for all the three surfactants. Surfactinshows minimum absorbance unit in highestdilution then followed by lipopeptide (Biosurfactant)supernatant then survanta i.e., 0.17, 0.24 and0.42AU respectively. Surfactin and Lipopeptide(Biosurfactant) produced from B.subtil isMTCC2423 and C.tropicalis MTCC230respectively, can be considered for RDS treatmentas it clear the primarily study of radiologicalproperty of drug by comparable study with FDAapproved drug survanta.

Keywards: Respiratory distress syndrome,Surfactin, Lipopeptide, Near IR, Far IR.

IntroductionLungs are lined by a surface active material

called the pulmonary surfactant which preventsalveolar collapse and maintains alveolar stability.Pulmonary surfactant (PS) is a complex mixtureof lipids and proteins that coats the interior surfaceof the vertebrate lung as a film. It consists of about90% lipids (mainly phospholipids) and 8–10%protein. The main component of native Lungsurfactant (LS) is dipalmitoyl-phosphatidylcholine(DPPC) an amphiphilic protein that were recentlyshown to play an important role in the surfaceactivity of LS (1,2). Lack of deficiency orinactivation of PS causes severe respiratorydisorders that can be lethal such as breathingproblems in newborn babies, pulmonaryhaemorrhage, pulmonary oedema, andPneumonia, respiratory distress syndrome (RDS)-a most common causes of death in prematureinfants (3,4).

However, in general probability for RDS ismore prominent in premature infant because lungsare under developed, so the lung surfactantproduction is less. In Most cases of RDS babieswere born before 37 weeks. If a premature babyis lacking surfactant, artificial surfactant may begiven for normal functioning (5-7).

Amount of surfactant present in fetal lungscan be estimated by measuring the amount ofsurfactants in amniotic fluid (8). Treatment ofneonates immediately after birth with exogenoussurfactant delivered endotracheally can be

Radiological: Pre-clinical Comparative study of Microbialderived Surfactants with Survanta for Treatment of

Respiratory Distress Syndrome (RDS)Ashish1, Jitendra Singh Parihar2, Neeraj Sharma3 and Mira Debnath (Das)4*

1, 4 School of Biochemical Engineering, 2, 3School of Biomedical EngineeringIndian Institute of Technology (B.H.U), Varanasi, India-221005

*For Correspondence - [email protected]

Comparative study of Microbial derived biosurfactants


effective if early delivery cannot be prevented (9).In the medical research, imaging and diagnosisvia a infra-red (IR) imaging has been increaseddue penetration in deep tissue imaging (10).Diagnosis of RDS noninvasively, primary test ischest radiography which is used as one of themost usual and accessible diagnostic tools inrespiratory distress syndrome, which has shownpromising results regarding the early diagnosis ofrespiratory distress (11). IR spectroscopy is oneof a standard clinical analyses test of amnioticfluid assays to assess fetal lung maturity (12).

In a clinical study and radiographic diagnosismatched upto 95% of cases (13)], In another studythe specificity and sensitivity of the radiographictest was 82.5% and 35.8% respectively (14) andin the same study author concluded thatdiagnostic value of RDS based on radiologistopinion were about one-third of all cases and haslow sensitivity. Although we have many options/methods to diagnose RDS but still there is not asingle non invasive method which can be 100%reliable.

In 1990, the United States Food and DrugAdministration (FDA) released colfoscerilpalmitate for use in preterm infants with RDS.Shortly thereafter, other types of surfactantsbecame available: Exosurf® (synthetic surfactant)and Survanta® (modified natural surfactant). Alsodomestically available are Curosurf® (naturalsurfactant) and Infrasurf® (natural surfactant) (15).Animal derived surfactants are mostly used asdrugs for the treatment of respiratory distresssyndrome (RDS) in neonates. They also haveseveral disadvantages such as they carry thepotential risk of transmission of infectious agentas well as exposure to foreign proteins, pro-inflammatory mediators and variability in a nativesurfactant proteins from batch to batch (16). Inthis study we have taken survanta as standard forthe comparative analysis because this is mostcommon one, have highest DPPC compositionand cost-effective.

The objective of present study was to docomparative study of microbial produced

surfactant with FDA approved drug survanta byusing Near IR and Far IR spectroscopy this isuseful for pre clinical test for RDS treatment. Limet al. (17) identified two spectral diagnosticwindows for deep tissue imaging in Near IR regionof the spectrum (700–900 nm) and for Far IRspectrum 1,200– 1,600-nm, these two IR spectrumregion used as “diagnostic window” of tissueimaging, where the absorption coefficients of bodyfluids are at their minimum (12).

In this study we analysed the Near IR andFar IR spectrums of three different surfactants(Survanta, surfactin, lipopeptide). Spectrums wereobtained from different concentrations of surfactantwere compared and analyzed. The results showedthat there is a specific set of frequencies for allthree surfactants and these set of frequencies canbe used to detect the presence of surfactant.Survanta (FDA approved drug used for RDStreatment) was used as a standard for thecomparative radiological study of surfactinproduced from B.subtilis MTCC 2423 andlipopeptide (Biosurfactant) produced fromC.tropicalis MTCC230. This study reveals thatmicrobial surfactant may be considered fortreatment of RDS in an infant.

Materials and MethodsWe analysed the spectrum of Survanta

(manufactured by: M/s AbbVie Inc. 1401, SheridanRoad, North Chicago, II, 60064, USA) , Surfactin(B.subtilis MTCC 2423) and Lipopeptide(C.tropicalis MTCC230) were produced in ProteinEngineering Laboratory, School of BiochemicalEngineering, IIT (BHU), Varanasi (18,19).A spectroscopic measurement was performed byusing Aventes Spectrometer system whichconsists of:1. Light source- AvaLight-HAL Tungsten

Halogen Light Source, The AvaLight-HAL isa compact stabilized halogen fan-cooled lightsource that can be used for the visible andthe near infrared range. Its wavelength rangeis 360-2000 nm.

2. Detectors: AvaSpec-2048 Fiber OpticSpectrometer, it is a 2048 pixel CCD

Ashish et al


Detector Array. Usable wavelength range is300-1100 nm. AvaSpec-NIR256-2.2TEC NIRLine Near-Infrared Fibre-optic Spectrometer,its wavelength range is 1000-2200 nm.

3. Cuvette sample holder4. Fiber optic cables5. Cuvette6. Micropipette7. Avasoft 8(software installed in a personal

computer) was used to process data receivedfrom above detectors.

Sample preparation: Samples of differentconcentration of each surfactant (survanta,surfactin and lipopeptide) were prepared. Survantawas obtained from pharma market of varanasi,india (manufactured by: M/s AbbVie Inc. 1401,Sheridan Road, North Chicago, II, 60064, USA),Surfactin was produced from B.subtilis MTCC2423 and lipopeptide (Biosurfactant) was producedfrom an adaptive strain of C.tropicalis MTCC230(18,19). There supernatants were used as a puresample for Near IR and Far IR spectrum studies.

Comparative Study: Radiological Comparativestudies of survanta, surfactin and lipopeptide(Biosurfactant) were performed by using Near IRspectrum of wavelength range is 300-1100 nm.Survanta act as a standard for the comparativestudy, further comparative studies were performedby using Far IR spectrum of wavelength range is1000-2200 nm.

Concentration Variation: Radiological study ofsurvanta, surfactin and lipopeptide (Biosurfactant)was performed at different concentrationmaintaining the final volume 2ml via a differentdilution rate with distilled water.

Fermented broth of Supernatant containingsurfactin and lipopeptide (Biosurfactant) used aspure sample then gradually increases the dilutionwith distilled water at dilution rate i.e., 50%,62.5%, 75%, 87.5% respectively. Pure survantais highly viscous so initial dilution rate was startedfrom 0.20ml survanta + 1.80ml distilled water, thengradually increases the dilution rate as same incase of surfactin and lipopeptide. All samples were

measured by using VIS-NIR spectrum ofwavelength range is 300-1100 nm and Far IRspectrum of wavelength range is 1000-2200 nm.

ResultsComparative Study: Comparative Studies

of surfactin and lipopeptide (Biosurfactant) withthe survanta were performed by using Near IRspectrum of wavelength range is 300-1100 nm asshown in Figure 1, result shows that theabsorbance peak frequencies for surfactin,lipopeptide (Biosurfactant) and survanta were396nm and 967nm, So the absorbance peak forsurfactin and lipopeptide (Biosurfactant) was fallin same wavelength as survanta shows,Absorbance units (AU) were different for eachsurfactant due to concentration difference.Similarly for Far IR spectrum, wavelength range1200nm-2200nm used for comparative study ofsurfactin and lipopeptide (Biosurfactant) with thesurvanta, Figure 2 shows that the least absorbancefrequency was obtained at 1249nm for all the threesurfactants. These results reveal that microbialproduced surfactin from B.subtilis MTCC2423 andlipopeptide (Biosurfactant) from an adaptive strainof C.tropicalis MTCC230 showing the sameSpectroscopic properties for Near IR and Far IRas shown by drug survanta. This result is verymuch helpful to pass preclinical trials for thedevelopment of drug used in RDS (respiratorydistress syndrome). Surfactin and lipopeptide(Biosurfactant) may considered as drug for thetreatment of respiratory distress syndrome (RDS)in premature infants, as it clears the primarilypreclinical test i.e., radiological study.

Further surfactin and lipopeptide(Biosurfactant) comparative studies with survantawere done on account of varying theconcentrations, absorbance peak of eachsurfactant shows the concentration and presenceof that surfactant so it’s important to measure theabsorbance peak by varying the concentrations.

Concentration Variation study: Results ofComparative study reveal that Surfactin andLipopeptide (Biosurfactant) showing the sameabsorbance spectrum of Near IR and Far IR as



Ashish et al


shown by survanta then their concentrationvariation studies were performed. Lipopeptide(Biosurfactant) produced from an adaptive strainC.tropicalis, was serially adapted from low to highconcentrations of hydrocarbons (petroleum waste)(18,19). Supernatant of fermented broth ofB.subtilis MTCC 2423 and C.tropicalis MTCC 230containing surfactin and Lipopeptide(Biosurfactant) respectively, These Supernatantswere used for analysing Near IR spectrometry andFar IR spectrometry with different dilutions.

Initially 2ml of supernatant was used for NearIR spectrum (range 300nm-1100nm), Supernatantcontaining Lipopeptide (Biosurfactant) showingabsorbance 1.35 AU at 396nm (Figure 3) andSupernatant containing Surfactin showabsorbance 0.59 AU at 369nm as (Figure 4),Absorbance Unit (AU) of 2ml supernatantcontaining the Lipopeptide (Biosurfactant) isnearly equal to pure drug survanta when dilutedas 0.20ml of survanta + 1.80ml of distilled waterat 369nm as shown in Figure 5.

To estimate the lowest absorbance unit foranalysing the sensitivity and efficacy of Near IRand Far IR spectrum for microbial surfactants,dilution was increases gradually for lipopeptide(Biosurfactant) and surfactin containingsupernatant, and also for survanta with the samedilution rate respectively.

Surfactin shows minimum absorbance unitin highest dilution then followed by lipopeptide(Biosurfactant) supernatant then survanta i.e.,0.17, 0.24 and 0.42AU respectively. Further FarIR spectrometry studies were also performed foreach surfactants (Lipopeptide, surfactin andsurvanta) for different dilution rate as similar incase of Near IR, all the surfactants shows leastabsorbance at 1250nm for Far infrared spectrumrange 1200nm-2200nm, this absorbance isshowing very small change with change inconcentration of Lipopeptide, surfactin andsurvanta as shown in Figure 6, 7, and 8respectively.

DiscussionNear IR and Far IR spectrum are the two

diagnostic windows considering how light interactswith living tissues, penetration of these IR intothe living tissues can helps to diagnose thatwhether the lung contains normal or sufficientsurfactants (20), this is one of most useful methodfor the determination of lung surfactant in neonates,as it can meet the standards of accuracy that arerequired for a specialized test such as amnioticfluid assays to assess fetal lung maturity. IRspectroscopic analysis having number of practicaladvantages like, No reagents are required, thereis generally no need to dilute very concentratedspecimens, very little sample is required (12).

Comparative Studies of surfactin andlipopeptide (Biosurfactant) with the survanta revealthat microbial produced surfactin from B.subtilisMTCC2423 and lipopeptide (Biosurfactant) froman adaptive strain of C.tropicalis MTCC230showing the same Spectroscopic properties forNear IR and Far IR as shown by drug survanta.This result may consider as to cleared preclinicaltrials for the development of drug used in RDS(respiratory distress syndrome). So, Surfactin andlipopeptide (Biosurfactant) produced fromB.subtilis MTCC 2423 and an adaptive strain ofC.tropicalis MTCC 230 respectively, mayconsidered as drug for the treatment of respiratorydistress syndrome (RDS) in premature infants,as it clears the primarily preclinical test i.e.,radiological study.

Further, surfactin and lipopeptide(Biosurfactant) comparative studies with survantawere done on account of varying theconcentrations, absorbance peak of eachsurfactant shows the concentration and presenceof that surfactant so it’s important to measure theabsorbance peak by varying the concentrations.This study was helpful to establish the sensitivityof NIR and Far IR absorbance spectrum with thesesurfactants, Different dilution rate shows differentabsorbance unit, as dilution rate increasesabsorbance unit decreases respectively.



Ashish et al


Results signifies that adaptive strainC.tropicalis MTCC 230 producing high amount oflipopeptide (Biosurfactant) then known strain ofB. subtilis MTCC 2423 producing surfactin andthe absorbance unit (AU) of Lipopeptide(Biosurfactant) is nearly equal to pure drugsurvanta at 369nm as shown in Fig. 5.

Absorbance of IR at particular wavelengthshows the presence and concentration ofsurfactant inside the lungs, microbial synthesizedsurfactants i.e., surfactin and lipopeptide(Biosurfactant) showing the absorbance at 396nmand 960nm for Near IR and least absorbance isshown at 1250nm for Far IR spectrum is as sameas survanta.

ConclusionThis study reveals that microbial surfactants

can be considered as drug for the treatment ofrespiratory distress syndrome (RDS) in neonates,as these surfactants are eco friendly and nonhazardous for body many of microbial synthesizedsurfactants are used for many diseases as a drug(21).

Surfactin and Lipopeptide (Biosurfactant)produced from B.subtilis MTCC2423 andC.tropicalis MTCC230 respectively, can beconsidered for RDS treatment as it clear theprimarily study of radiological property of drug bycomparable study with FDA approved drugsurvanta, showing the same IR pre clinicalspectrometric results. Animal derived surfactantsand chemically synthesized surfactants used forRDS treatment having some limitations like limitedscale of production when animally derived and mayhazardous when chemically synthesized overthese limitations microbial synthesisedsurfactants having some advantages, it can beproduced in large scale and found to be non-hazardous for human body. This work explore thepotential use of microbial synthesized lipopeptidefor the treatment of RDS as it passes the preclinical trial of radiological study and so thisinformation and data open the path for clinical andinvivo study of microbial derived surfactant for RDStreatment in neonates.

AcknowledgementsThe authors received the financing for theimplementation of this work: Indian Institute ofTechnology (BHU), Varanasi supported by Ministryof Human Research and Development (MHRD),India.

References1. Gerber, F., Krafft, M. P., Vandamme, T. F.,

Goldmann, M. and Fontaine, P. (2005).Preventing Crystallization of Phospholipidsin Monolayers: A New Approach to Lung-Surfactant Therapy. Angew. Chem. Int. Ed.,44:2749–2752.

2. Nguyen P. N., Waton, G., Vandamme, T.and Krafft, M. P. (2013). Behavior of anAdsorbed Phospholipid MonolayerSubmitted to Prolonged Periodical SurfaceDensity Variations. Angew. Chem. Int. Ed.,52:6404–6408.

3. Rubenfeld, G. D. et al. (2005). Incidence andoutcomes of acute lung injury. N. Engl. J.Med., 353(16):1685-93.

4. Liggins, G. C. and Howie, R. N. (1972). Acontrolled trial of antepartum glucocorticoidtreatment for prevention of the respiratorydistress syndrome in premature infants.Pediatrics., 50:515–25.

5. Kattwinkel, J., Bloom, B. T., Delmore, P.,Davis, C. L., Farrell, E., Friss, H., et al.(1993). Prophylactic administration of calflung surfactant extract is more effective thanearly treatment of respiratory distresssyndrome in neonates of 29 through 32weeks’ gestation. Pediatrics., 92(1):90-98.

6. Dunn, M. S., Shennan, A. T., Zayack, D.and Possmayer, F. (1991). Bovine surfactantreplacement therapy in neonates of lessthan 30 weeks’ gestation: a randomizedcontrolled trial of prophylaxis versustreatment. Pediatrics., 87(3):377-386.

7. Merritt, T. A., Hallman, M., Bloom, B. T.,Berry, C., Benirschke, K., Sahn, D., et al.



(1986). Prophylactic treatment of verypremature infants with human surfactant. N.Engl. J. Med., 315(13):785-790.

8. Haymond, S., Luzzi, V. I., Parvin, C. A. andGronowski, A. M. (2006). A DirectComparison between Lamellar Body Countsand Fluorescent Polarization Methods forPredicting Respiratory Distress Syndrome.Am. J. Clin. Pathol., 126:894-899.

9. Kendig, J. W., Notter, R. H., Cox, C.,Reubens, L. J., Davis, J. M., Maniscalco,W. M., et al. (1991). A comparison ofsurfactant as immediate prophylaxis and asrescue therapy in newborns of less than 30weeks’ gestation. N. Engl. J. Med., 324:865-71.

10. Aswathy, R. G., Yoshida, Y., Maekawa, T.and Kumar, D. S. (2010). Near-infraredquantum dots for deep tissue imaging. Anal.Bioanal. Chem., 397:1417–1435.

11. Armstrang, P., Wastie, M. and Rockall, A.(2004). Diagnosting imaging. 4th ed. UnitedState of America: Black well, 17-99.

12. Shaw, R. A. and Mantsch, H. H. (2008).Infrared Spectroscopy of Biological Fluidsin Clinical and Diagnostic Analysis.Biomedical Spectroscopy DOI:10.1002/9780470027318.a0106.

13. Kurl, S., Heinonen, K. M. and Kiekara, O.(1997). The First chest Radiography inneonates exhibiting respiratory distress atbirth. Clin. Pediatr. (phila)., 36(5):285-90.

14. Shahri, H. M. M., Naghibi, S., Mahdavi, E.and Khademi, G. (2014). Diagnostic Utilityof Chest X-rays in Neonatal Respiratory

Distress: Determining the Sensitivity andSpecificity. Int. J. Pediatr., 2(4.2):65-72.

15. Lawson, S. J. (2004). SurfactantAdministration in the Neonate. RcEducational Consulting Services, Inc.

16. Logan, J.W. and Moya, F. R. (2009). Animal-derived surfactants for the treatment andprevention of neonatal respiratory distresssyndrome: summary of clinical trials. Ther.Clin. Risk. Manag., 5: 251-260.

17. Lim, Y. T., Kim, S.; Nakayama, A., Stott, N.E., Bawendi, M. G. and Frangioni, V. J.(2003). Selection of Quantum DotWavelengths for Biomedical Assays andImaging. Molecular Imaging, 2:50-64.

18. Ashish., Neeraj, G. and Debnath. M. (2011).Characterization of biosurfactant productionby mutant strain of Candida tropicalis. Int.Proc. Chem. Biol. Environ., 6:133–136.

19. Verma, A., Gupta, N., Verma, S. K. andDas, M. D. (2015). Multifactorial Approachto Biosurfactant Production by AdaptiveStrain Candida tropicalis MTCC 230 in thePresence of Hydrocarbons. J. Surfact.Deterg.,18(1):145-153.

20. Brian, W., Leblond, P. F., Krishnaswamy,V. and Paulsen, K. D. (2010). Radiologicand Near-Infrared/Optical SpectroscopicImaging: Where Is the Synergy?. AJR. Am.J. Roentgenol., 195(2):321–332.

21. Gudina, E. J., Rangarajan, V., Sen, R. andRodrigues, L. R. (2013). Potentialtherapeutic applications of biosurfactants.Trends Pharmacol. Sci., 34(12):667–675.

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